CN101472940A - Droplet-based biochemistry - Google Patents

Abstract

A method of inoculating a culture medium including providing a droplet including a single cell type on a droplet actuator and inoculating a culture medium with the droplet. A method of providing a metabolically useful substance to a cell culture, including providing a droplet actuator including a cell culture droplet loaded thereon, the sample droplet including cells and a cell culture medium, and a second droplet comprising a metabolically useful substance. The method also includes conducting one or more droplet operations to combine the cell culture droplet with the second droplet on the droplet actuator. Related methods, droplet actuators, and systems are also provided.

Description

Biological chemistry based on droplet

Fund information

Work of the present invention to small part obtains the subsidy of XXX fund YYY number.United States Government enjoys certain rights and interests in the present invention.

Related application

The application relates to following U.S. Provisional Application and incorporates them into this paper by reference: 60/744,780, denomination of invention is ＂ Apparatus and Methods for Droplet-Based Protein Crystallization ＂, and the applying date is on April 13rd, 2006; 60/745,058, denomination of invention is ＂ Filler Fluids forDroplet-Based Microfluidics ＂, and the applying date is on April 18th, 2006; 60/745,049, denomination of invention is ＂ Apparatus and Methods for Droplet-Based Protein Crystallization ＂, and the applying date is on April 18th, 2006; 60/745,039, denomination of invention is ＂ Apparatus and Methods forDroplet-Based Blood Chemistry ＂, and the applying date is on April 18th, 2006; 60/745,073, denomination of invention is ＂ Apparatus and Methods for Droplet-Based PCR ＂, and the applying date is on April 18th, 2006; 60/745,059, denomination of invention is ＂ Apparatus and Methods for Droplet-BasedImmunoassay ＂, and the applying date is on April 18th, 2006; 60/745,914, denomination of invention is ＂ Apparatusand method for Manipulating Droplets with a Predetermined Number of Cells ＂, and the applying date is on April 28th, 2006; 60/745,950, denomination of invention is ＂ Apparatus and Methods ofSample Preparation for a Droplet Microactuator ＂, and the applying date is on April 28th, 2006; 60/746,797, denomination of invention is ＂ Portable Analyzer Using Droplet-Based Microfluidics ＂, and the applying date is on May 9th, 2006; 60/746,801, denomination of invention is ＂ Apparatus and Methods forDroplet-Based Immuno-PCR ＂, and the applying date is on May 9th, 2006; 60/745,054, denomination of invention is ＂ Droplet-Based Multi-Well Plate ＂, and the applying date is on April 18th, 2006; 60/806,400, denomination of invention is ＂ Droplet Microactuator Stamping Platform ＂, and the applying date is on June 30th, 2006; 60/806,412, denomination of invention is ＂ Systems and Methods for DropletMicroactuator Operations ＂, and the applying date is on June 30th, 2006; 60/807,104, denomination of invention is ＂ Method and Apparatus for Droplet-Based Nucleic Acid Amplification ＂, and the applying date is on July 12nd, 2006.

Technical field

The present invention relates to droplet microdrive (droplet microactuator) and utilize described droplet microdrive to use discrete droplets (discrete droplet) to carry out system, the apparatus and method of multiple scheme.The present invention can be used for, and for example, implements to carry out the scheme of biological chemistry and/or biology mensuration.The invention still further relates to the system and method that is used to implement based on the scheme of droplet.

Background technology

The ability of carrying out biological chemistry and other mensuration fast all is vital in a lot of fields.For example, quick and accurate diagnose infections disease is all most important for effective control individual disease and public health problems such as minimizing multidrug resistant pathogenic agent and the acquired infection of community.

The DNA cloning method of present PCR-based has a lot of shortcomings, comprises reagent expense height, labor intensive and is easy to take place crossed contamination.In addition, for cultivation, PCR measures and is difficult to measure simultaneously a plurality of species, virulence factor and drug resistance marker's thing.They are to responsive inadequately usually, and can not carry out quantitatively pathogenic agent with calculating.Therefore, this area need be used for the modifying device of detection of nucleic acids, and these devices can overcome above-mentioned limitation, and can also make this technology miniaturization and automatization, so that can minimize in these mensuration of sample collection field conduct and with training.

It is more and more general that nucleic acid sequencing becomes in multi-field, for example genome sequencing, diagnostics, pharmacogenomics and medical jurisprudence.But, Ang Gui sequencing device has hindered the order-checking field.The cheap high-throughput test macro of exploitation is most important for the popularization of genetics test and relevant many advantages thereof.Therefore, need new technology platform, so that can carry out nucleic acid sequencing fast and reliablely with reasonable price.The present invention provides the cheap sequencing system based on droplet at this.

Immunoassay are widely used in clinical diagnosis, and it only just constitutes in the U.S. and surpasses 3,000,000,000 dollars market.Immunoassay belong in the clinical labororatory conventional use have susceptibility and specific method most.Immunoassay utilize the high-affinity between its corresponding antibody of antigen and specific combination detects and the quantitative described antigen in the sample substrate.Heterogeneous immunoassay for example ELISA (enzyme-linked immunosorbent assay) belong to the most responsive and special clinical assays method, and have been widely used in antigen and the antibody of identifying a big class.For example, can carry out immunoassay waits and identifies cardiac marker, tumor marker, medicine, hormone and infectious diseases.

The sample that consumes is few, it is quicker to measure and fully automated is that any clinical analysers all needs three features updating.Although the breadboard immunoassay analyzer of prior art can provide good automatization and flux, their each tests all need obviously many sample size (comprising dead space) and very long minute.Minute is long and the analyser build is huge is difficult in the sample collection rig-site utilization it.

In addition, the performance of immunoassay has very big mutability, and this major part is because this technology depends on the operator, if cause between the different researchs even same research when having used more than one laboratory, its result is difficult to compare.Full automatic integrated analysis instrument can be eliminated operator's influence and can carry out stdn to the immunologic surveillance measurement result, therefore can greatly improve the analysis to measurement result.

Although the automatization immunoassay have obtained tangible progress, these analysers are very expensive, are difficult to burden for small throughput research.The key step that those end systems with Optical devices of automatization plate washing device, incubation device and integration still need the technician to carry out some immunoassay is for example prepared to contain the titer plate of antibody and is given the plate application of sample.This causes because of disturbing the personal errors that causes repeatedly by hand, and this is between different mensuration and the main source of measuring interior variation.

A lot of fields also need to carry out the sample collection on-the-spot test, for example in medical treatment, the relevant monitoring with bioterrorism of environment field.For example, sample collection scene (POC) test of the other hemanalysis of bed makes moderate progress, but it remains the key challenge of modern medical service.Ideally, poc testing should be able to make that the clinician can real-time diagnosis and implement emergency treatment technique, and need not large-scale experiment chamber facility.This area still needs to monitor synchronously with small samples at POC the chip lab of vim and vigour, metabolite, ionogen, enzyme, DNA, protein and cell.

The microfluidic control of liquid is the key condition of successful chip lab.Microfluid system can broadly be divided into based on Continuous Flow with based on the structure of discrete flow.As suggested in its title, continuous-flow system depends on the continuous flow of liquid in path, and the discrete flow system utilize path inside or the nonpassage structure in liquid droplets.Continuous-flow system common limitation is the physical property restriction of the path that is permanently fixed of the transfer of liquid.Employed transfer device is carried out pressure-driven or moves electricity by high-low pressure driving by the pump of outside usually.These methods relate to complicated path, and need the valve or the power supply of a large amount of subsystems such as outside.These limitation make and functional integration and the control that is difficult to realize height in the continuous-flow system of routine particularly are difficult to realize the handheld apparatus at sample collection scene.This area also needs the sample collection on-site test system that utilizes droplet to control, particularly can realize the system of many tests or polymorphic type test on single chip.

Summary of the invention

One aspect of the present invention relates to the washing based on droplet.An embodiment according to this aspect, method with the droplet of the material of the contacted concentration with reduction in surface is provided, and wherein said method comprises: (a) provide and comprise the material of initial concentration and initial amount and have the droplet contacted surface of initial volume; (b) carry out one or repeatedly droplet operation so that the droplet that provides in washing droplet and the step (a) is converged, to produce the droplet of merging; (c) carry out one or repeatedly droplet operation is so that be split as one group of droplet with the droplet of described merging, described one group of droplet comprises: (i) with the contacted droplet in described surface, the material concentration that described droplet has reduces with respect to described initial concentration; (ii) with the droplet of described surface isolation.

Another aspect of the present invention relates to based on the modification on the surface of droplet and washing.An embodiment according to this aspect, method with the droplet of the material of the contacted concentration with reduction in surface is provided, wherein said method comprises: the droplet microdrive (a) is provided, it comprises the surface, and described surface and the material that comprises initial concentration and initial amount and the droplet with initial volume contact; (b) carry out one or repeatedly droplet operation so that the droplet that provides in washing droplet and the step (a) is converged, to produce the droplet of merging; (c) carry out one or repeatedly droplet operation is so that be split as one group of droplet with the droplet of described merging, described one group of droplet comprises: (i) with the contacted droplet in described surface, the material concentration that described droplet has reduces with respect to described initial concentration; (ii) with the droplet of described surface isolation.According to another embodiment, the method for modifying the surface on the droplet microdrive is provided, wherein said method comprises enforcement one or repeatedly droplet operation, so that the droplet that comprises coating materials is contacted with described surface.According to another embodiment, the droplet microdrive is provided, it comprises and is immobilized onto its lip-deep sample or reagent, and it is provided so that the droplet on the described droplet microdrive can contact with described surface.According to another embodiment, the method that material is removed from this material institute bonded surface is provided, wherein said method comprises carries out one or repeatedly droplet operation is so that make droplet contact with described surface, and described droplet comprises the solution that is used for from the described material of described surperficial wash-out.

Another aspect of the present invention relates to nucleic acid amplifier, the system and method based on droplet.An embodiment according to this aspect provides the droplet microdrive, and it comprises: the base material that (a) comprises the electrode that is used to carry out the droplet operation; (b) one or more temperature-control device, it is used to heat and/or cool off the zone of droplet microdrive near being set at one or more described electrode, and is provided so that droplet can be transferred on described electrode in the described zone so that heating.

Another aspect of the present invention relates to based on the nucleic acid amplification method of droplet and equipment.An embodiment according to this aspect, the method of the nucleic acid of the biological sample that is used for increasing is provided, wherein said method comprises: the system that comprises the droplet microdrive (a) is provided, described droplet microdrive electronics is connected in and is controlled by the treater that can execute instruction, and described droplet microdrive comprises: the sample that (i) may comprise target nucleic acid; The base material that (ii) comprises the electrode that is used to carry out the droplet operation; (iii) one or more temperature-control device, it is used to heat the zone of droplet microdrive near being set at one or more described electrode, is used for heating in the described zone so that make droplet be transferred to; (b) utilize droplet to operate on the described droplet microdrive one or more amplifing reagent droplet and one or more sample droplet are merged, to produce amplification instant droplet (amplification-ready droplet); (c) the described amplification instant of thermal cycling droplet, when having target nucleic acid in the described amplification instant droplet, this is enough to make described target nucleic acid to be amplified.According to another embodiment, the method for amplification of nucleic acid on the droplet microdrive is provided, wherein said method comprises that repeatedly transfer volume is no more than the amplification instant droplet of 50nL by one or more heating zone on the base material.According to another embodiment, provide and under the temperature condition that raises, carried out the droplet operation repeatedly, wherein said method comprises: the droplet microdrive (a) is provided; (b) droplet that heats on it makes temperature surpass about 25 ℃ of droplets with the generation heating; (c) on the droplet of described heating, carry out the droplet operation.According to another embodiment, the droplet microdrive that uses fluorescent material to make is provided, it comprises the surveyed area that is used to detect from the fluorescent signal of droplet, and described surveyed area is coated with light absorption, low fluorescence or non-fluorescent material coating.

Another aspect of the present invention relates to the diagnostics based on droplet.An embodiment according to this aspect provides droplet microdrive system, and it comprises: the droplet microdrive that (a) is configured to carry out the droplet operation; (b) transmitter, it is constructed to have sensing relation with described droplet microdrive, so that the signal and/or the character of one or more droplet of described transmitter on can the described droplet microdrive of sensing.

Another aspect of the present invention relates to based on the avidity of droplet to be measured.An embodiment according to this aspect, the method of the target analyte in the test sample is provided, wherein said method comprises: (a) implement the droplet operation mensuration reagent based on avidity on the droplet microdrive is merged with the sample that may comprise described target analyte, to produce the signal that exists, do not exist and/or measure of representing analyte; (b) detect described signal, wherein said signal and the existence of analyte described in the sample, do not exist and/or measure corresponding.

Another aspect of the present invention relates to avidity determinator and the system based on droplet.An embodiment according to this aspect provides the droplet microdrive, and it comprises the antibody that is immobilized onto the surface.According to another embodiment, the droplet microdrive is provided, it comprises the droplet on the described droplet microdrive, and described droplet comprises antibody.Another aspect of the present invention relates to the fill fluid that is used for the droplet operation.An embodiment according to this aspect provides the droplet microdrive, and it comprises: (a) first base material, and it comprises the electrode that is configured to carry out the droplet operation on the surface of described base material; (b) second base material of a described surperficial segment distance of the described base material of distance, this distance is enough to limit internal volume between described first base material and second base material, and wherein said distance is enough to hold the droplet that is placed in the above space of first base material; (c) be arranged in the described internal volume and allow to use described electrode to realize that on described droplet droplet operates with respect to its set-up mode of droplet that described electrode is provided with.According to another embodiment, the droplet microdrive is provided, it comprises base material, and described base material comprises: the electrode that (a) is used to spray or distribute the droplet operation; (b) electromagnet, itself and droplet are fully approaching to impel all basically pearls to be retained in the droplet in described division or batch operation process.According to another embodiment, the droplet microdrive is provided, it comprises base material, described base material comprises the electrode that is used to carry out the droplet operation, wherein the described base material of at least a portion comprises the electrode that is used to carry out the droplet operation and lacks second base material, can carry out the droplet operation on the described base material of substantially parallel base material lacking like this.According to another embodiment, the droplet microdrive is provided, it comprises one or more electrode that is used to carry out the droplet operation, wherein comprises opening in one or more described electrode, is used to transmit light to the droplet that is positioned on the described electrode or the transmission light from described droplet.According to another embodiment, the droplet microdrive is provided, it comprises the electrode that is used to carry out the droplet operation of one or more substantial transparent, wherein the covered thing of one or more described electrode part covers, described overcover leaves window, is used to transmit light to the droplet that is positioned on the described electrode or the transmission light from described droplet.According to another embodiment, the droplet microdrive is provided, comprise opaque fill fluid and transparent droplet on it.

Another aspect of the present invention relates to the grain sorting based on droplet.An embodiment according to this aspect provides the droplet microdrive, and it comprises: (a) particle suspension; (b) electrode, it is set to be used for use and comprises the particulate droplet and carry out the droplet operation.According to another embodiment, the droplet microdrive is provided, it comprises droplet, comprises individual particle in the described droplet.According to another embodiment, the method for transfer particle is provided, wherein said method comprises providing and comprises described particulate droplet and described droplet is transferred on the droplet microdrive.According to another embodiment, the method for the droplet that comprises individual particle is provided, wherein said method comprises: the droplet that comprises particle suspension (a) is provided; (b) droplet from (a) distributes droplet so that the droplet of distribution to be provided; (c) whether the droplet of determining described distribution comprises individual particle and/or required grain type.

Definition

In this article, following term has given implication.

＂ starts ＂ when relating to one or more electrode, refers to the change of the electricity condition of realizing described one or more electrode, to produce the droplet operation.

＂ avidity ＂ refers to a kind of molecule to another kind of molecule or to magnetism in the specificity of substrate or the non-specific molecules, for example antibody is to its corresponding antigen or haptenic magnetism.

＂ analyte ＂ refers to the target material that is detected, and it may reside in the sample.Exemplary example comprises antigenicity substance, haptens, antibody, protein, peptide, amino acid, Nucleotide, nucleic acid, medicine, ion, salt, small molecules, cell.

＂ antibody ＂ refers to the polypeptide that epi-position or haptens are had avidity.Antibody can be polyclonal antibody, monoclonal antibody, antibody fragment and/or the through engineering approaches molecule that can combine with the right corresponding member of particular combination.Antibody can mark or is puted together in helping described antibody is directly or indirectly detected and/or quantitative molecule.

For the pearl on the droplet microdrive, ＂ pearl ＂ refer to can with the droplet microdrive on or near interactional any pearl of droplet or particle.Pearl can have diversified shape, for example spherical, roughly spherical, ovum shape, plate-like, cubes or other 3D shapes.Described pearl is passable, for example, can be transferred in the droplet on the droplet microdrive; Be constructed to make it possible to allow droplet on the described droplet microdrive and described pearl are interacted, take on the described droplet microdrive and/or make it to leave described droplet microdrive with respect to the droplet microdrive.Can adopt diversified material to make pearl, comprise for example resin and polymkeric substance.Pearl can have any suitable size, for example comprises microballon, particulate nano-beads and nano particle.In some cases, pearl has magnetic responsiveness; In other cases, pearl does not have magnetic responsiveness basically.For the magnetic responsiveness pearl, the magnetic responsiveness material can constitute a kind of component whole or only pearl of pearl basically.The rest part of described pearl can comprise for example polymer materials, coating and the part that allows to adhere to sorting reagent.The example of suitable magnetic responsiveness pearl can be referring to United States Patent (USP) publication No.2005-0260686, ＂ Multiplex flow assays preferably with magnetic particles as solidphase ＂, it is disclosed on November 24th, 2005, incorporates its full content about the instruction of magnetic responsiveness material and pearl into this paper by reference at this.

＂ dNTP ＂ refers to the deoxyribonucleotide triguaiacyl phosphate, and wherein Nucleotide is any Nucleotide, for example A, T, C, G or U.＂ ddNTP ＂ refers to the bi-deoxyribose nucleotide three phosphate, and wherein said Nucleotide is any Nucleotide, for example A, T, C, G or U.Be appreciated that except as otherwise noted, can substitute dNTP with ddNTP, and vice versa.

＂ droplet ＂ refers to the liquid of the certain volume on the droplet microdrive, and it is subjected to the restriction of fill fluid to small part.For example, droplet can be filled fluid fully and surround one or more surperficial restriction that maybe can be filled fluid and described droplet microdrive.Droplet can have diversified shape; Non-limiting instance comprises roughly plate-like, bullet shape (slug shaped), interception shape, spheroid, spherical, part compression sphere, semisphere, different shape avette, cylindric and that form in the droplet operating process, the shape of for example converging or dividing or contact and form by above-mentioned shape and one or more surface of droplet microdrive.

＂ droplet operation ＂ refers to any operation that the droplet on the droplet microdrive is made.Droplet is operated and can be comprised, for example: droplet is loaded in the droplet microdrive; Distribute one or more droplet from the source droplet; Droplet is divided, separates or is split as two or more droplets; Droplet is transferred to another place from a place along any direction; Two or more droplets are converged or merge into a droplet; The dilution droplet; Mix droplet; Stir droplet; Make little droplet deformation; Make droplet keep original position; The incubation droplet; The heating droplet; The evaporation droplet; The cooling droplet; Arrange droplet; Droplet is migrated out microdrive; Other droplet operations described herein; And/or above-mentioned any combination.Term ＂ converges ＂, ＂ and merges ＂ etc. and be used for describing from two or more droplets and produce a droplet.Should be appreciated that when this term related to two or more droplet, can use was enough to make described two or more droplet to merge into any droplet operative combination of a droplet.For example, can make it to contact, shift droplet B and make it to contact or shift droplet A and B and they are contacted with each other realize that ＂ makes droplet A and droplet B converge ＂ by shifting droplet A with the droplet A of static state with static droplet B.Term ＂ division ＂, ＂ separate ＂ and ＂ splits ＂ and is not intended to hint that the size of gained droplet has the quantity of any special (that is, the size of gained droplet can be identical or different) or gained droplet that any special (quantity of gained droplet can be 2,3,4,5 or more) arranged.Term ＂ mixing ＂ refers to a kind of droplet operation, and it causes the distribution of one or more component in the droplet more even.＂ loads the operation of ＂ droplet and comprises that microdialysis loading, pressure secondary load, robot loading, passive loading and pipettor load.

＂ electronics connection ＂ referred to herein as relations electricity or data between two or more components or the element.Therefore, the so-called first component electronics is connected in the situation of second component and is not intended to get rid of the possibility that extra component can come across between described first and second components and/or operationally be associated or be provided with described first and second components.In addition, the component of electrical connection can comprise wireless intermediate component in some cases.

When relating to user interface or analogue droplet microdrive figure as described herein, it can be visually differentiable " highlighting " component that refers to described user interface or figure, for example by changing the dimly visible of color, brightness, shade, shape or symbol, icon or other visual identifier.For example, the sign that holds or select the electrode on user interface or the figure with mouse can change the color of this electrode sign.Sound also can be followed the interaction with further help user and system of the project that highlighted.

＂ input unit ＂ this comprise widely might be used for information imported device and approach on computer system or the network.Example comprises pin type device, pen device, key board unit, key area device, touch panel device, touch panel device, arrangement of levers, commentaries on classics ball device, mouse apparatus, apparatus for reading of bar code, magnetic stripe reading device, infrared unit, speech recognition technology.

＂ magnetic responsiveness ＂ refers to the responsiveness to magnetic field.The example of magnetic responsiveness material comprises paramagnetic material, ferromagnetic material, ferrimagnetic material and metamagnetism material.The example of suitable paramagnetic material comprises iron, nickel and cobalt, and metal oxide Fe for example ₃O ₄, BaFe ₁₂O ₁₉, CoO, NiO, Mn ₂O ₃, Cr ₂O ₃, and CoMnP.

＂ take-off equipment ＂ this comprise widely might be used for device and the approach of exporting to user or another system with from the information or the data of computer system.Example comprises visual display unit, LED, printer, loud speaker, modulator-demodulator unit and transceiver.

＂ scheme ＂ refers to a series of step, includes but not limited to the droplet operation on one or more droplet microdrive.

For example when icon, field or virtual key, ＂ selects ＂ to refer to and provides input the user interaction element that shows on relating to user interface, and it causes carrying out the instruction relevant with target.Therefore, for example, select the sign of the electrode that shows on the droplet microdrive figure by pointing to and click the electrode sign, can cause carrying out start the required instruction of true electrode and/or carry out the required instruction of the instruction interpolation line code of starting true electrode for one group of indication with.Can use the combination of diversified input unit or input unit to realize selecting, for example mouse, control stick and/or keyboard.

When relating to molecule that for example antibody or analyte are immobilized onto the surface, ＂ surface ＂ refer to this molecule can be immobilized in still keep simultaneously it on and the droplet microdrive on any surface of the interactional ability of droplet.For example, described surface can be the surface on the described droplet microdrive, the top board of for example described droplet microdrive or the surface on the base plate; Top board or the extended surface of base plate from described droplet microdrive; Be positioned over the surface of the physical objects on the described droplet microdrive, the modes of emplacement of this physical objects allows the droplet on itself and the described droplet microdrive to interact; And/or be positioned over pearl on the described droplet microdrive, as be positioned in the droplet and/or be positioned in the droplet microdrive but as described in the droplet outside.

With regard to washing surface, ＂ washing ＂ refer to reduction from the contacting with described surface or be exposed to one or more amount of substance on described surface of the contacted droplet in described surface.The reduction of described amount of substance can be part or complete.Described material can be various materials; Example comprises the target material that is used for further analysis, and unnecessary material, for example sample component, pollutent and/or excessive reagent.The surface can be, for example, and the surface of the pearl on the surface of droplet microdrive or the droplet microdrive.In some embodiments, washing operation starts from and the contacted initial droplet in surface, and wherein said droplet comprises the material of initial total amount.Washing operation can adopt multiple droplet operation to carry out.Washing operation can produce and the contacted droplet in described surface, and the total amount of the described material that wherein said droplet has is lower than the original bulk of described material.In another embodiment, droplet operation can produce eguttate surface, wherein is lower than in the described initial droplet with the original bulk of the contacted described material in described surface with the total amount of the contacted described material in described surface or the total amount that is exposed to the described material on described surface or is exposed to the original bulk of the described material on described surface.Other embodiment is stated in this article in addition, and more other embodiment will be conspicuous on the disclosed basis of this specification sheets.

When given component for example layer, zone or base material be referred to herein as be placed in or be formed at another component " on " time, this given component can be directly on described another component, perhaps, also can there be intermediate component (for example, one or more coating, layer, interlayer, electrode or contactant).Will be further understood that term ＂ places ... on ＂ and ＂ exist ... the ＂ that go up to form is used interchangeably, and is used to describe given component and how places or to lay with respect to another component.Therefore, term ＂ places ... on ＂ and ＂ exist ... the ＂ that go up to form has no intention material transfer, the ad hoc approach depositing or make are introduced any restriction.

When any type of liquid (for example droplet or continuum, that no matter move or static) when being described to be positioned at electrode, array, matrix or surperficial ＂ last ＂, the ＂ of ＂ place or ＂ top ＂, this liquid can be direct contact described electrode/array/matrix/surface, or contacts with one or more layer or film between described liquid and described electrode/array/matrix/surface.

When droplet is described to be positioned at droplet microdrive ＂ and goes up ＂ or ＂ and load on the droplet microdrive ＂, should be appreciated that, the mode that this droplet is set on the described droplet microdrive helps to utilize described droplet microdrive to carry out the droplet operation on described droplet, the mode that this droplet is set on the described droplet microdrive helps character or the signal of sensing from described droplet, and/or described droplet has experienced the droplet operation on described droplet microdrive.

Description of drawings

Fig. 1 is the plan view from above that is used for according to the droplet microdrive of the amplification scheme of an embodiment of the invention;

Fig. 2 A and 2B are respectively the plan view from above according to the droplet microdrive of the single integrated well heater of having of different embodiments of the present invention and a plurality of integrated well heaters;

Fig. 3 is the plan view from above that is used for according to the droplet microdrive of the nucleic acid sequence analysis of an embodiment of the invention;

Figure 4 and 5 are to show the reactions steps of an exemplary embodiment of the present invention and the diagram of droplet operation;

Fig. 6 is the skeleton view that is used to carry out according to the droplet microdrive of the immunoassay of an embodiment of the invention;

Fig. 7 shows the diagram of carrying out the step measured based on the sandwich based on avidity of droplet according to an embodiment of the invention on the droplet microdrive;

Fig. 8 shows according to an embodiment of the invention to carry out the diagram of competitiveness based on the step of the mensuration of avidity on the droplet microdrive;

Fig. 9 is the skeleton view according to the biological fluid analyser of an embodiment of the invention;

Figure 10 is the lateral sectional view according to the droplet microdrive loading structure of an embodiment of the invention;

Figure 11-the 13rd shows that different embodiments according to the present invention utilize the diagram of the step of fixing of magnetic field and release magnetic responsiveness pearl;

Figure 14 shows the diagram of utilizing the step of immobilization of physical property obstacle and release pearl according to an embodiment of the invention;

Figure 15 is the diagram that shows the step of droplet microdrive surface being washed according to an embodiment of the invention;

Figure 16 A and Figure 16 B are respectively the lateral sectional view and the plan view from above of droplet microdrive that is used to shift droplet according to an embodiment of the invention;

Figure 17 is the skeleton view according to the biological fluid analyser of an embodiment of the invention;

Figure 18 is the diagram according to the droplet microdrive system of an embodiment of the invention;

Figure 19 A and 19B are the diagrams according to the portable hand-held analyser of an embodiment of the invention;

Figure 20 is the diagram according to the user interface of the droplet Controlling System of an embodiment of the invention; With

Figure 21 A-21D is the lateral sectional view of demonstration according to the structure of the different droplet microdrive sensor elements of different embodiments of the present invention.

Detailed Description Of The Invention

The invention provides and be used to implement one or more methods, devices and systems based on the biochemical measurement of droplet.For example, the invention provides and be used for amplification of nucleic acid, analysis of nucleic acids sequence, carry out methods, devices and systems based on the mensuration and/or the analysing body fluid component of avidity.

In some embodiments, the scheme of described system can comprise with any and can realize following in sequence one or more step of testing goal of the present invention: from the object extraction sample; Processing is used to be loaded into the sample on the droplet microdrive; Sample is loaded on the described droplet microdrive; Distribute one or more sample droplet of described sample to be used to transfer to described droplet microdrive; With one or more reagents loaded to described droplet microdrive; Distribute one or more reagent droplet to be used to transfer to described droplet microdrive; Shift one or more reagent droplet and/or one or more sample droplet, so that one or more reagent droplet is contacted with one or more sample droplet, thus the interaction of realization reagent and sample; The interactional influence of detection reagent and sample; Output is provided, will detects the result notification user of step.The example of the biochemical scheme of using droplet microdrive of the present invention hereinafter is discussed.

8.1 nucleic acid amplification

The invention provides the methods, devices and systems that are used for the nucleic acid in the amplification droplet on the droplet microdrive.Can in single droplet, from this droplet, exist a small amount of or or even one or more nucleic acid molecule of a large amount of copies of nucleic acid molecule preparation of single copy.Method of the present invention comprises the reactant that merges necessity generally with formation amplification instant droplet, and being enough to cause the temperature of target nucleic acid amplification that droplet is implemented thermal cycling, for example by polymerase chain reaction (PCR).In some embodiments, the droplet that comprises the target nucleic acid of amplification can be transferred to subsequently in the follow-up method, for example the method for detection method, further amplifying target nucleic acid and/or sequence measurement (for example referring to 8.2 joints).Amplification device can comprise the droplet microdrive and be enough to carry out the component of droplet operation, implement method of the present invention when having loaded suitable reagent with the described droplet microdrive of box lunch.System of the present invention can comprise described droplet microdrive and system components, and it is designed to help operation with the described droplet microdrive of software control to implement the solution of the present invention.

Fig. 1 has provided the exemplary droplet microdrive 100 that is used for amplification scheme of the present invention.In this embodiment, can provide a plurality of fluid port and/or pond, for example sample pool 102, PCR reagent pond 104 and primer sets pond 106.Also can provide heating region, for example low-temperature heat zone 108 and heat zone 110.The sample viewing area 112 that uses microscope for example or photomultiplier (PMT) also can be provided.

In one embodiment, the invention provides that droplet microdrive and/or system are configured and program turns to the amplification of implementing sample in amplification instant droplet, catch the nucleic acid of amplification subsequently.With before or after the magnetic responsiveness pearl contacts, it is single-chain nucleic acid that the nucleic acid that can handle amplification makes it sex change, so that described single-chain nucleic acid is incorporated into described magnetic responsiveness pearl.For example can use vitamin H-streptavidin system to realize combination, for example, wherein single-chain nucleic acid is by biotinylation, and described surface (for example pearl or droplet microdrive surface) bag is by covalent attachment streptavidin thereon.Can adopt washing scheme flush away amplifing reagent.Various additive method of the present invention, device, system and other aspects will be conspicuous by following description.

Be appreciated that an importance of the present invention relates to the sample that can use on various nucleic acid amplification reagent and/or the droplet microdrive and carries out the droplet operation.For example, the present invention includes:

(1) droplet microdrive comprises droplet on it, described droplet comprises any one or more reagent and/or the sample that is used to carry out nucleic acid amplification described herein;

(2) device of the present invention or system, it comprises described droplet microdrive;

(3) in described droplet microdrive or system, carry out droplet method of operating or utilize the method for described droplet microdrive or system operation droplet; And/or

(4) utilize described droplet microdrive or system to carry out method based on the sequential analysis scheme of droplet.

For example, described droplet operation can comprise following one or more operation: droplet is loaded in the droplet microdrive; Distribute one or more droplet from the source droplet; Droplet is divided, separates or is split as two or more droplets; Droplet is transferred to another place from a place along any direction; Two or more droplets are converged or merge into a droplet; The dilution droplet; Mix droplet; Stir droplet; Make little droplet deformation; Make droplet keep original position; The incubation droplet; The heating droplet; The evaporation droplet; The cooling droplet; Arrange droplet; Droplet is migrated out microdrive; Other droplet operations described herein; And/or above-mentioned any combination.Various additive method of the present invention, device, system and other aspects will be conspicuous by following description.

8.1.1 sample and specimen preparation

The sample that amplification method of the present invention uses comprises be used to increase nucleic acid-templated.Described nucleic acid-templated can be any kind, as genomic dna, RNA, plasmid, phage and/or artificial sequence.Described nucleic acid-templated can be any source, as complete organism, organ, tissue, cell, organoid (for example chloroplast(id), plastosome), synthetic nucleic acid source etc.In addition, template can be multiple origin, as pathology sample, forensic samples, extinct plants and animal sample etc.Biological samples can comprise, for example, whole blood, lymph liquid, serum, blood plasma, sweat, tear, saliva, phlegm, cerebrospinal fluid (CSF), amniotic fluid, seminal fluid, vaginal secretions, slurries, synovial membrane liquid, pericardial fluid, peritoneal fluid, hydrothorax, spill liquid, transudate, capsule liquid (cystic fluid), bile, urine, gastric juice, intestinal juice, faecal samples and swab or washing lotion (for example oral cavity, nasopharynx, eye, rectum, enteron aisle, vagina, epidermis etc.) and/or other biological is learned sample.

Can carry out various sample preparation steps prepares described nucleic acid-templated.In some cases, for example from plasmid or phage amplification, rough sample can use.In other cases, the big fragment of amplifying genom DNA for example, the template of preferred heights purifying.Sample preparation steps can on the described droplet microdrive or outside carry out.

System of the present invention can be configured and program turns to and can handle biological sample and comprise the nucleic acid-templated droplet that is used to increase with preparation.Some of this treating processes partly or entirely can on the described droplet microdrive or outside implement, for example use the pearl that the target organism is had the reagent of avidity that contains in conjunction with thereon that described target organism is separated from biological sample.Described droplet microdrive can be handled sample, and it is split as one or more discrete droplets, is used for carrying out subsequent operations on described droplet microdrive.

In some cases, can handle sample to reduce the viscosity in the follow-up droplet operating process.For example, can prepare sample on the described droplet microdrive or outside described droplet microdrive, itself and basic solution (for example, 10% KOH) mixed or mixes with sample dissolution and make it fully to flow and operate so that on the droplet microdrive, carry out droplet with reductive agent such as dithiothreitol (DTT) (DTT) or dithioerythritol (DTE).The example of the sample preparation technology that other are suitable can be referring to U.S. Patent application No.60/745,950, denomination of invention is ＂ Apparatus and Methods of Sample Preparation for a Droplet Microactuator ＂, and the applying date is on April 28th, 2006.

Comprise that described nucleic acid-templated droplet can merge so that amplification instant droplet to be provided with amplifing reagent, produces PCR instant droplet as merging with PCR reagent.According to selected reagent, amplification instant droplet can be used for isothermal duplication or thermal cycling with amplifying target nucleic acid.Amplified production can detect on the droplet microdrive and/or quantitatively in real time.By this way, the invention provides real-time quantitative on the chip increases and detects and the quantitative target nucleic acid in the sample.

Because almost 100% sample can be used for analyzing (not needing to fill pipeline or pump), therefore can analyze the sample volume (for example be less than about 100 μ L or be less than about 50 μ L or be less than about 25 μ L) of minute quantity.Most tests can use single small sample to carry out, and can at utmost save the reagent expense.

8.1.2 reagent

In amplification scheme of the present invention, all ingredients can with nucleic acid-templated merging to produce amplification instant droplet, for example PCR instant droplet.PCR reagent typically comprises primer, Nucleotide, polysaccharase and damping fluid.The form that these input reagent can be loaded into the independent reagent on the droplet microdrive respectively provides and utilizes the droplet operation to merge on described droplet microdrive.In addition, some or all these reagent can be used as reagent mixture to be provided, and it is loaded on the described droplet microdrive with the form that is pre-mixed.In one embodiment, all amplifing reagents are merged into single droplet, just this droplet only needs and the sample droplet merges and produces amplification instant droplet, as PCR instant droplet.

8.1.2.1 damping fluid

Reagent typically comprises damping fluid.Selected damping fluid helps amplified reaction.Any damping fluid that meets this function all is suitable.When nucleic acid to be amplified was DNA, damping fluid generally included magnesium ion.

In one embodiment, damping fluid comprises KCl, Tris and MgCl ₂Other suitable damping fluids can be referring to Chamberlain etc., Nucleic Acid Rerearch 16:1 1141-11156 (1988).For example, damping fluid can comprise about 500mM KCl, about 100mM Tris-HCl (pH 8.3) and about 15mMMgCl ₂In another example, damping fluid can comprise about 83mM (NH ₄) ₂SO ₄, about 335mMTris-HCl (pH8.8), about 33.5mM MgCl ₂, about 50mM beta-mercaptoethanol and about 34mM EDTA.Damping fluid also can comprise primer and/or polysaccharase.

In one embodiment, can be in some droplets successively or the parallel PCR that carries out, in these droplets, one or more damping fluid component concentrations is changed (for example in a series of droplets) so that improve or optimize the used damping fluid of specific reaction systemicly.Therefore, for example, can change following any one or more damping fluid component: KCl; Tris; MgCl ₂(NH ₄) ₂SO ₄Beta-mercaptoethanol; EDTA.In case identify best assay buffer condition, can use this optimized buffer liquid system to carry out PCR or can near described best assay buffer system, further optimize.

The present invention includes the droplet microdrive, comprise droplet on it, described droplet is damping fluid or comprises the damping fluid component that the present invention also comprises the system and/or the device of described droplet microdrive and carrying out the droplet operation on the droplet microdrive or the droplet on the droplet microdrive is carried out method of operating.Therefore, for example, the present invention includes the droplet microdrive, comprise droplet on it, described droplet comprises one or more following component: KCl, Tris, MgCl ₂((NH ₄) ₂SO ₄Beta-mercaptoethanol; EDTA.

In addition, the present invention includes the droplet microdrive, comprise droplet on it, described droplet comprises one or more said components, and described component concentrations is enough to help amplifying target nucleic acid.In addition, the present invention includes described droplet and polysaccharase, Nucleotide and/or one or more primer, their concentration is enough to help amplifying target nucleic acid.The present invention also is included in and carries out the droplet method of operating in described droplet microdrive, device and/or the system or use described droplet microdrive, device and/or system to operate the described any droplet of this section.For example, described droplet operation can comprise following one or more operation: droplet is loaded in the droplet microdrive; Distribute one or more droplet from the source droplet; Droplet is divided, separates or is split as two or more droplets; Droplet is transferred to another place from a place along any direction; Two or more droplets are converged or merge into a droplet; The dilution droplet; Mix droplet; Stir droplet; Make little droplet deformation; Make droplet keep original position; The incubation droplet; The heating droplet; The evaporation droplet; The cooling droplet; Arrange droplet; Droplet is migrated out microdrive; Other droplet operations described herein; And/or above-mentioned every arbitrary combination.

8.1.2.2 primer

The used reagent of amplification method of the present invention can comprise one or more primer.In typical method, use two kinds of primers to determine described nucleic acid-templated zone to be amplified.Typically, Xuan Ding primer sequence and length can guarantee effectively to duplicate and almost do not have mistake.The length of this type of primer is generally about 18-24 base.It is known selecting other conditions of effective primer.The example of suitable primer character comprises does not have inner secondary structure, 40-60%G/C content, rich G/C and the A/T regional balance distributes and 3 ' end does not have complementarity in order to avoid form primer dimer.Although there is not particular requirement, has the primer of one or more these character and be suitable for implementing the present invention.In addition, the melting temperature(Tm) of primer (Tm) is chosen as usually and makes that annealing temperature is about 55 to about 65 ℃ or about 62 to about 65 ℃.There is multiple common computer program to can be used for identifying primer with the character that is suitable for increasing.If use two kinds of primers, they use the concentration that equates usually.If nucleic acid to be amplified is RNA, then not necessarily need primer.

In some embodiments, use the degeneracy mixture of primer.For example, the aminoacid sequence of Yin Teding can be encoded by multiple possible codon, so mixture can comprise all possible sequence that covers all used codons combinations of target polypeptide.The codon bias that can be tested and appraised organism interested is also only included the normally used primer of this organism in and is simplified the degenerated primer mixture.

The primer that is provided can have any concentration that helps amplifying target nucleic acid.Concentration should be enough low, in order to avoid too much primer mispairing, the accumulation of non-specific product and/or formation primer dimer.Primer concentration also should be enough high, in order to avoid primer is exhausted before amplified reaction is finished.In some embodiments, primer concentration be about 0.1 μ M to about 1 μ M or about 0.1 μ M to about 0.6 μ M.

Primer can also be labeled.For example, can selectable marker so as to detect, location, quantitatively and/or separate the PCR product.For example, can use biotinylation detects and/or purifying so that utilize streptavidin to catch lip-deep biotinylated PCR product.In addition, streptavidin can be used to catch biotinylated PCR product with the magnetic responsiveness pearl is linked together.Also can use digoxigenin to detect the PCR product.Primer can, for example, carry out mark at its 5 ' end and/or inside, in addition, the Nucleotide of mark can be mixed the PCR product so as to detect, location, quantitatively and/or separate.

The present invention includes the droplet microdrive, comprise droplet on it, that described droplet comprises the mark that is used for amplifying target nucleic acid and/or unlabelled primer (for example concentration be about 0.1 μ M to about 1 μ M or about 0.1 μ M to about 0.6 μ M), its concentration is enough to help amplified reaction, and the present invention also comprises the system and/or the device of described droplet microdrive and carrying out the droplet operation on the droplet microdrive or the droplet on the droplet microdrive is carried out method of operating.As another example, the present invention includes the droplet microdrive, comprise droplet on it, described droplet comprises primer mark and/or unlabelled, primer concentration should be enough low, pile up to reduce or eliminate primer mispairing and non-specific product, and primer concentration also should be enough high, in order to avoid primer is exhausted before amplified reaction is finished.In another example, the present invention includes the droplet microdrive, comprise droplet on it, described droplet comprises primer mark and/or unlabelled, primer concentration be about 0.1 μ M to about 1 μ M or about 0.1 μ M to about 0.6 μ M.In addition, the present invention includes described droplet and polysaccharase, Nucleotide and/or damping fluid component, their concentration is chosen to be and helps amplifying target nucleic acid.In addition, present invention resides in and carry out the droplet method of operating in described droplet microdrive, device and/or the system or use described droplet microdrive, device and/or system to operate the described any droplet of this section.For example, described droplet operation can comprise following one or more operation: droplet is loaded in the droplet microdrive; Distribute one or more droplet from the source droplet; Droplet is divided, separates or is split as two or more droplets; Droplet is transferred to another place from a place along any direction; Two or more droplets are converged or merge into a droplet; The dilution droplet; Mix droplet; Stir droplet; Make little droplet deformation; Make droplet keep original position; The incubation droplet; The heating droplet; The evaporation droplet; The cooling droplet; Arrange droplet; Droplet is migrated out microdrive; Other droplet operations described herein; And/or above-mentioned every arbitrary combination.

8.1.2.3 Nucleotide

The used reagent of amplification method of the present invention comprises Nucleotide.The storage liquid of dNTP can obtain from the commercialization of multiple source.The common concentration of storage liquid is 100-300mM.Before storage liquid being introduced on described droplet microdrive and/or the described droplet microdrive, can adopt the droplet operation that described storage liquid and one or more damping fluid are merged to dilute described storage liquid.Final concentration in the PCR instant droplet typically is about 50 μ mol to about 200 μ mol.Four kinds of dNTP typically with etc. volumetric molar concentration provide.

The present invention can use the Nucleotide of various modifications.Example comprise designed to be used reduce that secondary structure changes, antipollution Nucleotide and radiolabeled Nucleotide, nonradioactive labeling's Nucleotide and the Nucleotide that is designed to promote random mutagenesis.Example is referring to McPherson etc., PCR, Taylor and Francis Group, 2006 (incorporating its full content into this paper by reference).

The present invention includes the droplet microdrive, comprise droplet on it, described droplet comprises the Nucleotide that is used for amplifying target nucleic acid, its concentration is enough to help amplified reaction, and the present invention also comprises the system and/or the device of described droplet microdrive and carrying out the droplet operation on the droplet microdrive or the droplet on the droplet microdrive is carried out method of operating.Therefore, for example, the present invention includes the droplet microdrive, comprise droplet on it, described droplet comprises one or more Nucleotide, its concentration for about 100mM to about 300mM (storage concentration) or about 50 μ mol about 200 μ mol (working concentration) extremely.In another example, the present invention includes the droplet microdrive, comprise droplet on it, described droplet comprises 1,2,3 or 4 kind of Nucleotide, the concentration of each for about 100mM to about 300mM or about 50 μ M about 200 μ M extremely.System of the present invention can be configured and program turns to the scheme of enforcement dilution storage nucleotide concentration so that the droplet that comprises the work nucleotide concentration to be provided.For example, system of the present invention can be configured and program turn to about 100mM to the dilution of the storage nucleotide concentration of about 300mM be the extremely schemes of the working solution of about 200 μ mol of about 50 μ mol.In addition, the present invention includes the droplet and polysaccharase, primer and/or the damping fluid component that contain Nucleotide, their concentration is chosen to be to be used to provide and helps amplifying target nucleic acid.In addition, present invention resides in and carry out the droplet method of operating in described droplet microdrive, device and/or the system or use described droplet microdrive, device and/or system to operate the described any droplet of this section.For example, described droplet operation can comprise following one or more operation: droplet is loaded in the droplet microdrive; Distribute one or more droplet from the source droplet; Droplet is divided, separates or is split as two or more droplets; Droplet is transferred to another place from a place along any direction; Two or more droplets are converged or merge into a droplet; The dilution droplet; Mix droplet; Stir droplet; Make little droplet deformation; Make droplet keep original position; The incubation droplet; The heating droplet; The evaporation droplet; The cooling droplet; Arrange droplet; Droplet is migrated out microdrive; Other droplet operations described herein; And/or above-mentioned every arbitrary combination.

8.1.2.4 PCR polysaccharase

PCR scheme based on droplet of the present invention can be used multiple PCR polysaccharase.Suitable polysaccharase has optimum activity at about 75 ℃ usually, and can still keep this activity behind the long-time incubation of the temperature that for example is higher than 95 ℃.Useful polysaccharase can comprise, for example, and DNA dependent dna-polymerases and/or RNA dependent dna-polymerases (reversed transcriptive enzyme).Can use various thermostable type polysaccharases, for example the Taq archaeal dna polymerase.Suitable example comprises AmpliTaq

Stoffel fragment or the like.Example with thermostable type polysaccharase of proofreading ability comprises

Tli,

Pfu, Pwo,

With KOD Hifi, archaeal dna polymerase and aforementioned every various exo ^-Form.In some cases, the polysaccharase prepared product can comprise the dyestuff that is used for determining or confirming the PCR reagent concentration.In some cases, system is configured and program turns to and detects this type of dyestuff and calculate reagent concentration based on the colorimetric estimation value.In some cases, the droplet of the present invention's use comprises the combination of thermostable type polysaccharase (for example Taq archaeal dna polymerase) and proofreading polysaccharase (for example Pwo archaeal dna polymerase).

The present invention includes the droplet microdrive, comprise droplet on it, described droplet comprises one or more polysaccharase, its concentration is enough to help amplified reaction, and the present invention also comprises the system and/or the device of described droplet microdrive and carrying out the droplet operation on the droplet microdrive or the droplet on the droplet microdrive is carried out method of operating.In addition, present invention resides in and carry out the droplet method of operating in described droplet microdrive, device and/or the system or use described droplet microdrive, device and/or system to operate the described any droplet of this section.For example, described droplet operation can comprise following one or more operation: droplet is loaded in the droplet microdrive; Distribute one or more droplet from the source droplet; Droplet is divided, separates or is split as two or more droplets; Droplet is transferred to another place from a place along any direction; Two or more droplets are converged or merge into a droplet; The dilution droplet; Mix droplet; Stir droplet; Make little droplet deformation; Make droplet keep original position; The incubation droplet; The heating droplet; The evaporation droplet; The cooling droplet; Arrange droplet; Droplet is migrated out microdrive; Other droplet operations described herein; And/or above-mentioned any combination.In addition, the present invention includes the droplet that contains polysaccharase and Nucleotide, primer and/or damping fluid on the droplet microdrive of the present invention, their concentration is selected as the condition that is enough to help amplifying target nucleic acid can be provided.

8.1.2.5 detect the product of amplification

In some embodiments, at some nucleic acid that detect amplification after taking turns amplification cycles.For example, can be in each working cycle or afterwards or after the predetermined wheel circulation, for example after per 2,3,4,5,6,7,8,9 or 10 circulations, the nucleic acid of quantitative amplification.Detection generally includes and utilizes droplet to operate to shift droplet to detection zone, at this place, and some aspect of sensor measurement droplet, for example physics, chemistry or electric aspect, these aspects are associated with amplification.System can be found to have reached desired signal level so that continue to carry out amplification cycles until detecting by sequencing.In one embodiment, amplification detection method is a fluorescence technique.

In addition, in some embodiments, the droplet that comprises the nucleic acid of amplification can be transferred and be used for further processing/detection.For example, in the diagnosis embodiment, the droplet that comprises the nucleic acid of amplification can be transferred to be used for detecting whether have a diagnosis target nucleic acid.Target nucleic acid for example can comprise the nucleic acid from pathogenic organisms, for example DNA or the RNA that is associated with parasite, bacterium, fungi or virus.Described droplet microdrive can be a component of integrated portable diagnostic platform.Described system can carry out full-automatic rapid detection to the infectious diseases group by PCR in real time.Described system can be by bed, Stat laboratory, doctor's office or field usage.

Fluoroscopic examination is suitable for detecting the nucleic acid of amplification.Photodiode (LED) and laser diode are suitable as excitation light source, because their volume is little, power saving and durable.LED is very attractive, because they are cheap, laser diode then has narrower spectral width, and they can be focused in very little spot diameter and can not send a large amount of light.

Except the reagent of having discussed, the reagent that can be used for nucleic acid amplification scheme of the present invention comprises various detection reagent, for example fluorescence and non-fluorescence dye and probe.For example, scheme can be used the reagent that is suitable for the TaqMan reaction, for example TaqMan probe; Be suitable for

The reagent of Green reaction, for example

Green; Be suitable for the reagent of molecular beacon reaction, for example molecular beacon probe; Be suitable for the reagent of scorpion type reaction (scorpion reaction), for example scorpion type probe (scorpion probe); Be suitable for the reagent of fluorescent DNA-combination dye type reaction, for example fluorescent probe; And/or be suitable for the reagent of LightUp scheme, for example LightUp probe.

The present invention includes the droplet microdrive, comprise droplet on it, described droplet comprises one or more detection reagent, for example any one or more above-mentioned probe, its concentration is enough to help amplified reaction, and the present invention also comprises the system and/or the device of described droplet microdrive and carrying out the droplet operation on the droplet microdrive or the droplet on the droplet microdrive is carried out method of operating.In addition, present invention resides in and carry out the droplet method of operating in described droplet microdrive, device and/or the system or use described droplet microdrive, device and/or system to operate the described any droplet of this section.For example, described droplet operation can comprise following one or more operation: droplet is loaded in the droplet microdrive; Distribute one or more droplet from the source droplet; Droplet is divided, separates or is split as two or more droplets; Droplet is transferred to another place from a place along any direction; Two or more droplets are converged or merge into a droplet; The dilution droplet; Mix droplet; Stir droplet; Make little droplet deformation; Make droplet keep original position; The incubation droplet; The heating droplet; The evaporation droplet; The cooling droplet; Arrange droplet; Droplet is migrated out microdrive; Other droplet operations described herein; And/or above-mentioned any combination.In addition, the present invention includes the droplet that contains Nucleotide and Nucleotide, primer, detection probes and/or damping fluid on the droplet microdrive of the present invention, their concentration is selected as the condition that is enough to help amplifying target nucleic acid can be provided.

In addition, the present invention includes amplification is detected and/or quantitative methods, this method comprises the signal (for example fluorescent signal) of measurement from the droplet on the droplet microdrive.Therefore, for example, this method can will (for example may comprise fluorescence dye

Green) droplet is exposed to the light source with a selected wavelength, and this selected wavelength causes described compound to send fluorescence, and measures the fluorescence that is produced.Can in the amplified reaction process, follow the tracks of the fluorescence that sends from droplet so that monitoring reaction for example uses

The Green compounds.System of the present invention is passable, for example, is turned to this fluorescence of detection and demonstrates corresponding figure or other output forms by program, so that the process of user's real-time follow-up PCR reaction.

In another example, the present invention includes method by the existence of following steps detection and/or quantifying target nucleic acid, adding has specific probe (for example TaqMan class probe) to the target nucleic acid in the amplification instant droplet that may comprise target nucleic acid, the described amplification instant of thermal cycling droplet, and detect by the described probe caused any fluorescent signal of degrading, wherein fluorescent signal is represented and is had described target nucleic acid in the described droplet.The present invention includes for example corresponding method of scorpion type probe and molecular beacon of other target-specific probes of using.

8.1.3 thermal cycling

In practice of the present invention, PCR instant droplet by thermal cycling so that implement the amplification of target nucleic acid.For some nucleic acid that effectively increases, may need the strict control thermal cycling.Hereinafter 8.8.6 joint has been discussed to carry out the example of the structure that the controlled thermal circulation designs on the droplet microdrive.Typically, each thermal cycling comprises at least two steps:

(1) droplet is heated to the temperature that the double-strandednucleic acid sex change that is enough to make in the droplet is a single stranded DNA (typically being about 90-100 ℃); With

(2) reduce the temperature of described droplet so that allow primer and its complementary sequence annealing (typically being about 50-75 ℃) in described nucleic acid-templated chain.

In some cases, thermal cycling also can comprise the 3rd step:

(3) regulate the droplet temperature so that promote the double-stranded sections of described nucleic acid to extend (typically being about 70-75 ℃) by mixing extra Nucleotide.

According to selected reagent, can so just not need the temperature regulation in the 3rd step allowing the primer temperature identical to carry out mixing of extra Nucleotide with described nucleic acid-templated chain annealed temperature.In different schemes of the present invention, also can add extra thermal cycling step.

The present invention allows a plurality of droplets are carried out thermal cycling abreast.Therefore, in different embodiments, on single droplet microdrive, carry out thermal cycling to surpassing 2,3,4,5,10,20,30,40,50,100 or 1000 amplification instant droplets abreast.In some embodiments, the amplification in these droplets of The real time measure abreast.

In one embodiment, each droplet that just experiences thermal cycling is placed near the transmitter, or be transferred to transmitter near so that can monitor in real time and the relevant signal that increases from described droplet.On behalf of the real-time information of amplification process, system can will export to the user.In addition, system can be set to allow to export when carrying out thermal cycling to a plurality of droplets are parallel, as the instant droplet is parallel to carry out thermal cycling to increasing above 2,3,4,5,10,20,30,40,50,100 or 1000 on single droplet microdrive, and the real-time information of amplification process is represented in system's output.

Method of the present invention also comprises temperature optimization step or scheme, is used to optimize the temperature and/or the time of sex change, annealing and/or extension.In this step, can use one or more heating zone to change the temperature of one or more heating steps.For example, described method can comprise optimization step or the scheme that is used to optimize annealing temperature.Can use different annealing temperatures that a series of droplets are carried out thermal cycling, subsequently amplification be detected quantitatively, determine that thus which test annealing temperature can produce optimal results.Thermal cycling subsequently can be carried out by this probe temperature.Similarly, can use the different time carry out thermal cycling to the annealing temperature of a series of droplets, subsequently amplification be detected quantitatively, definite thus which can produce optimal results in the annealing temperature of giving test duration.The time that thermal cycling subsequently can be selected carries out.In addition, can be successively or implement this type of prioritization scheme simultaneously so that determine to optimize temperature and the optimization time.Scheme optimization sex change and/or extend the temperature and time of step like can implementation of class.

In one embodiment, realize thermal cycling by the whole droplet microdrive of heating and cooling.This embodiment may further comprise the steps substantially:

(1) described droplet microdrive is heated to be enough to make double-stranded DNA (being present in the droplet on the described droplet microdrive) sex change be the temperature of single stranded DNA;

(2) temperature of described droplet microdrive is reduced to is enough to allow primer (being present in the droplet on the described droplet microdrive) and its complementary sequence annealed temperature on described nucleic acid-templated chain;

(3) randomly, adjust the temperature of described droplet microdrive so that by mixing the double-stranded sections (being present in the droplet on the described droplet microdrive) that extra Nucleotide extends described nucleic acid.

Thermal cycling scheme of the present invention can not cause moisture or other components in the PCR instant droplet obviously to lose.In addition, described thermal cycling scheme can not cause significantly crossed contamination between the droplet.In addition, described thermal cycling can obviously not destroy the ability that described droplet microdrive is proceeded the droplet operation.For example, in some cases, can continue to carry out the droplet operation at different sex change, annealing and/or elongating temperature.

In relevant embodiment, realize thermal cycling by the section or the zone of the described droplet microdrive of heating and cooling.This method may further comprise the steps substantially:

(1) section of described droplet microdrive or zone are heated to be enough to make double-stranded DNA (being present in the droplet on the described droplet microdrive) sex change be the temperature of single stranded DNA;

(2) temperature in the section of described droplet microdrive or zone is reduced to is enough to allow primer (being present in the droplet on the described droplet microdrive) and its complementary sequence annealed temperature on described nucleic acid-templated chain;

(3) randomly, adjust described droplet microdrive section or the zone temperature so that by mixing the double-stranded sections (being present in the droplet on the described droplet microdrive) that extra Nucleotide extends nucleic acid.

In one embodiment, use the droplet microdrive shown in Fig. 2 A to carry out this method with single integrated well heater 202.

In another embodiment, the zone of described droplet microdrive can remain on required temperature, and droplet is shifted by suitable temperature province to implement thermal cycling. and this method may further comprise the steps substantially:

(1) the instant droplet that will increase is transferred to the zone of droplet microdrive, and this zone keeps suitable temperature so that be single stranded DNA with the double-stranded DNA sex change in the droplet;

(2) the instant droplet that will increase is transferred to the zone of droplet microdrive, and this zone remains on primer and its complementary sequence annealed temperature on described nucleic acid-templated chain that is enough to allow in the droplet; With

(3) randomly, amplification instant droplet is transferred to the zone of droplet microdrive, this zone remains on to be enough to by mixing extra Nucleotide to promote or to optimize the temperature of the extension of the double-strandednucleic acid sections in the droplet.

In this embodiment, by repeating that droplet is implemented thermal cycling from the transfer step that a district moves to another district.In one embodiment, described droplet microdrive only provides a humidity province for each is temperature required, and by making each droplet rotation implement thermal cycling by suitable humidity province.In another embodiment, described droplet microdrive comprises two or more every kind humidity province.In another embodiment, described droplet microdrive comprises two or more one or more described humidity province.In addition, described droplet microdrive can comprise 2,3 or more a plurality of humidity province, and each can be heated to different specified temps.In one embodiment, use the droplet microdrive shown in Fig. 2 B to carry out this method with a plurality of integrated well heaters 204.

In addition, can use one or more well heater to set up the continuous temperature gradient in the zone of striding the droplet microdrive.In this embodiment, can use electrode matrix (electrode matrix), electrode path (electrodepath) or serial electrode path that droplet is positioned at suitable humidity province to implement required thermal cycling step.Can sentence by the electrode that droplet is transferred to correct position in the described thermograde and reach the target temperature and implement thermal cycling.Can realize variation of temperature by changing the position of droplet in described humidity province simply, for example, be used to optimize various sex change, annealing and/or extend step.

Thermal cycling can comprise uses various heating and/or refrigerating module to set up the target humidity province of sex change, annealing and/or extension.These heating and cooling modules can be set to help form suitable temperature ramp (temperature ramp) between the target humidity province.Therefore temperature that can be by changing specific heating and/or refrigerating module and/or the distance by selecting the heating and cooling module control described slope to realize having the target humidity province of suitable temperature ramp.Described droplet microdrive can have heating and/or refrigerating module, and the temperature range of described module and gap are chosen to be potential target humidity province and the temperature ramp that is used to produce predetermined group.Can have various heating and/or refrigerating module between the target humidity province to adjust the slope between each district.

In one embodiment, described droplet microdrive comprises a series of independent regulation formula heating units.Can regulate the temperature of each heating unit, so that suitable heating slope is provided during to next target humidity province by a target humidity province at droplet.In addition, the distance in the time of can selecting the target temperature between the heating unit is so that help to form suitable temperature ramp and/or so that prevention is overheated because of causing near the interaction between the heating unit of placement.These class methods require the multiple scheme of different target humidity provinces and characteristics of ramps that handiness is provided for carrying out separately.For example, in the heating unit of a series of or matrix, the target humidity province can and/or can be separated so that they are separated by bigger distance by one or more heating unit at approaching heating unit place.So, can change distance to satisfy the temperature requirements of various scheme demands.System of the present invention can select best heating unit to set up the target humidity province that has suitable or best temperature ramp between the heating zone.

Method of the present invention can comprise sex change, the annealing that is used to optimize thermal cycling and/or extend the temperature in stage and/or temperature optimization step or the scheme of time.For example, described method can comprise optimization step or the scheme that is used to optimize annealing temperature.Can set up a series of heating zone, wherein, amplification instant droplet be circulated to determine which annealing temperature produces optimum by different annealing temperatures.Can carry out follow-up thermal cycling in described optimum temps.Similarly, can adopt following scheme that a series of amplification instant droplets are circulated, in the described scheme, the time of annealing stage is changed to determine which section period can produce optimum under the fixed temperature by systematicness.Can use described Best Times section to carry out follow-up thermal cycling.In addition, can be successively or implement this type of scheme simultaneously so that determine optimum temps and the Best Times section.But scheme is to optimize sex change and/or to extend step like the implementation of class.Prioritization scheme can be successively or parallel carrying out.

Similarly, method of the present invention can comprise temperature optimization step or scheme, and it uses temperature and/or the time of humidity province to optimize sex change, annealing and/or to extend of a plurality of independent heating.For example, can set up a series of heating zone, the instant droplet that will increase passes through from wherein shifting.Described district can comprise to start sex change, annealing and/or to extend to the temperature of purpose.In specific one group of droplet, can change the temperature of one of described sex change, annealing and/or the extension area of one group of PCR instant droplet, and other two temperature keep constant systemicly.Can test a plurality of droplet groups, so that can change each temperature parameter as required.Can be in turn and/or test one or more described a plurality of droplet groups abreast.The variation of Heating Zone Temperature, for example, can be controlled (temperature and/or the position of control droplet in adding thermal gradient of for example controlling heating unit) by treater, use is turned to the software of implementing prioritization scheme by pre-program and/or uses by the software of user by user interface control.Can optimize the time in described each stage of thermal cycling in a similar manner.Prioritization scheme can in turn or abreast carry out.

In addition, the temperature condition that present invention resides in rising uses down PCR instant droplet to carry out one or droplet method of operating repeatedly on the droplet microdrive, and for example temperature is higher than about 70,75,80,85,90,95 or 100 ℃.For example, described droplet operation can comprise: droplet is loaded in the droplet microdrive; Distribute one or more droplet from the source droplet; Droplet is divided, separates or is split as two or more droplets; Droplet is transferred to another place from a place along any direction; Two or more droplets are converged or merge into a droplet; The dilution droplet; Mix droplet; Stir droplet; Make little droplet deformation; Make droplet keep original position; The incubation droplet; The heating droplet; The evaporation droplet; The cooling droplet; Arrange droplet; Droplet is migrated out microdrive; Other droplet operations described herein; And/or above-mentioned any combination.Moreover, the present invention includes by between two or more humidity provinces of droplet microdrive, shifting the method that droplet heats and/or cool off droplet.In addition, the present invention includes the method for heating and/or cooling droplet, wherein, between two or more humidity provinces of droplet microdrive, shift droplet when the scope of described humidity province during at about 40 ℃ to about 120 ℃.The present invention also comprises the method for heating and/or cooling droplet, wherein when the scope of described humidity province during at about 40 ℃ to about 120 ℃, between two or more humidity provinces of droplet microdrive, shift droplet, so that reach the target temperature, wherein the described target temperature of at least a portion is to be higher than about 70,75,80,85,90,95 or 100 ℃ temperature.

The present invention includes droplet microdrive or droplet microdrive system, it has one or more input pond, described input pond has loaded the reagent that is used to carry out biochemical reaction, the reagent that for example is used for nucleic acid amplification scheme, the mensuration scheme based on avidity, order-checking scheme and is used to analyze the scheme of biological fluid.For example, one or more pond can comprise the reagent that is used to provide damping fluid, primer, Nucleotide, polysaccharase and carries out other reagent of PCR reaction.In one embodiment, one or more pond comprises damping fluid, and it comprises two or more reagent that are used to carry out the PCR reaction, and wherein said reagent is selected from primer, Nucleotide, polysaccharase and other PCR reagent.In another embodiment, one or more described pond comprises droplet, and described droplet comprises and carry out all required reagent of PCR reaction, like this when with comprise nucleic acid-templated sample droplet merging after, what obtain is the ready-made droplet that is used for the PCR thermal cycling.The present invention also comprises droplet microdrive or droplet microdrive system, and it has one or more input pond that has loaded the sample that is used to carry out the PCR reaction.

8.1.4 amplification scheme

It will be understood by those in the art that according to content disclosed herein, many variations still belong to scope of the present invention.Generally, described scheme comprises two or more droplets that comprise PCR reagent and template of merging with generation PCR instant droplet, and in the selected temperature that helps amplifying target nucleic acid described PCR instant droplet is carried out thermal cycling.

In the upstream, described scheme can comprise various sample preparation steps, so that be provided for the ready-made nucleic acid-templated of pcr amplification.For example, can before PCR, carry out the reverse transcription of RNA to provide the stable DNA that is used to increase nucleic acid-templated.Therefore, in one embodiment, the invention provides preparation and comprise the method for the nucleic acid-templated droplet of DNA, wherein said method comprises that the reverse transcription that carries out based on the RNA of droplet is described nucleic acid-templated to produce.

Can adopt ＂ warm start ＂ method will react that the formation of primer dimer minimizes in the set-up procedure.By limit polymerization enzymic activity before the PCR circulation, can reduce non-specific amplification and increase target output.Common heat start PCR method comprises chemically modified, cured barrier approach and uses the antibody at Taq to suppress.

In the downstream, described scheme can comprise various subsequent steps, and the nucleic acid that increased of order-checking for example is as adopting tetra-sodium order-checking (pyrosequencing) method or adopting the fragment of capillary electrophoresis separation amplification.

Also can in the PCR process, use various special technology.For example, can use the primer that has with described nucleic acid-templated incomplete complementary sequence to carry out vitro mutagenesis based on droplet.Therefore, for example, the present invention can be included in the method for implementing vitro mutagenesis in the droplet on the droplet microdrive, wherein said method comprises two or more droplets of merging, described droplet comprises PCR reagent and the selected primer that is used for mutagenesis, the mutant form of the described target nucleic acid that presents in an amount at least sufficient to help to increase.In addition, the target nucleic acid of mutant form can be transferred and be used for downstream processing, as to as described in the target nucleic acid of mutant form check order to confirm required sudden change.

Aspect medical diagnosis of the present invention, can use molecular label for example digoxigenin (DIG) or biotin labeled dUTP to detect specific sequence.The PCR product of mark can, for example, as hybridization probe, or use capture probe to detect.

In many schemes, need handle one or more contrast droplet simultaneously to determine the quality or the verity of reaction.Therefore, for example,, can carry out thermal cycling to one or more PCR instant droplet that does not contain the sample template, and otherwise processing mode is identical with the droplet that comprises the sample template in order to guarantee not pollute.The nucleic acid representative that detects amplification in the contrast droplet exists pollutes.Other contrast droplet can comprise the mould material of known quantity or concentration or the fluorescence dye of known quantity or concentration.The contrast droplet can be handled simultaneously, handle before it and/or afterwards with real sample droplet on same droplet microdrive with described sample droplet.

Described system can carry out independently customization based on software to the reaction scheme of each sample or mensuration and condition.This point combines with the upgradability of this platform, guarantees the ability of described system to be extended to large-scale target nucleic acid.

Present invention resides in and carry out the droplet method of operating on the amplifing reagent.For example, present invention resides in and carry out one or droplet method of operating repeatedly on the droplet that comprises damping fluid, primer, Nucleotide, polysaccharase and/or other PCR reagent.The present invention comprises that also the damping fluid droplet that comprises one or more primer that uses on the droplet microdrive carries out one or droplet method of operating repeatedly.The present invention comprises that also the damping fluid droplet that comprises one or more Nucleotide that uses on the droplet microdrive carries out one or droplet method of operating repeatedly.The present invention comprises that also the damping fluid droplet that comprises one or more polysaccharase such as archaeal dna polymerase that uses on the droplet microdrive carries out one or droplet method of operating repeatedly.The present invention comprises that also the damping fluid droplet that comprises one or more reversed transcriptive enzyme that uses on the droplet microdrive carries out one or droplet method of operating repeatedly.In another embodiment, the present invention includes the damping fluid droplet that uses on the droplet microdrive and carry out one or droplet method of operating repeatedly, described damping fluid droplet comprises 2,3,4 or more kinds of PCR reagent, and described reagent is selected from the category that comprises primer, Nucleotide, polysaccharase and other PCR reagent.In addition, the present invention includes and use PCR instant droplet to carry out one or droplet method of operating repeatedly, described PCR instant droplet comprises one or more cushion, primer, Nucleotide, polysaccharase and comprises the nucleic acid-templated of target nucleic acid sequence.

The content that also can be used for analyzing RNA based on the amplification scheme of droplet.In some embodiments, RNA is a primary target nucleic acid.Can use the double buffering system, wherein for reverse transcription (RT) step that produces cDNA from viral RNA provides a kind of cushion, and selected different cushion is used for the DNA cloning step.In relevant embodiment, use single cushion method, the cushion that wherein provides can be suitable for this two kinds of reactions, does not need one of them is optimized.

In one embodiment, can on the droplet microdrive, implement the change with the gene expression dose of quantitatively relevant carcinobiology marker, as vascular endothelial growth factor (VEGF) and cyclin-dependent kinase inhibitor p21 (Cip1) and p27 (Kip1) based on the PCR of droplet.For example, the cell in the cleavable droplet, no matter its be suspend or be incorporated into the surface.Can use pearl, for example few (dT) magnetic responsiveness pearl catches poly (A) mRNA of release in droplet.Reagent can be available from the mRNA DIRECTMicro Kit of Dynal Biotech.Can mix or stir droplet with enhancing lysis, and increase poly (A) mRNA that is captured on the pearl.Can the RNA-DNA duplex be unwind with the mRNA of wash-out from few (dT) magnetic responsiveness pearl by heating.Suitable temperature depends on the length of chain.Can use the described washing scheme of other parts of this paper to wash pearl based on droplet.Can adopt scheme based on droplet, with at the suitable primer of target gene (for example VEGF, p21 (Cip1) and p27 (Kip1)) at the enterprising performing PCR of described droplet microdrive (for example qRT-PCR).Also can use the RNA amplification of the technology implementation of Van Gelder and Eberwine based on droplet.

The invention provides DNA rolling circle amplification based on droplet.In rolling the ring method, on the droplet microdrive, heat the damping fluid droplet that contains dsDNA and reach the temperature (typically being about 95 ℃) that is enough to cause the dsDNA sex change.The incubation time can be about 1 to about 10 minutes in some cases.But the droplet that will comprise the cyclisation probe merges with the droplet that comprises denatured DNA, but so that make cyclisation probe and target dsDNA efficient temperature (for example about 60 ℃) and contain polysaccharase (for example yellow wriggle shape mould (T.flavus) archaeal dna polymerase) and the damping fluid of suitable ligase enzyme (for example Ampligase dna ligase) in anneal and be connected.In some cases, but incubation is less than about 45 minutes.With the gained droplet with roll ring primer, damping fluid,

29 archaeal dna polymerases merge down in the temperature (for example about 31 ℃) that reduces.In some cases, the incubation time can be about 2 to about 30 minutes.Available

29 archaeal dna polymerases mix vitamin H catching streptavidin pearl or lip-deep amplicon, and observe with fluorescent probe.

The invention provides DNA strand displacement amplification (SDA) based on droplet.In this embodiment, will comprise that the segmental damping fluid droplet of the dsDNA with target-specific amplimer heats on the droplet microdrive, reach the temperature (typically being about 95 ℃) that is enough to make the dsDNA sex change.In some cases, the incubation time can be less than about 4 minutes.Then droplet is cooled to annealing temperature (for example about 37 ℃) so that annealing.In some cases, annealing time can be less than about 4 minutes.By the droplet operation described droplet and the droplet that comprises restriction enzyme and exo (-) KJenow polysaccharase are merged.On described droplet microdrive the gained droplet is carried out the isothermal incubation, temperature is enough to cause DNA cloning (for example about 37 ℃).In some cases, the incubation time can be about 1 to about 5 hours.Can use, for example, fluorescent probe or chain specific molecular beacon detect amplification.

The invention provides amplification or NASBA based on the transcriptive intermediate of the RNA of droplet.In this embodiment, the droplet that will comprise target nucleic acid on described droplet microdrive is heated to the temperature (for example about 95 ℃) that is enough to make the target sex change.In some cases, the sex change time can be less than about 4,3 or 2 minutes.Then droplet is cooled to suitable temperature (for example about 41 ℃), and utilizes droplet operation and the droplet that comprises sub-target primer 1 of T7 rna polymerase promoter and target primer 2 to merge.The gained droplet merges by droplet operation and the droplet that comprises reversed transcriptive enzyme, RNAse H and T7 RNA polymerase.Then the temperature of described droplet being adjusted to is enough to the temperature that causes the RNA amplicon to be amplified.In some cases, proliferation time is sustainable about 60 minutes.

One aspect of the present invention is the droplet microdrive with base material of being used for fixing nucleic acid.In one aspect, described base material is the auri material.Another aspect is the droplet microdrive, and it comprises the base material of being used for fixing nucleic acid and is used for the reagent of described dna immobilization in described base material.Another aspect is the droplet microdrive again, it comprise being used for fixing nucleic acid base material, be used for reagent and the nucleic acid samples of described dna immobilization in described base material.These reagent and sample can, for example, be stored in the pond on the described droplet microdrive and/or pond or other containers (for example tube) that described droplet microdrive is outer in.In yet another aspect, the present invention relates to nucleic acid samples is immobilized onto the method for base material, it comprises implements the droplet operation so that will comprise the droplet of nucleic acid samples contacts with described base material, and thus described nucleic acid samples is deposited on the described base material.

8.1.5 downstream analysis

In some embodiments, the target nucleic acid that comprises amplification can be transferred to the downstream further analyzes.For example, droplet can be transferred to the downstream analyzes by the microgel electrophoresis.The microgel electrophoresis can on the described droplet microdrive or outside carry out.

In one embodiment, use two-dimentional microgel electrophoresis apparatus, for example Mohanty etc. (sees with the described device of U.S. electrophoresis association annual meeting (the American Electrophoresis Society Annual Meeting) Http:// aiche.confex.com/aiche/2005/techprogram/P30621.HTM).

In some embodiments, will comprise that the droplet of the nucleic acid of amplification contacts with the droplet that comprises reagent, described reagent is enough to see that the nucleic acid clone of described amplification goes in the suitable carriers.Can select carrier to be used for, for example, gene library and/or in cell, express.

8.2 nucleic acid sequence analysis

The invention provides the methods, devices and systems that are used for carrying out in droplet microdrive system based on the nucleic acid sequence analysis of droplet, described droplet microdrive system has avoided and the relevant problem of the needed mixture that becomes increasingly complex of the method for prior art.

Figure 3 shows that exemplary droplet microdrive 300, it is suitable for nucleic acid sequence analysis.In this embodiment, can provide a plurality of fluid port or pond, for example DNA input pond 302, DNA reagent pond 304, primer sets pond 306, Nucleotide (for example dA, dC, dG and dT) pond 308 and tetra-sodium sequencing primer pond 310.Washing buffer liquid pool 312 and waste liquid district 314, thermal cycling district 316 and imaging area 318 also can be provided.Various thermal cyclings district embodiment can utilize the well heater of multiple structure, and for example this paper is at the described well heater of other parts.Imaging area 318 can use, for example, and photomultiplier (PMT).

Figure 4 and 5 have shown the reactions steps and the droplet operation of illustrative embodiments of the present invention.Can be as required (on the described droplet microdrive or outside) the amplification of nucleic acid sample, be used for analyzing with the nucleic acid that obtains enough concentration.Nucleic acid samples can be introduced the droplet microdrive and be immobilized onto solid support.Can use the little actuation techniques of droplet will be used to make nucleic acid denaturation to give immobilized nucleic acid as strand, initiation and the agent transfer of progressively extending nucleic acid.Importantly, can for example be used for further processing, analysis and/or waste liquid and dispose with the droplet that comprises reaction product from immobilized nucleic acid call away to.Importantly, in some embodiments, detection can separate on time and space with the extension building-up reactions to be carried out.Only this ability can reduce or eliminate the problem that some degraded byproduct that art methods causes is piled up.Another advantage of the present invention is to detect near transmitter, also therefore improves the susceptibility of analyzing with the efficient that improves the light collection.

The present invention can comprise droplet microdrive or droplet microdrive system, and it has one or more input pond, has wherein loaded the reagent that is used to the scheme of checking order.For example, one or more pond can comprise the reagent that is used to carry out tetra-sodium order-checking scheme.The present invention also can comprise droplet microdrive or droplet microdrive system, and it has one or more input pond, has wherein loaded the sample that is used to carry out tetra-sodium order-checking scheme.

Be appreciated that an importance of the present invention comprises that can use each sequential analysis reagent and/or sample to carry out droplet operates on the droplet microdrive.For example, the present invention can comprise:

(1) droplet microdrive comprises droplet on it, described droplet comprises any one or more reagent and/or the sample that is used to carry out sequential analysis described herein;

(2) device of the present invention or system, it comprises described droplet microdrive;

(3) in described droplet microdrive or system, carry out droplet method of operating or utilize the method for described droplet microdrive or system operation droplet; And/or

(4) utilize described droplet microdrive or system to carry out method based on the sequential analysis scheme of droplet.

For example, described droplet operation can comprise following one or more operation: droplet is loaded in the droplet microdrive; Distribute one or more droplet from the source droplet; Droplet is divided, separates or is split as two or more droplets; Droplet is transferred to another place from a place along any direction; Two or more droplets are converged or merge into a droplet; The dilution droplet; Mix droplet; Stir droplet; Make little droplet deformation; Make droplet keep original position; The incubation droplet; The heating droplet; The evaporation droplet; The cooling droplet; Arrange droplet; Droplet is migrated out microdrive; Other droplet operations described herein; And/or above-mentioned any combination.Various additive method of the present invention, device, system and other aspects will be conspicuous by following description.

8.2.1 sample amplification

Nucleic acid sequence analysis typically begins with the sample of the nucleic acid analyte that comprises amplification or with the sample that comprises the nucleic acid analyte that is used to increase.For the latter, can use standard technique to increase and/or as on the droplet microdrive, using amplification as described in 8.1 joints based on droplet.Can on the droplet microdrive that is used to carry out to increase on the same droplet microdrive of sequential analysis scheme and/or is separating, increase.In some embodiments, use the second droplet microdrive that is connected with the fluid mode of communicating with sequential analysis droplet microdrive.

8.2.2 dna immobilization

As shown in Figure 4, the nucleic acid samples of described amplification can be immobilized onto in the described droplet microdrive, so that the reagent droplet is contacted with immobilized nucleic acid.Can carry out immobilization, the pearl that for example makes and randomly comprise the magnetic responsiveness material on the surface of described droplet microdrive or other surfaces in the described microdrive by polymkeric substance, fluoropolymer resin.

Can be used for carrying out this type of base material that adheres to and comprise glass, gold, polyacrylamide gel, polypyrrole, tetrafluoroethylene (Teflon) and optical fiber.The form of material can be, for example, and film, particle, matrix or pearl.In one embodiment, described base material comprises the magnetic responsiveness pearl.Described droplet microdrive can comprise magnet or electromagnet, is used to produce magnetic field to control (for example immobilization, release or mobile) described magnetic responsiveness pearl.For example, can use magnetic stirring magnetic responsiveness pearl and the magnetic responsiveness pearl is immobilized onto microdrive to strengthen washing step (seeing 8.6 joints).

Can use multiple technologies that molecule such as DNA are immobilized onto the surface.Example comprises that those are used for the chemical process that oligonucleotide is attached to the chemical process of microarray surface and is used for solid phase synthesis technique.

Can and be adsorbed in golden base material with nucleic acid samples mercaptanization.For example, can oxygen can be with nucleic acid mercaptanization between the non-one-tenth bridge of phosphodiester part Nucleotide by replacing with sulphur, as referring to United States Patent (USP) 5,472,881, Beebe etc., denomination of invention is ＂ Thiol Labeling of DNA for Attachment to Gold Surfaces ＂, incorporates its full content into this paper by reference.The droplet that one or more can be comprised the nucleic acid of mercaptanization is transferred to gold surface on described droplet microdrive, at this place, the nucleic acid samples of mercaptanization will be deposited on described gold surface.The droplet of the DNA that comprises mercaptanization is contacted with described gold surface, and the time of contact is enough to make between sulphur and the gold and forms covalent linkage.Can utilize electricity to drive the surface-area that the increase of (Electroactuation) technology has the droplet of gold.

Can use the plain ester of water-soluble biological or use biotinyl phosphoramidite reagent with 5 of DNA sample '-the end biotinylation.Biotinylated DNA can coated streptavidin base material catch.Therefore, the transferable droplet of biotinylated DNA sample that comprises makes it to contact the streptavidin surface, can catch thus and the described DNA of immobilization.

The chemical process that single stranded DNA is immobilized onto base material is disclosed (S.Taira etc., ＂ Immobilization of single-stranded DNA by self-assembled polymer on goldsubstrate for a DNA chip, ＂ Biotechnol Bioeng.2005 Mar 30; 89 (7): 835-8).In the method, Thioctic Acid (TA) is covalently attached to poly-(hydrochloric acid allylamine) side chain (PAH), so that polymkeric substance is immobilized onto gold surface by self-assembly.The N-hydroxy-succinamide ester can easily be immobilized onto on the gold surface of TA-PAH-bag quilt for terminal probe strand (ss) DNA.This surface can cover polyacrylic acid, and the latter and TA-PAH form ion complex, to reduce cationic charge.

Shown in Fig. 4 step 3, with denaturing agent such as NaOH solution-treated double-strandednucleic acid to produce the strand sample.Show among the figure that this step occurs in after the immobilization step; But, being appreciated that can be before immobilization, in the process or afterwards, makes it to contact the double-strandednucleic acid sample and realizes sex change by shifting denaturing agent.Also can double-stranded complex body pyrolysis chain be carried out sex change by heated sample.

One aspect of the present invention is the droplet microdrive, and it has the base material of being used for fixing nucleic acid.Another aspect is the droplet microdrive, and it comprises the base material of being used for fixing nucleic acid and is used for the reagent of described dna immobilization in described base material.Another aspect is the droplet microdrive again, and it comprises the base material of being used for fixing nucleic acid and is used for reagent and the nucleic acid samples of described dna immobilization in described base material.In these reagent and sample for example can be stored in the pond on the described droplet microdrive and/or described droplet microdrive the is outer pond or container (for example in the tube).In an embodiment of pond charging assembly, reagent and/or sample pool are (for example, in tubule or syringe) can be connected with the fluid mode of communicating with droplet microdrive pond, so that can flow to or directly be pushed described droplet microdrive pond from the liquid of tubule.Of the present invention this is upgradeable on the one hand, and like this, the quantity of pond and pond charging assembly can increase as required, as required for many reagent provide notch, so that carry out required scheme.Reagent/specimen pond and reagent/specimen be carried in 8.8.4 joint and the further discussion of 8.8.5.1 joint.

8.2.3 the Nucleotide that polysaccharase is facilitated mixes

Shown in the step 4 and 5 of Fig. 4, immobilized strand Nucleotide sample is initiated, and produces double-stranded sections.Can make it to contact immobilized sample and realize causing by for example shifting the droplet comprise primer.

There are under the situation of polysaccharase the sample of initiation and deoxynucleotide triguaiacyl phosphate (dNTP) reaction.This reaction can make it to contact with immobilized sample and realizes by shifting one or more droplet that comprises deoxynucleotide triguaiacyl phosphate (dNTP) and polysaccharase.If first base complementrity in dNTP and the described nucleic acid samples strand part, but polysaccharase catalysis is mixed it in DNA chain.Each incident of mixing all follows tetra-sodium (PPi) to discharge, and the amount of release is consistent with the amount of the Nucleotide that mixes.Therefore can determine the incident of mixing by measuring the PPi that discharges.The interpolation of dNTP typically be every next, in the damping fluid that each leisure separates.Along with the order according to chosen in advance or sequencing obtains various Nucleotide, Nucleotide mixes along each immobilized template and carries out successively.

Can use the polysaccharase of multiple natural and improvement.The polysaccharase of improvement comprises for example, having the native sequences of interpolation, insertion or replacement, the ability in the sample that the polysaccharase that obtains has kept promoting Nucleotide to be incorporated into initiation.Polysaccharase can be an excision enzyme defective type polysaccharase.Also can use the big fragment or the Klenow fragment of archaeal dna polymerase.

Can select dATP or ddATP analogue, described analogue does not disturb enzymatic PPi detection reaction, is incorporated in the DNA chain that is prolonging and can disturb normal base pairing but can be aggregated enzyme.The example of suitable analogue comprises deoxidation or two deoxidation ATP's [1-sulfo-] triguaiacyl phosphate (or α-thio triphosphates ester) analogue, as Desoxyadenosine [1-sulfo-] triguaiacyl phosphate or be also referred to as Desoxyadenosine α-thio triphosphates ester (dATP α S).These and other analogue can be referring to United States Patent (USP) 6,210,891, Nyren, etc., denomination of invention is ＂ Method of sequencing DNA ＂, incorporates its full content about this type of analogue into this paper by reference.

Can include single strand binding protein in, by reducing the folding sequence length that can be checked order that prolongs of strand sample.Therefore, for example, the present invention includes the droplet microdrive, it comprises the droplet that comprises single strand binding protein.Described droplet can be the amplification instant droplet that comprises single strand binding protein.Present invention resides in and carry out the droplet method of operating on the droplet that comprises single strand binding protein.

8.2.4 detecting Nucleotide mixes

Determine whether particular bases has been mixed target site and comprised and quantitatively mix the PPi that discharges in the reaction process.When each dNTP adds in the nucleic acid chains that is prolonging in the polymeric enzyme reaction process, all can discharge PPi.The PPi of Shi Fanging can detect with Enzymology method in this case, for example by produce light (hereinafter further discussing) in luciferase-luciferin reaction.Along with continuing of reaction, complementary strand is assembled, and can pass through signal peak definite kernel nucleotide sequence.But this system's generating routine, as shown in Figure 5.

8.2.4.1 PPi is converted into ATP

In some cases, but indirect detection is mixed the PPi that discharges in the incident.For example, can be under the condition that has the enzyme catalyzer the quantitative ATP that produces of APS and realize that PPi is quantitative.When having adenosine 5 ' phosphorus sulfuric ester (APS), the ATP sulfurylase is converted into ATP with PPi quantitatively.Therefore, in one embodiment, PPi can be converted into ATP, and can measure the amount of ATP so that the amount of the dNTP that determines to be mixed in the reaction process.

8.2.4.2 quantitative ATP

In case PPi is converted into ATP, can quantitative ATP mixing with mensuration dNTP.As shown in Figure 5, the luciferin that ATP drives the luciferase mediation transforms to the oxygenate luciferin, and the latter produces visible light, and the amount of generation and the amount of ATP are proportional.The light that the catalytic reaction of luciferase produces can detect by for example electric charge coupling devices (CCD) camera, photorectifier and/or photomultiplier (PMT).Optical signal is proportional with the quantity of the Nucleotide that mixes.Detected signal can be exchanged into the system output visual with the corresponding user of result.

Release with luciferin-luciferase reaction detection PPi is known.For example, Nyren and Lundin (Anal.Biochem., 151,504-509,1985, incorporate its full content into this paper by reference) method based on the continuous monitoring PPi release of ATP sulfurylase and luciferase is disclosed, this method is called enzymatic light-emitting inorganic tetra-sodium detection assay method (Enzymatic Luminometric Inorganic PyrophosphateDetection Assay, ＂ ELIDA ＂).Using the PPi in the droplet on the ELIDA method detection droplet microdrive is one aspect of the present invention.Can improve this method, for example use the luciferase have more thermostability (Kaliyama etc., 1994, Biosci.Biotech.Biochem., 58,1170-1171 incorporates its full content into this paper by reference).The example that is used for the suitable detection enzyme of PPi detection reaction is ATP sulfurylase and luciferase.

Some art methods is used Nucleotide degrading enzyme, apyrase for example, and uncorporated dNTP and excessive ATP degrade.After treating degraded fully, add another kind of dNTP again.Owing to be reflected in the single solution and carry out, therefore the unwanted product that carries out along with order-checking continues to pile up.An importance of the present invention is that it does not need the Nucleotide degradation step.Therefore, although some aspect of the present invention can comprise the Nucleotide degradation step, in other respects, system and method for the present invention has omitted the Nucleotide degradation step especially.Therefore, for example, can under the situation of the Nucleotide degrading enzyme that does not have obviously to measure, realize analyzing, as not have the obviously apyrase of amount.

In addition, the inventor thinks, under the situation that does not have tangible byproduct to pile up, mix/transform/detection reaction makes the result have more predictability.Therefore, for example,,, be difficult to distinguish the concrete quantity of mixing usually along with the increase of the single length of nucleotides of this section if there is one section single Nucleotide in the sample.The inventor thinks that the clear characteristic that reaction of the present invention has can make longer single Nucleotide section have higher accuracy and repeatability.Therefore, for example, the inventor thinks, method of the present invention can accurately detect has 6,7,8,910,11,12,13,14,15 or the single Nucleotide section of polynucleotide more, and accuracy can reach 90,95,99 or 99.9%.

8.2.5 droplet operation scheme

Be appreciated that except such scheme, also can use multiple droplet operation scheme to carry out sequential analysis of the present invention.Therefore, for example, can mix reaction with dNTP in a reaction and be converted into ATP, or described reaction can progressively be carried out: (1) mixes dNTP to discharge PPi, and (2) are converted into ATP with PPi subsequently.Carry out the transferable single droplet that comprises required reagent (dNTP, polysaccharase, ATP sulfurylase and APS) of system together if mix with step of converting.Therefore, can with mix dNTP to immobilized sample, discharge PPi and make PPi and required all reagent of APS reaction generation ATP place pond on the described droplet microdrive, as the single source reagent of every kind of Nucleotide.The source reagent of various Nucleotide contacts with immobilized sample serially, and reaction is taken place, and produces ATP existing under the situation of complementary dNTP.

Separately carry out if mix with step of converting.The source that primer, polysaccharase, dNTP, sulfurylase and APS can separate certainly provides as the reagent droplet that separates, and described reagent droplet combines and carries out reaction of the present invention.Perhaps, can provide some or all these reagent with the form of tetra-sodium sequencing reagent droplet from single source, tetra-sodium sequencing reagent droplet contacts with immobilized sample, to carry out reaction of the present invention.For example, the droplet that comprises dNTP and polysaccharase is contacted with immobilized sample,, discharge PPi thus if complementation then base can be mixed.The droplet that may comprise PPi is then removed from immobilized sample place, and merges with the droplet that comprises ATP sulfurylase and APS, to produce ATP.Perhaps, comprise that the droplet of ATP sulfurylase and APS can merge with the droplet that may comprise PPi under the situation that has the immobilization sample, to produce ATP.

In addition, though Fig. 4 show cause step 4 and mix/step of converting 5 is to take place successively, be appreciated that they can be separated farther or they can be merged in a single step.In other words, various specific reagent can perhaps can provide any combination of plurality of reagents in single droplet or in a series of droplet in the reaction in reasonable time is added to one or more droplet.Therefore, in one embodiment, described droplet microdrive shifts one or more droplet and makes it to contact with the strand sample, wherein, the following reagent (or their function equivalent) that one or more droplet comprises together together or provides with any combination and any order in the droplet that separates: primer, polysaccharase and dNTP, consequently the hybridization of primer and sample forms double-stranded part, the any complementary dNTP of polysaccharase catalysis is incorporated into and the described double-stranded partly target site of the first contiguous strand base position, thereby discharges PPi.

In another embodiment, described droplet microdrive shifts one or more droplet and makes it to contact the strand sample, and the following reagent (or their function equivalent) that provides with any combination and any order is provided described one or more droplet together: primer, polysaccharase, dNTP, sulfurylase and APS, consequently the hybridization of primer and sample forms double-stranded part, the any complementary dNTP of polysaccharase catalysis is incorporated into and the described double-stranded partly target site of the first contiguous strand base position, thereby discharge PPi, and the ATP sulfurylase is converted into ATP with any PPi existing under the situation of APS.Determine mixing of base by the quantitative PPi that discharges.

The droplet that can may comprise ATP by the little actuation techniques of droplet with comprise reagent for example the droplet of luciferase and luciferin converge so that detect any ATP.Similarly, can converge with the droplet that comprises luciferase by the droplet that the little actuation techniques of droplet will comprise luciferin and may comprise ATP and be used to detect any ATP.In addition, can will comprise that luciferase also may comprise the droplet of ATP and comprise that the droplet bout of luciferin is used to detect any ATP by the little actuation techniques of droplet.

Can converge the droplet that is used for detection reaction under the situation of immobilization sample existing or do not have.For example, luciferase/luciferin droplet can converge under the situation that has the immobilization sample with the droplet that may comprise ATP.Perhaps, the droplet that may comprise ATP can separate with treating the immobilization sample that converges with luciferase/luciferin droplet.In any situation, can near transmitter, converge the droplet that comprises the reagent that produces optical signal, so that make detected light quantity maximization.

Remove from the immobilization sample for the treatment of to converge with the luciferase droplet if may comprise the droplet of ATP, this transfer step can comprise incubation step, is used for detecting in fluorescent reaction to produce ATP substantially.In other words, this incubation can carry out in transfer process, and perhaps droplet can temporarily store, in order to carrying out incubation before the fluorescent reaction.Described droplet microdrive can comprise the incubation district that is used for this purpose.This incubation district can comprise or not comprise the heating unit that is used for temperature in the control region.This incubation district can comprise or not comprise the wall of the rest part of separating this district and described droplet microdrive.This incubation district can comprise that electrod-array is transferred to droplet inside and outside this district helping.This district is upgradeable, and can comprise electrode, is used for shifting and storing in this incubation district tens of, hundreds of or more droplet.

When enforcement is of the present invention, one or more detection reagent can be got rid of outside the polymeric enzyme reaction step, in the polymeric enzyme reaction process, will not send any signal like this.For example, in one embodiment, do not add the detection enzyme in the reaction mixture of polymerase step.On the contrary, the droplet that will be used to carry out polymerase step is removed from the immobilization sample, then in the scope of transmitter with comprise that the droplet that detects enzyme converges, and is used to detect the signal from any reaction that takes place.

Therefore the reaction mixture that is used for polymeric enzyme reaction can comprise at least a dNTP, polysaccharase, APS and ATP sulfurylase, and randomly comprises luciferin, lacks the luciferase of any obvious amount simultaneously.So, dNTP mixes reaction and can separate with detection reaction.Therefore can carry out detection reaction under the situation of transmitter existing, as shown in Figure 5, so that make the detection signal maximization.For example, if detection signal comprises light, can in being used to detect the ranges of sensors that produces the light that reaction sends by any gained light, carry out detection reaction.

In addition, detection reaction can parallelly be carried out with the reaction of mixing subsequently, accelerates order-checking speed thus.Similarly, can in the droplet that separates, mix reaction, incubation abreast and before mix the output of reaction and the output of previous incubation is detected, accelerate order-checking speed thus.

Different with some method of prior art is that described droplet microdrive method of the present invention has been avoided the substance transfer effect.Reagent can directly contact with each sample, does not need the reagent stream between various product.If use the magnetic responsiveness pearl, available magnetic field holds them in original position and/or they are transferred to another place from one in droplet.Reagent can directly be transferred to sample and need not to contact other samples from reagent source, and can potentially not cause crossed contamination.Because of droplet is filled fluid isolation, can avoid the disperse of by product.Also can avoid using the hole of the filling pearl (for example stablizing pearl) that may stray light detects.Can apply in each time and use the washing lotion that contains apyrase between the Nucleotide, but this is not necessary in practice of the present invention, and in some embodiments, avoid especially doing like this.

Respectively organize novel agent between the immobilization sample applying, can wash, for example wash with damping fluid to it.Various washing schemes can be referring to 8.6 joints.

8.2.6 use

Nucleic acid amplification of the present invention and sequence measurement, device and system have purposes widely, for example are used for scientific research, medical science and veterinary science diagnosis, pharmacogenomics, gene order-checking, gene expression profile, detection sequence variations, medical jurisprudence and environmental testing.Because the portability that described droplet microdrive of the present invention is brought checks order as needing in the various uses as mentioned herein, examining order can be carried out at the sample collection scene.

In one embodiment, the invention provides the influenza test analysis.In this embodiment, system can receive the biological sample as the input thing, handles the target influenza nucleic acids that sample is used to increase with preparation, uses the solution of the present invention to increase, and detects whether there is influenza nucleic acid.Biological sample can, for example, collect from Nasopharyngeal swabs.

In another embodiment, the invention provides the respiratory tract infection analysis.In this embodiment, system can receive the biological sample as the input thing, handle sample with preparation be used to increase from the common respiratory pathogen nucleic acid of bacterium, virus and/or fungi for example, use the solution of the present invention to increase, and detect whether there is target respiratory pathogen nucleic acid.In the extension form that described respiratory tract infection is analyzed, described analysis can comprise that the test atypia infects, and for example those involve immunologic hypofunction patient's infection.Biological sample can, for example, from Nasopharyngeal swabs.The example of the respiratory pathogen that suitable use respiratory tract infection analysis of the present invention detects comprises: streptococcus pneumoniae (S.pneumoniae), hemophilus influenza (H.influenzae), legionella, chlamydozoan, mycoplasma; Virus is influenza virus, RSV, coronavirus, parainfluenza virus, adenovirus, sex change Pneumovirinae, bocavirus (bocavirus), Hantaan virus for example; With fungi for example lung sac worm, aspergillus tubigensis, cryptococcus.

For the order-checking of longer nucleic acid, as full genome, can be overlapped less fragment (for example 100-1000bp, 200-900bp or 300-800bp) with sample breakage, for example digestion by Restriction Enzyme.These less fragments can use system and method for the present invention to analyze.The result can rebuild long sequence through combination and editor, and this for example is tested and appraised and mates by the segmental lap of order-checking and realizes.This analyzes these fragments in a parallel manner, so that accelerate order-checking.Each template can check order repeatedly with best accuracy.So, whole karyomit(e) even whole genome can accurately be checked order.

Can determine the gene of in particular organization, transcribing by extracting from the mRNA of cell or tissue.Use reversed transcriptive enzyme the mRNA copy can be DNA (cDNA).The cDNAs that obtains can be cloned, and can produce EST from clone's end in cDNA library through method order-checking of the present invention, and the latter provides the express spectra that extracts the tissue of mRNA from it.The RNA spectrum in some cases can be relevant with morbid state, and can be by order-checking as diagnostic tool.But also sequence rna virus.

In case obtain the reference sequence (for example being considered to the gene of involved in diseases) of interesting areas, can check order again and identify the sequence variations that exists between Different Individual or the closely-related species by the sub-fraction of known array being carried out selectivity.Variation can be, for example, and SNP; The difference (insertion/disappearance) of size; Copy number difference (repetition) and reorganization (inversion, transposition).

For example can check order from the colony of the organism of water, soil and/or atmospheric sample.For example, present microbiological most knowledge are still from the indivedual species that cause disease, or the simple culture of conduct is grown under the laboratory condition and the indivedual species that therefore are easy to study easily.Can study colony, member, function and the relation of this type of organism by order-checking.Therefore can be from the quantitative analysis that can not obtain sequence and gene diversity by the organism that routine techniques is cultivated.

System and method of the present invention can be used for providing the diagnosis and the treatment of genome specificity.For example, system can be used for identifying and the more or less favourable related yielding characteristics of treatment result.The result can be used for instructing the treatment decision-making.Similarly, the intracellular sudden change that system and method for the present invention can be used for identifying infectious organisms or has genetics damage or change, for example cancer and other neoplastic diseases, and this information can be used for instructing or confirm the treatment decision-making.Infectious organisms can comprise, for example, and virus, bacterium, parasite or fungi.The invention provides, for example, be used for the cheap fast system and method that detects antibiotic-resistant bacteria or virus (for example new resistance HIV strain), this is the vital component that tackles these pathogenic organisms bodies.

System and method of the present invention can be used for genetics test, for example identifies the intravital DNA sections that works of object or can be used for predicting the DNA sections of specified disease in specified disease.For example, if in the family a kind of marker of form in having the people of certain disease far more than those blood relations of this disease not, promptly provable exist chain.These class methods are proved to be success in Huntington disease, cystic fibrosis, mammary cancer and other diseases.Therefore, for example, system and method for the present invention can be used for that those exist only in the identified gene (gene dosage is enough to cause disease) suffers from the patient or tends to take place sudden change among this disease patient.In another embodiment, system and method of the present invention is used for identifying the genetics variation in following situation: (1) statistics shows that certain sequence is present in the diseased individuals probably, and (2) statistics shows that this sequence is not present in the individuality of not suffering from this disease probably.Similarly, system and method for the present invention is used to identify that the situation of genetics variation is: statistics show have positive test result the people probably certain disease, and the people with negative test result can not get should disease.In addition, system and method for the present invention can be used for the sequenced dna sections to identify one or more SNP.

System and method of the present invention can be used for clinical trial.For example, can check order from the nucleic acid that participates in the trier, disease can compare the variation of the genetics in adverse events and the test group, to identify participant's subgroup that one or more adverse events has high susceptibility takes place.According to the severity of the concrete adverse events of being studied, have the object that correlated inheritance is learned variation, for example, can obtain closer observation, accept further protectiveness treatment and/or withdraw from test fully.Similarly, can compare the genetics variation in result of treatment and the test group, to identify participant's subgroup that more may obtain the positive treatment result.Can and/or lack improperly adverse events and define target colony based on positive findings.Can correspondingly mark product.The doctor can test its patient and whether have relevant genetics variation, and is that the acceptable colony of pharmacology subgroup is opened the product prescription to it is treated only.

The present invention also can be used for medical jurisprudence and identifies that for example: identify possible suspect, its DNA can be complementary with the evidence of staying the crime scene; Make by the guilty person's proof of innocence of misidentification; Identify victim of crime and disaster victim; Establish parent and other family relationships; Auxiliary wildlife official identifies endangered species and protected taxa; But detect the bacterium and the other biological body of polluted air, water, soil and food; Transplanting the type of joining that carries out donor organ and receptor in the works; Determine domestic animal kind or family; With distinguish the consumer's goods for example caviare and the true and false vinous; The food that identified gene is engineered.

Other application examples comprise: measure related between genetics variation and the object result in the medicament research and development process, for example curative effect, untoward reaction spectrum, pharmacokinetics and/or pharmacodynamics; The gene expression characteristics spectrum of analytic target is to distinguish effective pharmacological agent according to genotypic variation; The examination disease susceptibility makes object can adopt measure to monitor, to treat, to avoid or to alleviate the severity of heredopathia; How examination reduces the ADR quantity in the patient colony; For being unsafe medicine in the appraisable colony of heredity subgroup, how examination can use this medicine; Treat with monitoring gene.

8.3 mensuration based on avidity

The invention provides methods, devices and systems, its be used to carry out based on droplet, based on the mensuration of avidity, for example based on the mensuration of avidity.These assay methods comprise wherein utilizes compound that droplet operation will have a binding affinity to analyte and described analyte maybe may comprise the sample of described analyte any assay method that contacts.For example, can in droplet, provide described analyte is had the compound of binding affinity, and transfer make it the contact analysis thing, described analyte is present in another droplet on the droplet microdrive or is immobilized on the surface of droplet microdrive.As another example, the compound itself that described analyte is had a binding affinity can be immobilized onto on the surface of droplet microdrive and/or be immobilized onto on the surface that is included in the pearl on the droplet microdrive, and can will comprise that analyte may comprise that maybe the droplet of described analyte contacts with described immobilized antibody.

Be appreciated that the present invention contains the multiple possible mensuration scheme based on avidity.Based on the example of the mensuration form of avidity comprise direct mensuration based on avidity, based on the indirect measurement of avidity and based on the competitive assay of avidity.These assay methods can be used for detecting whether there is target analyte, and in some cases, also can be used for quantitatively being present in the described target analyte in the sample.In competitive assay, will comprise that the droplet of the antibody of sample antibody and mark contacts with the surface.Described sample antibody combines with the antibody competition of mark and is adsorbed in described lip-deep antigen.A kind of variation of this method comprises by intermediate link antigen is connected in described surface.For example, described connector can be an antibody.Catching bridging measure to use the droplet that comprises sample antibody will be adsorbed in described lip-deep antigen to be connected with antigen in the solution.Another kind method relates to uses biotinylated antigen and bag by the solid phase of streptavidin.The antigen that another kind method relates on making described sample antibody and being immobilized onto described solid phase combines.Can use the second antibody of isotype specific marker to detect generation bonded antibody.Can adopt the excessive antibody of droplet scheme flush away of the present invention.

Being appreciated that an importance of the present invention relates to can use every kind of required mensuration reagent and/or sample based on avidity to carry out the droplet operation on the droplet microdrive.For example, the present invention includes:

(1) droplet microdrive comprises droplet on it, described droplet comprises any one or more reagent and/or the sample that is used to carry out based on the mensuration of avidity described herein;

(2) device of the present invention or system, it comprises described droplet microdrive;

(3) in described droplet microdrive or system, carry out droplet method of operating or utilize the method for described droplet microdrive or system operation droplet; And/or

(4) utilize described droplet microdrive or system to carry out method for measuring based on avidity based on droplet.

For example, described droplet operation can comprise following one or more operation: droplet is loaded in the droplet microdrive; Distribute one or more droplet from the source droplet; Droplet is divided, separates or is split as two or more droplets; Droplet is transferred to another place from a place along any direction; Two or more droplets are converged or merge into a droplet; The dilution droplet; Mix droplet; Stir droplet; Make little droplet deformation; Make droplet keep original position; The incubation droplet; The heating droplet; The evaporation droplet; The cooling droplet; Arrange droplet; Droplet is migrated out microdrive; Other droplet operations described herein; And/or above-mentioned any combination.Various additive method of the present invention, device, system and other aspects will be conspicuous by following description.

8.3.1 sample and specimen preparation

The invention provides the mensuration based on avidity based on droplet, it can be used for detecting multiple analytes.For example, any analyte that can specificity be incorporated into affinity molecules (for example antibody) all is fit to use detecting of system of the present invention.Can analyze one or more target analyte in the single sample.Analyte can be, for example, and biological analyte or synthesis analysis thing.Example comprises the analyte of following classification: the analyte in the analyte of the analyte of the analyte of human origin, the analyte of animal-origin, plant origin, the analyte of bacterial origin, viral source and spore source.Analyte can comprise, for example, and protein, peptide, small molecules and various biological molecule, for example carbohydrate, lipid or the like.In one embodiment, sample is contact immobilized antibody (for example being immobilized onto the antibody on the pearl) earlier, then described immobilized antibody is incorporated on the described droplet microdrive.

What Fig. 6 showed is a kind of exemplary droplet microdrive 600 that is suitable for carrying out immunoassay.This embodiment can comprise two kinds of base materials, the first base material 601a and the second base material 601b, and they are separated by in substantially parallel mode, and a space between two parties is provided.Can in described space between two parties, provide a plurality of fluid port or pond, for example washing buffer liquid pool 602, sample pool 604, primary antibody pond 606, secondary antibodies pond 608 and immobilized antibody (for example being immobilized onto the antibody on the pearl) pond 610.Also can provide waste liquid district 612, and magnet 614, interact between the magnetic field of its modes of emplacement permission magnet and the magnetic responsiveness component in described space between two parties.In this concrete embodiment, transfer electrode 616 provides on described first base material.

8.3.2 the sandwich based on avidity is measured

In one embodiment, the invention provides on the droplet microdrive, carry out, measuring based on droplet based on the sandwich of avidity.Be appreciated that the present invention contains the multiple scheme measured based on the sandwich of avidity of being used to carry out.Following scheme based on droplet is with the basis that is illustrated as of Fig. 7, and it only is an example, and it has no intention to limit the scope of the invention:

(1) will have specific antibody (primary antibody) to target analyte and be immobilized onto the surface;

(2) the described immobilized antibody of washing is as using the washing scheme based on droplet, to remove excessive antibody;

(3) with the droplet operation described immobilized antibody is exposed to the sample droplet that may comprise described target analyte, described target analyte has binding affinity to described immobilized antibody;

(4) washing described immobilized antibody-target analyte mixture, as using washing scheme based on droplet, with remove described sample droplet not in conjunction with component;

(5) described immobilized antibody (may comprise the described target analyte of bonded with it now) is exposed to the droplet that comprises report (secondary) antibody;

(6) the excessive report antibody of flush away is as using the washing scheme based on droplet;

(7) randomly, carry out extra step and provide measurable parameter or signal with product;

(8) measure described measurable parameter or signal; With

(9) provide the output of representing described signal.

Can adopt droplet to operate in and carry out any one or more aforesaid step on the described droplet microdrive of the application.

8.3.2.1 immobilized antibody

Primary antibody is immobilized onto the surface.This surface can be, for example, and the surface of described droplet microdrive or the surface of pearl, for example magnetic responsiveness pearl, non-magnetic responsiveness pearl or particle, for example nano particle.

Can on described droplet microdrive, use scheme based on droplet with antibody immobilization in the surface.For example, can antibody immobilization be introduced described droplet microdrive in the reagent on surface, it is separated into discrete droplets and shift and make it to contact described surface so that deposition with being used for.If described surface is the surface of one or more pearl, described pearl can be introduced described droplet microdrive, be separated into the damping fluid droplet, be transferred on the described droplet microdrive, and converge with one or more droplet that comprises reagent, described reagent is used for described droplet is immobilized onto the surface of described pearl.

Perhaps, antibody can immobilization outside described droplet microdrive.For example, antibody can be immobilized onto earlier on the pearl, is incorporated into described droplet microdrive then.Have multiple commercial protein (for example streptavidin) bag quilt with pearl antibody sandwich.In another embodiment, antibody can be immobilized onto on the surface of described droplet microdrive, as carrying out in the process of droplet microdrive as described in producing.

In addition, but treat surface so that immobilized antibody.For example, the surface that is provided can comprise that antagonist has the part of avidity or the bound fraction that is connected with antibody.Antibody, it randomly comprises bound fraction, can contact with the surface, thus with antibody immobilization in the surface.For example, can with wrap in advance by the pearl of streptavidin introduce described droplet microdrive.With comprise described bag by the droplet of pearl of streptavidin carry out the droplet operation, and make this type of droplet that contains pearl contact the droplet that one or more comprises biotinylated antibody with the droplet operation, thus antibody is connected in pearl.As another example, described droplet microdrive can comprise on the little driving of droplet path by the surface of streptavidin, so just, the operation of available droplet makes the droplet contact bag that comprises biotinylated antibody by the surface of streptavidin, and thus with antibody immobilization in described surface.In another example, optionally capture antibody is immobilized onto the surface of described droplet microdrive, as by picture on surface is drawn so that can immobilized antibody, or by optical active substance being directly connected in antibody or streptavidin, then by using write-through etching system (direct-write light system) or making the described antibody of device selectivity immobilization that exposes light with light hovel or other selectivity.

Known multiple technologies can be with antibodies in the surface.For example, available G protein activation surface makes antibody molecule can pass through its Fc structural domain combination with it, is used for acquisition target and stay the variable region.By activatory antibody and 1, the reaction of 3-diaminopropanes link coupled polystyrene aldehyde radical/sulfuric ester latex (1,3-diaminopropane coupled, polystyrene aldehyde/sulfate latex) can be covalently bonded in antibody the latex surface.By incubation antibody with put together damping fluid (30mM Na ₂CO ₃, 70mMNaHCO ₃, pH 9.5), the available antibodies bag is not had the white polystyrene latex pearl of sulfuric ester (Surfactant-free sulfate white polystyrene latex bead) of surfactant.Biotinylated antibody can be captured to bag by on the base material of streptavidin.Antibody can be covalently bonded in the surface of the modification of described droplet microdrive, as silane or mercaptan layer.Antibody can be covalently bonded in the surface (for example in assembling process or utilize droplet operation that antibody is deposited on the surface) of the modification of described droplet microdrive, for example with hydride modified surface or mercaptan surface.

8.3.2.2 target analyte is incorporated into immobilized antibody

The sample droplet contacted with described immobilized antibody be incorporated into described immobilized antibody with any target analyte that allows to exist in the sample.Can utilize droplet transition of operation sample droplet make it with its on adhere to described antibody the surface contact and realize this step, described surface for example be comprise by antibody pearl droplet or on it immobilization the surface of described droplet microdrive of described antibody.In an alternative embodiment, the surface is the surface of pearl, and pearl contacts with sample earlier, is introduced into described droplet microdrive then.Can wash pearl earlier, be introduced into described droplet microdrive then.Antibodies is from the analyte of described sample droplet.Can accelerate cohesive process by improving substance transfer speed.Several examples of accelerating substance transfer comprise the high speed transfer droplet so that can have the pearl and the target analyte of antibody or replenish target analyte for the surface of immobilized antibody by thorough mixing.Other means comprise by electricity controls that the droplet original position is stirred the droplet of incubation or by multiple external device (ED) piezoelectricity startup method for example.If without any substance transfer, means, binding events depends on diffusion to take place, and this will need the longer time, therefore can prolong minute.In some embodiments, can be on described droplet microdrive described immobilized antibody (for example described pearl or surface) be implemented the washing scheme, as referring to 8.6 joints, to remove excess sample or other materials.

8.3.2.3 the report antibodies is in target analyte

After the washing (for example described pearl or surface), comprise the droplet that the different epi-positions on the analyte are had the report antibody of avidity and can contact the washed immobilized antibody that may contain captive analyte.The antibody conjugates of mark comprise be connected in reporter molecules (for example Geigers) but antibody, enzyme (for example color change, chemoluminescence, fluorescence, chemoluminescence or electrochemistry), chemiluminescent molecule or fluorescence molecule that can the catalysis detection reaction.According to employed reporter molecules, can be subsequently the described immobilized antibody (for example pearl or other surfaces) that comprises analyte and report antibody be implemented the washing scheme, as referring to 8.6 joints, to remove excessive report antibody.

8.3.2.4 produce and detect measurable parameter

Can report that antibody is quantitative to bonded by detecting the signal of facilitating by described report antibody.For example, signal can be radioactivity, color change, chemoluminescence, fluorescence, chemoluminescence, Raman spectrum, light scattering method, particle/pearl gathering, surface plasma body resonant vibration, Raman spectrum effect or the like.Detection can be direct or indirect, can by detection be incorporated into analyte antibody amount or undertaken by the amount that detects unconjugated antibody.

Needs are further reacted to produce the method for signal,, can make the droplet contact antibody-antigen-antibody complex that comprises required additional reactant, to promote described further reaction as non-fluorescence-causing substance is converted into fluorescence-causing substance.

Allowed to report after the antibodies analyte, can adopt the excessive report antibody of washing scheme flush away, and utilized the droplet operation to make the droplet that comprises required report reactant contact described immobilized antibody.In one embodiment, described report antibody labeling can catalysis produce the enzyme (for example horseradish peroxidase (HRP) or alkaline phosphatase (ALP)) of the reaction of measurable parameter.For example, but the HRP catalyzing hydrogen peroxide produces electrochemical signals, and it can detect by measuring curtage.Can by catalytic by HRP be that the fluorescent reaction of substrate detects bonded antibody with Amplex Red and hydrogen peroxide.Another example uses the NBT of alkaline phosphatase mediation to the purple first

(formazan) conversion, it can detect by colorimetric method in droplet.In another method, chemical luminous substrate is luminol,3-aminophthalic acid cyclic hydrazide or can be produced chemiluminescence signal by HRP catalysis from the Ps-atto of Lumigen for example.The example of the detection method that other are suitable is discussed (for example seeing the 8.3.5 joint) in other parts.

In one embodiment, detect step in the droplet on described droplet microdrive existing under the situation of transmitter, so that strengthen or maximization catches self-reacting signal.In another embodiment, leave transmitter and react, and with droplet operation droplet is transferred to the transmitter place and detects.

8.3.2.5 alternative sandwich measuring method

Be appreciated that multiple alternative method is feasible.For example, not necessarily need carry out described step, as can be before immobilized antibody as described in analyte is exposed to or will report that simultaneously antibodies is in analyte according to above-mentioned order.In another method, the combination of capture antibody, analyte and report antibody all can be carried out simultaneously, they is provided to catch the site and with after scouring then.In certain methods, for example surperficial enhancement type resonance Raman scattering can not need washing.

8.3.3 competitive assay based on avidity

In one embodiment, the invention provides the competitive assay that on the droplet microdrive, carries out based on avidity.Be used for based on the analyte of the competitive assay of avidity typically those because of too little can not according to sandwich measure need be in conjunction with the analyte of two kinds of antibody.Be appreciated that the present invention contains multiple scheme of carrying out based on the competitive assay of avidity.Scheme based on droplet given below is with the basis that is illustrated as of Fig. 8, and it only is used for illustrative purposes, and has no intention to limit the scope of the invention:

(1) will have specific antibody immobilization in the surface to target analyte;

(2) the described immobilized antibody of washing is as using the washing scheme based on droplet, to remove excessive antibody;

(3) provide the sample droplet that may comprise target analyte and comprise the report analysis thing;

(4) described immobilized antibody is exposed to the target analyte/report analysis thing droplet of merging, so that described report analysis thing and any target analyte competition binding site;

(5) the washing base material is to remove unconjugated analyte and report analysis thing;

(6) randomly, carry out extra step to produce measurable parameter or signal; With

(7) quantitative bonded report analysis thing, the amount of wherein said report analysis thing and the amount of described target analyte are inversely proportional to.

Can on the described droplet microdrive of the application, use droplet to control technology and carry out any one or more aforesaid step.Each step further goes through at follow-up chapters and sections.

8.3.3.1 immobilized antibody

Can save described immobilized antibody according to 8.4.1.1.

8.3.3.2 competitive combination

The droplet that will comprise the sample that may comprise target analyte merges with the droplet that comprises described report analysis thing, makes the droplet of merging contact described immobilized antibody.Perhaps, can in loading the process of described droplet microdrive, carry out mixing of target and reporter molecules.

Described report analysis thing can will report that nucleic acid is incorporated into analyte and prepares by any of multiple conjugation methods.In one embodiment, the analyte part is by molecular modification, and the biological example element has produced the secondary site of catching like this, being used for fixing streptavidin transmitter DNA mixture.Vitamin H and analyte must not combine undue interference its with the combining of capture antibody.In some cases, biotinylated material can be competed comparably with the analyte from specimen.Biotinylated analyte can take place before or after biotinylated analyte is by described immobilized antibody capture with combining of reporter molecules.

For example, in one embodiment, the droplet of the biotinylated analyte by will having known quantity mixes with the sample droplet of the unmodified analyte that may contain unknown quantity to be measured.The droplet that will comprise biotinylated analyte merges with the droplet that may comprise target analyte.The droplet that merges contacts with described immobilized antibody, so that biotinylated analyte combines described immobilized antibody with target analyte (if present) competition.The amount of the biotinylated analyte of institute's bonded is inversely proportional to the amount of testing the analyte in the droplet.Described immobilized antibody (pearl or surface as described) can carry out the washing scheme subsequently, as referring to 8.6 the joint, to remove excessive report analysis thing.

8.3.3.3 detect described report analysis thing

After the washing, have the streptavidin-vitamin H-droplet of reporter molecules mixture and be added to the droplet of the pearl that comprises washing, perhaps contact with the surface that comprises described immobilized antibody.Streptavidin-vitamin H-reporter molecules mixture in conjunction with any by the biotinylated analyte of the described antibody capture on the described pearl.

8.3.3.4 alternative competitive assay method

Competitive assay based on avidity described herein only is an example that is adapted at the droplet microdrive scheme implemented on the described droplet microdrive of the present invention.Multiple possible alternative form is contained in the present invention.For example, described step is not limited to given order.Can merge by droplet and one or more droplet that comprises target analyte/report analysis thing that will comprise free antibodies, thereby elder generation in target analyte/report analysis thing, contacts being used for fixing with antibody with described surface with antibodies subsequently before antibody immobilization.Described report analysis thing can merge on described droplet microdrive by the droplet that will comprise these two kinds of reagent with report nucleic acid and be puted together.The droplet that comprises described report analysis thing can merge with comprising the droplet of reporting nucleic acid, so that realized puting together before or after described report analysis thing is exposed to capture antibody.

In another embodiment, the droplet of the analyte of the enzyme labelling by will containing known quantity mixes with the sample droplet that contains target analyte, further mixes being at war with property mensuration then with the droplet that contains antibody.What compete is the analyte of mark and the binding site between the target analyte.The activity of enzyme reduces after the analyte of enzyme labelling and the antibodies, can monitor this by the change event (for example absorption) of number of different types, so that the concentration of quantitative sample target test thing.For example, containing the droplet that is marked with glucose-6-phosphate dehydrogenase (G6PD) (G6P-DH) vancomycin can mix with the sample droplet that contains unlabelled vancomycin, further mixes with the droplet that contains with vancomycin has reactive antibody, G-6-P and a Reduced nicotinamide-adenine dinucleotide (NAD) then.The activity of G6P-DH reduces behind the binding antibody.G6P-DH is with NAD ⁺Be converted into NADH, cause changing with the absorbance that spectrophotometry is measured at the 340nm place.After in described droplet microdrive, calibrating, can determine the concentration of vancomycin in each unknown sample by the absorbance that obtains in the working curve that stores and the sample determination.Other analytes that can adopt identical method to detect comprise valproic acid, tobramycin, gentamicin and caffeine.

8.3.4 other are based on the mensuration scheme of avidity

Competitive assay based on avidity described herein only is an example that is adapted at the droplet microdrive scheme implemented on the described droplet microdrive of the present invention.Multiple possible alternative form is contained in the present invention.For example, described droplet microdrive of the present invention system can make single sample is carried out a plurality ofly carrying out single mensuration or its combination based on avidity based on the mensuration of avidity or to a plurality of samples simultaneously.In addition, can carry out with other tests based on the mensuration of avidity, for example PCR and/or immunity-PCR.

According to the instruction of this specification sheets, can use based on the polytype mensuration of the scheme implementation of droplet.Example comprises immune precipitation determination; Immunoradiometric assay; The mensuration based on avidity of heterogeneous enzyme labelling, wherein quantitatively the combination of antibody and not binding constituents need these two kinds of compositions of physical sepn; The mensuration based on avidity of uniformity (non-separation) enzyme labelling, it does not need these two kinds of compositions of physical sepn, because can functionally distinguish unconjugated and bonded antibody.For immune precipitation determination, comprise that the droplet of the reagent that is used to carry out immune precipitation determination merges to carry out immune precipitation determination on the droplet microdrive.Can use light scattering detector to detect immunoprecipitation.

Although most methods of being discussed till now all relate to the immobilization of antibody or analyte, immobilization is not to be that all immunoassay based on droplet of the present invention are necessary.For example, the present invention includes uniformity based on the multiple immunoassay of the enzyme of droplet, wherein the antibody of mark comprises and the enzyme that is inactivated after primary antibody combines.Enzymic activity is roughly proportional with analyte concentration.This method is included in substantially on the droplet microdrive to merge and is used to carry out based on the droplet of the multiple immunoassay of enzyme of droplet and measures the gained enzymic activity.

In another method, the light scattering characteristic of antigen/antibody mixture changes behind binding events, and can monitor this change by the scattering of light change (for example by turbidity measurement) that detects in the reaction droplet on the described droplet microdrive, to identify and/or quantitative capturing events.For example, can utilize the droplet operation that the physiology sample droplet that contains immunoglobulin (Ig) (for example IgA, IgG and IgM (after specimen preparation comprises dilution and adds polymkeric substance)) or lipoprotein (for example ApoAl, ApoB (after specimen preparation comprises dilution and adds polymkeric substance or surfactant)) on the described droplet microdrive is merged with the droplet that contains corresponding antibodies, after binding events takes place, the turbidity that causes the droplet of merging changes, and this change can be monitored by spectrophotometry.Several examples of other analytes that can measure by this technology comprise: α ₁-antitrypsin (AAT), Transferrins,iron complexes, prealbumin, haptoglobin, complement C3 and complement C4.

Be suitable for another kind of immunoassay of the present invention and comprise CA, it can droplet form carry out.The droplet that contains analyte with contain the droplet that combines capture antibody or antigenic particle (for example latex particle) and mix.If there is target analyte in the sample, latex particle promptly begins aggegation together, and can be by measuring absorbancy quantitatively.

Native system provides the mensuration based on avidity of multi-path.In one embodiment, system can use immunoassay to detect 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 or more kinds of analyte.In some high throughput analysis, system provide 96,384,1536 or the multi-path of greater amt analyte detect, it can serial mode carries out or parallel mode carries out.Analyte can comprise, for example, and the analyte in natural or non-natural source.In another embodiment, the present invention can implement to detect based on the mensuration scheme of avidity one or more analyte of any 2,3,4 or 5 kinds that come from following classification: the analyte of the analyte of bacterial origin, the analyte of viral source, originated from fungus, archon analyte and small molecules toxin analysis thing.

System can be turned to as required replication to increase single target result's confidence level by program.Importantly, described droplet microdrive system can be turned to by program the positive findings property confirmed is measured to reduce false-positive possibility again.System can also and be configured to allow to store the sample of testing by sequencing on described droplet microdrive and be used for follow-up additional experiments chamber test.

The important advantage of isomery of the present invention is that described droplet microdrive can generate working curve rapidly and accurately.Described droplet microdrive can be accurately and can repeatedly be distributed the solution droplet of the check analysis thing with concentration known, and can be by this type of droplet and damping fluid droplet being merged and being diluted, so that a series of contrast droplets with different concns check analysis thing to be provided.These contrast droplets can carry out with the same scheme of sample droplet to produce working curve.Working curve can be used for determining the amount of analyte in the sample droplet.

8.3.5 be used for other measuring methods based on the mensuration of avidity

Multiple detection method can be used for the mensuration based on avidity based on droplet of the present invention.Selected method can based on droplet based on the mensuration of avidity in produce signal directly or indirectly.Can by be placed on contact with droplet or with its very approaching sensor detecting to described signal.Be suitable for comprising the signal that following material produces: radioisotopic tracer, fluorescent marker, luminous marker, electroluminescent marker, particulate, nano particle, enzyme reaction, aggegation compound, Raman active dyestuff, electroactive marker and influence conductive marker based on the example of the signal of the mensuration of avidity.The example of suitable radioisotopic tracer comprises: ⁵⁷Co, ³H, ³⁵P, ³⁵S and ¹²⁵I.In one embodiment, radioisotopic tracer be used on the droplet microdrive scintillation proximity assay (scintillation proximity assay, SPA).SPA can detect binding events and need not washing step.Radioactively labelled substance can, for example, be incorporated in the competitive analysis thing of competitive assay, or be incorporated in the secondary antibodies that sandwich measures.The radioactively labelled substance that discharges α or weak beta-particle is preferred.SPA carries out near fluorophore, and described fluorophore is launched light after being exposed to radioactively labelled substance.For example, fluorophore can be provided in the pearl, the combining antigen or primary antibody surface or connected in the nano particle of antigen or primary antibody of described droplet microdrive.The example of suitable luminous marker comprises acridinium ester, rhodamine, two evil diketone, acridine, phenanthridiniums and various isoluminol derivative.The example of suitable fluorescent marker comprises fluorescein and Eu ³⁺The example of suitable enzyme labelling thing comprises that those produce the enzyme labelling thing of visible, coloured, fluorescence and/or luminous product from suitable substrate.For example, suitable enzyme can comprise penicillinase, horseradish peroxidase, beta-galactosidase enzymes, urease, desaminase and alkaline phosphatase.The example of suitable nano particle comprises metal nanoparticle.The relevant further information that is suitable for the detection method of the mensuration based on avidity of the present invention is seen 8.11 joints.

8.3.6 sample size and finding speed

Use the digital micro-fluid platform greatly degree reduce the volume and the expense of equipment, mainly be to be to make all liquid processing capacity to minimize.In some embodiments, measure the sample and the reagent that use and be less than used percentum of present method or some thousandths of, but have identical susceptibility and specificity.In one embodiment, typically to use drop volumes be 1 μ L or still less or 100 receive and rise or sample still less carries out mensuration based on avidity in described system.

Other advantages comprise, used miniature form has faster kinetics and multi-path has higher flux owing to measure, and can reduce the used time of result that obtains.For example, in one embodiment, described system implementation can detect 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 or more kinds of analytes being less than based on the mensuration scheme of avidity in about 60,50,40,30,20,15,10,5 or 2 minutes time.

8.3.7 application based on the mensuration of avidity based on droplet

As mentioned above, the mensuration based on avidity of the present invention can be used for detecting multiple molecule.Any molecule that is incorporated into affinity molecules (for example antibody) with avidity all is suitable analytes.Analyte can comprise, for example, and biomolecules or synthetic molecule.Biology can comprise, for example, and from the molecule of plant, animal, microorganism and virus.The synthetic molecule can comprise, for example, and industry byproduct, pollutent and medicine.Analyte can comprise that there are the analyte of organism in toxin or representative, as infectious diseases, or is used for the toxin of bioterrorism or biological warfare.The example of this type of organism comprises anthrax, bird flu, sausage poisoning, Hantaan virus, l, pneumonic plague, smallpox, tularaemia and hemorrhagic fever virus (VHF).Other examples comprise the analyte relevant with the immunogenicity monitoring of anticancer disease vaccine, chronic infectious disease (for example HIV, malaria, hepatitis C, moniliosis), immunology (for example irritated, autoimmunization), incretology (Tiroidina, non-Tiroidina), drug test (for example drug abuse, curative drug) and the detection of biological terrorism factor (for example anthrax, smallpox).

In one embodiment, immunoassay is used for specimen and whether has organism, for example bacterium or virus.In some embodiments, system can realize very responsive detection, even can detect individual cells.In addition, some embodiments can comprise PCR typing of bacteria or variation.

Mensuration based on avidity of the present invention also can be used for detecting chemical, biological or explosive hazardous articles.For example, the trace explosive substance that can be used for existing in the test sample at the antibody of explosive substance.Therefore, the present invention also is provided for representative in area of examination and has biological, chemical or the chemical analyte of explosive weapon or the method for biological analyte.

In one embodiment, the present invention can detect the multiple analytes in the single droplet.According to the present invention, a kind of mode that realizes this purpose relate to on the described droplet microdrive spatially the different analytes of separated position use different antibody.For example, described droplet microdrive can comprise a plurality of surfaces, and each surface comprises a specific antibody.Can control the single sample droplet and make it to contact simultaneously these antibody, perhaps when described droplet is transferred by antibody regions, contact these antibody successively.Mensuration scheme based on avidity of the present invention can be used for detecting whether have an analyte that is incorporated into the antibody in any described various zones.Similarly, can use different markers to detect different analytes in the same space zone simultaneously, in single droplet, detect multiple analytes thus.In another embodiment, described droplet microdrive comprises the pearl that is separated out on the space, and each pearl or each group pearl have antibody a kind of or one group of uniqueness.Can utilize droplet to operate and control the sample droplet and/or contain the droplet of pearl so that the sample droplet contacts with each described pearl or every group of described pearl.

8.4 immuno-PCR

The invention provides the immuno-PCR (pick) based on droplet, it is used for selectivity and detects the analyte that only exists with the trace level.This invention made up use based on detection antibody on the platform of droplet various based on avidity the mensuration means and use the nucleic acid chains thing that serves as a mark.Adopt the amplification technique this nucleic acid chains (for example seeing 8.1 joints) that increases.

Being appreciated that an importance of the present invention relates to can use various required iPCR reagent and/or the sample on the droplet microdrive to carry out droplet operation.For example, the present invention includes:

(1) droplet microdrive comprises droplet on it, described droplet comprises any one or more reagent and/or the sample that is used to carry out the iPCR scheme described herein;

(2) device of the present invention or system, it comprises described droplet microdrive;

(3) in described droplet microdrive or system, carry out droplet method of operating or utilize the method for described droplet microdrive or system operation droplet; And/or

(4) utilize described droplet microdrive or system to carry out method for measuring based on avidity based on droplet.

For example, described droplet operation can comprise following one or more operation: droplet is loaded in the droplet microdrive; Distribute one or more droplet from the source droplet; Droplet is divided, separates or is split as two or more droplets; Droplet is transferred to another place from a place along any direction; Two or more droplets are converged or merge into a droplet; The dilution droplet; Mix droplet; Stir droplet; Make little droplet deformation; Make droplet keep original position; The incubation droplet; The heating droplet; The evaporation droplet; The cooling droplet; Arrange droplet; Droplet is migrated out microdrive; Other droplet operations described herein; And/or above-mentioned any combination.Various additive method of the present invention, device, system and other aspects will be conspicuous by following description.

8.4.1 sandwich iPCR

In one embodiment, the invention provides the sandwich iPCR that on the droplet microdrive, carries out based on droplet.Generally, described sandwich iPCR comprises:

(1) will have specific antibody immobilization in the surface to target analyte;

(2) the described immobilized antibody of washing is as using the washing scheme based on droplet;

(3) described immobilized antibody is exposed to the sample droplet that may comprise described target analyte;

(4) the described immobilized antibody of washing is as using the washing scheme based on droplet;

(5) described immobilized antibody (may comprise the described target analyte of bonded with it now) is exposed to comprises the washing of puting together in the second antibody of nucleic acid molecule;

(6) the described immobilized antibody of washing is as using the washing scheme based on droplet;

(7) the described nucleic acid of amplification and detect described amplification (example if any) so that determine the existence of acquisition target analyte and it is carried out quantitatively.

Can on the described droplet microdrive of the application, use droplet to control technology and carry out any one or more aforesaid step.Each step further goes through at follow-up chapters and sections.

8.4.1.1 immobilized antibody

Primary antibody is immobilized onto the surface.Described surface can be, for example, and the surface of described droplet microdrive or the surface of pearl, for example magnetic responsiveness pearl.Can on described droplet microdrive, use scheme to realize antibody immobilization in the surface based on droplet.For example, can antibody immobilization be introduced described droplet microdrive in the reagent on surface, it is separated into discrete droplets and shift and make it to contact described surface so that deposition with being used for.If described surface is the surface of one or more pearl, described pearl can be introduced described droplet microdrive, be allocated in the damping fluid, shift as droplet, and converge with one or more droplet that comprises reagent, described reagent is used for described droplet is immobilized onto the surface of described pearl.

Known multiple technologies can be with antibodies in the surface.For example, available G protein activation surface makes antibody molecule can pass through its Fc structural domain combination with it, is used for acquisition target and stay the variable region.By activatory antibody and 1, the reaction of 3-diaminopropanes link coupled polystyrene aldehyde radical/sulfuric ester latex can be covalently bonded in antibody the latex surface.By incubation antibody with put together damping fluid (30mM Na ₂CO ₃, 70mMNaHCO ₃, pH 9.5), the available antibodies bag is not had the white polystyrene latex pearl of sulfuric ester of surfactant.Biotinylated antibody can be captured to bag by on the base material of streptavidin.Also can carry out the immobilization that light instructs, for example referring to of the description of this specification sheets in other parts.

8.4.1.2 target analyte is incorporated into immobilized antibody

The sample droplet contacted with described immobilized antibody be incorporated into described immobilized antibody with any target analyte that allows to exist in the sample.Can by shift the sample droplet make it with its on immobilization the surface of described droplet microdrive of described antibody contact and realize this step.In another embodiment, can by with the sample droplet with comprise it on immobilization the droplet of pearl of described antibody contact and realize this step.Described antibodies is in the analyte from described sample droplet.Can implement the washing scheme to described immobilized antibody (for example described pearl or surface), as saving referring to 8.6.

8.4.1.3 antibody-NA is incorporated into target analyte

After the washing (for example described pearl or surface), can make to comprise the washed immobilized antibody that may contain captive analyte of droplet contact that the different epi-positions on the analyte is had the antibody-NA conjugate of avidity.Antibody-NA conjugate comprises the nucleic acid molecule that is connected in described antibody.Described nucleic acid molecule is with acting on the nucleic acid-templated of amplification.Can implement the washing scheme to described immobilized antibody (for example described pearl or surface), as saving referring to 8.6.

8.4.1.4 amplification of nucleic acid

Amplification of nucleic acid is for example referring to 8.1 joints.Measure the amount of the amplified production that produces, detect as adopting real-time fluorescence, electrochemistry and/or electrochemiluminescence detect.The amount of the PCR product that produces is relevant with the amount of bonded antibody-DNA, and the latter is depended on the amount of the analyte that exists in the described sample droplet conversely.

8.4.1.5 alternative method

In an alternative embodiment, usually with these steps come out of order be based on the mensuration of avidity subsequently to carry out nucleic acid amplification, the latter produces optical signalling or electrical signal.In this order, can carry out immunoassay and monitor nucleic acid amplification.

8.4.2 competitive iPCR

In one embodiment, the invention provides the competitive iPCR that on the droplet microdrive, carries out.The analyte that competitive iPCR is detected typically those because of too little can not be according to sandwich mensuration need be in conjunction with the analyte of two kinds of antibody.Generally, competitive iPCR comprises:

(1) will have specific antibody immobilization in the surface to target analyte;

(2) may comprise that the sample droplet of target analyte and the droplet that comprises the report analysis thing merge;

(3) described immobilized antibody is exposed to the target analyte/report analysis thing droplet of merging, so that described report analysis thing and any target analyte competition binding site;

(4) the washing base material is to remove unconjugated analyte;

(5) bonded report analysis thing is connected in report nucleic acid; With

(6) process of amplification report nucleic acid and monitoring amplification to be determining the amount of unconjugated report analysis thing, and the amount of the amount of bonded report analysis thing and the target analyte in the sample is inversely proportional to.

Can on the described droplet microdrive of the application, use droplet to control technology and carry out any one or more aforesaid step.Each step further goes through at follow-up chapters and sections.

8.4.2.1 immobilized antibody

Can save described immobilized antibody according to 8.4.1.1.

8.4.2.2 competitive combination

The droplet that will comprise the sample that may comprise target analyte merges with the droplet that comprises described report analysis thing, and makes the droplet of merging contact described immobilized antibody.

Described report analysis thing can will report that nucleic acid is incorporated into analyte and prepares by any of multiple conjugation methods.In one embodiment, the analyte part is by molecular modification, and the biological example element has produced the secondary site of catching like this, being used for fixing streptavidin-DNA mixture.Vitamin H and analyte must not combine undue interference its with the combining of primary capture antibody.In some cases, biotinylated material can be competed comparably with the analyte from sample.Biotinylated analyte can take place before or after biotinylated analyte is by described immobilized antibody capture with combining of nucleic acid of report.

For example, in one embodiment, the droplet of the biotinylated analyte by will having known quantity mixes with the sample droplet of the unmodified analyte that may contain unknown quantity to be measured.The droplet that will comprise biotinylated analyte merges with the droplet that may comprise target analyte.The droplet that merges contacts with described immobilized antibody, so that biotinylated analyte combines described immobilized antibody with target analyte (if present) competition.The amount of the analyte in the amount of the biotinylated analyte of institute's bonded and the described sample droplet is inversely proportional to.

8.4.2.3 bind nucleic acid reporter molecules

After the washing, the droplet with streptavidin-vitamin H-report nucleic acid complexes is added to droplet or the surface of the pearl that comprises washing.Streptavidin-vitamin H-report nucleic acid complexes in conjunction with any by the biotinylated analyte of the described antibody capture on the described pearl.

8.4.2.4 amplification of nucleic acid

After the washing, determine the amount of immobilized streptavidin-vitamin H-report nucleic acid complexes by the described report nucleic acid that increases.The amount of analyte is inversely proportional in amplified signal and the original sample.Can as described in 8.1 joints, increase.Measure the existence and the amount of the amplified production that is produced, carry out as adopting the real-time fluorescence detection method.Amount by the amount of metathetical report analysis thing and the target analyte in the sample is proportional.

8.4.2.5 alternative method

Described step is not limited to given order.For example, can merge by droplet and one or more droplet that comprises target analyte/report analysis thing that will comprise free antibodies, thereby elder generation in target analyte/report analysis thing, contacts being used for fixing with antibody with described surface with antibodies subsequently before antibody immobilization.Described report analysis thing can merge on described droplet microdrive by the droplet that will comprise these two kinds of reagent with report nucleic acid and be puted together.The droplet that comprises described report analysis thing can merge with comprising the droplet of reporting nucleic acid, so that realized puting together before or after described report analysis thing is exposed to capture antibody.Multiple possible alternative form is contained in the present invention.

8.4.3 sample and specimen preparation

Can use the scheme of the iPCR based on droplet of the present invention to detect multiple analytes.Can analyze one or more target analyte in the single sample.Analyte can be, for example, and biological analyte or synthesis analysis thing.For example, in one embodiment, analyte is selected from following classification: analyte, archon analyte and the small molecules toxin analysis thing in the analyte of bacterial origin, the analyte of viral source, spore source.In one embodiment, target analyte comprises that there are the analyte of special organism in toxin or representative, as infectious diseases, or is used for the toxin of bioterrorism or biological warfare.The example of this type of organism comprises anthrax, bird flu, sausage poisoning, Hantaan virus, l, pneumonic plague, smallpox, tularaemia and hemorrhagic fever virus (VHF).

8.4.4 immuno-PCR scheme

System of the present invention can make that carrying out panimmunity PCR on single sample simultaneously measures.In addition, immuno-PCR is measured and can be carried out with other tests, for example PCR and/or based on the mensuration of avidity.

System provides multi-path to detect.In one embodiment, system can detect 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 or more kinds of analyte.Analyte can comprise, for example, and the analyte in natural or non-natural source.In another embodiment, the present invention can detect one or more analyte of any 1,3,4 or 5 kinds that come from following classification: analyte, archon analyte and the small molecules toxin analysis thing in the analyte of bacterial origin, the analyte of viral source, spore source.

System can be turned to by program and implement extra mensuration as required to increase single target result's confidence level.Importantly, described droplet microdrive system can be turned to by program the positive findings property confirmed is measured to reduce false-positive possibility again.System can also and be configured to allow to store the sample of testing by sequencing on described droplet microdrive and be used for follow-up additional experiments chamber test.

In operation, the present invention analyzes and provides the result at a terrific speed.For example, in one embodiment, system can detect 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 or more kinds of analyte being less than in about 60,50,40,30,20,15 or 10 minutes time.In another embodiment, system can detect and come from any 2 of following classification, 3,4, or 5 kinds 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50 or more kinds of analyte: the analyte of bacterial origin, the analyte of viral source, the analyte in spore source, the archon analyte, with small molecules toxin analysis thing, and all test is being less than about 60,50,40,30,20, finish in 15 or 10 minutes time.

8.4.5 use

IPCR provided by the invention measures and can be used for detecting the varied molecule that exists with minute quantity.For example, this system can be used for monitoring chemical, biological or explosive hazardous articles.For example, the trace explosive substance that can be used for existing in the test sample at the antibody of explosive substance.Therefore, the present invention also is provided for representative in area of examination and has biological, chemical or the chemical analyte of explosive weapon or the method for biological analyte.

In one embodiment, the iPCR technology is used for specimen and whether has organism, for example bacterium or virus.In some embodiments, system even can realize single living body detection.In addition, some embodiments can comprise PCR typing of bacteria or variation.

8.5 analysis biological fluid

The invention provides methods, devices and systems, it is used for analyzing blood, various blood ingredient and other biological and learns liquid.Fig. 9 and 17 is depicted as the exemplary design of biological fluid analyser.

Fig. 9 has shown an embodiment of biological fluid analyser 900.In this embodiment, can provide the various modules that biological fluid is analyzed that are used to carry out, for example, detect metabolite (for example glucose, lactic acid, blood urea nitrogen and creatinine), ionogen (K for example ⁺, Cl ^-, and Na ⁺), protein and enzyme.These different modules can comprise current module 902, current potential module 904, optical module 906 and electric guide module 908.

Figure 17 shows that another embodiment of biological fluid analyser 1700.In this embodiment, can provide a plurality of fluid port or pond, for example antibody pond 1701 (for example being bacteriocidin, spore antibody, bacterium AB-DNA, spore AB-DNA, archon antibody, small molecular antibody, a-protein b form dna and small molecules SB-A DNA), PCR primer pond 1702 and PCR reagent pond 1703.Also but the sampling port one 704, and sample pool 1705, washing soln district 1706 and waste liquid pool 1707.Available other zones comprise hot humidity province 1708, cold humidity province 1709 and detector district 1710.

Be appreciated that an importance of the present invention comprises that can use various required biological fluid analytic samples and reagent to carry out droplet operates on the droplet microdrive.For example, the present invention includes:

(1) droplet microdrive comprises droplet on it, described droplet comprises any one or more reagent and/or the sample that this type of biological fluid is analyzed that be used to carry out described herein;

(2) device of the present invention or system, it comprises described droplet microdrive;

(3) in described droplet microdrive or system, carry out droplet method of operating or utilize the method for described droplet microdrive or system operation droplet; And/or

(4) utilize described droplet microdrive or system to carry out method for measuring based on avidity based on droplet.

For example, described droplet operation can comprise following one or more operation: droplet is loaded in the droplet microdrive; Distribute one or more droplet from the source droplet; Droplet is divided, separates or is split as two or more droplets; Droplet is transferred to another place from a place along any direction; Two or more droplets are converged or merge into a droplet; The dilution droplet; Mix droplet; Stir droplet; Make little droplet deformation; Make droplet keep original position; The incubation droplet; The heating droplet; The evaporation droplet; The cooling droplet; Arrange droplet; Droplet is migrated out microdrive; Other droplet operations described herein; And/or above-mentioned any combination.Various additive method of the present invention, device, system and other aspects will be conspicuous by following description.

8.5.1 sample and specimen preparation

The suitable sample example that can be used for described droplet microdrive of the present invention comprises whole blood, serum and blood plasma and various component thereof.Can use vein, artery or capillary blood.Other sample examples that can use the present invention to analyze comprise cerebrospinal fluid (CSF), urine, saliva, sweat, tear, amniotic fluid, hydrothorax, milk, capsule liquid, synovial membrane liquid, ight soil and seminal fluid.

Can extract serum and/or blood plasma from whole blood on the described droplet microdrive and/or before introducing described droplet microdrive.Figure 10 has provided the example of the loading structure 1000 that is used for this purpose.In this embodiment, 1002 closed units 1004 of flowing through enter sample addition zone 1006 to liquid from the pond, locate it at this and contact with film 1008.Penetrant enters permeate flow path 10 10, by this place, in auxiliary down the flow into droplet microdrive pond 1012 of pressure source 1014 by path 10 16 applied pressures.

This device volume is little, compares with the volume that the conventionally test method is required, and this has significantly reduced sample volume, and sample volume all is important factors in many cases.For example, in one embodiment, typical sample volume for about 1nL to about 100mL or about 10nL about 10mL or about 1 μ L about 10 μ L extremely extremely.

8.5.2 analyte

The invention provides multi-usage droplet microdrive system, it can rise the blood of droplet or approximately carry out array test on any physiology sample of～0.5 μ L at single sub-micro.The example of suitable tester comprises metabolite (for example glucose, creatinine, lactic acid, blood urea nitrogen), ionogen/element (for example K+, Na+, Cl-, P, Mg, Li, Ca, Fe), gas (for example pH, pCO ₂, NH ₃), enzyme (alkaline phosphatase (ALP), aspartic transaminase (AST), alanine aminotransferase (ALT), serum lactic dehydrogenase (LDH), lipase, creatine kinase, creatine kinase mb), protein (albumin, C-reactive protein (CRP), microdose urine protein, urinary albumin, cerebrospinal fluid protein, total serum protein) and pcv.Other analytes comprise glycolated hemoglobin (AIc), oxyphorase, uric acid, triglyceride level, cholesterol, high-density lipoprotein (HDL) (HDL) and low-density lipoprotein (LDL).

8.5.2.1 metabolite

The present invention can be used for carrying out the plurality of enzymes coupling assay method that detects based on electrochemistry or light, for example is used to measure glucose, blood urea nitrogen, creatinine and lactic acid.In some embodiments, during the enzyme coupling carried out on described droplet microdrive is measured, by to H ₂O ₂Carry out current detecting and measure glucose, lactic acid and creatinine.

In some embodiments, the present invention includes the electrochemistry module, it has the electrode that is used for metabolite (for example glucose, lactic acid, blood urea nitrogen, creatinine) is carried out electric current and/or current potential detection.

8.5.2.2 ionogen

For the various ions (for example ammonia, bromine, cadmium, calcium, chlorine, copper, prussiate, fluorine, fluoroboric acid root, iodine, lead, nitrate radical, perchlorate, potassium, silver/sulfide, magnesium, iron, lithium, phosphorus, sodium, surfactant, thiocyanate ion) in the quantitative sample, for example the biological sample of Chu Liing can improve described droplet microdrive and make it to comprise ion specific electrode (ISE).Transferable sample droplet makes it to contact the ISE that is used to detect desired ion.In addition, before the detection, can make standard substance droplet contact ISE, be used to calibrate.In some embodiments, the present invention includes the electrochemistry module, it has and is used for ionogen (K for example ⁺Cl ^-Na ⁺) carry out the electrode that electric current, current potential and/or electricity are led detection.

ISE can be included into and be used to detect electrolytical described droplet microdrive.For example, ISE can be used as the component of top and/or bottom substrate and/or be exposed to the top and/or bottom substrate between the spatial component or be associated with single base material droplet microdrive.They can be integrated with transfer electrode.ISE is set to utilize droplet to operate usually makes the droplet on the described droplet microdrive contact with ISE.Can use multiple technologies to prepare ion specific electrode on the described droplet microdrive.Example comprises screen printing and photolithography, etching and lift-off technology.

As concrete limiting examples, but silk screen printing Ag/AgCl is to provide working electrode and the reference electrode with KCl salt bridge.The suitable ionophore that is used for making the PVC of ion selective membrane comprise methyl monensin (methyl monensin), the valinomycin that is used for K+ that is used for Na+, the aliquat that is used for Cl-and, be used for the trilaurylamine (Tridodecylamine of pH.Can distribute and/or spraying (spray-coating) (for example, heat/ultrasonic wave printing) preparation ion selective membrane by trace.

Ionogen but detection of biological imitates in the product.Concrete limiting examples comprises the example that whole blood, blood plasma and serum and other parts of this specification sheets are mentioned.

8.5.2.3 gas

For the gas that quantitatively exists (pCO for example ₂, pO ₂) or pH, can use various special electrodes.As limiting examples, the carbonic acid gas microprobe can be integrated into droplet microdrive of the present invention and be used for detection and/or quantitative carbonic acid gas.Described microprobe can be arranged so that and can carry out the droplet operation so that make the droplet on the described droplet microdrive contact described microprobe.In addition, for the purpose of calibrating, before the detection, can utilize the droplet operation to make the standard droplet contact the carbonic acid gas microprobe.Corresponding method is suitable for detecting oxygen and/or definite pH.In some embodiments, described droplet microdrive can comprise and being used for vim and vigour (pCO for example ₂, pO ₂, pH) carry out the electrode that electric current, current potential and/or electricity are led detection.

As concrete limiting examples, the pH electrode that is made by gold-quinhydrone electrode can be immersed by NaHCO ₃, the internal solids ionogen that makes of NaCl and sucrose binding substances and prepare Severinghaus type CO ₂Transmitter.But deposition gases permeability polydimethylsiloxane film on it.Digital adjustment electronics (for example high input impedance amplifier) can be connected in potential electrode.

Can be in any droplet on the described droplet microdrive detected gas.Concrete limiting examples comprises the droplet that those comprise the biological sample that whole blood, blood plasma and/or serum and other parts of this specification sheets are mentioned.

8.5.2.4 enzyme

In some embodiments, the present invention includes chemical luminescent detecting, it is used to detect enzyme, for example the enzyme of liver.Use a series of a plurality of enzymatic steps based on droplet, the mensuration of ALT and AST can be reduced to the final step that produces hydrogen peroxide, and the latter can pass through absorbancy, luminous, fluorescence or electrochemical method quantitative assay.

8.5.2.5 serum protein

Can use colorimetric estimation to come total protein in the test sample.The example of suitable colorimetric method comprises: biuret method, Lowry method, dihomocinchonine acid (bicinchoninic acid, BCA) assay method and Bradford assay method.Biuret Method generally includes the droplet that makes the contact of sample droplet comprise bivalent cupric ion.Bivalent cupric ion and protein form coloured mixture.The Lowry reaction method is based on amplifying biuret reaction by merging with Folin reagent droplet.A kind of version of Lowry assay method use dihomocinchonine acid (BCA) droplet in case detect by bivalent cupric ion under alkaline condition with droplet in proteins react and the univalent copper ion that produces.The Bradford assay method comprises sample and the droplet that comprises the blue dyestuff of coomassie is merged to form coloured mixture.In various situations, for example can use the LED/ photoelectric diode device shown in Figure 21 A to monitor absorbancy.Can use technology described herein in any sample droplet, to detect total protein, comprise that use is based on fluorescence and/or chemiluminescent optics method for sensing and use the determination techniques based on avidity described herein.The concrete limiting examples that can be used for determining the sample of total protein comprises biological sample, for example whole blood, blood plasma and serum, and the sample mentioned of other parts of this specification sheets.

8.5.2.6 pcv

Can use the red corpuscle in the quantitative sample droplet of multiple technologies.For example, can calculate content of hemoglobin by the absorbancy of measuring the 805nm place.Can calculate Oxyhemoglobins by the absorbancy of measuring the 650nm place.Obtain a result by the absorbance measurement value of comparative sample and the absorbance measurement value of a series of known standard things.

In some embodiments, the present invention includes the electrochemistry module, it has and is used for pcv is carried out the electrode that electric current, current potential and/or electricity are led detection.Described droplet microdrive can comprise conductance element, and it has the pair of electrodes that is used for pcv is carried out the AC conductance measurement.For bi base material droplet microdrive, electrode can place on one or two base material.Electrode can be provided so that and can carry out the droplet operation so that make the droplet on the described droplet microdrive contact described electrode.

8.5.3 multiple analyte analyser

The example of suitable analyte is glucose, creatinine, lactic acid, BUN, K ⁺, Na ⁺, Cl ^-, pH, pCO ₂, ALP, total protein and pcv.In one embodiment, on single droplet microdrive, analyze 2,3,4,5,6,7,8,9,10,11,12 or more kinds of analyte.Preferably, these analytes are selected from glucose, creatinine, lactic acid, BUN, K ⁺, Na ⁺, Cl ^-, pH, pCO ₂, ALP, total protein and pcv.In one embodiment, on single droplet microdrive, analyze glucose, creatinine, lactic acid, BUN, K ⁺, Na ⁺, CI ^-, pH, pCO ₂, ALP, total protein and pcv.The example of the analyte that gas is suitable comprises calcium, bilirubin, albumin, clotting time, ALT and AST.

In some embodiments, the mensuration of parallel processing 2,3,4,5,6,7,8,9,10,11,12 or more kinds of analytes in droplet microdrive of the present invention system, promptly to one or more processing of this analyte and/or detect step with to one or more processing of another kind of analyte and/or detect step and on single droplet microdrive, realize simultaneously.Droplet microdrive system can implement 2,3,4,5,6,7,8,9,10,11,12 or more multinomial colorimetric estimation, with the identical or different types of analytes of parallel detection.Described droplet microdrive system can implement 2,3,4,5,6,7,8,9,10,11,12 or more multinomial chemical luminescent detecting, with the identical or different types of analytes of parallel detection.Described droplet microdrive can implement 2,3,4,5,6,7,8,9,10,11,12 or more multinomial current measurement, with the identical or different types of analytes of parallel detection.Described droplet microdrive can implement 2,3,4,5,6,7,8,9,10,11,12 or more multinomial potential measurement, with the identical or different types of analytes of parallel detection.Described droplet microdrive can implement 2,3,4,5,6,7,8,9,10,11,12 or more multinomial fluorometric assay, with the identical or different types of analytes of parallel detection.Described droplet microdrive can comprise 2,3,4,5,6,7,8,9,10,11,12 or more multinomial electric conductance determination, with the identical or different types of analytes of parallel detection.Described droplet microdrive will comprise droplet or pond, and described droplet or pond comprise the reagent that is used to implement every kind of described scheme.Described droplet micro-drive device and/or system will comprise the required detected components of detection step that is used to implement described scheme.

In addition, described droplet microdrive can comprise 2,3,4,5,6,7,8,9,10,11,12 or more kinds of analyte of parallel processing, and described system can be to these analytes enforcement 1,2,3,4,5,6 or more kinds of mensuration scheme.Described droplet microdrive can comprise 2,3,4,5,6,7,8,9,10,11,12 or more kinds of analyte of parallel processing, and can be to these analytes enforcement 1,2,3,4,5,6 or more kinds of mensuration scheme, wherein said mensuration is selected from colorimetric estimation, chemical luminescent detecting, fluorometric assay, current measurement, potential measurement and electric conductance determination.Described droplet microdrive will comprise droplet or pond, and described droplet or pond comprise the reagent that is used to implement every kind of described scheme.Described droplet micro-drive device and/or system will comprise the required detected components of detection step that is used to implement described scheme.

In addition, be used for nucleic acid amplification, nucleic acid sequencing, based on mensuration, the cell of avidity handle, pearl is handled and the various schemes of washing and detection of analytes scheme also can be incorporated into single droplet microdrive system.In one embodiment, single droplet microdrive comprises reagent and the detected components that is used to carry out nucleic acid amplification and nucleic acid sequencing.In another embodiment, single droplet microdrive comprises and is used to carry out reagent and the detected components of nucleic acid amplification with the pathogenic agent that detects blood propagation, and the reagent and the detected components that are used to carry out one or more other mensuration, described other are measured from those types described herein and/or types of analytes.In another embodiment, the droplet microdrive comprises the component that is used for manipulation cell and is used to carry out component and reagent based on the mensuration of avidity.

In brief, the invention enables droplet microdrive system not only can with higher flux and significantly lower sample volume carry out the routine operation of centralab's chemical analyzer, and also provide better functional by integrating hematology, pathology, molecular diagnostics, cytology, microbiology and serology in identical platform.

In one embodiment, the invention provides the droplet operational module, it has integrated optical detection module and the Electrochemical Detection module that is used to analyze vim and vigour, ionogen, enzyme, protein and metabolite.

An embodiment of the invention use modular design to separate the manufacturing processed of independent optimization.For example, can on a base material, make all electrochemistry components, on another base material, make all microfluid electrodes, and making all electronic installations on another base material again.Can form disposable sandwich droplet microdrive between electrochemistry module and droplet operational module, it can be coupled to and be used for the reusable electronic module that data are obtained and analyzed.Can in analyser, make up the optical detection module.

8.5.4 biological fluid analyzing and testing

Biological fluid analysis described herein can be used multiple detection method, as saving referring to 8.1,8.2,8.3,8.4,8.5 and 8.11.

8.6 surface and surface washing scheme

Various scheme of the present invention needs the surface of immobilized reagent.For example, can use the surface to catch or the target component of immobilization droplet, for example cell, other pearls, particulate, nano particle, antibody, protein, peptide, nucleic acid, small molecules and/or other chemical compositions.The surface that is used for this type of purpose can comprise, for example, and the surface on pearl, particulate, nano particle, film, physical objects and/or droplet microdrive surface.Various schemes need washing step, wherein remove unconjugated material from one or more surface.

Make the sample droplet of the target component that comprises that one or more is used to catch contact the surface that this type of target is had avidity by the droplet operation.Can use washing scheme of the present invention remove from described surface described sample droplet not in conjunction with component.For example, can make one or more droplet that comprises one or more target component contact one or more surface, make one or more target component be immobilized onto or be trapped in described one or more surface by the droplet scheme.Implement the washing scheme and remove not binding substance from described one or more surface.Similarly, can make one or more droplet that comprises one or more target component contact one or more pearl, make one or more target component be immobilized onto or be trapped in described one or more pearl by the droplet scheme.Implement the washing scheme and remove not binding substance from described one or more pearl.

Washing generally includes and makes one or more washing droplet contact immobilized surface.When can being included in surface in contact, washing stirs droplet.Washing droplet can comprise, for example, and water, deionized water, salts solution, acidic solution, basic solution, washing agent solution and/or damping fluid.

Washing scheme of the present invention causes not, and binding substance is efficiently removed from described surface.In one embodiment, the invention provides a kind of method, it provides and surperficial contacted droplet, and described droplet has the material concentration of reduction.This method generally includes and provides the surface that contacts with droplet, droplet to comprise the described material of initial concentration and have initial volume; Carry out one or repeatedly droplet operation converge droplet so that will wash droplet and described droplet with the generation merging; With carry out one or repeatedly droplet operation is so that be split as one group of droplet with the droplet of described merging, described one group of droplet comprises: (i) with the contacted droplet in described surface, with respect to described initial concentration, it has the material concentration of reduction and the amount of substance of reduction; (ii) with the droplet of described surface isolation.

Method of the present invention can produce and the contacted droplet in described surface, and with respect to initial concentration, it has the amount of substance of reduction or the amount of substance that obviously reduces.In some embodiments, the volume of gained droplet and described initial volume are roughly the same.In some embodiments, but the repeated washing step meets or exceeds the predetermined maximum of one or more component until the gained droplet.Described predetermined amount can be represented the abundant reduction with respect to described initial concentration.In some cases, the gained droplet can not have described component basically.For example, in some embodiments, the reduction of described amount surpasses 99%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999%, 99.999999% in mole.

Method of the present invention can produce and the contacted droplet in described surface, and with respect to initial concentration, it has the concentration of material of reduction or the concentration of the material that obviously reduces.In some embodiments, the volume of gained droplet and described initial volume are roughly the same.In some embodiments, but the repeated washing step meets or exceeds the predetermined peak concentration of one or more component until the gained droplet.Described predetermined concentration boundary can be represented the abundant reduction with respect to described initial concentration.In some cases, the gained droplet can not have described component basically.For example, in some embodiments, the reduction of described concentration surpasses 99%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999%, 99.999999% in mole.

8.6.1 washing pearl

For the scheme of using pearl, can utilize droplet to operate droplet and one or more washing droplet with pearl are merged.Then, when (for example by physical method or magnetism method) keeps pearl, can utilize the droplet operation that the described droplet that converges is split as two or more droplets: one or more droplet with pearl does not have the obviously droplet of the pearl of amount with one or more.In one embodiment, utilize the droplet operation that the described droplet that converges is split as a droplet and the droplet that does not have the pearl of obvious amount with pearl.

Usually, carry out the washing scheme causes enough integuments to be preserved for carrying out required mensuration and can not cause detrimental effect improperly to measurement result at every turn.In some embodiments, split the described droplet that converges at every turn cause keeping pearl more than 90%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999% or 99.999999%.In other embodiments, remove in order to make that the concentration of material and/or amount reach predetermined reduction and each washing scheme of carrying out causes keeping the pearl more than 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, 99.99999% or 99.999999%.In other embodiments, the amount that calculates the pearl of reservation is also correspondingly adjusted the result.

In some embodiments, can wash pearl in the pond, the droplet that wherein will contain pearl merges with the washing droplet, keep pearl (for example by magnet, physical structure, electrostatic force), and the droplet that utilizes the droplet operation will lack pearl distributes in described pond.For example, can wash pearl by dilution and allocation strategy, wherein lavation buffer solution be joined and dilute content in the pond, use magnet that the magnetic responsiveness pearl is positioned in the described pond, and most solution are distributed, and repeat this circulation until reaching acceptable washing level in described pond.

8.6.1.1 washing magnetic responsiveness pearl

Limiting examples shown in Figure 11 relates to utilizes magnetic field immobilization magnetic responsiveness pearl.Can discharge immobilized magnetic responsiveness pearl by reducing or eliminating magnetic field.Pearl can may further comprise the steps the washing magnetic responsiveness usually:

(1) utilize droplet operation will comprise magnetic responsiveness pearl 1102 and not the droplet 1101 of binding substance 1103 place near the magnet 1104;

(2) utilizing the droplet operation will wash droplet 1106 merges with the droplet 1101 that comprises described magnetic responsiveness pearl 1102;

(3) by applying magnetic field with pearl 1102 immobilizations;

(4) the part or all of described droplet that utilizes droplet operation to remove to surround described pearl with generation comprise having of described pearl reduce concentration not in conjunction with the droplet 1108 of target material and comprise not droplet 1110 in conjunction with the target material;

(5) discharge described pearl 1102 by removing magnetic field;

(6) repeating step (2) to (3) or (2) to (4) are until the purifying that reaches predetermined extent.

By this way, binding substance not, for example pollutent, byproduct or excess reagent can separate with pearl.Each circulation produces the droplet that comprises pearl and have the useless material of level reduction.Step (5) is not that each circulation all needs; But, it can be used for strengthening washing by discharging the pollutent that can be caught in the immobilized pearl.Can carry out described step according to different order, can put upside down as step (2) and (3).Can utilize droplet described herein to operate in the step of the scheme of washing on the droplet microdrive.

Figure 12 shows that another embodiment, it can comprise top board 1201, base plate 1202, electrode 1203 and magnet 1204.This embodiment can comprise usually:

(1) utilize droplet operation with bullet 1205 with comprise magnetic responsiveness pearl 1207 and not the droplet 1206 of binding substance 1208 near magnet 1204, merge;

After (2,3) pearl 1207 immobilizations, utilize the bullet 1210 of the merging of droplet transition of operation gained to pass described pearl 1207 so that binding substance 1208 is not separated from described pearl 1207;

(4) utilize droplet operation tell described merging bullet 1210 a part with generation comprise have that concentration reduces not in conjunction with the part 1212 of the pearl of target material and comprise not part 1214 in conjunction with the target material;

(5) as required repeating step (1)-(4) so that binding substance does not reach required reduction.

In a relevant method, can add extra washing droplet when passing described immobilized pearl and/or bullet continues additional described bullet by being transferred at described bullet.This method is sustainable carries out reaching required reduction until binding substance not.

Figure 13 shows that an alternative embodiment, it also can comprise top board 1301, base plate 1302, electrode 1303 and magnet 1304.In this embodiment, moving magnet 1304, for example along the A1 direction, so that pearl 1305 and the not binding substance 1306 in the merging bullet 1307 are separated, rather than mobile bullet 1307.Similar method both relates to, and the also mobile bullet of moving magnet separates (not shown) with realization.Another method relates to and uses a plurality of magnets to come mobile pearl (not shown) again.

In the embodiment that uses the magnetic responsiveness pearl, the inventor finds, though apply magnetic field immobilization temporarily pearl, mobile pearl and/or positioning bead, causes described pearl that deleterious gathering takes place sometimes.In one embodiment, including surfactant in prevents or reduces pearl and assemble.The example that is suitable for the surfactant of this purpose comprises:

Triton X-100.Selected surfactant and consumption thereof should reduce or eliminate the pearl gathering and non-specific adsorption is minimized, and do not cause target analyte or reagent obviously to be lost in droplet simultaneously.

The method of the reunion of another elimination or minimizing pearl relates to the bigger pearl of using lesser amt.Can use any amount of one or repeatedly can be placed into pearl in the droplet in the droplet operation.In some embodiments, the quantity of magnetic responsiveness pearl can be in 1 scope to hundreds of thousands of.For example, in one embodiment, the present invention uses 1 to 100 magnetic responsiveness pearl/droplet.For example, the present invention uses 1,2,3,4,5,6,7,8,9,10...100 magnetic responsiveness pearl/droplet.In one embodiment, the quantity of magnetic responsiveness pearl is 1 to 10.Use the magnetic responsiveness pearl of lesser amt to allow to use bigger pearl.For example, in one embodiment, the present invention uses 1 to 100 magnetic responsiveness pearl/droplet, and the mean diameter of wherein said pearl is about 25 to about 100 microns.At another embodiment extremely, the present invention uses 1 to 10 magnetic responsiveness pearl/droplet, and the mean diameter of wherein said pearl is about 50 to about 100 microns.

8.6.1.2 wash non-magnetic responsiveness pearl

A similar method can be used the pearl that does not have magnetic responsiveness or do not have obvious magnetic responsiveness.As shown in figure 14, be different from and use magnetic field fixing pearl 1401, can use physical barrier 1402 to remove the part or all of described droplet 1403 that surrounds pearl 1401.Physical barrier 1402 can comprise, for example, and film, sieve and/or from the protuberance of described droplet microdrive (for example from top board 1404 and/or base plate 1405).If use the physical barrier 1402 (projection or object) that is attached to top board 1404 and/or base plate 1405, it should be set to allow to use one or more electrode 1406 to shift, but can prevent that pearl 1401 from and then shifting, for example use to shift to droplet and reserve enough spatial from the projection of top board and/or have the projection of one or more opening, described opening allows droplet to shift by this opening and prevents that simultaneously pearl from and then shifting.

8.6.2 washing droplet microdrive surface

Figure 15 shows that the example of the method on washing droplet microdrive surface.In this limiting examples, surface 1501 is positioned at the inboard of top board 1502.In the method, (1) makes and comprises sample droplet 1503 surface in contacts 1501 (utilizing the droplet operation) that surface component 1505 had the target material 1504 of avidity, causes partly or entirely being immobilized of (2) described target material.(3) utilize droplet operation will wash droplet 1506 and described sample droplet 1503 merges to produce the washing-sample droplet 1507 of merging.(4) utilize then droplet operation with the washing-sample droplet of described merging be split as contact with described surface and comprise reduce concentration not in conjunction with the part 1508 of target material and from described surface isolation and comprise not part 1509 in conjunction with the target material.Repeating step (3) and (4) are not so that binding substance reaches required reduction as required.

8.7 cell is handled

Various scheme of the present invention can be used the droplet that comprises cell.Droplet can comprise substratum and/or the grown cell culture that is used to keep cell viability.

In some cases, the present invention uses the droplet of the cell with pre-determined quantity.For example, in some embodiments, the present invention uses the droplet that comprises individual cells.For example, unicellular droplet can be used for producing the pure cell colony of clone and/or is used to study the experiment of unicellular reaction to particular stimulation.Be split as one or more inferior droplet by being dispensed to from the droplet of cell suspension, the droplet of the cell with pre-determined quantity can be provided from the droplet transferring path of cell suspension or network and/or by having cellulous droplet.Suspension can provide or be stored in the droplet microdrive pond by external source.Can analyze droplet determining the cell quantity in each droplet, and the droplet with previously selected cell quantity can be sent to the downstream and be used for further processing.Droplet itself with distribution of a plurality of cells can merge with one or more damping fluid droplet, and is split as two or more inferior droplets, and analyzes its release and have individual cells.

To make the sorting decision based on the droplet analysis.For example, can make to use up to propagate and identify droplet with pre-determined quantity cell.Can make the sorting decision based on the measured value of the light of propagating.Other embodiments can utilize Automated Image Analysis and/or multicolor fluorescence and/or scattering analysis.Against regulation droplet can be sent sample pool back to and be done another time trial or be transferred to waste liquid pool.

The droplet that meets cell count regulation is transferred to droplet microdrive pond and/or transfer is used for sorting and/or enrichment.Figure 16 A and 16B have provided a kind of method that is used to provide the pond with enrichment of cell content.In this embodiment, distribute droplets 1602 and be transferred to pond 1606 on the droplet microdrive 1600 from cell suspension 1604 according to its character.

In other embodiments, can further control droplet, as be used for its inner contained cell of sorting as discrete droplets.Comprise that the droplet of the cell of pre-determined quantity can be used as the input of various mensuration schemes described herein.In some embodiments, gravity is not as the prime mover that shifts droplet.

In a concrete embodiment, can be on described droplet microdrive separating tumor cell.For example, can be from fine needle aspiration thing (FNA) isolated cell of microlitre level.In another embodiment, but the analytic sample cancer cells in blood stem cell, medullary cell, GI washing lotion and the freeze thawing sample for example.

Can use antibody to realize the immunocapture of relevant cell, for example anti--cytokeratin pearl can be used for catching relevant cell from sample and is introduced into described droplet microdrive then and/or catches relevant cell from the droplet on the droplet microdrive.By droplet is moved back and forth or in the pond whirlpool vibrate described droplet, can on described droplet microdrive, strengthen in conjunction with or reduce the incubation time.Can separate and wash pearl according to other parts of this paper are described.Target cell can be released in the suspension in the droplet on the described droplet microdrive.

Can use the pond (on-chip reservoir) of cell apportioning method described herein on chip to distribute the cell of equal amts for each droplet.Can be with in droplet five equilibrium with cell pond to a plurality of chips.Incubation cell in can the pond on chip.Can estimate cell viability, for example can use resazurin as the fluorescence oxidation-reduction indicator.Viable cell is converted into the resorufin that sends red fluorescence with the resazurin dyestuff of non-fluorescent.Nonactive cell does not send fluorescence.Cell can be dispensed in the pond on the chip and use the nucleic acid of method described herein amplification from described cell.

8.8 droplet microdrive structure and operation

The outstanding droplet microdrive that comprises by treater control of system of the present invention.For example, for example can be turned on the control droplet microdrive by program be that droplet is controlled to treater.The structure of droplet microdrive has multiple possibility.Fig. 1,2,6,9 and 17 has provided multiple example.The example that can be configured to the component in the droplet microdrive of the present invention comprises: various fill fluids, and it can be loaded on the described droplet microdrive; The liquid load maintainer is used for fill fluid, sample and/or reagent are incorporated into described droplet microdrive; Pond and/or treating pond are for example imported in various ponds; The droplet distributor gears; Temperature-control device is used to control the temperature of the droplet on described droplet microdrive, fill fluid and/or the droplet microdrive; Produce component with magnetic field, be used to control the magnetic responsiveness pearl on the droplet microdrive.This section is discussed these and other aspect and their purposes in system of the present invention of described droplet microdrive.

8.8.1 droplet microdrive

Described system utilizes the droplet microdrive.Described droplet microdrive comprises the base material with one or more electrode, and described electrode is set to be used to carry out one or repeatedly droplet operation.In some embodiments, described droplet microdrive can comprise one or more array, path or the network of this type of electrode.Can utilize multiple electrical characteristic to realize the droplet operation.Example comprises electricity wetting (electrowetting) and electrophoresis.

In one embodiment, described droplet microdrive comprises two or more electrodes that are connected with base material, and comprises and be used to the device that makes that described electrode starts/stops.For example, but the electrode electronics be connected in and be controlled by one group of manual switch and/or controller.Therefore described droplet microdrive can implement the droplet operation, and for example distribute, divide, shift, converge, mix, stir, or the like.In one embodiment, adopt the driving of electric field mediation to realize that droplet controls.But the electrode electronics is connected in the device that is used to control with described droplet microdrive electrical communication.

Basic droplet microdrive comprises electrode path or array.In some embodiments, described droplet microdrive comprises the base material of two parallel separately certain intervals and is positioned at one of described base material or the electrod-array on both.One of described base material or both can be plates.One of described base material or both can PCB, glass and/or semiconductor material are made as base material.If the base material following material that is PCB is the example of suitable material: Mitsui BN-300; Arlon 11N; Nelco N4000-6 and N5000-30/32; Isola FR406, particularly IS620; Fluoropolymer series (, therefore being suitable for fluoroscopic examination) because of its background fluorescence is low; Polyimide series.Multiple material also is suitable as the dielectric component of base material.Example comprises: vapor deposition dielectric medium, for example parylene C (particularly on glass) and parylene N; Tetrafluoroethylene AF; Cytop; With solderable surface solder mask (soldermasks), but fluid light imaging-type solderable surface solder mask (liquid photoimageable soldermasks) (for example on PCB) for example is as Taiyo PSR4000 series, Taiyo PSR AUS series (having the good thermal property that is suitable for the temperature control purposes) and Probimer 8165 (having the good thermal property that is suitable for the temperature control purposes); Dry film solderable surface solder mask, for example those in the Dupont Vacrel series; With the film dielectric medium, for example polyimide film (Kapton), polyethylene and fluoropolymer such as FEP, PTFE.Base material partly or entirely also can comprise hydrophobic coating.Suitable example comprises tetrafluoroethylene AF; Cytop; The coating of Fluoropel series; Silane coating; Fluorine silicomethane (fluorosilane) coating; With 3M Novec electronics coating.

If described droplet microdrive comprises two plates, then droplet can be put into two spaces between the plate.The space that surrounds droplet typically comprises fill fluid.Described droplet microdrive can use diversified droplet of fluid to carry out the droplet operation, but the conductivity fluid is preferred.

The surface of described droplet microdrive typically is coated with hydrophobic coating.If be used in thermal cycling, selected hydrophobic coating should be able to be resisted the thermal stresses in the long-time thermal cycling operating process.The example of suitable heat-stable material comprises: the solderable surface solder mask for example

8165, it is used for car industry and has outstanding heat shock resistance; And the pcb board material, Mitsui BN-300 for example, its high temperature resistance and thermal distortion.

Droplet shifts along the path or the network of control electrode and carries out.Array or path comprise electrical communication, are used for electrode is electrically connected on external circuit.Array or path can comprise electrical communication, are used for special electrodes is electrically connected.Electrode is controlled by external circuit by treater.Can realize the droplet operation by giving electrode application voltage.Although preferred voltage is according to the variation of dielectric thickness, be 2-100 and thickness situation for specific inductivity at 1nm to 10mm, the preferable range of energy per unit area is about 300 little joules/square metres to about 300000 little joules/square metres.The scope of preferred trigger voltage for about 1mV to about 50kV or about IV about 10kV or about 5V about 1000V or about 10V about 300V extremely extremely extremely.

Typically, electrode is started by voltage relay.Described droplet microdrive moves by directly controlling discrete droplets, controls as using electric field.For example, contiguous droplet with pressurization electrode of ground connection encirclement electrode will shift so that himself is alignd with the pressurization electrode, and promptly droplet will be transferred to the position of this electrode.A series of continuous transfers are with path or the network transitions of droplet along control electrode.Except shifting, other operation comprises converging, divide, mix and distributing of droplet, and these can carry out in an identical manner by the pattern of this disease voltage starting.

It is noted that electrode can start by number of ways.For example, can be by applying DC current potential starting electrode.Similarly, can be by applying AC current potential starting electrode, the electrode of described like this startup has the AC current potential and the electrode that do not start has ground wire or other reference potentials.On the other hand, can make it to become the anti-phase current potential that applies then by starting electrode repeatedly.Realize the AC pattern by the polarity of using software to change output fast.

In some embodiments, droplet operating structure and technology that the present invention uses can be referring to United States Patent (USP)s 6,911,132, denomination of invention is ＂ Apparatus for Manipulating Droplets by Electrowetting-Based Techniques ＂, the day for announcing is on June 28th, 2005, authorizes Pamula etc.; U.S. Patent application No.11/343,284, denomination of invention is ＂ Apparatuses and Methods for ManipulatingDroplets on a Printed Circuit Board ＂, the applying date is on January 30th, 2006; United States Patent (USP) 6,773,566, denomination of invention is ＂ Electrostatic Actuators for Microfluidics and Methods forUsing Same ＂, and the day for announcing is on August 10th, 2004 and 6,565,727, denomination of invention is ＂ Actuatorsfor Microfluidics Without Moving Parts ＂, and the day for announcing is on January 24th, 2000, all authorizes Shenderov etc.; United States Patent (USP) publication No.20060254933, denomination of invention is ＂ Device fortransporting liquid and system for analyzing ＂, open day is on November 16th, 2006, Adachi etc. incorporate the content of above-mentioned document into the application by reference because of its instruction has related to a structure technology that is used to carry out the droplet operation.

Droplet operation can be carried out fast, typically, its average linear velocity that relates to for about 0.01cm/s to about 100cm/s or about 0.1cm/s extremely about 10cm/s, about 0.5cm/s about 1.5cm/s extremely more preferably.In addition, typically, droplet is controlled control frequency for about 1Hz to about 100KHz, extremely about 10KHz, about 25Hz about 100Hz extremely more preferably of about 10Hz preferably.Except fast, use the droplet of described droplet microdrive to control and go back pin-point accuracy, and can on single droplet microdrive, control a plurality of droplets independently and side by side.

Discrete droplets operation need not to use the Continuous Flow structure, and has avoided all the various shortcomings with the Continuous Flow structurally associated.For example, do not fill pipeline and be full of the pond, therefore can almost 100% utilize sample and reagent owing to there is liquid to be wasted in.In addition, as mentioned above, the droplet motion can be exceedingly fast.In some cases, described droplet microdrive can be replenished by the Continuous Flow component, comprises that therefore these combined methods of discrete droplets operation and Continuous Flow element belong to category of the present invention.The Continuous Flow component can be controlled by controller.But, in some other embodiment, droplet microdrive of the present invention and/or method of the present invention will avoid using various Continuous Flow elements especially.For example, in some embodiments, one or more following component is excluded from droplet microdrive of the present invention and/or method: the microchannel; Fixing microchannel; The microchannel network; Pump; External pump; Valve; High-voltage power supply; The centrifugal force element; Moving portion.

The driving of electric field mediation also need not to use other droplet operations, and has avoided all various shortcomings relevant with this type of technology.Be appreciated that, described droplet microdrive can be controlled technology complementation or replenished by other droplets, for example electricity (for example static driven, two-dimensional electrophoresis), magnetic, heat (for example hot Marangoni effect, hot kapillary), machinery (for example surface acoustic wave, Micropump, wriggling), light (for example photoelectricity is wetting, optical tweezers (opticaltweezers)) and chemistry (for example chemical gradient) device.If use these technology, but relevant hardware also electronics be connected in and be controlled by described controller.But, in other embodiments, droplet microdrive of the present invention will be got rid of one or more these droplet operative techniquies especially.

Described droplet microdrive can be highly integrated the form manufacturing and use minimum device drives.For example, droplet microdrive and equipment have added together little of several cubic inches of sizes.Described droplet microdrive only needs electric energy in a small amount, and can, for example, easily use battery-operated.Described droplet microdrive can use minimum droplet to carry out the droplet operation.Droplet typically is about 1fL to about 1mL, extremely about 1 μ L, further more preferably about 10nL about 1 μ L extremely of about 100pL more preferably.

Use discrete droplets to handle on the chip again but not use Continuous Flow that some important advantages are provided.Owing to do not need to expand sample liquids and be used to fill pipeline or pump, in fact all samples liquid all can be used for analyzing, and can analyze the sample (for example be less than about 100 μ L or be less than about 50 μ L or be less than about 25 μ L) of minimum volume.Also having same advantage aspect the use reagent, wherein reduce the cost that reagent consumption can reduce analysis.Use discrete small volume droplet also to allow in very little substrate (footprint), to carry out a large amount of reactions (for example greater than 10/cm ²Or greater than 100/cm ²Or greater than 1,000/cm ²Or greater than 10,000/cm ²).

Various component of the present invention can be used as the component of described droplet microdrive and is included into.In fact, total system of the present invention can be integrated droplet microdrive.In some embodiments, described droplet microdrive comprises various transmitters and the device that is used for the transmitter electronics is connected in external circuit.In other embodiments, described droplet microdrive comprises well heater and/or magnetic field producing component and the device that is used for described element is connected in external circuit.In addition, the form with pond or droplet comprises that the droplet microdrive of any one or more reagent described herein also is one aspect of the present invention.

Can arrange in electrode that optical window is to strengthen the ability of carrying out optical detection on chip.If form electrode in the opaque material on transparent substrate, window can be set on electrode pass base material to allow light.Perhaps, if electrode materials is transparent, can overcover be set so that eliminate stray light.In addition, can be diffraction grating with described aperture arrangement.Also can use the second electric wetting layer that the adaptive optics window is set.For example, can use opaque oil (for example dying black oil) to be provided with provisional and optical window movably to transparent droplet.

8.8.2 tube

In some embodiments, the present invention includes the tube that is used to be connected in the droplet microdrive.Although be appreciated that tube is optional to practice the present invention, it is very convenient in some cases.If any, tube can comprise the device that is used for the path of described droplet microdrive or network are electrically connected on the treater treater of system (for example of the present invention droplet microdrive).In this embodiment, electrical communication: electrode-tube-treater wherein can have extra element between this three.In another embodiment, tube can comprise the device that is used for physically being connected in described droplet microdrive.In this embodiment, electrical communication can be: electrode-treater-tube.Perhaps, tube can not have electric component fully.

If any, tube can comprise the pond that is used for one or more reagent (for example pre-loaded reagent).Described droplet microdrive can be constructed to make it possible to set up fluid passage between the inside of described tube pond and described droplet microdrive, is used to make reagent, sample and/or fill fluid from described tube to flow to described droplet microdrive.For example, can be before tube be connected in analyser, afterwards or in the process a pre-loaded tube pond is assigned in the described droplet microdrive.Tube can be sealing, independently and/or disposable.It can with or do not provide with the droplet microdrive.The repeatability that this type of can be used for guaranteeing condition determination allows to handle safely or dispose infectivity or hazardous material, and/or reduces the crossed contamination between each wheel.The tube can comprise, for example, the parts of plastics of mechanical workout.It can be attached to described droplet microdrive and make up with it and provide.

Selected tube material can be used for storing reagent and degraded or pollution that reagent can not take place.In addition, they also should be selected as can and guaranteeing in stable operation under the hot conditions to adapt with real-time chemical process.They can comprise, for example, and the molded plastics component.In some embodiments, the disposable test tube of sealing can increase operator's security and help to dispose safely.

The various components of described droplet microdrive system can place on the described tube.For example, have, the inside visible top board that comprises described droplet microdrive can be used as the component of described tube and provides.Various transmitters also can be used as component and are placed on the described tube.

8.8.3 fill fluid

Described droplet microdrive of the present invention comprises one or more freedom (that is fluid-fluid) interface.Example comprises liquid-liquid or liquid-gas interface.Typically, carry out chemical process mutually, and the conduct of secondary phase is with droplet fill fluid separated from one another at elementary (droplet).Secondaryly can be mutually, for example, liquid, gel and/or gas.If the secondary liquid that comprises mutually, this liquid and described elementary liquid phase are fully immiscible, so that described droplet microdrive can carry out one or more droplet operation.

Be noted that described droplet microdrive can comprise two or more phases.For example, in one embodiment, described droplet microdrive can move on the basis of water,oil,gas three-phase system.In a relevant embodiment, described droplet microdrive can move on the basis of the oily three-phase system of water-first oil-second, and for example system comprises the water-based droplet, and it is surrounded by silicone oil, and the latter is surrounded by fluorosilicon oil again.Usually, three-phase system can comprise immiscible or immiscible substantially three kinds of components mutually.

In another embodiment, oily or another kind of immiscible liquid can be used as and is used for the wetting droplet sealing agent of electricity.For example, move through gas/oily interface by making droplet, can be in oilcan with the droplet capsulation.Each droplet will have its oneself local oil bath, and the space between the capsulation droplet is aerification or the third immiscible liquid then.One of advantage of the present invention is that this method can minimize the substance transfer between the droplet in the system by separating in the oil phase, keeps the advantageous characteristic of oil aspect evaporation and surperficial dirt simultaneously.This method also can be used for promoting electric wetting with the immiscible non-electric wettable liquid of electric wettable liquid.In the embodiment under this design, can select crosslinkable (by UV, heat, wet or chemical process) immiscible liquid to be used for the drug conveying synthetic use with the liquid capsule that generation has solid shell.

In addition, may need in some applications or must for example carry out some operation in the oil at immiscible liquid and in gas, carry out other operation.The present invention includes mixing system, wherein not only at gas but also in immiscible liquid filling fluids such as oils, carry out droplet and control.For example, sample can be handled then to be transferred in the air dielectric part in oil and evaporate so that analyze by MS subsequently.Conversely, can collect sample in air then handles with the droplet in the oil.Therefore, described droplet microdrive can comprise that transferring path is used for droplet is moved to the droplet microdrive that opens at atmosphere or comprise the gaseous state fill fluid from the droplet microdrive surface that is full of fill fluid.

Fill fluid can be any type of fluid, and wherein described droplet microdrive can carry out one or repeatedly droplet operation under correct situation.It should be noted that some fill fluid can be solid or highly viscous fluid under given conditions, for example in the attention process, but when operation, their are changed into fluid, as by heating.In the operating process of described droplet microdrive, fill fluid can be liquid or gas.Suitable liquid filling fluidic example includes, but not limited to silicone oil; Fluorosilicon oil; Hydrocarbon polymer comprises for example alkane, for example decane, undecane, dodecane, tridecane, the tetradecane, pentadecane, n-Hexadecane; Aliphatic hydrocarbon and aralkyl hydrocarbon, for example dodecane, n-Hexadecane and hexanaphthene, hydrocarbon oils, mineral oil, paraffin oil; Halogenated oil, for example fluorocarbon and perfluorocarbon (for example 3M Fluorinert liquid); The aforesaid oil mixt of any same classification; Any different classes of aforesaid oil mixt.The example of suitable gas fill fluid includes, but not limited to air, argon gas, nitrogen, carbonic acid gas, oxygen, humidifying air, any rare gas element.In one embodiment, elementary is the aqueous solution mutually, and secondary be mutually with water to immiscible air or oil.In another embodiment, fill fluid comprises the spatial gas of the encirclement described droplet of filling between plate.Preferred fill fluid is low viscous oil, for example silicone oil.Other suitable fluidic examples can be referring to U.S. Patent application No.60/736,399, denomination of invention is ＂ Filler-Fluids for Droplet-Based Microfluidics ＂, and the applying date is on November 14th, 2005, incorporates its full content into this paper by reference.Can select fluid significantly evaporation to occur to prevent droplet.

Can select in the solution of the present invention employed fluidic can not form improperly bleed fill fluid and/or reagent of bubble, reagent with the smooth implementation the solution of the present invention and adhere to described droplet microdrive surface.

In some embodiments of the present invention, can select fill fluid to reduce or to prevent the evaporation of the employed sample of the solution of the present invention, reagent or other droplets.Can select fill fluid with the evaporation that prevents the employed sample of the solution of the present invention, reagent or other droplets and become very few so that be difficult to carry out further valid function.Similarly, can select fill fluid to cause material generation hazardous property in the droplet to concentrate so that the use of described droplet is caused detrimentally affect improperly with the evaporation that prevents the employed sample of the solution of the present invention, reagent or other droplets.In addition, can select fill fluid to pass the border of phase, with the volume of keeping droplet and/or guarantee reliable microfluidic procedures and/or measurement result to reduce or to prevent that material from shifting from the employed sample of the solution of the present invention, reagent or other droplets.Blendability mutually causes droplet that shrinkage (or expansion) takes place mutually sometimes.In order to prevent or reduce this problem that one or more of system can carry out saturated to reduce shrinkage or expansion mutually through the another kind of equilibrium concentration mutually.Therefore, for example, can use the saturated fill fluid of solvent that is used for the employed sample of the solution of the present invention, reagent or other droplets of equilibrium concentration, and/or the employed sample of one or more the solution of the present invention, reagent or other droplets can use the fill fluid of equilibrium concentration to carry out saturated.

In some embodiments, select the liquid filling fluid so that contacting between droplet and the droplet microdrive surface minimizes.That is to say, can have liquid membrane between droplet and the surface, it prevents the material contact in the droplet and adheres to coated surface.This method helps to prevent the dirt on surface and the relevant interference that transfer causes to droplet.For example, some protein that has been found that the high density in the water-based droplet is bonded at hydrophobic surface easily and damages these surperficial hydrophobic propertys; And if be immersed in the minimized oil of contact that can make between two surfaces, then same droplet is removable to be passed identical surface and perceptible protein adherence can not occur.This method also can help avoid because of the crossed contamination between the droplet that is received to cause by second kind of droplet from a kind of deposited material of droplet.In similar embodiment, can use film between droplet and the droplet microdrive surface by preventing that the frictional force sample physical property between the droplet and surface interacts to lubricate droplet in the droplet operating process.

In one embodiment, the invention provides liquid filling stream layer thin layer coating in the system that other parts are filled by gas.For example, the invention provides microfluid system, it comprises system open or sealing, and this system comprises and be formed on the lip-deep thin layer fill fluid of droplet microdrive, oil for example, and other parts of wherein said system are filled by gas.Oil has enough thickness so that the lubricated of droplet microdrive surface is provided and is infected with and by droplet microdrive surface being infected with droplet.Preferably, select oil so that the substance transfer between droplet and the oil phase is minimized.An advantage of this method is to reduce the hangover in the described droplet microdrive.In some embodiments, can handle described surface with the fill fluid coated surface by when operation in air.This method also can be used for load operation, avoids oil gas to steep the means that are absorbed in a large amount of fill fluids simultaneously as the lubrication that keeps oil.

Handling tetrafluoroethylene AF surface with silicone oil can provide the filled with silicone oil fluidic to lubricate benefit, also is like this when promptly box lunch is operated in air.This method can be used for using the oil lubrication layer to fill described droplet microdrive, subsequently again with air displacement so that load sample and can not introduce bubble is introduced oil subsequently again to prevent sample evaporation.Therefore, according to the type of pending microfluidic process, the system of each type is all helpful.

In another embodiment, can the different step in scheme exchange fill fluid fully.For example, can in the application of sample process, introduce the gas fill fluid preventing to bring into air filled cavity, but then the pumping liquid fill fluid in case the fluid stopping evacuator body.Can dissimilar fill fluids be pumped into or pumping out system according to pending concrete determination step.

In another embodiment, can in individual system, use multiple fill fluid.For example, can select to have the droplet microdrive in gas separated fill area and fluid filled district.Can between different fill fluids district, separate the droplet operation of particular type.

Can be selected to make it to be complementary to prevent refraction to it based on the specific refractory power of fill fluid through near the light described droplet or the described droplet with droplet.Perhaps, can select specific refractory power to be different from the fill fluid of droplet so that provide contrast medium for the opticmeasurement or the optical control of particular type.Can select specific refractory power to be lower than elementary fluidic fill fluid and propagate so that light can pass through elementary fluid by total internal reflection.Elementary phase can comprise the highly droplet of elongation, and it can be used as ＂ light pipe ＂ light is transmitted between two positions, for example is used to promote optical analysis.

Can be selected it based on the color of fill fluid, to help directly or indirectly to manifest droplet, for example by providing contrast between employed sample, reagent and/or other droplets and the described fill fluid in the solution of the present invention.This method can strengthen out of phase manifesting, and for example is used for droplet and fill fluid or air filled cavity are distinguished.In optical application, the difference absorbancy of two kinds of phases can be used for adjusting the color through the light of described system.As another example, for the application of in droplet, carrying out fluorescence measurement, may wish that oil comprises the molecule of the light that absorbs emission wavelength, dyestuff for example will be so that will be close to mutual minimum interference between the reaction that takes place in the droplet.

Can select to have the fill fluid of special thermal property, it can make the droplet thermal insulation or heat is conducted from droplet.For example, in amplification scheme of the present invention, may need thermal conductivity or low heat capacity fill fluid so that change temperature fast.For the application of the stable temperature of needs, can use thermal insulation or high heat capacity fill fluid to keep temperature-stable.

Can undergo phase transition in the time of can selecting fill fluid to provide suitable stimulation with box lunch.For example, can use cured sample fill fluid (for example paraffin or octadecane), wherein described fill fluid be become liquid form by solid by heating.Reduce temperature and can make that fill fluid reverts to solid, droplet just can be contained in the solid substrate like this.The liquid phase capsulation can be helped the storage of the employed sample of the solution of the present invention, reagent or other droplets and operation and/or allow after using described droplet microdrive, can safely and conveniently dispose material in solid.Fill fluid can be used as solid be stored on the described droplet microdrive, based in the pond of tube or elsewhere, and can heat to allow described fluid to flow to and fill described droplet microdrive.Perhaps Xuan Ding immiscible fill fluid can be crosslinkable (by UV, heat, wet or chemical process), is positioned at the liquid capsule of solid shell with generation.

Selected fill fluid can have specific gas permeability or saturation characteristic.In some applications, but the reaction oxygen consumed or other gas that take place in the droplet need be replenished by gas contained in the fill fluid or the gas by its transfer.For example, some fluorizated oil has the gas permeability that can be used for this type of purpose.Perhaps, can select fill fluid, for example, be used to keep the anaerobic condition in the droplet so that get rid of some gas from droplet.Selected fill fluid can have blendability or the interior mutually separation of droplet to a certain degree.Usually, wish to lack fully between droplet and the fill fluid or lack blendability basically fully, but some application may have benefited from the blendability of certain limited extent between the phase or the separation of the particular molecule between the phase, uses as liquid-liquid extraction.Gas in being dissolved in fill fluid may be in problematic some embodiment, before use or may need to provide the device that fill fluid is outgased in the process.For example, can fill fluid be outgased by incubation, heating, spraying under vacuum condition or by centrifugal.

Selected fill fluid can have special surface or interfacial tension mutually or with described droplet microdrive surface with droplet.Surfactant can join in the fill fluid so that stablize the liquid film that may exist between droplet phase and the solid phase.The example of suitable surfactant comprises that nonionic hangs down HLB (hydrophile-lyophile balance) surfactant.HLB preferably is less than about 10 or be less than about 5.Suitable example comprises: Triton X-15 (HLB=4.9); Span 85 (HLB 1.8); Span 65 (2.1); Span 83 (3.7); Span 80 (4.3); Span 60 (4.7); With the fluorizated surfactant.

Selected surfactant and the amount that is provided thereof preferably (1) are compared with the corresponding droplet microdrive of no described surfactant, can carry out more droplet operation on described droplet microdrive; Or (2) compare with the corresponding droplet microdrive of no described surfactant, can carry out one or repeatedly droplet operation on described droplet microdrive; Or (3) compare with the corresponding droplet microdrive of no described surfactant, can make one on the described droplet microdrive or repeatedly the droplet operation is more reliable.In relevant example, selected surfactant and the amount that is provided thereof are preferably such that, compare with droplet operation on the corresponding droplet microdrive of no described surfactant, can be on described droplet microdrive the droplet that comprises one or more particular agent or mixture be carried out one or repeatedly droplet operation or make that described droplet operation is more reliable the same droplet that comprises one or more particular agent or mixture.In another relevant example, selected surfactant and the amount that is provided thereof are preferably such that, compare with droplet operation on the corresponding droplet microdrive of no described surfactant, can be on described droplet microdrive one or more droplet that comprises amphipathic molecule be carried out one or repeatedly droplet operation or make that described droplet operation is more reliable the same droplet that comprises amphipathic molecule.

One preferred embodiment in, described surfactant add in the fill fluid amount about 0.001 to about 10%w/w about 0.001 to about 1%w/w or about scope of 0.001 to about 0.1%w/w in.For example, in one embodiment, fill fluid is a 2cSt silicone oil, and described surfactant is Triton X-15, the amount of described Triton X-15 about 0.001 to about 10%w/w about 0.001 to about 1%w/w or about scope of 0.001 to about 0.1%w/w in.Solid-liquid interface tension force can be adjusted to and be used to control the moistening of the lip-deep fill fluid of described droplet microdrive, for example, be used to control formation, thickness or the performance of the fill fluid thin layer between droplet and the droplet microdrive surface, or be used for control and fill described fluid or the moistening performance of described fluidic when described droplet microdrive empties described fluid for described droplet microdrive.

By coating surfactant to fill fluid, the inventor finds that the concentration of compatible protein matter solution can be enhanced 3 more than the order of magnitude, from mg/L to mg/mL.The inventor can use new fill fluid to distribute the also lysozyme soln of the 75mg/mL of transferase 12 5nL droplet reliably.For example, fill fluid can be the silicone oil that scribbles surfactant, for example Triton X-15.Preferably, surfactant is the lipotropy surfactant.In one embodiment, we are 0.1% (w/w) Triton X-15, and promptly a kind of lipotropy surfactant adds oil supply, can form the high concentration protein droplet like this or such droplet is distributed in the pond from chip.For all consistency fluids, droplet shifts rapidly (typically being about 3-10cm/sec) and reliable (〉 25,000 and operates).In one embodiment, fill fluid comprises the surfactant dopant, and the protein concn that its amount causes being dispensed to reliably on the described droplet microdrive increases.

Selected fill fluid can have special viscosity or volatility.For example, low viscosity fluid (for example 0.65cSt. silicone oil) helps to shift droplet, and the fill fluid loss that low volatility fill fluid (for example 2,5 or 10cSt. silicone oil) is suitable for avoiding evaporating and causes is particularly carried out in the situation of nucleic acid amplification at high temperature.In some applications, the fill fluid evaporation may be wished, therefore the volatility filtered fluid can be selected.Selected fill fluid can have special temperature-viscosity dependence, because the viscosity influence hydrokinetics of fill fluid and temperature on the described droplet microdrive can change.In nucleic acid amplification scheme of the present invention, select fill fluid so that any viscosity-modifying that thermal cycling causes all can not cause infringement improperly to implementing the required droplet operation of amplification scheme.

Selected fill fluid can have special electrical characteristic.For example, some application comprises the electric wetting non-conducting fill fluid (for example silicone oil) that is beneficial to use.Perhaps can select dielectric Constant will enter system from the electric energy coupling of outer electrode with control.Some can use non-conducting fill fluid as electrical insulator or dielectric medium in using, and wherein droplet only swims in the electrode top and do not have physics to contact with electrode.For example, in electrowetting system, can use fill fluid (for example silicone oil) layer between droplet and the electrode that droplet is carried out Electrostatic Control.Fill fluid can be by deionization to reduce conductivity.

Selected fill fluid can have special density mutually with respect to droplet.Density variation between the two-phase can be used to control or the buoyancy of developmental function on droplet.The example that can be used for this biphasic system on the one hand of the present invention comprises water/silicone oil, water/Flourinert, He Shui/fluorosilicon oil.If one is buoyant mutually, can in vertical configuration, use this effect to pass through the means of another phase mutually as transfer one so.For example, waste liquid or collection hole can be positioned at the top or the bottom of described droplet microdrive, can be by simply droplet being released in suitable point and allowing them floating or sink to the target point of destination and droplet is delivered to this pond at this place.This method is applicable to removing reagent from the droplet microdrive, and the fluid that for example removes the nucleic acid that contains amplification is used for other processing.Density variation also can be used as control or handles means that contact between droplet and the droplet microdrive surface.For example, can discharge the droplet that does not contact top board usually makes it sinking or is floated to this surface to contact with it.Also density variation and buoyant effect can be used for sensing and use, wherein detect the motion of droplet and make it and be associated with the variation of position, direction or acceleration.

Selected fill fluid should have material compatibility with described droplet microdrive surface.For example, but some fill fluid etch, dissolving, pollute, be adsorbed to or otherwise incompatible with some droplet microdrive material.For example, the fluorizated hydrocarbon polymer, Fluorinert for example may be incompatible with tetrafluoroethylene AF or Cytop surface, because they can dissolve these materials, and silicone oil may be incompatible with the PDMS surface, because these materials tend are in mutual dissolving.

Selected fill fluid will have the biological chemistry consistency with sample that is used for the solution of the present invention and reagent.

The present invention can comprise introducing or the round-robin device that is used to control described droplet microdrive, tube and/or intrasystem fill fluid.In a kind of operating method, in the process of initial droplet microdrive operation, inject a fill fluid.Can use syringe, dropper, suction to move device, kapillary, pipe or other devices and provide fill fluid from external source.Perhaps, can provide fill fluid from the pond that is positioned at described droplet microdrive assembly or tube inside.For example, fluid can place air-tight pouch, through puncture or extruding and with described fluid transfer to the described droplet microdrive.

In another kind of operator scheme, can be provided for one or more fill fluid repeatedly being introduced described droplet microdrive or making it at described droplet microdrive at the round-robin device.Can provide the secondary fluid treatment system so that inject fluid or it is removed from described droplet microdrive.But applying pressure, gravity or other for example install the use temperature gradient and fill fluid is transferred in the described droplet microdrive or from its transfer goes out.This system can be used for following purpose:

(1) replenishes the loss that fill fluid evaporates in time or leaks.Can use the slow steady flow of fill fluid or periodically injection and any loss of supplying the fill fluid volume.

(2) provide the ＂ fill fluid of ＂ cleaning to reduce the pollution between the droplet constantly or periodically.Can clean fill fluid by whole replacements or the filter or the bed that cycle through the sorbing material of removable pollutent.

(3) provide the device that droplet is transferred to the waste liquid place.For example, after measuring end, can discharge droplet and make it to flow to outlet the means that provide ＂ to wash the described droplet microdrive of ＂ with fill fluid.Flushable described droplet microdrive is so that reset the state of described droplet microdrive, in order to carrying out extra mensuration.

(4) when different step needs different fluid, the exchange fill fluid, for example with the air replaced oil so that dry droplet or replace another kind of oil with a kind of oil.

(5) provide by heating or cooling round-robin fluid in described droplet microdrive to control the device of droplet temperature.For example, can in described droplet microdrive, circulate by the fill fluid that makes controlled temperature and carry out PCR in the droplet that is containing suitable PCR reagent (for example primer, Nucleotide and polysaccharase) to carry out thermal cycling.The temperature of the fill fluid that enters and leave described droplet microdrive be can directly measure, and the temperature of fill fluid and the temperature control that flow velocity provides the best to give described droplet microdrive inside adjusted.

Use the regional area or even the independent unitary fill fluid of fill fluid can for each droplet.For example but water-based droplet capsulation is in the independent shell of fluidic, oil for example, and it moves with this droplet.Each this type of droplet will have its oneself partial fluid thus bathes, and the third immiscible fluid, for example air or fluorosilicon oil are filled in the space between the droplet of capsulation.Can use this method the substance transfer between the droplet in the system to be minimized, keep the advantageous characteristic of oil aspect evaporation and surperficial dirt simultaneously by separating in the oil phase.By simply droplet being moved through oily interface,, can produce oilcan when droplet causes the oil that squeezes out a unit when expanding along described interface.

Can use mixing system, the different zones of wherein said droplet microdrive is filled different fluids.For example, sample can be handled then to be transferred in the air part in oil and evaporate so that analyze by MS subsequently.Conversely, can collect sample in air then handles in oil.

Can use the magnetic responsiveness pearl to move oil phase on the droplet microdrive and the material between the water.Usually, water-soluble compound or material trend towards staying in the droplet, can not pass oil-water interface in a large number, and oily soluble compounds or material are stayed in the lipotropy fill fluid.When described material is attached to the magnetic responsiveness pearl, can use magnetic field to move pearl and accompanying material passes oil-water boundary.Need to select that oil and glassware for drinking water are had the pearl of enough avidity so that they can easily pass separation surface.This operation can be used for drying or condensed matter, also can be used for promoting washing and/or dilution.For example, can from a droplet, remove and be transferred to another droplet by means of the material that fill fluid will be incorporated into the magnetic responsiveness pearl.

Fill fluid capable of circulation by described droplet microdrive with reduce in each wheel process and/or between pollution.Fill fluid is sustainable or periodically flow through described droplet microdrive, so that fresh fill fluid is constantly replenished to described droplet microdrive.Except being used to remove the oil that contaminated thing pollutes, also can when end of run, use this technology so that from array, remove droplet, can remove voltage, make droplet be released, and along with oil flow to the outside of described droplet microdrive and/or enter waste liquid pool.

8.8.4 the droplet microdrive loads

Described droplet microdrive generally includes one or more input port, droplet microdrive as described in being used for one or more fill fluid, reagent and/or sample (for example be used to carry out the reagent and/or the sample of described scheme of other parts of this paper and/or mensuration, as referring to 8.1,8.2,8.3,8.4 and/or 8.5 joints) introduced.In some embodiments, use conventional robot technology by input port load sample or reagent.In an alternative embodiment, by plug cock sample separation in the long glass capillary of pre-filling type or reagent droplet, this device can be caught sample or reagent droplet when being connected in described droplet microdrive, enter input port and send it on the described droplet microdrive along with droplet is pumped out kapillary.Another loading technique relates to and is molded into reagent on the described droplet microdrive in advance and makes its drying, as using high speed reagent mold pressing or printing method.Another kind method relates to uses direct plate-to-droplet microdrive interface, wherein by applying pressure impel content by the input port that aligns with the hole content of plate (as 1536 or 384 or 96 orifice plates) is transferred to abreast as described on the droplet microdrive.The loaded with hardware electronics can be connected in and be controlled by described controller in some embodiments.

8.8.5 pond

Described droplet microdrive comprises multiple pond, for example imports pond and/or treating pond.

8.8.5.1 input pond

In some embodiments, described droplet microdrive comprises one or more input pond that is communicated with one or more input port fluid, typically is communicated with the direct fluid of input port.The input pond is used to distribute droplet (for example reagent droplet or sample droplet) as the pond that stores a large amount of sources materials (for example reagent or sample).Therefore, the input pond can be used as, for example, and sample well or reagent wells.

The input pond generally includes the hole wall that one or more limits internal space and opening.The internal space that hole wall limited separates at least in part by the rest part of hole wall with described droplet microdrive inside.Described hole be fit to and will introduce the port contiguous (with any direction, as vertical or side direction) in input pond from the fluid of the outside of described droplet microdrive.Can in hole wall, provide one or more opening so that be communicated with, be used to distribute droplet to enter this internal volume with the described internal volume fluid of described droplet microdrive.Opening can allow fluid to flow into or be transferred in the described internal volume of described droplet microdrive, arrives on the path or network of electrode.Input Chi Yeke comprises one or more outlet (vent), introduces described hole by described port or opening or when removing in the hole, can replace from the fill fluid in input pond with convenient fluid.

The input pond can further comprise one or more plane control electrode in top board or the base plate, the spatial neighbor that described electrode and hole wall limited or be positioned at wherein.The plane electrode electronics is connected in and is controlled by controller.One preferred embodiment in, plane electrode has two or more branches or radial line, like this, exists under the fluidic situation, the start-up control electrode can apply ＂ pulling force ＂ by convection cell in the droplet distribution process, and the direction of pulling force is opposite with the direction that droplet distributes usually.In some cases, the shape of electrode produces multidirectional amount pulling force, and the direction of the average vector that it has is opposite with the direction that droplet is assigned with usually.

Hole wall for example can be formed by the projection from top board or base plate, and/or can form material by wall and be deposited on the surface of top board or base plate and form.For example, hole wall can be formed by deposition and solderable surface solder mask material or the polymeric liner cushion material of medelling in described surface.In some embodiments, the source of continuous or semicontinuous sample or reagent stream is connected with one or more described input port in the mode that fluid is communicated with.

It should be noted, distribute, in some embodiments, carry out the droplet distribution and can not use the pond that physically limits although can carry out droplet from the pond that is limited.Can be from the droplet distribution process (for example by electric wetting power or pass through hydrophilic surface) source droplet of being limited distribute.

8.8.5.2 treating pond

Described droplet microdrive can also comprise one or more treatment zone or pond.These zones or pond be with acting on the position of implementing various droplet treatment steps, for example mix, heating, incubation, cooling, dilution, titration and or the like.Described droplet microdrive comprises one or more control electrode path or network, and it is enough to droplet is transferred to one or more treatment zone or pond from one or more input port.In some cases, treatment zone only is the component or the district of these paths or network.In other embodiments, treatment zone is the treating pond that limits.The structure in this type of pond is for example, identical with above-mentioned input pond substantially.But, treating pond typically directly is not communicated with the input port fluid, that is, along one or more control electrode path or network carry out droplet and shift and need add reagent or sample to treating pond.In some cases, treating pond comprises that the path in pond or network are so that carry out the droplet operation in treating pond.

8.8.5.3 droplet operation

Described droplet microdrive can carry out multiple droplet operation to droplet.Example comprises: droplet is loaded in the droplet microdrive; Distribute one or more droplet from the source droplet; Droplet is divided, separates or is split as two or more droplets; Droplet is transferred to another place from a place along any direction; Two or more droplets are converged or merge into a droplet; The dilution droplet; Mix droplet; Stir droplet; Make little droplet deformation; Make droplet keep original position; The incubation droplet; The heating droplet; The evaporation droplet; The cooling droplet; Arrange droplet; Droplet is migrated out microdrive; Other droplet operations as herein described; And/or above-mentioned every arbitrary combination.

Droplet distributes the process that the fluid of comparatively large vol is divided into less droplet that refers to.Can use at fluid interface, input pond and treating pond and distribute.Form droplet by providing voltage to cause near the fluid pool electrode to stretch out fluid ＂ finger piece ＂ from described pond.When fluid front end arrived end electrodes, the voltage of target was withdrawn from, and this causes in the fluid withdrawal pond, and the electrode place stays the droplet of new formation endways simultaneously.As previously mentioned, one or more electrode in the pond also can be provided the droplet of voltage to help separately to distribute from big fluid.Because droplet is consistent with the shape of electrode, and electrode is a fixed, therefore can reach outstanding precision and accuracy.Droplet distributes to be controlled by controller.In some embodiments, the present invention uses described droplet distribution pattern of following document and/or technology: United States Patent (USP) 6,911,132, denomination of invention is ＂ Apparatus for Manipulating Droplets by Electrowetting-Based Techniques ＂, and the day for announcing is on June 28th, 2005; U.S. Patent application No.11/343,284, denomination of invention is ＂ Apparatuses and Methods for Manipulating Droplets on a Printed Circuit Board ＂, the applying date is on January 30th, 2006; United States Patent (USP) 6,773,566, denomination of invention is ＂ ElectrostaticActuators for Microfluidics and Methods for Using Same ＂, and the day for announcing is on August 10th, 2004 and 6,565,727, denomination of invention is ＂ Actuators for Microfluidics Without MovingParts ＂, and the day for announcing is on January 24th, 2000, all authorize Shenderov etc., incorporate the content of above-mentioned document into the application by reference.

In some embodiments, the droplet operation is mediated by electrowetting technology.In other embodiments, the droplet operation is mediated by electrophoretic technique.In other embodiments, the droplet operation is by electrowetting technology and electrophoretic technique mediation.

In one embodiment, can be used in combination electricity wettingly separates with electrophoresis.Can make the wetting little driving of electricity consumption produce passage so that carry out electrophoresis; So that the conveying sample is caught the sample composition from described passage to passage or after electrophoretic separation.For example, in order to form passage, can make the wetting little droplet deformation of separating medium (elongation) that makes of electricity consumption become subsequently elongated shape.In some cases, passage can polymerization, as using the UV polymerization.In other cases, can form passage by utilizing the droplet operation that droplet is added in the microchannel with physical boundary.In relevant embodiment, can be by also subsequently it being sent back to the useful length that the input aperture increases electrophoresis path with the interested composition of round-robin mode in droplet is caught in the exit.Adopt identical principle, can carry out the separation of a series of progressively refinements.Can also use multiple different separating medium simultaneously and realize separation.

Droplet division or droplet split and are usually directed to droplet is divided into two or more inferior droplets.In some cases, the droplet that obtains has the same relatively size.

Transfer relates to droplet is moved to another position from a position along any direction.Droplet can shift or do three-dimensional the transfer in the plane.Be appreciated that the operation of various droplets, for example distribute and/or divide that can comprise transfer element, droplet is transferable another droplet that leaves on this element.

Converge to relate to two or more droplets are merged into single droplet.In some cases, droplet of a size is relatively converged mutually.In other cases, droplet can converge in the bigger droplet, for example the more volume that exists in droplet and the pond is merged.

The mixing droplet relates to various droplets to be controlled, and for example shifts or stirs, and causes the distribution of component in droplet to have uniformity more.Mix in the embodiment at one, be out of shape fast and periodically in position by the droplet that starts and stop electrode, make to be placed on the electric wetting electrode, induced fluid flow in droplet, thus promote to mix.Can utilize the frequency dependence effect for example mechanical resonance adjust blended quality and speed.Shift droplet from the teeth outwards with needs and be used for the blended technology and compare, this method will be mixed required area and be reduced to minimum.This mixed program can use under the situation that does not have top board.Owing to have the spatial advantage of saving, this plan makes it possible to the mixing simplified at the reacting hole place, and this only needs an electrode.

Reagent or sample from the pond can be assigned as discrete droplets, be used to be transferred to other positions on the described droplet microdrive.

The present invention includes the droplet operation of using the droplet that comprises pearl.This paper has described various these generic operations in other parts.In one embodiment, use pearl being easy to carry out the droplet operation on the reagent that disturbs droplet to operate.For example, some protein is easy to be incorporated into the surface of droplet microdrive and/or separates fill fluid.This compounds can be immobilized onto on the wetting ability pearl to promote to use the droplet operation of described compound.Described compound can be incorporated into pearl, and described pearl can be contained in the droplet, and droplet is carried out the droplet operation.

In a kind of special batch operation, utilize coagulation of blood separation of serum from whole blood.Whole blood is loaded on the chip and merge with the droplet that comprises coagulant.After condensing, from sample, distribute droplet.Because cell and thrombocyte are limited in original position, from sample, distribute the fluid that comes only to contain serum.

8.8.6 thermal control

Described droplet microdrive of the present invention can comprise the device of the temperature in the zone that is used to control described droplet microdrive or droplet microdrive.Wherein, thermal control can be used for the scheme of various needs heating or cooling step.Example comprises the various mensuration schemes that need the amplification of thermal cycling scheme and need incubation step.

8.8.6.1 thermal control design

Generally, can provide thermal control by three kinds of approach: (1) is to the thermal control of whole droplet microdrive; (2) to the thermal control in the zone of droplet microdrive, wherein use the well heater that contacts or be close to it with controlled district; (3) to the thermal control in the zone of droplet microdrive, wherein use the well heater be incorporated in the described droplet microdrive (for example to be positioned at the base material that comprises electrode path or array and/or to be positioned at the top board of described droplet microdrive, if present).The combination of preceding method also is feasible.Figure 2 shows that two kinds of methods that discussed the front.

In integrated well heater method, use the thermal control system that is integrated directly in the described droplet microdrive to produce and the controlled temperature district.Can use by directly integrating thermal control so that at utmost improve speed, flux and the quality of the amplified reaction on the described droplet microdrive at the film heating element of making on the described droplet microdrive.Because heat (thermal mass) is very little, so droplet can carry out thermal cycling at a terrific speed.By heating unit is positioned near the position of droplet and reduce well heater and droplet between parasitic heat loss enhancing thermal control.Heating unit can be incorporated in the top board and/or base plate of described droplet microdrive.

Heating unit is incorporated into also makes it possible in described droplet microdrive to use a plurality of different humidity provinces on the described droplet microdrive.The a plurality of steps that need differing temps in this feasible analysis, for example specimen preparation and thermal cycling can be carried out on the different piece of described droplet microdrive simultaneously.Droplet can be shifted by physical property or ＂ shuttles back and forth ＂ between the zone with different fixed temperatures, to carry out the thermal cycling part of amplified reaction.This method can provide reaction faster, because the heating and cooling of whole humidity province no longer are rate-limiting factors.Otherwise, reach the required time of balance in case heating and cooling speed depends on the temperature that shifts required time of droplet and droplet arrival described district back droplet temperature and described district between each district, and expect that both are all very fast.Another advantage is that reactions steps can be the ＂ that lines up of ＂ but not ＂ ＂ in batch, and this allows higher flexibility of operation.For example, discrete samples can be added in the described droplet microdrive continuously, but not carries at single time point.

Can use single well heater droplet to be carried out thermal cycling or with circulation pattern (flow-through mode) droplet carried out thermal cycling by the differing temps district that makes droplet cycle through the heating unit generation to become batch mode.Become the key difference between batch mode and the circulation pattern to be, in becoming batch mode, thermal control is to realize by the temperature that changes described well heater, and in circulation pattern, thermal cycling is to realize by shift droplet between different steady temperature districts.In batch in the ＂ method, use the thin film heater of the single integration on the described droplet microdrive that the static droplet that is positioned at heater zone is carried out thermal cycling at ＂.In ＂ circulation ＂ method, on described droplet microdrive, produced two kinds of different fixed temperature districts, and carried out thermal cycling between these two districts by droplet is shuttled back and forth.

At ＂ in batch under the ＂ situation, can be located immediately near the droplet thin film heater by use and make the heat and the further minimum heat losses of well heater itself.Because described heat comprises droplet itself, and is all so little, therefore can realize temperature change fast.Passive cooling (in fill fluid) is same fast, because compare with total heat, it is minimum to be input to intrasystem total energy.

For ＂ circulation ＂ heating, need bigger heat, because this helps equilibrium temperature, and because heater temperature no longer changes after in a single day arriving its setting point, therefore slower ramp velocity is acceptable.For example, can use the module well heater of described droplet microdrive outside to move the system for the distribution of commodities, more accurately and easier control, but, in principle, arbitrary type heater all can be used for implementing arbitrary method to the module well heater than thin film heater.

In another embodiment, flow through by the fill fluid that makes heating or circulate in around chip and the droplet and controlled temperature.

Described droplet microdrive layout is upgradeable, thus the droplet microdrive can comprise minority for example 1 heating zone to dozens of, hundreds of and even more heating zone.

8.8.6.2 heater types

Well heater can be formed by the thin layer conducting membrane.The example of suitable film comprises Pt well heater metal wire and transparent indium-Xi-oxide compound (ITO).ITO can show better and is beneficial to Real Time Observation by droplet.That also can use long-range placement is used for thermoregulator conventional thermopair (TC).In one embodiment, can use thin metal (for example copper) path in the PCB base material between fluid and long-range TC, to set up thermopair joint (thermal junction) closely.In addition, determine sample temperature by the thermistor or the infrared sensor monitoring copper path that use the surface to place.Use an advantage of thermistor be they enough little (2 x 2mm), can directly be welded on the described droplet microdrive, and use the advantage of IR to be that it is contactless method, can connect by simplifying interface.Because the thermal conductivity of copper is than FR-4 base material high at least 700 times (350-390W/m-K is than 0.3-0.5W/m-K), the temperature of Cu path can accurately be represented the fluid internal temperature.Well heater can be incorporated on described droplet microdrive base plate and/or the top board (if present) and on the bottom surface and/or end face of arbitrary plate, or is incorporated into the inside configuration of arbitrary plate.

In a circulation embodiment, can provide the thermograde of reduction by using well heater, stride the continuous temperature gradient (for example from 100 to 50 ℃) of described droplet microdrive with generation.Use continuous gradient to eliminate the steep thermograde that appears at the heater module edge.Controlled thermograde allows to implement to have the scheme of any amount temperature spot, therefore obvious intensifier functional also.In addition, each reaction can be carried out according to the hot scheme of customization, and only needs the temperature of two or more modules of thermal conditioning.Droplet will be transferred to and remain on the correct position between the well heater, so that reach the target temperature.When droplet is transferred to check point, can use fluorescent optical sensor to come the fluorescence of imaging droplet.Can change the bound temperature of target temperature by the position that changes droplet.

In some embodiments, place the well heater of droplet top can cover droplet, therefore disturb real-time optical to measure.In this type of situation, droplet can be transferred to preferred optical detection position (that is check point) from the well heater below.Purpose for the droplet microdrive detects for example detects by fluorescent quantitation, and droplet can periodically be transferred to check point from the well heater below.When a humidity province was circulated to another, droplet can be sent near the transmitter at droplet.

8.8.7 the droplet microdrive is made

Can use routine to be used on the droplet microdrive producing conductivity and interconnect the standard micro-fabrication technology of structure and/or use printed circuit board (PCB) that (printed-circuit board, PCB) manufacturing technology prepares the droplet microdrive.Suitable PCB technology comprises disclosed those technology of following document: U.S. Patent application No.11/343,284, denomination of invention is ＂ Apparatuses and Methods for Manipulating Dropletson a Printed Circuit Board ＂, the applying date is on January 30th, 2006, incorporates its full content into this paper by reference.Use these technology can be with the described droplet microdrive of extremely low cost mass production.The low-cost manufacturing makes it possible to produce the droplet microdrive economically, or even disposable disposal type.Therefore, the invention provides a kind of method, wherein the droplet microdrive is offered the user as the component of the disposable cartridges that is used for system of the present invention.

Also can adopt conventional microlithography (microlithography) technology to implement design on glass or silicon, this technology can produce the parts more much smaller than the typical component in the PCB technology.Even, for example, have 1 for one, 572,864 ponds, pond distances are that 70 μ m, pool volume are the droplet microdrive of 3fL, and required lithography component sizes minimum is～0.5 μ m, fully within the limit of power of the conventional microlithography printing technology in present semi-conductor industry.

8.9 system

Can use droplet microdrive system for example shown in Figure 180 to realize that fluid loads.Can use droplet Controlling System 1801 to carry out the step of fluid loading scheme.Write out the executable instruction of a sets of computer, be loaded on and be used to carry out in the controller of loading scheme.Also can use the integration system that comprises droplet Controlling System 1801 and scheme executive system 1802.Droplet Controlling System 1801 allows the user to control droplet microdrive system function, for example operation of the droplet of fluid loading scheme and transmitter operation.Scheme executive system 1802 allows the user to carry out the software program of control droplet microdrive system function, and for example droplet is operated and the fluid load operation.The present invention also is provided for carrying out the method or the computer-useable instructions of fluid loading method or scheme.This platform has the sequencing handiness, allows the rapid Optimum assay method and allows to implement performing step with good conditionsi.For example, if be subjected to fc-specific test FC result's triggering, can carry out calibration, confirm test or extra control.In some embodiments, system can integrate sample preparation steps.Operation has increased portability on the automatization of system and the droplet microdrive, and makes it possible to measure quickly and by the personnel that accept minimum training, has reduced personal errors thus.

Further exhibition Figure 18, at high level, each system of system of the present invention typically comprises treater or controller 1803, droplet microdrive 1804, transmitter or detector 1805, input unit 1806, take-off equipment 1807 and software.U.S. Patent application No.60/806,412, denomination of invention is ＂ Systemsand Methods for Droplet Microactuator Operations ＂, the applying date is on June 30th, 2006, incorporate its full content into this paper by reference, the document discloses droplet microdrive system, and it can use with droplet microdrive of the present invention aspect is collaborative.The droplet Controlling System comprises the droplet control software, and it moves on computer 1808 and is turned to the droplet control interface that demonstration is used to control droplet microdrive system function by program.The scheme executive system comprises the scheme executive software, and it is turned to by program and helps to carry out a set of computer-executable instructions or computer-useable instructions, is used to control droplet microdrive system function and loads to carry out fluid.

8.9.1 controller

System of the present invention can comprise controller 1803.Controller is used to provide processing power, for example stores, analyzes and or executive software instruction.Controller for example can be made up of the digital signal processor with internal memory (DSP), microcontroller or special purpose integrated circuit (ASIC).The example of suitable dsp processor is an Analog Devices Blackfin dsp processor.

The controller electronics is connected in various hardware components of the present invention, for example described droplet microdrive, any transmitter and any input and/or take-off equipment.Controller can be configured and program turns to data and/or the power supply aspect of controlling these devices.For example, for described droplet microdrive, controller is controlled droplet and is controlled by starting/stop electrode.These aspects of the present invention are further discussed at 8.8 joints.

Controller further electronics is connected in independently computer system, and described computer system comprises treater, input and output device, data storage medium and other components.This set is specially adapted to following droplet Controlling System, and computer system is wherein turned to by program and handles droplet control user interface.In this set, the treater of computer system can receive input and instruction is transferred to controller by user interface, so as for example to start/stop electrode, read electrode, internal memory and/or transmitter and or the like.

In the scheme executive system, the software that is used for Controlling System can directly load in the controller and by controller to be carried out so that controller is controlled described droplet microdrive system function.In this embodiment, system can move automatically, as portable or hand held system.

8.9.2 droplet microdrive

System can comprise droplet microdrive 1804, as described in 8.8 joints.Described droplet microdrive electronics is connected in treater, makes the various operations of the described droplet microdrive of treater may command, for example droplet manipulation operation.

8.9.3 transmitter

Various embodiment of the present invention has used transmitter or detector 1805.Transmitter can comprise the transmitter that is connected in described droplet microdrive, is used to measure the parameters of interest on the described droplet microdrive, the fluorescence or the luminous intensity of the position that reaction product may be on for example described droplet microdrive.Transmitter also can comprise the transmitter of Monitoring systems state, for example droplet microdrive plug in sensor, cover lock transmitter, environment temperature sensor or the like.Output from each transmitter can be located in specific core position, and treater must only be inquired about the position that is positioned to obtain the reading from transmitter.Transmitter places with respect to described droplet microdrive and/or electronics is connected in described droplet microdrive, makes transmitter can detect the signal from described droplet microdrive, for example electricity or optical signal.Transmitter goes through in other parts of this specification sheets, as sees 8.11 joints.

8.9.4 input and output device (s)

System of the present invention also comprises various input units 1806 and take-off equipment 1807.In some embodiments, for example the scheme executive system can use man-machine interface (HMI) controller to control some input and output device.

8.9.5 software

Various system of the present invention comprises software.The software that is provided on the storage media is one aspect of the present invention.Suitable storage media example comprises magneticstorage, optical memory, phase transition internal memory, holographic memory, molecular memory storer, battery or electrical condenser-back SRAM and flash memories.Software can be installed in internal memory and/or the treater.The system that software of the present invention is arranged in internal memory and/or treater and/or the storage media also is one aspect of the present invention.

Software of the present invention can use various programming languages to write, for example Visual C, Java and/or Python.System can comprise interpretive routine, is used for becoming intermediate language to carry out for treater with controlling from the droplet of High-Level Language with other instruction translations.Perhaps, the software translating that can use compiler to write according to the present invention becomes machine language.The software interpreter and the compiler itself that are used for language of the present invention are new aspects of the present invention.Therefore, data-carrier store, internal memory and the treater that contains the form of ownership of described interpretive routine and/or compiler is aspect of the present invention.

System can be turned to the diversified scheme that any amount of droplet is controlled that relates to of carrying out by program.A plurality of droplets can be controlled on single droplet microdrive independently and side by side.Can independently control a plurality of droplets with parallel mode makes it possible to complex scenario is carried out as a series of basic microfluid instructions.System is upgradeable, and tens of, hundreds of, the thousands of or more droplet that can control each droplet microdrive is in a parallel manner controlled.The maximum value of each control electrode on the droplet microdrive for example, at any time all can be set in the droplet operation.

System can by program turn to make the user can input order to carry into execution a plan.Can and adjust existing program according to the monitoring of user's needs.Can implement complex scenario, wherein how the result of one or more step decision selects one or more subsequent step.For example, wherein the positive droplet of certain measuring result can be transferred and be used for further processing, and the negative droplet of result then is dropped, or vice versa.

8.9.6 it is portable

With reference to Figure 19 A and 19B, in some embodiments, the analyser that provides is a mancarried device, and for example handheld apparatus 1900.Figure 19 A is depicted as the outside of handheld apparatus 1900, and Figure 19 B is depicted as the groove 1902 that is used to insert droplet microdrive (not shown), is used for optical pickocff 1904 and the cover lock 1906 of sensing from the optical signalling of described droplet microdrive, and is that the latter can be connected in that system opens with the indication lid or close.Can predict, hand-held analyzer also can be a desktop assembly.The portability of droplet microdrive of the present invention system helps to realize treatment scene or the on-the-spot purposes of sample collection under the various conditions such as (alarm reaction group, unscheduled event, disaster, battlefield, bioterrorism places or the like) of outpatient service, Operation theatre, emergency room, little laboratory and open air, be used for quick diagnosis, so that strive for the chance of circling round fast for emergency situation.

8.10 user interface

The droplet Controlling System comprises the droplet control software, and it is turned to by program and shows that being used for controlling on the droplet microdrive droplet operates, the control transmitter, if any, the droplet of other hardware relevant with droplet Controlling System control interface with control.System also can comprise software, is used to produce one group of software or computer-useable instructions so that control droplet microdrive system function, for example droplet operation and/or transmitter operation.

As shown in figure 20, system can comprise user interface 2000.Description about user interface can be in conjunction with U.S. Patent application No.60/806,412, denomination of invention is ＂ Systems and Methods for DropletMicroactuator Operations ＂, and the applying date is on June 30th, 2006, incorporates its full content into this paper by reference.User interface can show Figure 200 1 of droplet microdrive, is preferably interactive figure.Figure can with droplet microdrive direct interaction so that the droplet of controlling on the described droplet microdrive carries out fluid loading scheme of the present invention.Can use this figure by Virtualization Mode, be used to control the sub-routine of droplet microdrive function and related hardware with the virtual droplet 2011 of sequencing mode control with generation and record.

8.10.1 droplet Controlling System and user interface

The droplet Controlling System comprises the droplet control software.The droplet control software is turned to by program and shows that being used for controlling on the droplet microdrive droplet operates, the control transmitter, if any, the droplet of other hardware relevant with droplet Controlling System control interface with control.The droplet control software allows the user to control droplet on the droplet microdrive by the user interface of software-driven.As mentioned above, the example at this interface is presented among Figure 20.Wherein, user interface can allow the user to browse the information of droplet microdrive.User interface also is convenient to the function that user input instruction is controlled for example relevant transmitter of described droplet microdrive and relative unit thereof etc.

For control droplet operation on the droplet microdrive, software is configured to by sequencing and system, and for example, driving control on the droplet microdrive and reference electrode are to carry out the droplet operation.The droplet operation, it was further discussed at above 8.8 joints, can be undertaken by giving selected electrode application voltage.Software and system can be constructed to allow software to be installed on the treater, so that control the startup of selected electrode by the operation of controlling the rly. that is associated with electrode.

As shown in figure 20, user interface 2000, it is presented on the take-off equipment, can be turned to picture specification or the Figure 200 1 that shows the design of droplet microdrive by program.Figure 200 1 can be based on matrix or other layout drawings of the position that limits each control electrode and/or pond.The component of figure can be distinguished by outward appearance, for example by shape, color, brightness, icon or the like.For example, in the figure that Figure 20 provides, the droplet that stops to be controlled electrode 2002 can be shown as first kind of color (for example grey), and the droplet of startup controls electrode and pond 2003 can be shown as second kind of color (for example red), and the pond 2004 that stops can be shown as the third color (for example blue).

In a simple embodiment, matrix limits in control documents, and it has indicated the row and column in each electrode and/or pond.When control documents was installed, system read defined matrix and show corresponding matrix diagram on user interface.

This interface can show the information of component among the figure, and these also can be stored in the control documents.In one embodiment, system shows the otherwise information of the component that marks of electronics of the mouse indication, selected or user.The information that shows can comprise, for example, following information partly or entirely:

-component type is controlled electrode, reagent pond, sample pool or the like as droplet;

-electrical connection information is as electrode numbering, grounding wire, outlet line quantity or the like;

-proximity relations is arranged as polygonal electrodes;

-representative geometry is used for figure is presented to user interface;

-design mark and/or other suggestions;

-part number;

The position of-Lie and/or row.

System also can write down the startup history of each electrode, and the user can follow the tracks of the number of times that electrode had been activated like this.Can pass through, for example, mouse-pointing or selected electrode and show historical information.System can be turned to acceptance shows all electrode historical informations simultaneously from user's indication input by program.

For ease of user interaction, mouse-pointing or selected electrode 2002 or other components also can cause described electrode or other components highlighted on described droplet microdrive figure.This ability makes that the user who is directly controlling the operation of droplet microdrive can be before reality be selected and started described droplet microdrive component, by look back the information of each possible step with the described droplet microdrive of mouse-pointing component.System can be turned to the component that highlights mouse-pointing by different way and selected component by program, and the user can be distinguished both like this.

System can comprise the device 2007 that is used for select operating mode for the user, for example selects virtual or the sequencing pattern, wherein can write the program that is used to control the droplet microdrive; And operator scheme, wherein on the droplet microdrive, directly control droplet.

System can comprise the device 2012 of selecting to be used for showing the design of droplet microdrive for the user.Perhaps, the data that indicate the design of described droplet microdrive can be used as after described droplet microdrive assembly or tube and system's coupling, can supply system access described droplet microdrive assembly or tin component and include in.

It should be noted, in some designs, can be connected in same electric output terminal more than one electrode.This type of design can be simplified the design of described droplet microdrive.In this type of design, select or mouse-pointing will cause selecting, highlight from an electrode in the total group and starts all electrodes in this group.

Therefore, in one embodiment, system is made that by sequencing system starts this electrode when the user selectes the electrode 2002 that stops on microdrive Figure 200 1.For example, system is by sequencing and be constructed so that, can cause corresponding true electrode application voltage on the droplet microdrive in the sign of clicking electrode on the figure, starts selected electrode thus.So, the user can use this interface directly to control droplet on the droplet microdrive.

The electrode that the droplet Controlling System can allow the user to pass through to click successively a series of vicinities shifts droplet.Similarly, system can allow the user to shift droplet by the droplet 2011 on the selected virtual indicating meter and with the dummy electrodes place that droplet is dragged to desired location on the droplet microdrive figure.In addition, system can allow the user to pass through droplet 2011 on the selected virtual indicating meter, clicks the dummy electrodes place of desired location on the droplet microdrive figure then and shifts droplet.Can control other droplet microdrive components similarly by user interface.

System can be turned to by program and show that electronics is connected in the sign of the electric control line 2005 of droplet microdrive component, like this, when the user with mouse-pointing and/or when selecting a component, system highlights its electrical signal that is activated as being located in the result of this component.

But Virtual Monitoring droplet microdrive is as using microscope and video capture device.User interface can be turned to the real-time image of demonstration from the described droplet microdrive of video capture device by program.In addition, described droplet microdrive figure may be superimposed on the described real-time droplet microdrive image, make when the user by user interface during with the interaction of droplet microdrive, the droplet that can observe on the droplet microdrive is operated.

Similarly, system can be turned to the virtual droplet 2011 that shows on the droplet microdrive figure by program, its demonstration be the real behavior that is controlled by the droplet on the droplet microdrive of system, and/or system can be turned to the virtual droplet 2011 that shows on the droplet microdrive figure by program, its demonstration be the anticipatory behavior of the droplet on the droplet microdrive, even the droplet microdrive directly is not controlled by system at this moment.

System can also be turned to by program and realize that ＂ oppositely exports ＂ 2006 operations.In typical operation, droplet is connected with voltage to ground/ground wire regularly.Oppositely export in the ＂ operation at ＂, signal is reverse, makes droplet be in high-voltage, and starts by electrode is made as ground potential.In other words, the ＂ polarity of signal of oppositely having exported the ＂ operation change.

System also can promote to produce one group of software or computer-useable instructions, is used to control the droplet operation on the described droplet microdrive and other functions that are used to control droplet microdrive and relevant hardware.Software instruction can comprise, for example, and the instruction of implementing processing and analytic sample and exporting the scheme of analytical results.System can promote coding, is used to control the function of droplet microdrive and relevant component, transmitter component for example, and need not to interact with actual droplet microdrive.

System can comprise, for example, allows the user to produce and has one group of device for the program of the instruction of droplet microdrive execution.The example of suitable instruction comprises:

-＂ on ＂ is used to differentiate electrode to be started;

-＂ frequency ＂ sets the speed that described step is performed, the time course that starts/stop as electrode;

-＂ wait ＂ allows instruction to suspend one section preset time;

-＂ loop ＂, the step in the cycling program;

-＂ voltage ＂ sets the voltage that puts on output terminal.

Instruction can be a bytecode instruction, and it comprises that carrying out droplet controls and the otherwise instruction of Controlling System.The instruction that system is prepared can the assembly language record and compilation be syllabified code.Syllabified code can be installed in the system of the present invention, for example in the scheme executive system, is used for carrying out.System can comprise software interpreter, is used for translating being used for the language for example carried out in the scheme executive system.

One preferred embodiment in, system shows a series of button or icon 2008, it can be used for adding, insert, upgrade, change or deletes instruction from sub-routine.Suitable, button or icon can be used to import the parameter that and instruction is associated with field 2009.For example, by clicking ＂ add ＂ button, can add order at the sub-routine end.By clicking ＂ insert ＂ button, can order inner insertion of sub-routine.By clicking ＂ modify ＂ button, the order in the sub-routine can be changed.By clicking ＂ delete ＂ button, order can be deleted.In addition, display field 2010 is editable, and it can be included into and be used for reading, input and/or edit code.

System can show the excitation formula execution of the sub-routine on the droplet microdrive figure, and it exports the effect of the selected command sequence of vision demonstration to the user.In other words, in the excitation formula execution pattern, the step of software execution subroutine, but do not send electrical signal to the droplet microdrive.In preferred excitation mode, the droplet that is excited is presented on the screen so that to the actual effect of this program of instruction manual.So, the user can easily find out the problem of sub-routine and need not to interact with the droplet microdrive.

8.10.2 scheme executive system and user interface

The invention provides the scheme executive system.Described scheme executive system comprises the scheme executive software, and it is turned to by program and helps to carry out one group of software instruction, is used to add droplet operation on carrying object, the control droplet microdrive and/or other functions of droplet microdrive and related hardware.The scheme executive system can carry into execution a plan on erection system, portable typically or hand held system.

The scheme executive system is constructed to control droplet microdrive and any relevant component.Pre-programmed instructions can be loaded on comes Controlling System and any relevant component in the controller.The scheme executive system can comprise various components, so that allow the user input to be provided and to obtain output from treater to treater.Can use the HMI plate to promote man-machine interface.The HMI plate typically comprises controller and various electronics component, for example is used for bus and port that the input and output device is connected with the treater electronics.

8.11 transmitter

Droplet microdrive and turnkey are drawn together the transmitter that is used to measure droplet character, for example physical properties, chemical property and electrical property.In some embodiments, transmitter will comprise sensing element, and it is set to droplet and/or from the signal interaction of droplet; Inverting element, its output of autobiography sensor in the future is converted to measurable signal; Be used to transmit signals to the device of treater.Treater can be the discernible output of user with conversion of signals.

Sensor element can be the component of droplet microdrive, as be placed on top board or the base plate, be placed in the top board of droplet microdrive and the internal space between the base plate or be fabricated to of droplet microdrive and integrate component, as the integration component of top board or base plate.In other embodiments, sensor element can be positioned at the outside of droplet microdrive, but is arranged in the system, makes transmitter can receive from the signal on the droplet microdrive, as signal from the droplet on the droplet microdrive.For example, the sensor element of sensing photon can be set to receive the photon from the droplet on the droplet microdrive.If system has the top board that can send from the photon of droplet, transmitter can be set to be used for the sensing photon with the top board vicinity.If the top board of system can not send the photon from droplet, the window that can transmit photon is provided then can for this top board, and transmitter can be set to be used for the sensing photon with the window vicinity.

Figure 21 A-21D has provided the example of sensor arrangement, and wherein transmitter can be associated with base plate 2102, top board 2104 and electrode 2106.Figure 21 A has illustrated optical pickocff, and it can comprise the device that uses a kind of LED of comprising 2108 and photorectifier 2110, is used to monitor absorbancy.Figure 21 B has illustrated luminescence sensor, and it can comprise and uses photomultiplier (PMT) 2112.Figure 21 C has illustrated potentiometric sensor 2114, and it typically works based on measuring the current potential under the no current situation.Figure 21 D has illustrated current sensor 2116, and it typically works by produce electric current when applying voltage between two electrodes.

Importantly, as described in other parts of this specification sheets, the droplet microdrive can be independent component, and it can be connected it by the user with system.If transmitter is positioned at the outside of droplet microdrive, in some embodiments, these transmitters can align, make when it is connected in droplet microdrive system, sensing element is suitably alignd so that detect signal from the droplet microdrive, as window suitable on photon sensor and the droplet microdrive and/or with the droplet microdrive on suitable aligned in position, these windows or position are will carry out the place of sensing step in the droplet scheme process.

In different embodiments, droplet microdrive and/or system and transmitter component are constructed to make it possible to implement the sensing of one or more type.The example of suitable sensing type comprises physics sensing, electrochemical sensing and optics sensing.

8.11.1 physical method

Droplet microdrive of the present invention and/or system can comprise one or more physical sensors, and they are set to be used for the character of the droplet on the sensing droplet microdrive.The example of physics sensing comprises the size (for example carrying out thermal measurement by the substrate to droplet) of temperature and droplet.

8.11.2 electrochemical method

Droplet microdrive of the present invention system uses multiple optical detecting method.Droplet microdrive of the present invention and/or system can comprise one or more electrochemical sensor, and they are set to be used for the character of the droplet on the sensing droplet microdrive.Suitable example electrochemical sensing type comprises potentiometric sensor, current sensor, voltammetric sensor and conductivity sensor.The various components of transmitter (for example electrode, counter electrode, reference electrode or the like) can be provided at same or the base material that separates on, and be set to allow to contact droplet on the droplet microdrive.For example, the droplet microdrive comprises in the embodiment of two substantially parallel base materials therein, and the various components of sensor module can be arranged on one of base material or both.In some embodiments, can use circuit is measurable voltage with amplification of signal.The all respects of these methods are discussed hereinafter.

8.11.2.1 amperometric sensor

Droplet micro-drive device or system can comprise amperometric sensor and power supply, and they are set to allow droplet on the droplet microdrive to be transferred to power supply and transmitter to contact, so that detect the electric current of the droplet of flowing through.

8.11.2.2 potential measurement transmitter

Droplet micro-drive device or system can comprise potential measurement electrode and reference electrode, and they are set to allow droplet on the droplet microdrive to be transferred to and measure electrode and reference electrode contacts, so that measure the equilibrium electrode potential of droplet.

8.11.3 optical means

Droplet microdrive of the present invention system uses various optical detecting methods.

Droplet microdrive of the present invention and/or system can comprise one or more optical pickocff, and they are set to be used for the character of the droplet on the sensing droplet microdrive.The example of optics sensing comprises absorbancy, chemoluminescence and fluorescence.In some cases, optical pickocff can be with suitable light source, as is used for fluorescence excitation or carries out absorbance measurement.These transmitters that provide can be used as the component that is placed on the droplet microdrive and/or as the integrated part of droplet microdrive, as using semiconductor fabrication.

Optical pickocff can comprise the various optics that are used for the detection optical signal, and can be connected with the various presentation managers that are used to analyze optical image.For example, can detect the size of droplet by the image of handling droplet.Similarly, by obtaining droplet size at the bottom of the hot radical of measuring droplet.Also can make power demand sensor measure droplet size, as impedance by the substrate of measurement droplet.

In some cases, can improve the surface of droplet microdrive to strengthen the optics sensing.For example, can use electrode to promote the optical detecting of droplet with reflective surface will coating.The use reflecting electrode increases the path length of absorbance measurement and adapts with reflection spectrometry.For the autofluorescence base material, for example PCB can use the droplet microdrive surface that is coated with non-fluoresent coating that non-fluoroscopic examination district is provided.

The all respects of these methods are in a few joint discussion down.

8.11.3.1 optical sensor

Droplet micro-drive device or system can comprise the absorbance detection component, it comprises light source and optical sensor, they are set to allow the droplet on the droplet microdrive is transferred to described light source and optical sensor contiguous, so that by light or the energy of light sensors through droplet.

Droplet micro-drive device or system can comprise the chemiluminescence detection component, it comprises optical sensor (for example photorectifier avalanche photodide photomultiplier) or photon sensor (for example photon counting photomultiplier), they are set to allow the droplet on the droplet microdrive is transferred to described optical sensor or photon sensor contiguous, so that detect the photon that is discharged by the chemical substance in the droplet by optical sensor or photon sensor.

8.11.3.2 fluorescent optical sensor

Droplet micro-drive device or system can comprise the fluoroscopic examination component, it comprises excitation light source with suitable spectral filter (if necessary) and optical sensor (photorectifier for example, avalanche photodide, photomultiplier) or photon sensor (for example photon counting photomultiplier), transmitter has suitable spectral filter and dichroic mirror (if necessary), described component is set to allow the droplet on the droplet microdrive is transferred to and described excitation light source and optical sensor or photon sensor vicinity, so that detect the photon that is discharged by the fluorescent substance in the droplet by optical sensor or photon sensor.

8.11.3.3 surface plasma body resonant vibration

In another embodiment, use surface plasma body resonant vibration (SPR) sensing detects the interaction between antibody and any target analyte.The SPR sensing can be used for detecting and quantitatively this type of interaction.Typically, a kind of interactant of interactant centering (that is, antibody or analyte) is immobilized in the active gold surface of SPR-on the glass baseplate.Can use this interactant of method immobilization, shift wherein that droplet makes it to contact gold surface so that above interactant is deposited on based on droplet.The transferable droplet of another kind of interactant that comprises makes it to contact described immobilized interactant, makes described another kind of interactant be incorporated into described immobilized interactant thus.When light (for example visible light or near infrared light) shone glass baseplate and when shining gold surface near the angle of surface plasma body resonant vibration condition and wavelength, the optical reflectivity of gold according to the biomolecules that exists in biomolecules that exists on the gold surface or the thin layer coating above the gold very responsive variation takes place.Because it relates near the conduction electron of gold surface by efficiently, jointly excite, so optic response can be extremely sensitive.By monitoring the change of this reflectivity, can observe and volumetric soiutions phase interactant and immobilized interactant between combination degree.The present invention also comprises the droplet microdrive, comprises gold surface and electrode path or network on it, and electrode is set to allow to carry out droplet and controls, and described droplet operation is enough to make droplet contact gold surface.In addition, the present invention includes system, it comprises this type of droplet microdrive and also comprises the light source that can light shine described gold surface that the angle of described light and wavelength are near the surface plasma body resonant vibration condition.Similarly, the present invention includes system, it comprises this droplet microdrive and further comprises the device of the reflectance varies that is used to detect gold surface.In addition, the present invention includes droplet micro-drive device and/or system, it has loading being enough to thereon and carries out the reagent of some or all steps of SPR scheme.

8.11.3.4 Raman spectrum

In one embodiment, droplet microdrive and/or system comprise the Raman spectrum detectivity.Generally, this ability comprises light source, Raman signal detection surface and the laman spectrophotometer that produces Raman signal.

The light source that produces Raman signal can be, for example, and monochromatic ray, as laser source with the excitation wavelength in the visible wavelength range.Light source is set to shine the Raman signal detection surface on the droplet microdrive.The surface can be, for example, and the particulate surface on the surface of droplet microdrive and/or the droplet microdrive.For example, the surface can be the particulate surface in the droplet on the droplet microdrive.The droplet microdrive can have use and comprise that this type of particulate droplet carries out the droplet operation so that realize the ability of various use Raman signal detection method schemes.

The Raman signal detection surface can comprise any surface that is suitable for Raman scattering.Example comprises gold or silver surface.The surface can be coarse.In some cases, the droplet microdrive can comprise a plurality of exemplary metallic surfaces (for example surface of droplet microdrive, pearl, particle, nano particle or the like), the surface of different Raman reporter molecules that comprised mark.Can identify antibody or the analyte that is incorporated into the surface by the characteristic Raman spectrum of Raman reporter molecules.The Raman detection surface can be, for example, and the coating on electrode, the electrode or any chip surface upper strata.In operation, utilize the droplet operation that droplet is positioned on the Raman detection surface, and with laser beam irradiation.Collect from the scattered light on illuminated surface with spectrometer.In another embodiment, the Raman detection surface is the particle in the droplet on the droplet microdrive.Described particle can be, for example, and nano particle, for example silver or gold nano grain.For example, silver nano-grain can be prepared as the form of single dispersoid suspension, and it can utilize droplet to control on the droplet microdrive.In some embodiments, particle can be assembled cluster, can use the gathering additive, for example inorganic salt such as sodium-chlor or SODIUMNITRATE, acid nitric acid or hydrochloric acid or organic amine poly-L-Lysine for example for example.Can utilize the droplet operation that these gathering additives are merged with comprising sample and particulate droplet, as utilize the droplet operation to merge the droplet that comprises the droplet of assembling additive and comprise particle and sample.The droplet microdrive surface of selecting to be associated with the Raman spectrum district is so that the background fluorescence signal minimizes.

Laman spectrophotometer is set to be used to detect the Raman diffused light from the sample droplet emission.Laman spectrophotometer can be integrated with the droplet microdrive, is arranged on the outside of droplet microdrive, and its set-up mode makes it can detect the Raman diffused light of the sample droplet emission from the droplet microdrive.

In operation, provide the droplet microdrive, have the Raman detection surface on it.Utilize the droplet operation that analyte is associated with the Raman detection surface.To produce the light source irradiation surface of Raman signal.Detect and the related Raman scattering optical signal of expection signal, so that determine the character and/or the amount of analyte.

In another embodiment, adopt surperficial enhancement type Raman scattering (SERS) to detect interaction between antibody and any target analyte.Generally, this method relates to the binding events that is mediated by analyte in the monitoring sample droplet, described droplet comprises described analyte, specific binding members, Raman-activity mark's thing, and contact with the surface, the surface of pearl or droplet microdrive for example, and it can induced surface enhancement type Raman light scattering.The sample droplet is illuminated by radiation, and described radiation is enough to cause that the Raman-activity mark's thing in the test mixing thing launches detectable Raman spectrum.Difference in the detected surperficial enhancement type raman scattering spectrum depends on the amount of the analyte that exists in the test mixing thing.Can determine the existence and/or the amount of analyte in the sample droplet by the monitoring raman scattering spectrum.The present invention includes droplet micro-drive device and/or system, it has loading reagent thereon, and described reagent is enough to carry out some or all steps of SERS scheme.

In relevant embodiment, the invention provides by the part binding events of the analyte-mediation on the monitoring droplet microdrive and determine the existence of analyte in the sample droplet or the method for amount.This method comprises analyte and the antibody response that is connected in Raman-active tag substantially.This reaction uses the droplet on the droplet microdrive to carry out, and carries out under permission antibody and analyte (if present) specificity bonded condition, to produce first mixture in the sample droplet.Successively or simultaneously, the contact of first mixture can induced surface enhancement type Raman light scattering and is had the surface of the connection antibody that is specific to described analyte thereon, to form second mixture.Described second mixture is illuminated by radiation, and described radiation is enough to cause the Raman-detectable Raman spectrum of activity mark's deposits yields in the mixture.Difference in the enhancement type raman scattering spectrum of surface is represented the existence and/or the amount of analyte in the test mixing thing.

Kinds of surface can be suitable for the SERS scheme based on droplet of the present invention.Example comprises metal island (metal islands) that textured metal electrode, aggregation, film, different shape learn, near the semicontinuous film of percolation threshold and the nanostructured metal film of vacuum hydro-extraction.Therefore, the present invention includes the droplet microdrive, it comprises the SERS base material.The droplet microdrive suitably is provided with, and makes droplet extremely to contact with the SERS base material along electrode path or network transitions.

In DNA detection method of the present invention, can use the Raman labels thing.Marker can be non-sequence-specific insert or the specific marker thing that is covalently attached to the unique probes sequence.Electronegative marker may need to use the charge neutralization agent, spermine for example, and to promote combining of marker and electronegative surface, the latter for example is the silver-colored nano-beads with Citrate trianion upper layer.Also can use aggregating agent prepared therefrom to improve signal.Spermine also can be used as aggregating agent prepared therefrom.

8.11.3.5 multisensor ability

Preferred sensor is to be used to detect absorbancy, fluorescence, chemiluminescent transmitter and current potential, electric current and conductivity sensor.Droplet micro-drive device of the present invention and/or system comprise one or more these detectivities.In one embodiment, the droplet microdrive includes and help to carry out 2 or the component of multiple these detection methods on single droplet microdrive.In another embodiment, the droplet microdrive comprises a kind of detection module, but system is turned to by program and can use this module to carry out more than a kind of test.In this embodiment, need the sample droplet of handling well of test to be moved to test position successively.Therefore, if use single-sensor, a plurality of samples can form multichannel on check point.

8.11.4 sensor electronics

Detectivity can sensor board the form of one or more component provide.Sensor board can comprise one or more transmitter.Sensor board can comprise that extra being used to adjust or amplify from the electronic circuit of the signal of droplet for example amplifier, A/D converter, sensing circuit or the like.Sensor board can comprise the outer component of other droplet microdrives of controlling elements or detection scheme, and for example control is used for the motor of mobile system component.

In one embodiment, sensor board comprises servomotor controller, is used to control move to Magnetic Field Source near the droplet operating surface and near the servomotor from removing the droplet operating surface, applies/remove magnetic field for thus the droplet microdrive.This embodiment can be used for controlling the magnetic responsiveness material.Sensor board also can comprise source element and communication device, includes but not limited to, is used for the transmitter component of described plate or control component electronics are connected in the element of treater.

The optical detection position can comprise that special coating, electrode design or other help the parts of optical detection.For example, check point can comprise special pad and/or be convenient to coating as the background surface of optical detecting.

In some embodiments, for example nucleic acid amplification is used, and preferred optical detecting method is a fluorescent quantitation.In this embodiment, check point can be selected so that cover the microdrive base material or be deposited on the background fluorescence that exists in the coating on the microdrive base material.For example, in one embodiment, microdrive is made up of substrate for printed circuit board, and check point is made up of the gold pad, and described pad is the background fluorescence that transmitter has covered base material, and this droplet that helps to be opposite on the pad carries out fluorometric assay.Pad can form in the metal level that directly is arranged on the base material, perhaps forms in the metal level that is arranged on the middle layer of arranging on base material.

Preferably, metal level (pulvilliform is formed in wherein) should be disposed in any top that presents the layer of obvious background fluorescence.In one embodiment, pad directly is arranged on the substrate for printed circuit board, and this base material is formed in the same metal level at the electrode with the control droplet.In this embodiment, dielectric materials (it also can present background fluorescence) can be arranged in the metal level top, but it is optionally removed from detecting pad (but not control electrode place).

Therefore, cover this combination of fluorescent material that is positioned at the pad below by fluorescent material above the selective removal detecting pad and optics and realize low background fluorescence check point.Preferably, pad is designed to other droplet microdrive function of minimal level of interference.In the above-described embodiment, pad can be formed in the same metal level with control electrode, but separates with control electrode, and different with it aspect electric.Therefore fill up

8.11.5 detection method

The invention provides the multiple signal of sensed/detected droplet or the method for feature of being used for.This specification sheets has been described a lot of these methods in other parts.This section is described the extra method that can be used under the various situations.

The advantage of droplet microdrive method of the present invention comprises the reactions steps that can make in the particular assay method connection of uncoupling.Many biochemical measurements use the common end reaction to produce color, light or other detectable amounts.It is on-the-spot combined in detection with the droplet that contains end reaction reagent to use the scheme based on droplet of the present invention will measure droplet.The connection of uncoupling of determination step allows each step to be separated to be optimized, and when one of reactions steps is rate-limiting step, step in time greater flexibility separately is provided.For example, chemical luminescent detecting has better result at alkaline pH usually.For the mensuration of acid pH, assaying reaction can be finished at acid pH earlier for top condition, and the luminous aspect of reaction then can be carried out at alkaline pH.

Droplet microdrive of the present invention can be used for studying the speed kinetic reaction.Can periodically from react ongoing pond, distribute the sample droplet.Droplet can individually be analyzed to determine the time course of reaction subsequently.Can carry out real-time analysis to droplet, or be used for post analysis with another kind of reagent mix.Can make also that electricity consumption is wetting to come the short mix droplet so that the research fast reaction kinetics.

Can measure the means of the change of droplet viscosity with the inner chemical response behaviour of droplet that judges.For example, can in droplet of blood, add coagulant, shift droplet subsequently and monitor the easness that droplet shifts.The high more then droplet of the degree of condensing shifts difficulty all the more, can be detected and be used as the method for masurement of degree that condenses to this.

Preferred sensor comprises and is used for sensing optical signalling for example absorbancy, fluorescence and chemiluminescent optical pickocff and be used for for example electrochemical sensor of current potential character, electric current character, electric conductivity matter of sensing electrochemical properties.Therefore, droplet microdrive of the present invention system comprises the component that is set to help to detect one or more these character.In one embodiment, on one or more droplet on the single droplet microdrive, detect 2 or more kinds of these character, perhaps can use single droplet microdrive system to realize these purposes.In another embodiment, the droplet microdrive comprises a kind of transmitter of particular type, and system is turned to by program and uses described transmitter to carry out one or more test.In this embodiment, need the sample droplet of handling well of test to be moved to test position successively, promptly move to enough place near the transmitter of necessity so that can detect.Therefore, a plurality of samples can form multichannel on check point, be used to use the detection of single-sensor.Make in this way, a plurality of sensor types can be provided on the single droplet microdrive.

In some embodiments, droplet microdrive system can be constructed to droplet or the sample deposition position to droplet microdrive outside is used for detecting.For example, the droplet deposition that comprises (maybe may comprise) analyte can be used for the MALDI-TOF analysis to base material.

Droplet can periodically be shifted by near the common check point the right sensors so that monitor a plurality of reactions simultaneously.For example, the droplet microdrive can comprise that two or more connect the interior height of circulation PCR reaction chamber and the ＂ road ＂ in low temperature district.Single detector is positioned at the point of crossing in these roads.Arrange the current time of droplet properly, make droplet successively by described check point.

The assay method example that is adapted at implementing in the scheme based on droplet of the present invention on the droplet microdrive of the present invention comprises optical detecting, for example absorbance measurement, fluorometric assay, bioluminescence assay and chemical luminescent detecting; And electrochemical gaging, for example potential measurement, current measurement and electric conductance determination.For example, can use the various combinations of one or more aforementioned type to identify and/or quantitatively one or more analyte, for example protein, enzyme, nucleic acid, metabolite, ionogen, gas (for example vim and vigour) and pcv.System of the present invention can be turned in the various combinations of carrying out these types on single droplet microdrive by program.

In one embodiment, single droplet microdrive or system comprise and carry out 2,3,4,5,6 or the detectivity of more kinds of dissimilar mensuration.For example, other components of droplet micro-drive device, system and/or droplet microdrive system can be dividually or are comprised one or more detected components together, for example are used for the component of current measurement, potential measurement, electric conductance determination, absorbancy, chemoluminescence, fluorescence and/or temperature.In addition, droplet microdrive system can be turned to by program and implement to be used for to carry out 2,3,4,5,6 or the mensuration scheme of more kinds of dissimilar mensuration on same or a plurality of samples or sample type.

In the droplet microdrive, droplet controls component and detected components in some embodiments can be by being provided in the connection of uncoupling on the base material separately.Similarly, various detected components can be used as the part of droplet micro-drive device or system and provide, but on different droplet microdrives.Therefore, for example, transmitter can be on tube, and the droplet microdrive is connected with tube.Coupling is provided so that suitable component is aligned so that detect when the droplet microdrive is connected in tin.Therefore, for example, photon sensor can align window or other transparent substrates make suitably to be positioned over when tin last when the droplet microdrive, the photon that goes out from the droplet emission on the droplet microdrive can pass through window or base material quilt sensor detecting to.Similarly, if it is essential that light source sends fluorescence for the molecule in the droplet on the droplet microdrive, then light source can be placed on tube or other components of droplet micro-drive device or system and alignment, makes light source can arrive droplet to produce required fluorescence.

In one embodiment, the droplet microdrive comprises and is used to carry out electrochemical electrode.Electrode can be arranged on the electric wetting base material, so that to carrying out electrochemical measurement with the contacted droplet of electrode.In bi base material droplet microdrive, can on arbitrary or two kinds of base materials, be formed for carrying out electrochemical electrode.In some embodiments, transfer electrode and electrochemical measuring electrode are provided on the different substrate materials.Electrode can comprise the film that is used to make up the ion selectivity analysis.

8.12 additive method

The present invention includes method, wherein propose or provide the component of desktop system (bench-top system) to client for the transaction reason.In one embodiment, the component that is suggested or is provided for client does not comprise PC.Software of the present invention can offer the user on storage media or but automatic network is downloaded, for example the internet.The user can obtain other components of system, and these components are connected in PC, and software is installed on PC, and therefore assembles system of the present invention.

The present invention includes method, wherein use desktop system to produce the code that is used to carry into execution a plan.Code is uploaded to system separately, for example portable or hand held system, and it is for concluding the business former thereby being suggested or being provided for client.But user's using system carries into execution a plan.

The present invention also comprises method, wherein realizes sequencing and/or system's control by network remote, for example by telephone system or internet.Therefore, for example, system can be sold to the user, and the programmer can be connected to system so that Controlling System by means of the user interface that shows by the internet, and the using system generating routine is installed to program in the system and/or repair procedure in system.As another example, the present invention includes a kind of method, wherein the long-distance user uses the droplet microdrive by network, and carries out one or more droplet and control in system.

8.13 test kit

Another aspect of the present invention is a test kit, droplet microdrive or tube that it comprises reagent, sample collection device and/or is used to carry out method of the present invention.

8.14 sum up

The front is described some embodiments in detail with reference to accompanying drawing, and these accompanying drawings are for example understood embodiments more of the present invention.Other embodiments with different structure and operation do not depart from the scope of the present invention.

It will be understood by those in the art that the present invention to can be used as method, system or computer program and implement.Therefore, form of the present invention can be the embodiment of devices at full hardware embodiment, full software embodiment (comprising firmware, resident software, microcode or the like) or integration software and hardware aspect, and they all can be called as ＂ circuit ＂, ＂ module ＂ or the ＂ of ＂ system generally at this.In addition, form of the present invention can be the computer program that has on the computer available storage media of the computer available program code that is included in the medium.

Can use any suitable computer available medium.Computer available or computer-readable medium can be, such as but not limited to, electronics, magnetic, optics, electromagnetism, infrared or semiconductor system, unit or propagation medium.The example of computer-readable medium (non exhaustive tabulation) can comprise following some or all more specifically: the electrical communication with one or more line, portable computer diskette, hard disk, random access memory (RAM), read-only storage (ROM), erasable programmable read only memory (EPROM) (EPROM or flash memory), optical fiber, portable compact disc read-only storage (CD-ROM), optical storage, transmission medium is for example supported the transmission medium of internet or local area network, or magnetic memory apparatus.Note, computer available or computer-readable medium can or even paper or another kind of suitable medium, be printed on program on it, because program can be caught electronically, for example by optical scanning paper or other media, compile then, translate or handle, if necessary, be stored in the computer memory then with other suitable manner.With regard to presents, computer available or computer-readable medium can be anyly to contain, store, transmit, propagate or branching program is used for by instruction execution system, equipment or device uses or and instruction executive system, equipment or device are associated and use medium.

Be used to implement the computer program code of operation of the present invention can object oriented programming languages for example Java, Smalltalk, C++ or similar language throughout write.But, for example ＂ C ＂ programming language or similar programming language write to be used to implement programming language that the computer program code of operation of the present invention also can be conventional.Program code is all moving on the subscriber computer, is partly moving on the subscriber computer, moving on the subscriber computer and partly moving on the remote computer or all moving on remote computer or server as isolated software package, part.For latter event, remote computer can be connected in subscriber computer by Local Area Network or wide Local Area Network (WAN), maybe can be connected to outer computer (for example, but by internet internet usage service provider).

Method according to the embodiment of the present invention, equipment (system) and computer program have been described the present invention with reference to flowchart text and/or functional diagram.Should be appreciated that each square in flowchart text and/or the functional diagram and the combination of the square in flowchart text and/or the functional diagram can be implemented by computer program instructions.Computer program instructions can be provided for treater, special purpose computer or other programmable data treatment facilities of multi-purpose computer, to produce machine, make and be used for the pointed function/action of one or more square of implementing procedure figure and/or functional diagram by the instruction generation device of carrying out by the treater of computer or other programmable data treatment facilities.

These computer program instructions also can be stored in the computer-readable storer, it instructs computer or other programmable data treatment facilities according to the ad hoc fashion functionating, make that be stored in instruction in the computer-readable storer produces and make article, it comprises the command device of function/action that one or more square of implementing procedure figure and/or functional diagram is pointed.

Computer program instructions also can be installed on computer or other programmable data treatment facilities so that the sequence of operations step is able to carry out on computer or other programmable equipment, so that produce computer-implemented method, make the instruction of on computer or other programmable equipment, carrying out be provided for the step of the pointed function/action of one or more square of implementing procedure figure and/or functional diagram.

For convenience of the reader, this specification sheets is split as a plurality of chapters and sections.Subhead should not be understood that it is restriction to scope of the present invention.

Should be appreciated that various details of the present invention can change and not depart from the scope of the present invention.In addition, the explanation of front is only used for setting forth the present invention, and has no intention to limit the present invention, and the present invention is limited by appended claims.

Claims (529)

1. the method with the droplet of the material of the contacted concentration with reduction in surface is provided, and described method comprises:

(a) provide and comprise the material of initial concentration and initial amount and have the contacted surface of droplet of initial volume;

(b) carry out one or repeatedly droplet operation so that the droplet that provides in washing droplet and the step (a) is converged, to produce the droplet of merging; With

(c) carry out one or repeatedly droplet operation is so that be split as one group of droplet with the droplet of described merging, described one group of droplet comprises:

(i) with the contacted droplet in described surface, the material concentration that described droplet has reduces with respect to described initial concentration; With

(ii) with the droplet of described surface isolation.

2. the process of claim 1 wherein that step 1 (c) produces and the contacted droplet in described surface, the described amount of substance that described droplet has reduces with respect to described initial amount.

3. the process of claim 1 wherein that step 1 (c) produces and the contacted droplet in described surface, the described amount of substance that described droplet has fully reduces with respect to described initial amount.

4. the process of claim 1 wherein that step 1 (c) produces and the contacted droplet in described surface, the concentration of the described material that described droplet has fully reduces with respect to described initial concentration.

5. the process of claim 1 wherein that step 1 (c) produces and the contacted droplet in described surface, volume that described droplet has and described original volume are roughly the same.

6. the method for claim 1 comprises that also repeating step 1 (a) to 1 (c) one or more component in 1 (c) droplet (i) meets or exceeds the predetermined concentration boundary.

7. the method for claim 1 comprises that also repeating step 1 (a) to 1 (c) one or more component in 1 (c) droplet (i) meets or exceeds predetermined maximum.

8. the method for claim 1 comprises that also repeating step 1 (a) to 1 (c) one or more component in 1 (c) droplet (i) meets or exceeds predetermined amount and concentration.

9. the method for claim 6, wherein said predetermined concentration fully reduces with respect to described initial concentration.

10. the method for claim 6, wherein said predetermined concentration reduces by 99% with respect to described initial concentration.

11. the method for claim 6, wherein said predetermined concentration reduces by 99.9% with respect to described initial concentration.

12. the method for claim 6, wherein said predetermined concentration reduces by 99.99% with respect to described initial concentration.

13. the method for claim 6, wherein said predetermined concentration reduces by 99.999% with respect to described initial concentration.

14. the method for claim 7, wherein said predetermined amount fully reduces with respect to described initial amount.

15. the method for claim 7, wherein said predetermined amount reduces by 99% with respect to described initial amount.

16. the method for claim 7, wherein said predetermined amount reduces by 99.9% with respect to described initial amount.

17. the method for claim 7, wherein said predetermined amount reduces by 99.99% with respect to described initial amount.

18. the method for claim 7, wherein said predetermined amount reduces by 99.999% with respect to described initial amount.

19. the process of claim 1 wherein that step 1 (b) comprises that making two or more that droplet that provides in droplets and the step 1 (a) is provided converges, to produce the droplet that merges.

20. the process of claim 1 wherein that described surface comprises the surface of one or more pearl.

21. the process of claim 1 wherein that the droplet that provides in the step 1 (a) comprises the sample droplet.

22. the process of claim 1 wherein that the droplet that provides in the step 1 (a) comprises the reagent droplet.

23. the process of claim 1 wherein that the droplet that provides in the step 1 (a) comprises biological sample.

24. the method for claim 23, wherein said biological sample comprises through preparation and non-sample through preparation, and described sample is selected from: whole blood, lymph liquid, serum, blood plasma, sweat, tear, saliva, phlegm, cerebrospinal fluid, amniotic fluid, seminal fluid, vaginal secretions, slurries, synovial membrane liquid, pericardial fluid, peritoneal fluid, hydrothorax, spill liquid, transudate, capsule liquid, bile, urine, gastric juice, intestinal juice, faecal samples, liquefaction tissue, organism, biology swab and biology washing lotion liquefy.

25. the process of claim 1 wherein that the droplet that provides in the step 1 (a) comprises the nucleic acid of amplification.

26. the process of claim 1 wherein that described washing droplet comprises solution, described solution is selected from: water, deionized water, salts solution, acidic solution, basic solution, washing agent solution and damping fluid.

27. the process of claim 1 wherein that step 1 (b) and/or 1 (c) comprise one or repeatedly droplet operation, it is selected from: droplet is loaded in the droplet microdrive; Distribute one or more droplet from the source droplet; Droplet is divided, separates or is split as two or more droplets; Droplet is transferred to another place from a place along any direction; Two or more droplets are converged or merge into a droplet; The dilution droplet; Mix droplet; Stir droplet; Make little droplet deformation; Make droplet keep original position; The incubation droplet; The heating droplet; The evaporation droplet; The cooling droplet; Arrange droplet; Droplet is migrated out microdrive; Other droplet operations described herein; With above-mentioned any combination.

28. the method for claim 27, wherein step 1 (b) and/or 1 (c) are the electric field mediations.

29. the method for claim 27, wherein step 1 (b) and/or 1 (c) are the electrode mediations.

30. the method for claim 27, wherein step 1 (b) and/or 1 (c) are electric wetting mediations.

31. the method for claim 27, wherein step 1 (b) and/or 1 (c) are the two-dimensional electrophoresis mediations.

32. the method for claim 27, wherein step 1 (b) and/or 1 (c) implement on the droplet microdrive.

33. the method for claim 32, wherein:

(a) described droplet microdrive comprises two kinds of base materials of relative to each other placing, and its distance is enough to limit the space between described two kinds of base materials; With

(b) a kind of electrode that is used to carry out the droplet operation that comprises is only arranged in described two kinds of base materials.

34. the method for claim 32, wherein:

(a) described droplet microdrive comprises two kinds of base materials of relative to each other placing, and its distance is enough to limit the space between described two kinds of base materials; With

(b) described two kinds of base materials all comprise the electrode that is used to carry out the droplet operation.

35. the method for claim 32, wherein:

(a) described droplet microdrive comprises two kinds of base materials of relative to each other placing, and its distance is enough to limit the space between described two kinds of base materials;

(b) one of described two kinds of base materials comprise the electrode that is used to carry out the droplet operation; With

(c) another kind in described two kinds of base materials comprises one or more reference electrode.

36. the method for claim 32, wherein said droplet microdrive comprises base material, and described base material comprises:

(a) be used to carry out the electrode that droplet is operated; With

(b) one or more reference electrode.

37. the method for claim 27, wherein step 1 (b) and/or 1 (c) implement on the droplet microdrive.

38. the method for claim 32, wherein:

(a) described droplet microdrive comprises two kinds of base materials of relative to each other placing, and its distance is enough to limit the space between described two kinds of base materials;

(b) one of described two kinds of base materials or both comprise the electrode that is used for one or more droplet character of sensing; With

(c) described method comprises the character of the described droplet of sensing.

39. the method with the droplet of the material of the contacted concentration with reduction in surface is provided, and described method comprises:

(a) provide the droplet microdrive, it comprises the surface, and described surface and the material that comprises initial concentration and initial amount and the droplet with initial volume contact;

(b) carry out one or repeatedly droplet operation so that the droplet that provides in washing droplet and the step (a) is converged, to produce the droplet of merging; With

(c) carry out one or repeatedly droplet operation is so that be split as one group of droplet with the droplet of described merging, described one group of droplet comprises:

(i) with the contacted droplet in described surface, the material concentration that described droplet has reduces with respect to described initial concentration; With

(ii) with the droplet of described surface isolation.

40. the method for claim 39, wherein:

(a) described droplet microdrive comprises:

(i) first base material comprises and is configured to handle the electrode that is positioned at the droplet on the described substrate surface; With

(ii) place with respect to described first base material and with the surface of described first base material second base material at a distance of a segment distance, this distance is enough to limit the space between described first base material and second base material; With

(b) described surface comprise first base material surface, second base material the surface and/or be positioned at described space and/or be exposed to described spatial surface.

41. the method for claim 40, wherein said second base material be set to described first base material be substantially parallel relation.

42. the method for claim 40, wherein said surface comprises the surface of one or more pearl.

43. comprising diameter, the method for claim 42, wherein said one or more pearl be about 0.1nm one or more pearl to about 25mm.

44. the method for claim 42, wherein said one or more pearl are contained in the droplet on the droplet microdrive.

45. the method for claim 42 also is included in step 39 (c) and attracts one or more described pearl before.

46. the method for claim 42 also is included in one or more described pearl of step 39 (c) immobilization before.

47. the method for claim 42 also is included in one or more described pearl of reservation in step 39 (c) process.

48. the method for claim 48 is included in also that step 39 (c) discharges afterwards or resuspended one or more described pearl.

49. the method for claim 48 also is included in step 39 (c) and stirs one or more described pearl afterwards.

50. the method for claim 48 also is included in step 39 (c) and stirs one or more described pearl with ultrasonic generator afterwards.

51. the method for claim 46, wherein said immobilization step realizes by the transfer of using one or more described pearl in the operating process of physical objects blocking-up droplet.

52. the method for claim 46, wherein:

(a) one or more described pearl is non-magnetic responsiveness; With

(b) described immobilization step realizes by the transfer of using non-magnetic responsiveness pearl described in the operating process of physical objects blocking-up droplet.

53. the method for claim 46, wherein:

(a) one or more described pearl is a magnetic responsiveness; With

(b) described immobilization step realizes by described magnetic responsiveness pearl or particle are exposed to magnetic field.

54. the method for claim 53, wherein step 53 (b) comprises that the droplet that comprises described pearl is positioned to be used to implement near the device in magnetic field, described device is positioned near one or more described electrode, and wherein the intensity in magnetic field and the degree of approach are enough to immobilization magnetic responsiveness pearl in carrying out washing scheme process.

55. the method for claim 54, the wherein said device that is used to implement magnetic field comprise the device of the power supply of electromagnet and control electromagnet.

56. the method for claim 54, the wherein said device that is used to implement magnetic field comprise magnet and move to described magnet near described one or more described electrode and with near the device of described magnet from removing described one or more described electrode.

57. the method for claim 53, wherein step 39 (c) produces one group of droplet, and described one group of droplet comprises:

(a) comprise the droplet of all described pearls basically, and the concentration of its described material that has reduces with respect to described initial concentration; With

(b) be substantially free of the droplet of described pearl.

58. the method for claim 57, wherein the droplet of 57 (a) comprises the pearl more than 99% in the droplet of described merging.

59. the method for claim 57, wherein the droplet of 57 (a) comprises the pearl more than 99.9% in the droplet of described merging.

60. the method for claim 57, wherein the droplet of 57 (a) comprises the pearl more than 99.999% in the droplet of described merging.

61. the method for claim 53, wherein step 39 (c) is the electrode mediation.

62. the method for claim 53, wherein step 39 (c) is electric wetting mediation.

63. the method for claim 53, wherein step 39 (c) is the two-dimensional electrophoresis mediation.

64. the method for claim 46, wherein said immobilization step realizes by the motion of pearl described in the operating process of physical property blocking-up droplet.

65. the method for claim 64, wherein said droplet operation comprises division, shifts and/or distributes.

66. the method for claim 64, wherein step 39 (c) produces one group of droplet, and described one group of droplet comprises:

(a) comprise the droplet of all described pearls basically, and the concentration of its described material that has reduces with respect to described initial concentration; With

(b) be substantially free of the droplet of described pearl.

67. the method for claim 66, wherein the droplet of 66 (a) comprises the pearl more than 99% in the droplet of described merging.

68. the method for claim 66, wherein the droplet of 66 (a) comprises the pearl more than 99.9% in the droplet of described merging.

69. the method for claim 66, wherein the droplet of 66 (a) comprises the pearl more than 99.999% in the droplet of described merging.

70. the method for claim 66, wherein step 39 (c) is the electric field mediation.

71. the method for claim 66, wherein step 39 (c) is the electrode mediation.

72. the method for claim 66, wherein step 39 (c) is electric wetting mediation.

73. the method for claim 66, wherein step 39 (c) is the two-dimensional electrophoresis mediation.

74. the method for claim 39, wherein said material comprises one or more pollutent.

75. the method for claim 39, wherein said material comprises one or more analyte.

76. the method for claim 40, wherein said surface comprises first component, and second component that provides in the droplet of described first component to 39 (a) has specificity or non-specific avidity.

77. the method for claim 76, wherein said avidity comprises the avidity that is used for non-specific adsorption.

78. the method for claim 76, wherein said avidity comprises the avidity that is used for specific adsorption.

79. the method for claim 76, wherein said first component comprises antigen binding domain.

80. the method for claim 76, wherein said first component comprises the antibodies district.

81. the method for claim 76, wherein said first component comprises antibody.

82. the method for claim 76, wherein said first component comprises two or more antibody, and every kind of antibody has avidity to the second different components.

83. the method for claim 76, wherein said first component comprises analyte.

84. the method for claim 76, wherein said first component comprises two or more analytes, and every kind of analyte has avidity to the second different components.

85. the method for claim 76, wherein said first component comprises antigen.

86. the method for claim 76, wherein said first component comprises nucleic acid.

87. the method for claim 76, wherein said first component comprises biotin compound or streptavidin compound.

88. the method for claim 76, wherein said second component comprises antigen binding domain.

89. the method for claim 76, wherein said second component comprises antibody.

90. the method for claim 76, wherein said second component comprises two or more antibody, and each has avidity to the second dissimilar components.

91. the method for claim 76, wherein said second component comprises analyte.

92. the method for claim 76, wherein said second component comprises two or more analytes, and each has avidity to the second dissimilar components.

93. the method for claim 76, wherein said second component comprises antigen.

94. the method for claim 76, wherein said second component comprises nucleic acid.

95. the method for claim 76, wherein said second component comprises the nucleic acid of amplification.

96. the method for claim 76, wherein said second component comprises biotin compound or streptavidin compound.

97. the method for claim 76, wherein said second component comprises pollutent.

98. the method for claim 76, wherein:

(a) described first component comprises the compound in conjunction with described second component; With

(b) described second component comprises the compound in conjunction with described first component.

99. modify the method on the surface on the droplet microdrive, described method comprises enforcement one or repeatedly droplet operation, so that the droplet that comprises coating materials is contacted with described surface.

100. the method for claim 99, wherein said droplet operation is selected from: droplet is loaded in the droplet microdrive; Distribute one or more droplet from the source droplet; Droplet is divided, separates or is split as two or more droplets; Droplet is transferred to another place from a place along any direction; Two or more droplets are converged or merge into a droplet; The dilution droplet; Mix droplet; Stir droplet; Make little droplet deformation; Make droplet keep original position; The incubation droplet; The heating droplet; The evaporation droplet; The cooling droplet; Arrange droplet; Droplet is migrated out microdrive; Other droplet operations described herein; With above-mentioned any combination.

101. the method for claim 100, wherein said droplet operation is the electric field mediation.

102. the method for claim 100, wherein said droplet operation is the electrode mediation.

103. the method for claim 100, wherein said droplet operation are electric wetting mediations.

104. the method for claim 100, wherein said droplet operation is the two-dimensional electrophoresis mediation.

105. the method for claim 99, wherein said coating materials comprises the material that is selected from protein, peptide, antibody, antigen binding domain, antigen and nucleic acid.

106. a droplet microdrive, it comprises and is immobilized onto its lip-deep sample or reagent, and is provided so that the droplet on the described droplet microdrive can contact described surface.

107. the droplet microdrive of claim 106, wherein said surface is provided so that at droplet described in the operating process can contact described surface.

108. the droplet microdrive of claim 106, wherein said sample or reagent are selected from protein and peptide.

109. the droplet microdrive of claim 106, wherein said sample or reagent are selected from antibody and antigen.

110. the droplet microdrive of claim 106, wherein said sample or reagent are selected from nucleic acid.

111. with the method that material removes from this material institute bonded surface, described method comprises carries out one or repeatedly droplet operation is so that droplet contacts with described surface, described droplet comprises the solution that is used for from the described material of described surperficial wash-out.

112. the method for claim 111, wherein said material is selected from protein, peptide, antibody, antigen binding domain, antigen and nucleic acid.

113. the method for claim 111, wherein said surface comprises the surface of described droplet microdrive.

114. the method for claim 111, wherein said surface comprises the surface of one or more pearl.

115. the droplet microdrive, it comprises:

(a) comprise the base material of the electrode that is used to carry out the droplet operation; With

(b) one or more temperature-control device, it is used to heat and/or cool off the zone of droplet microdrive near being set at one or more described electrode, and is provided so that droplet can be transferred on described electrode in the described zone so that heating.

116. the droplet microdrive of claim 115, wherein said temperature-control device comprises well heater.

117. the droplet microdrive of claim 116, wherein said temperature-control device comprises thin film heater.

118. the droplet microdrive of claim 115, wherein said temperature-control device comprises one or more cooling element.

119. the droplet microdrive of claim 115, wherein said cooling element comprises thermoelectronic cooler.

120. the droplet microdrive of claim 115, wherein said cooling element comprise Pa Er card device.

121. the droplet microdrive of claim 115, wherein the zone of droplet microdrive is set at is enough to the annealed temperature.

122. the droplet microdrive of claim 115, wherein the zone of droplet microdrive is set at the temperature that is enough to sex change.

123. the droplet microdrive of claim 115, wherein the zone of droplet microdrive is set at the temperature that is enough to extend.

124. the droplet microdrive of claim 115, wherein one or repeatedly described droplet operation be electric wetting mediation.

125. the droplet microdrive of claim 115, wherein one or repeatedly described droplet operation be the two-dimensional electrophoresis mediation.

126. the droplet microdrive of claim 116, wherein said temperature-control device comprises at least two well heaters:

(a) at least one well heater, near the temperature of the nucleic acid generation sex change it is set at and is enough to make on the described droplet microdrive in the droplet; With

(b) at least one well heater, near the nucleic acid it is set at and is enough to make on the described droplet microdrive in the droplet takes place to extend and/or the annealed temperature.

127. the droplet microdrive of claim 115, also comprise the device that is used to implement magnetic field, described device is positioned near one or more described electrode, and wherein the intensity in magnetic field and the degree of approach are enough to the magnetic responsiveness pearl in the immobilization droplet in implementing the droplet operating process.

128. the droplet microdrive of claim 127, the wherein said device that is used to implement magnetic field comprise the device of the power supply of electromagnet and control electromagnet.

129. the droplet microdrive of claim 127, the wherein said device that is used to implement magnetic field comprise magnet and move to described magnet near described one or more described electrode and with near the device of described magnet from removing described one or more described electrode.

130. the droplet microdrive of claim 115, comprise and be positioned near one or more droplet microdrive pond of one or more described electrode, it is provided so that electrode can distribute droplet from the liquid the described droplet microdrive pond, wherein said pond comprises reagent, and wherein every kind of reagent comprises one or more component that is selected from in next group: cushion, primer, Nucleotide, polysaccharase, reversed transcriptive enzyme and other nucleic acid amplification reagent.

131. the droplet microdrive of claim 115 also comprises loading amplification instant droplet thereon.

132. the droplet microdrive of claim 131, the volume of wherein said amplification instant droplet at about 1nL to the scope of about 10 μ L.

133. the droplet microdrive of claim 131, the volume of wherein said amplification instant droplet at about 10nL to the scope of about 1 μ L.

134. the droplet microdrive of claim 131, the volume of wherein said amplification instant droplet is less than about 100nL.

135. comprise the system of the droplet microdrive of claim 115, it comprises treater, described treater electronics is connected in and is configured to control:

(a) described electrode; With

(b) temperature of one or more described one or more temperature-control device.

136. the system of claim 135, it is turned to by the temperature of controlling described temperature-control device the step of implementing nucleic acid amplification thermal cycling scheme near the droplet that places the temperature-control device by program.

137. the system of claim 136, wherein said nucleic acid amplification thermal cycling scheme comprises PCR thermal cycling scheme.

138. the system of claim 136, wherein said nucleic acid amplification thermal cycling scheme comprises RT-PCR thermal cycling scheme.

139. the system of claim 135, it is turned to by droplet being moved near one or more temperature-control device and droplet being removed the step of implementing nucleic acid amplification thermal cycling scheme near one or more temperature-control device by program.

140. the system of claim 139, wherein said nucleic acid amplification thermal cycling scheme comprises PCR thermal cycling scheme.

141. the system of claim 135, it is turned to by program and implements the droplet operation so that one or more sample droplet and one or more amplifing reagent droplet are merged into one or more amplification instant droplet with suitable ratio on described droplet microdrive.

142. comprise the system of the droplet microdrive of claim 127, it is turned to the droplet operation of implementing to be used for based on the washing scheme of droplet, wherein washing magnetic responsiveness pearl on described droplet microdrive by program.

143. comprise the system of the droplet microdrive of claim 127, it is turned to by program and implements to be used for the droplet operation of one or more material from described magnetic responsiveness pearl wash-out.

144. comprise the system of the droplet microdrive of claim 130, it is turned to by program:

(a) distribute the reagent droplet from each described one or more pond;

(b) the reagent droplet of the described distribution of transfer;

(c) the reagent droplet of merging distribution and one or more sample droplet that may comprise target nucleic acid are to produce one or more amplification instant droplet; With

(d) on described one or more amplification instant droplet, implement to be enough to increase and be present in the thermal cycling scheme of the target nucleic acid in the described sample droplet.

145. the system of claim 144, wherein said thermal cycling scheme comprises determination step, wherein the target nucleic acid that quantitatively is amplified after the circulation of pre-determined quantity.

146. being turned to by program, the system of claim 145, wherein said system when detecting the target nucleic acid of predetermined amount, stop thermal cycling.

147. being turned to by program, the system of claim 145, wherein said system when not detecting the target nucleic acid of predetermined amount after the circulation at pre-determined quantity, stop thermal cycling.

148. the system of claim 144, wherein said amplification instant droplet has carried out thermal cycling scheme 144 (d), and described system is further turned in the thermal cycling process by program or shifts described amplification instant droplet afterwards and is used for further processing.

149. the system of claim 148, wherein said further processing comprises one or more target nucleic acid of detection.

150. the system of claim 149, wherein said system are turned to by program the user's output that indicates whether to exist one or more target nucleic acid are provided.

151. the system of claim 149, wherein said target nucleic acid diagnosis nucleic acid.

152. the system of claim 149, wherein said target nucleic acid comprises diagnosis and uses nucleic acid, and described diagnosis represents to exist pathogenic organisms with the existence of nucleic acid.

153. comprise the tube of the droplet microdrive of claim 130, described tube comprises one or more pond and is used to set up from one or more pond to the device of the fluid passage in one or more droplet microdrive pond.

154. the tube of claim 153 comprises one or more nucleic acid amplification reagent that is contained in advance in described one or more pond.

155. the tube of claim 154, wherein said reagent is selected from: cushion, primer, Nucleotide, polysaccharase and other nucleic acid amplification reagent.

156. the method for the droplet microdrive tube that assembling is used to operate, described method comprise that the tube with claim 153 is connected in the droplet microdrive.

157. load the method for the droplet microdrive tube be used to operate, described method comprises that the liquid that makes from the tube of claim 153 flow to droplet microdrive pond by described fluid passage.

158. the method for the nucleic acid in the amplification biological sample, described method comprises:

(a) provide the system that comprises the droplet microdrive, described droplet microdrive electronics is connected in and is controlled by the treater that can execute instruction, and described droplet microdrive comprises:

(i) may comprise the sample of target nucleic acid;

The base material that (ii) comprises the electrode that is used to carry out the droplet operation; With

(iii) one or more temperature-control device, it is set near the zone that is used to heat the droplet microdrive one or more described electrode, makes droplet can be transferred in the described zone so that heating;

(b) adopt droplet to operate in and merge one or more amplifing reagent droplet and one or more sample droplet on the described droplet microdrive, to produce amplification instant droplet; With

(c) described amplification instant droplet is carried out thermal cycling, it is enough to make the target nucleic acid that exists in the described amplification instant droplet to be amplified.

159. the method for claim 158, wherein step 158 (b) comprising:

(a) will be transferred to from one or more amplifing reagent droplet in pond on the described droplet microdrive;

(b) will be transferred to from one or more sample droplet in pond on the described droplet microdrive; With

(c) converge one or more amplifing reagent droplet and one or more sample droplet to produce amplification instant droplet.

160. the method for claim 159 also comprises:

(a) by making one or more amplifing reagent droplet flow in the pond on the described droplet microdrive in the pond that described one or more amplifing reagent droplet is loaded on the described droplet microdrive from the place of described droplet microdrive outside; And/or

(b) by one or more amplifing reagent droplet is flow in the pond on the described droplet microdrive in the pond that described one or more sample droplet is loaded on the described droplet microdrive from the place of described droplet microdrive outside.

161. the method for claim 158 also comprises:

(a) by one or more amplifing reagent droplet is flow on the described droplet microdrive from the place of described droplet microdrive outside described one or more amplifing reagent droplet is loaded on the described droplet microdrive; And/or

(b) by one or more sample droplet is flow on the described droplet microdrive from the place of described droplet microdrive outside described one or more sample droplet is loaded on the described droplet microdrive.

162. the method for claim 158, the volume of wherein said amplification instant droplet at about 1nL to the scope of about 10 μ L.

163. the method for claim 158, the volume of wherein said amplification instant droplet at about 1nL to the scope of about 1 μ L.

164. the method for claim 158, the volume of wherein said amplification instant droplet is less than about 100nL.

165. the method for claim 158 also is included in the thermal cycling process or detects the target nucleic acid that whether has amplification in the described amplification instant droplet afterwards.

166. the method for claim 158 also is included in the thermal cycling process and distributes droplet from described amplification instant droplet.

167. the method for claim 158 also is included in the thermal cycling process or afterwards the target nucleic acid that increases in the described amplification instant droplet is carried out quantitatively.

168. the method for claim 167 also comprises the amount of target nucleic acid in the described amplification instant droplet of inferring before the thermal cycling.

169. the method for claim 158, wherein step 158 (c) realizes near the amplification instant droplet described well heater by the temperature that changes well heater.

170. the method for claim 158, wherein step 158 (c) is by moving to described amplification instant droplet near one or more well heater and realizing from removing near one or more well heater.

171. the method for claim 158 comprises that also the dna immobilization that carries out to amplification has the droplet operation of selecting on the surface of avidity in the nucleic acid to described amplification.

172. the method for claim 171, wherein said surface comprises the surface of described droplet microdrive.

173. the method for claim 171, wherein said surface comprises one or more pearl.

174. the method for claim 171, wherein said droplet operation comprise that the target nucleic acid with described amplification places under the sex change condition so that it became strand before the described surface of contact.

175. the method for claim 171, the described amplification instant droplet that wherein said droplet operation will comprise the target nucleic acid of amplification converges with the droplet that comprises described pearl.

176. the method for claim 171, the target nucleic acid of wherein said amplification is by biotinylation, and described pearl comprises bonded streptavidin with it.

177. the method for claim 171 also comprises and carries out the droplet operation, described droplet operation is enough to:

(a) nucleic acid of the described amplification of sex change is to provide single-chain nucleic acid; With

(b) described single-chain nucleic acid is contacted with the base material that described single-chain nucleic acid is had avidity, thus described single-chain nucleic acid is immobilized onto on the described base material.

178. the method for claim 171, also comprise enforcement based on the washing scheme of droplet in case make described amplification instant droplet not in conjunction with component from described surface isolation.

179. the method for claim 178, wherein said washing scheme based on droplet comprise carry out droplet operation so that:

(a) the washing droplet is contacted with described surface; With

(b) make described washing droplet from described surface isolation, binding substance is not taken away from described surface thus.

180. the method for claim 173, also comprise enforcement based on the washing scheme of droplet so that make not separating from described pearl of described amplification instant droplet in conjunction with component.

181. the method for claim 180, wherein said washing scheme based on droplet comprises:

(a) described pearl is immobilized onto in the described droplet;

(b) carrying out the droplet operation makes washing droplet and the droplet that comprises described pearl converge the droplet that converges with generation; With

(c) carry out the droplet operation droplet is separated from the described droplet that converges, binding substance is not taken away from described pearl thus.

182. the method for claim 181 also comprises repeating step (a) to (c), until reaching the not preset value of binding substance.

183. the method for claim 181 also comprises step (d), this step (d) comprises the described immobilized pearl of release.

184. the method for claim 183 also comprises repeating step (a) value (d), until reaching the not preset value of binding substance.

185. the method for claim 181, wherein said pearl have magnetic responsiveness and described immobilization step realizes by using the described magnetic responsiveness pearl in the described droplet of magnet immobilization.

186. the method for claim 181, wherein said pearl do not realize by the transfer of using this type of pearl in the operating process of physical objects blocking-up droplet in response to magnetic field and described immobilization step basically.

187. the method for claim 180, wherein said washing scheme based on droplet comprises:

(a) carrying out the droplet operation makes washing droplet and the droplet that comprises described pearl converge the droplet that converges with generation;

(b) described pearl is immobilized onto in the described droplet that converges; With

(c) carry out the droplet operation droplet is separated from the described droplet that converges, binding substance is not taken away from described pearl thus.

188. the method for claim 187 also comprises repeating step (a) to (c), until reaching the not preset value of binding substance.

189. the method for claim 187 also comprises step (d), described step (d) comprises the described immobilized pearl of release.

190. the method for claim 189 also comprises repeating step (a) to (d), until reaching the not preset value of binding substance.

191. the method for claim 187, wherein said pearl have magnetic responsiveness and described immobilization step realizes by using the described magnetic responsiveness pearl in the described droplet of magnet immobilization.

192. the method for claim 187, wherein said pearl do not have magnetic responsiveness and described immobilization step realizes by the transfer of using magnetic responsiveness pearl described in the operating process of physical objects blocking-up droplet.

193. the method for claim 180 comprises that also being embodied as execution discharges the elution protocol of immobilized nucleic acid and selected droplet operation from described surface.

194. the method for claim 158 also is included in the target nucleic acid of quantitative amplification after the circulation of pre-determined quantity.

195. the method for claim 158 also is included in and stops described thermal cycling under the following situation:

(a) detect the target nucleic acid of predetermined amount; Or

(b) after the circulation of pre-determined quantity, do not detect the target nucleic acid of predetermined amount.

196. the method for claim 158 comprises that also the droplet that shifts the nucleic acid that comprises amplification is used for further processing.

197. the method for claim 158 also comprises and detects one or more target nucleic acid.

198. the method for claim 197 also comprises the user's output that indicates whether to exist one or more target nucleic acid is provided.

199. the method for claim 197, wherein said target nucleic acid comprise diagnosis nucleic acid.

200. the method for claim 158, wherein step 0 is carried out to the droplet microdrive by making from one or more reagent and/or one or more sample flow in the place of described droplet microdrive outside.

201. the method for claim 200, wherein said outside pond are positioned on the tube that is connected with described droplet microdrive, the fluid passage on its mode of connection has been set up from described outside pond to described droplet microdrive.

202. the method for claim 200, wherein said outside pond are positioned on the tube that is connected with described droplet microdrive, the fluid passage in the pond on its mode of connection has been set up from described outside pond to described droplet microdrive.

203. the method for amplification of nucleic acid on the droplet microdrive, described method comprise amplification instant droplet is shifted repeatedly by one or more heating zone on the base material, the volume of described amplification instant droplet is no more than 50nL.

204. the method for claim 203, wherein said transfer comprise the transfer of electric field mediation.

205. the method for claim 203, wherein said transfer comprise the transfer of electrode mediation.

206. the method for claim 203, wherein said transfer comprises the transfer of electric wetting mediation.

207. the method for claim 203, wherein said transfer comprise the transfer of two-dimensional electrophoresis mediation.

208. the method for claim 203, wherein said amplification instant droplet is substantially free of surfactant.

209. carry out the droplet method of operating under the temperature condition that raises, described method comprises:

(a) provide the droplet microdrive;

(b) droplet that heats on it makes temperature surpass about 25 ℃ of droplets with the generation heating; With

(c) on the droplet of described heating, carry out the droplet operation.

210. the method for claim 209, wherein step 209 (b) comprises that the described droplet of heating makes temperature surpass about 30 ℃.

211. the method for claim 209, wherein step 209 (b) comprises that the described droplet of heating makes temperature surpass about 50 ℃.

212. the method for claim 209, wherein step 209 (b) comprises that the described droplet of heating makes temperature surpass about 75 ℃.

213. the method for claim 209, wherein said droplet operation is the electric field mediation.

214. the method for claim 209, wherein said droplet operation is the electrode mediation.

215. the method for claim 209, wherein said droplet operation are electric wetting mediations.

216. the method for claim 209, wherein said droplet operation is the two-dimensional electrophoresis mediation.

217. the method for claim 209, wherein said droplet comprise based on the droplet in the incubation step of the mensuration scheme of droplet.

218. the method for claim 213, wherein said mensuration scheme based on droplet comprises the mensuration scheme based on avidity.

219. the droplet microdrive, it is made by fluorescent material and comprises the surveyed area that is used to detect from the fluorescent signal of droplet, and this surveyed area is coated with light absorption, low fluorescence or non-fluorescent material coating.

220. the droplet microdrive of claim 219, wherein said fluorescent material comprises dielectric materials.

221. the droplet microdrive of claim 219, wherein said coating comprises metallic coating.

222. the droplet microdrive of claim 219, wherein said coating comprises copper coating.

223. the droplet microdrive of claim 219, wherein said coating comprises black coating.

224. the droplet microdrive of claim 219, wherein said coating comprises copper coating, and described copper coating comprises coating golden surface layer thereon.

225. droplet microdrive system, it comprises:

(a) be configured to carry out the droplet microdrive that droplet is operated; With

(b) be constructed to have the transmitter of sensing relation, the signal and/or the character of one or more droplet of described transmitter on can the described droplet microdrive of sensing with described droplet microdrive.

226. the system of claim 225, wherein said system also comprises one or more treater, and described treater electronics is connected in described droplet microdrive and is turned to by program:

(a) the droplet operation on the described droplet microdrive of control; With

(b) processing is from the electronic signal of one or more described transmitter.

227. the system of claim 225, wherein said transmitter comprises selected and is configured to the physical properties of the droplet on the described droplet microdrive of sensing and/or the transmitter of signal.

228. the system of claim 227, wherein said physical properties comprises temperature.

229. the system of claim 227, wherein said physical properties comprises droplet size.

230. the system of claim 225, wherein said transmitter comprises selected and is configured to the electrochemical properties of the droplet on the described droplet microdrive of sensing and/or the transmitter of signal.

231. the system of claim 230, wherein said transmitter comprises potentiometric sensor.

232. the system of claim 230, wherein said transmitter comprises current sensor.

233. the system of claim 230, wherein said transmitter comprises conductivity sensor.

234. the system of claim 230, wherein said transmitter comprises ion specific electrode.

235. the system of claim 230, wherein said transmitter comprises pH electrode.

236. the system of claim 230, wherein said transmitter comprises thermistor.

237. the system of claim 230, wherein said transmitter comprises RTD.

238. the system of claim 230, wherein said transmitter comprises thermopair.

239. the system of claim 230, wherein said transmitter comprises radiation sensor.

240. the system of claim 225, wherein said transmitter comprises selected and is configured to the optical property of the droplet on the described droplet microdrive of sensing and/or the transmitter of signal.

241. the system of claim 240, wherein:

(a) described system also comprises excitation light source, and it is used for causing thus that with having the fluorescent substance of the droplet of photoconduction on described droplet microdrive of an excitation wavelength this type of material sends fluorescence; With

(b) described transmitter is configured to the fluorescence of sensing from the fluorescent substance in the described droplet.

242. the system of claim 240, wherein:

(a) described droplet comprises biological fluid;

(b) described system also comprises light source, and it is used to make light to pass droplet; With

(c) described transmitter is constructed to make sensing from described droplet.

243. the system of claim 240, wherein said transmitter is configured to noclilucence or the chemoluminescence of sensing from the droplet on the described droplet microdrive.

244. the system of claim 240, wherein said transmitter is configured to the scattered light of sensing from the droplet on the described droplet microdrive.

245. the system of claim 240, wherein said transmitter is configured to the turbidity of the droplet on the described droplet microdrive of sensing.

246. the system of claim 240, wherein said transmitter comprises sensing element, and described sensing element is selected from optical sensor and photon sensor.

247. the system of claim 240, wherein said transmitter comprises sensing element, and described sensing element is selected from photorectifier, avalanche photodide, photomultiplier and photon counting photomultiplier.

248. the system of claim 240, wherein:

(a) described droplet microdrive:

(i) comprise surface plasma body resonant vibration (SPR) surface, its luminous reflectance factor that has exists biomolecules to change because of described SPR surface; With

(ii) be configured to carry out the droplet operation, so that the sample droplet is contacted with described SPR surface; With

(b) described system comprises:

(i) light source is used for photoconduction to described SPR surface; With

(ii) transmitter, it is used to detect the change of the reflectivity on described surface plasma body resonant vibration surface.

249. the system of claim 240, wherein:

(a) described droplet microdrive:

(i) comprise surface and/or the material that produces Raman signal; With

(ii) be configured to carry out the droplet operation, so that droplet is contacted with described Raman signal detection surface and/or material; With

(b) described system comprises:

(i) light source of generation Raman signal, it is used to shine Raman signal detection surface and/or material on the described droplet microdrive; With

(ii) laman spectrophotometer, it is used to detect the scattered light from illuminated surface and/or material.

250. the system of claim 225, it comprises two or more types transmitter.

251. the system of claim 225, it is turned to enforcement by program and single droplet is successively the droplet operation of passing a plurality of transmitters.

252. the system of claim 225, it is turned to enforcement by program and a plurality of droplets is successively the droplet operation of passing single-sensor.

253. the system of claim 225, it is turned to enforcement by program and a plurality of droplets is the droplet operation of passing a plurality of transmitters.

254. the system of claim 225, it is turned to enforcement by program a plurality of droplets are the droplet operation of passing a plurality of transmitters in substantially parallel mode.

255. the system of claim 225, it is turned to enforcement by program and a plurality of droplets is the droplet operation of passing a plurality of transmitters that are used to carry out single type.

256. the system of claim 225, it is turned to enforcement by program and a plurality of droplets is the droplet operation of passing a plurality of transmitters that are used to carry out multiple type.

257. the system of claim 225, wherein said system are turned to one or more scheme based on droplet of implementing to be used to carry out based on the mensuration of droplet by program.

258. the system of claim 225, wherein said system are turned to two or more different schemes based on droplet of implementing to be used to carry out based on the mensuration of droplet by program.

To implement to be used to carry out with the single sample droplet be two or more different schemes based on droplet of the multiple test of beginning 259. the system of claim 257, wherein said system are turned to by program.

260. the system of claim 257, wherein said system are turned to two or more different schemes based on droplet of implementing to be used to carry out a plurality of sample droplet tests by program.

261. the system of claim 257, wherein said scheme based on droplet comprises the scheme that is used for human diagnostic test.

262. the system of claim 257, wherein said scheme based on droplet comprises the scheme that is used for amplification of nucleic acid.

263. the system of claim 257, wherein said scheme based on droplet comprise the scheme that is used to carry out based on the mensuration of avidity.

264. the system of claim 263, wherein said mensuration based on avidity comprises the competitive assay based on avidity.

265. comprising based on the sandwich of avidity, the system of claim 263, wherein said mensuration based on avidity measure.

266. the system of claim 263, wherein said mensuration based on avidity comprises the direct mensuration based on avidity.

267. the system of claim 257, wherein said scheme based on droplet comprises the scheme of utilizing the sample droplet, and described sample droplet comprises the liquid that is selected from as in next group: whole blood, serum, blood plasma, sweat, tear, saliva, phlegm, cerebrospinal fluid, amniotic fluid, seminal fluid, vaginal secretions, slurries, synovial membrane liquid, pericardial fluid, peritoneal fluid, hydrothorax, spill liquid, transudate, capsule liquid, bile, urine, gastric juice, intestinal juice, faecal samples, swab and washing lotion.

268. the system of claim 257, wherein said scheme based on droplet comprise one or repeatedly droplet operation.

269. the system of claim 268, wherein one or repeatedly described droplet operation be electric wetting mediation.

270. the system of claim 268, wherein one or repeatedly described droplet operation be the two-dimensional electrophoresis mediation.

271. the system of claim 268, wherein said droplet microdrive comprises the electrode that is used to carry out the droplet operation.

272. the system of claim 225, wherein said droplet microdrive comprises the electrode of one or more electric current that is used to detect the droplet on the described droplet microdrive, current potential and/or electric conductivity matter.

273. the system of claim 272, wherein said droplet microdrive comprises:

(a) first base material comprises and is configured to handle the electrode that is positioned at the droplet on the described substrate surface; With

(b) be associated with described first base material place and with the surface of described first base material second base material at a distance of a segment distance, this distance limits the space between described first base material and second base material, wherein said distance is enough to hold the droplet that is placed in the described space.

274. the system of claim 273, wherein said second base material comprises the electrode that one or more is used to detect droplet character.

275. the system of claim 273, wherein said first and second base materials respectively comprise the electrode that one or more is used to detect droplet character.

276. the system of claim 273, wherein:

(a) described second base material comprises one or more reference electrode; With

(b) described first and/or second base material comprises the electrode that one or more is used to detect droplet character.

277. the system of claim 272, wherein:

(a) described droplet microdrive comprises base material, and described base material comprises the electrode that is used to carry out the droplet operation; With

(b) the described base material of at least a portion comprises the electrode that is used to carry out the droplet operation and lacks second base material, can carry out the droplet operation like this on the base material that lacks second base material.

278. the system of claim 272, wherein:

(a) described droplet microdrive comprises base material, and described base material comprises the electrode that is used to carry out the droplet operation; With

(b) the described base material of at least a portion:

(i) comprise the electrode that is used to carry out the droplet operation; With

(ii) comprise second base material that is placed on apart from described first base material, one segment distance, wherein said distance is enough to allow to carry out the droplet operation on described first base material, and need not to contact between described droplet and described second base material.

279. the system of claim 273, wherein:

(a) described first and/or second base material comprises the electrode of the electric current character that is used to detect the droplet on the described droplet microdrive; With

(b) described system is turned to the scheme based on droplet of implementing to be used for detecting by electric current testing the component of described sample droplet by program.

280. the system of claim 273, wherein:

(a) described first and/or second base material comprises the electrode of the current potential character that is used to detect the droplet on the described droplet microdrive; With

(b) described system is turned to the scheme based on droplet of implementing to be used for detecting by the current potential detection method component of described sample droplet by program.

281. the system of claim 273, wherein:

(a) described first and/or second base material comprises the electrode of the electric conductivity matter that is used to detect the droplet on the described droplet microdrive; With

(b) described system is turned to the scheme based on droplet of implementing to be used for detecting by conductance detection the component of described sample droplet by program.

282. the system of claim 273, wherein first or second base material comprises two or more types electrode of the group that is selected from electrode.

283. the system of claim 225, wherein said droplet microdrive is configured and program turn to carry out 2 or more a plurality of be the test of beginning with the single sample droplet, the scope of described droplet is from about 1nL about 10 μ L extremely.

284. the system of claim 225, wherein said droplet microdrive is configured and program turns to and carries out 5 or more a plurality ofly rises the test that the sample droplet is beginning with single sub-micro, and the scope of described droplet is from about 1nL about 10 μ L extremely.

285. the system of claim 225, wherein said droplet microdrive is configured and program turns to and carries out 10 or more a plurality ofly rises the test that the sample droplet is beginning with single sub-micro, and the scope of described droplet is from about 1nL about 10 μ L extremely.

286. the system of claim 225 comprises:

(a) optical pickocff; With

(b) be used for described droplet microdrive is connected in the device of described system, described device aligns the surveyed area on the described droplet microdrive with described optical receiver assembly.

287. the system of claim 225 comprises:

(a) optical pickocff;

(b) tube, it comprises the pond that is used for one or more liquid;

(c) be used for described tube is connected in described droplet microdrive so that set up the device of fluid passage between described tube pond and described droplet microdrive; With

(d) be used for described tube is connected in the device of described system, described device aligns the surveyed area on the described droplet microdrive with described optical receiver assembly.

288. the system of claim 225, wherein said tube comprises mechanical mechanism, motor drive mechanism, pressure source and/or vacuum source, is used to promote liquid and passes through following fluid passage:

(a) from described droplet microdrive to described tube; And/or

(b) from described tube to described droplet microdrive.

289. the system of claim 272, wherein:

(a) described droplet microdrive comprises:

(i) first base material, it comprises the electrode that is configured to carry out the droplet operation on the surface of described base material;

(ii) apart from second base material of surface one segment distance of described first base material, this distance limits the space between described first base material and second base material, and wherein said distance is enough to hold the droplet that is placed in the above space of first base material; With

The surveyed area that (iii) is associated with described electrode and is provided with makes that the droplet on the described droplet microdrive can be transferred in the described surveyed area; With

(b) described system comprises:

(i) optical pickocff; With

(ii) be used for described droplet microdrive is connected in the device of described system, described device aligns the described surveyed area on the described droplet microdrive with described optical receiver assembly, to allow to detect optical property or the signal from the droplet in the described surveyed area.

290. the system of claim 272, wherein said system is constructed to mancarried device.

291. the system of claim 272, wherein said system is constructed to handheld apparatus.

292. the method for the target analyte in the test sample, described method comprises:

(a) carry out the droplet operation mensuration reagent based on avidity on the droplet microdrive is merged with the sample that may comprise described target analyte, to produce the signal that exists, do not exist and/or measure of representing analyte; With

(b) detect described signal, wherein said signal and the existence of analyte described in the sample, do not exist and/or measure corresponding.

293. the method for claim 292, wherein one or repeatedly described droplet operation on the droplet microdrive, implement.

294. the method for claim 292 comprises the droplet operation of electric field mediation.

295. the method for claim 292 comprises the droplet operation of electrode mediation.

296. the method for claim 292 comprises the droplet operation of electric wetting mediation.

297. the method for claim 292 comprises the droplet operation of two-dimensional electrophoresis mediation.

298. the method for claim 292, wherein said mensuration reagent based on avidity comprises immunoassay reagent.

299. the method for claim 292, wherein said detection step comprises the signal of detection from the droplet on the droplet microdrive.

300. the method for claim 292, wherein said detection step comprises the signal of detection from the surface on the droplet microdrive.

301. the method for claim 300, wherein said surface comprises the surface of pearl.

302. the method for claim 301, wherein said pearl is a magnetic responsiveness.

303. the method for claim 301, wherein said pearl do not have magnetic responsiveness basically.

304. the method for claim 300, wherein said surface comprises the surface of described droplet microdrive.

305. the method for claim 300, wherein:

(a) described surface comprises flicker molecule and capture molecules; With

(b) operation of described droplet comprises making and comprises the radiolabeled sample droplet that capture molecules is had the molecule of avidity and contact with described surface, make that when radiolabeled molecule is incorporated into capture molecules radioactive grain and described flicker interaction of molecules are to produce light.

306. the method for claim 305, wherein said surface comprise the surface of pearl or the surface of described droplet microdrive.

307. the method for claim 300, wherein:

(a) described surface comprises the textured metal film, and described textured metal film comprises capture antibody in its surface; With

(b) described droplet microdrive comprises electrode, and described electrode is configured to shift droplet and makes it to contact described textured metal film.

308. the method for claim 307, wherein said textured metal film comprises silverskin or golden film.

309. the method for claim 300, wherein said surface markers has the Raman reporter molecules.

310. the method for claim 300, wherein said signal comprises Raman spectrum.

311. the method for claim 298, wherein measured signal is corresponding to the binding site that is not occupied on the antibody.

312. the method for claim 298, wherein measured signal is corresponding to the binding site that is occupied on the antibody.

313. the method for claim 298, wherein said mensuration reagent based on avidity comprise the antibody that is immobilized onto the surface.

314. the method for claim 313, wherein said surface comprise the solid phase that is positioned at the droplet on the described droplet microdrive.

315. the method for claim 313, wherein said surface comprises the surface of described droplet microdrive.

316. the method for claim 313, wherein said surface comprise the pearl that is positioned at the droplet on the described droplet microdrive.

317. the method for claim 316, wherein said pearl are the magnetic responsiveness pearls.

318. the method for claim 316, wherein said pearl do not have magnetic responsiveness basically.

319. the method for claim 316, wherein said mensuration reagent based on avidity comprises the analyte tracer agent of mark.

320. the method for claim 319, the tracer agent of wherein said mark only produce signal when being incorporated into described avidity reagent.

321. the method for claim 319, the tracer agent of wherein said mark only produce signal when not being incorporated into described avidity reagent.

322. the method for claim 316, wherein said droplet operation realizes competitive mensuration scheme based on avidity.

323. the method for claim 298, wherein said mensuration reagent based on avidity comprises antibody, and described antibody has selectivity to target humidity province analyte.

324. the method for claim 298, wherein said mensuration reagent based on avidity comprises two or more antibody, and defined epitope or the zone to described target analyte has selectivity separately.

325. the method for claim 298, wherein said mensuration reagent based on avidity comprises two or more antibody, and these antibody comprise that described target analyte is had optionally first antibody and described first antibody is had optionally second antibody.

326. the method for claim 316, the wherein said antibody that comprises mark based on the mensuration reagent of avidity.

327. the method for claim 326, the antibody of wherein said mark comprises radio isotope.

328. the method for claim 316, but wherein said mensuration reagent based on avidity comprises the antibody of puting together in enzyme that can the catalysis detection reaction.

329. the method for claim 327, but wherein said detection reaction produces colorimetric, fluorescence and/or luminosity or signal.

330. the method for claim 326, wherein said droplet operation comprises antibody that is used to make the mark that is incorporated into described analyte and the isolating washing scheme based on droplet of antibody that is not incorporated into the mark of described analyte.

331. the method for claim 330, the wherein said antibody that is incorporated into the mark of described analyte also is incorporated into the surface, and described washing scheme based on droplet comprises:

(a) remove the droplet of the antibody that comprises the mark that is not incorporated into described analyte from described surface; With

(b) add the droplet of the antibody that does not comprise the mark that is not incorporated into described analyte for described surface.

332. the method for claim 331, wherein said surface comprise with the droplet microdrive on the surface of the contacted solid phase of droplet.

333. the method for claim 331, wherein said surface comprises the surface of droplet microdrive.

334. the method for claim 298, wherein said mensuration reagent based on avidity comprise the reagent that is used for being selected from as the type of next group: sandwich mensuration, immunoassay, immunoassay, catch bridging measure, directly the glass catch assay, indirectly the glass catch assay, to micromolecular mensuration, to micromolecular noncompetitive measure, the environmental analysis thing is measured and free analyte is measured.

335. the method for claim 334 is included in and carries out two or more described types on the droplet microdrive simultaneously.

336. the method for claim 334 is included in and carries out three kinds or more kinds of described type on the droplet microdrive simultaneously.

337. the method for claim 298 also comprises and carries out the droplet operation to set up calibration curve.

338. the method for claim 298, wherein:

(a) operation of described droplet makes the detection antibody of capture antibody and mark contact with described sample so that produce capture antibody-analyte-detection antibody complex under the situation of described analyte existing;

(b) use based on the washing scheme of droplet described capture antibody-analyte-detection antibody complex is separated with unreacted components and/or unreacted excessive antibody in the described sample; With

(c) described detection step comprises the signal of detection from described capture antibody-analyte-detection antibody complex.

339. the method for claim 298, the sample that wherein said droplet is manipulated is from being selected from as the source in next group: whole blood, lymph liquid, serum, blood plasma, sweat, tear, saliva, phlegm, cerebrospinal fluid, amniotic fluid, seminal fluid, vaginal secretions, slurries, synovial membrane liquid, pericardial fluid, peritoneal fluid, hydrothorax, spill liquid, transudate, capsule liquid, bile, urine, gastric juice, intestinal juice, faecal samples, liquefaction tissue, organism, biology swab and biology washing lotion liquefy.

340. the method for claim 298, wherein said mensuration reagent based on avidity comprises antibody.

341. the method for claim 340, wherein said antibody immobilization is on the surface.

342. the method for claim 341, wherein said surface comprises the surface of pearl.

343. the method for claim 342, wherein said pearl is a magnetic responsiveness.

344. the method for claim 342, wherein said pearl do not have magnetic responsiveness basically.

345. the method for claim 341, wherein said surface comprises the surface of described droplet microdrive.

346. the method for claim 341, the operation of wherein said droplet comprise with antibody immobilization on the surface to produce immobilized antibody.

347. the method for claim 346, wherein said droplet operation comprise the surface of the antibody in the droplet with described droplet microdrive contacted, this is enough to cause described antibody immobilization on described surface.

348. the method for claim 346, wherein said droplet operation comprise the antibody in the droplet is contacted with the surface of pearl, this is enough to cause described antibody immobilization on described surface.

349. the method for claim 348 wherein realizes described contact by the droplet operation, described droplet operation comprises that the droplet that will comprise described antibody merges with the droplet that comprises pearl.

350. the method for claim 346, wherein said droplet operation comprises that also the described surface of washing is to remove excessive antibody.

351. the method for claim 346, wherein said droplet operation also comprises the sample of sampling droplet form.

352. the method for claim 351, wherein said droplet operation comprises that also the antibody that will be immobilized onto the surface is exposed to the sample droplet that may comprise described target analyte.

353. the method for claim 352, the operation of wherein said droplet also comprises the described surface of washing, removes thus or reduces not amount in conjunction with sample material.

354. also comprising described immobilized antibody is exposed to, the method for claim 341, the operation of wherein said droplet comprise the droplet of reporting antibody.

355. the method for claim 341, wherein said droplet operation also comprises the report antibody that flush away is excessive.

356. the method for claim 341, wherein said droplet operation also comprises carries out extra step to produce measurable parameter or signal.

357. the method for claim 356, wherein said measurable parameter or signal comprise the electrochemical properties of the droplet on the droplet microdrive.

358. the method for claim 357, wherein said electrical property comprise electric current character, current potential character and/or the electric conductivity matter of the droplet on the described droplet microdrive.

359. the method for claim 356, wherein said measurable parameter or signal comprise the optical property of the droplet on the droplet microdrive.

360. the method for claim 359, wherein said optical property comprises absorption, chemoluminescence and/or fluorescence.

361. the method for claim 359, wherein said optical property comprises Raman spectrum.

362. the method for claim 356, wherein said measurable parameter or signal comprise the physical parameter of the droplet on the droplet microdrive.

363. the method for claim 362, wherein said physical parameter comprises temperature.

364. the method for claim 362, wherein said physical parameter comprises the emission radioactive particulate.

365. the method for claim 340, wherein said droplet operation also comprises:

(a) described immobilized antibody is exposed to the sample droplet that may comprise described target analyte, described target analyte has binding affinity to described immobilized antibody;

(b) washing described immobilized antibody-target analyte mixture remove thus described sample droplet not in conjunction with component;

(c) described immobilized antibody is exposed to comprises the droplet of reporting antibody;

(d) the excessive report antibody of flush away; With

(e) carry out extra step to produce measurable parameter or signal.

366. the method for claim 365 also comprises the output of the electron production that the described signal of representative is provided.

367. the method for claim 340, wherein said droplet operation also comprises provides the sample droplet that may comprise target analyte and comprise the report analysis thing.

368. the method for claim 367, wherein said droplet operation also comprise described immobilized antibody is exposed to described sample droplet, so that described report analysis thing and any target analyte competition binding site.

369. the method for claim 368, wherein said droplet operation comprises that also the described base material of washing is to remove unconjugated analyte and report analysis thing.

370. the method for claim 369 also comprises and carries out extra droplet operation to produce measurable parameter or signal.

371. the method for claim 369 also comprises quantitative bonded report analysis thing, wherein the amount of the amount of report analysis thing and target analyte is inversely proportional to.

372. the droplet microdrive, it comprises the antibody that is immobilized onto the surface.

373. the droplet microdrive of claim 372, the droplet on the described droplet microdrive of wherein said surface contact.

374. the droplet microdrive of claim 372 comprises the electrode that is used to carry out the droplet operation.

375. the droplet microdrive of claim 372 comprises the reagent that is used to implement the immunoassay scheme.

376. the droplet microdrive of claim 372 comprises the sample that is used to implement the immunoassay scheme.

377. the droplet microdrive of claim 376, the volume of sample is no more than about 1mL.

378. the droplet microdrive of claim 376, the volume of sample are no more than about 100 μ L.

379. the droplet microdrive of claim 376, the volume of sample are no more than about 50 μ L.

380. the droplet microdrive of claim 372 comprises the reagent and the sample that are used to implement the immunoassay scheme.

381. the droplet microdrive of claim 372, wherein said surface comprises the surface of pearl.

382. the droplet microdrive of claim 381, wherein said pearl is hydrophilic.

383. the droplet microdrive of claim 381, wherein said pearl is a magnetic responsiveness.

384. the droplet microdrive of claim 383 comprises that also the droplet that is used on described droplet microdrive produces the device in magnetic field, the intensity in described magnetic field is enough to the position of the described pearl of wide limits in the droplet microdrive.

385. the droplet microdrive of claim 384, the wherein said device that is used to produce magnetic field comprise magnet and are used for moving to described magnet near the described droplet microdrive and near the electric assembly from removing the described droplet microdrive.

386. the droplet microdrive of claim 384, the wherein said device that is used to produce magnetic field comprises electromagnet.

387. the droplet microdrive of claim 381, wherein said pearl do not have magnetic responsiveness basically.

388. the droplet microdrive of claim 372, wherein said surface comprises the surface of described droplet microdrive.

389. the droplet microdrive of claim 372 also comprises one or more well heater, described well heater is set to be used to heat one or more droplet on the described droplet microdrive.

390. the droplet microdrive of claim 389 comprises isolated two or more well heaters, so that keep different humidity provinces on described droplet microdrive.

391. the droplet microdrive of claim 389, wherein one or more described well heater comprises thin film heater.

392. the droplet microdrive of claim 389, wherein one or more described well heater comprises the module well heater.

393. the droplet microdrive of claim 389, wherein one or more well heater maintains the temperature that is fit to carry out the incubation step in the immunoassay with at least a portion of described droplet microdrive.

394. the droplet microdrive of claim 373, wherein said droplet are filled fluid and surround.

395. the droplet microdrive of claim 394, fill fluid comprises silicone oil.

396. the droplet microdrive of claim 394, fill fluid comprise and the immiscible oil of described droplet that described oil comprises surfactant.

397. the droplet microdrive of claim 396, wherein said surfactant comprises the lipotropy surfactant.

398. the droplet microdrive of claim 396, wherein said surfactant comprises TritonX-15.

399. the droplet microdrive of claim 394, fill fluid comprises silicone oil, and described silicone oil comprises surfactant.

400. the droplet microdrive of claim 399, wherein said surfactant comprises the lipotropy surfactant.

401. the droplet microdrive of claim 399, wherein said surfactant comprises TritonX-15.

402. the droplet microdrive of claim 389 comprises one or more pond, each comprises sample preparation reagent, based on the mensuration reagent and/or the contrast agents of avidity.

403. complexes, it comprises the droplet microdrive that places the claim 372 in the packing.

404. the complexes of claim 403, wherein said droplet microdrive comprise one or more pre-loaded one or more pond based on the mensuration reagent of avidity.

405. the complexes of claim 403, wherein said droplet microdrive comprise one or more pre-loaded one or more pond based on the mensuration contrast solution of avidity.

406. the complexes of claim 404 wherein are chosen to be based on the amount of the mensuration reagent of avidity and are used to use the sample volume that is no more than about 1mL to analyze.

407. the complexes of claim 404 wherein are chosen to be based on the amount of the mensuration reagent of avidity and are used to use the sample volume that is no more than about 100 μ L to measure.

408. the complexes of claim 404 wherein are chosen to be based on the amount of the mensuration reagent of avidity and are used to use the sample volume that is no more than about 50 μ L to measure.

409. a system, it comprises:

(a) the droplet microdrive of claim 372; With

(b) treater, it is turned to by program and carries out droplet operation, and described droplet operation comprises to be made droplet contact described surface and/or removes droplet from described surface.

410. the system of claim 409, also comprise one or more transmitter, described transmitter electronics is connected in described treater and is set to be used for signal or the character of sensing from droplet on the described droplet microdrive and/or surface, and wherein said treater is turned to by program and handles one or more signal from described transmitter.

411. the system of claim 410, wherein said transmitter comprises selected and is configured to the physical properties of the droplet on the described droplet microdrive of sensing and/or the transmitter of signal.

412. the system of claim 411, wherein said physical properties comprises temperature.

413. the system of claim 411, wherein said physical properties comprises droplet size.

414. the system of claim 410, wherein said transmitter comprises selected and is configured to the electrochemical properties of the droplet on the described droplet microdrive of sensing and/or the transmitter of signal.

415. the system of claim 414, wherein said transmitter comprises potentiometric sensor.

416. the system of claim 414, wherein said transmitter comprises current sensor.

417. the system of claim 414, wherein said transmitter comprises conductivity sensor.

418. the system of claim 414, wherein said transmitter comprises ion specific electrode.

419. the system of claim 414, wherein said transmitter comprises pH electrode.

420. the system of claim 410, wherein said transmitter comprises selected and is configured to the optical property of the droplet on the described droplet microdrive of sensing and/or the transmitter of signal.

421. the system of claim 409, it is turned to the droplet operation that is used to implement the immunoassay scheme by program.

422. the system of claim 409, it is turned on the droplet microdrive by program one or more sample droplet and one or more mensuration reagent droplet based on avidity is merged.

423. the system of claim 409, it is turned to by program and implements two or more dissimilar mensuration based on avidity.

424. the system of claim 409, it is turned in substantially parallel mode by program and implements two or more mensuration based on avidity.

425. the system of claim 409, it is turned in substantially parallel mode by program and implement mensuration based on avidity on two or more different sample.

426. the system of claim 409, it is turned in substantially parallel mode by program and implement mensuration based on avidity on two or more different sample type.

427. the system of claim 409, it is constructed to portable or handheld apparatus.

428. the system of claim 409 also comprises the wireless connections that are used to receive or launch data.

429. the system of claim 409, it is further turned to one or more amplification scheme of enforcement by program.

430. the system of claim 409, it is further turned to by program and implements one or more metabolite detection scheme.

431. the system of claim 409, it is by sequencing and be configured to distribution and the transfer droplet, and described droplet comprises the magnetic bead of antibody sandwich.

432. the system of claim 409, it is by sequencing and be configured to distribution and the transfer droplet, and described droplet comprises protein or poly saccharide peptide standard product.

433. the system of claim 409, it is by sequencing and be configured to distribution and the transfer droplet, and described droplet comprises the antibody of peroxidase conjugated.

434. the system of claim 409, it is by sequencing and be configured to carry out droplet operation, is used to carry out based on the mensuration of avidity existing or measure with the protein of determining to comprise interested epi-position or peptide.

435. a droplet microdrive, it comprises the droplet on the described droplet microdrive, and described droplet comprises antibody.

436. a droplet microdrive, it comprises:

(a) first base material, it comprises the electrode that is configured to carry out the droplet operation on the surface of described base material;

(b) second base material of a described surperficial segment distance of the described base material of distance, this distance is enough to limit internal volume between described first base material and second base material, and wherein said distance is enough to hold the droplet that is placed in the above space of first base material; With

(c) be arranged in the described internal volume and allow to use described electrode to realize that on described droplet droplet operates with respect to its set-up mode of droplet that described electrode is provided with.

437. the droplet microdrive of claim 436, wherein said droplet operation are electric wetting mediations.

438. the droplet microdrive of claim 436, wherein said droplet operation is the two-dimensional electrophoresis mediation.

439. the droplet microdrive of claim 436, wherein said fill fluid comprises oil.

440. the droplet microdrive of claim 436, wherein said fill fluid comprises one or more silicone oil.

441. the droplet microdrive of claim 436, wherein said fill fluid comprises one or more fluorosilicon oil.

442. the droplet microdrive of claim 436, wherein said fill fluid comprises one or more hydrocarbon.

443. the droplet microdrive of claim 436, wherein said fill fluid comprises one or more alkane.

444. the droplet microdrive of claim 436, wherein said fill fluid comprise the component that is selected from as in next group: decane, undecane, dodecane, tridecane, the tetradecane, pentadecane, n-Hexadecane.

445. the droplet microdrive of claim 436, wherein said fill fluid comprises the component that is selected from the aralkyl hydro carbons.

446. the droplet microdrive of claim 436, wherein said fill fluid comprises mineral oil.

447. the droplet microdrive of claim 436, wherein said fill fluid comprises paraffin oil.

448. the droplet microdrive of claim 436, wherein said fill fluid is solid-state when about 25 ℃ room temperature.

449. the droplet microdrive of claim 436, wherein said fill fluid is liquid based on the service temperature of the mensuration of avidity the time, and is solid-state when being lower than the temperature of this service temperature.

450. the droplet microdrive of claim 436 also comprises and surrounds described fill fluid or contacted with it second fill fluid.

451. the droplet microdrive of claim 436, wherein said fill fluid has color, and this color helps directly or indirectly to observe described droplet.

452. the droplet microdrive of claim 436, wherein said fill fluid comprises dyestuff, and described dyestuff absorbs light from one or more reaction in one or more droplet to reduce or eliminate the phase mutual interference between the reaction in the described droplet.

453. the droplet microdrive of claim 436, wherein said fill fluid comprises surfactant.

454. the droplet microdrive of claim 453, the amount of wherein selecting described surfactant is to stablize the liquid film between described droplet and the solid phase.

455. the droplet microdrive of claim 453, wherein said surfactant comprise that non-ionic type hangs down hydrophile-lyophile balance (HLB) surfactant.

456. the droplet microdrive of claim 455, wherein said HLB is lower than about 10.

457. the droplet microdrive of claim 455, wherein said HLB is lower than about 5.

458. the droplet microdrive of claim 455, wherein said surfactant is selected from TritonX-15; Span 85; Span 65; Span 83; Span 80; Span 60; With the fluorizated surfactant.

459. the droplet microdrive of claim 455, wherein said droplet comprises whole blood, and the HLB of described surfactant is lower than about 2.

460. the droplet microdrive of claim 455, wherein said surfactant comprises TritonX-15.

461. the droplet microdrive of claim 455, wherein said oil comprises silicone oil, and described surfactant comprises Triton X-15.

462. the droplet microdrive of claim 455, wherein said surfactant is selected and be adjusted into, and compares with the corresponding droplet microdrive of no described surfactant, can carry out more droplet operation on described droplet microdrive.

463. the droplet microdrive of claim 455, wherein said surfactant is selected and be adjusted into, and compares with the corresponding droplet microdrive of no described surfactant, can carry out one or repeatedly droplet operation on described droplet microdrive.

464. the droplet microdrive of claim 455, wherein said surfactant is selected and be adjusted into, compare with the corresponding droplet microdrive of no described surfactant, can make one on the described droplet microdrive or repeatedly the droplet operation is more reliable.

465. the droplet microdrive of claim 455, wherein said surfactant is selected and be adjusted into, compare with droplet operation on the corresponding droplet microdrive of no described surfactant, can be on described droplet microdrive the droplet that comprises one or more particular agent or mixture be carried out one or repeatedly droplet operation or make that described droplet operation is more reliable the same droplet that comprises one or more particular agent or mixture.

466. the droplet microdrive of claim 455, wherein said surfactant is selected and be adjusted into, compare with droplet operation on the corresponding droplet microdrive of no described surfactant, can be on described droplet microdrive one or more droplet that comprises amphipathic molecule be carried out one or repeatedly droplet operation or make that described droplet operation is more reliable the same droplet that comprises amphipathic molecule.

467. the droplet microdrive of claim 455, wherein said surfactant is selected and be adjusted into, for the corresponding droplet microdrive of no described surfactant, help to increase the proteinic concentration that can be distributed in described droplet microdrive reliably.

468. the droplet microdrive of claim 455, the amount of wherein said surfactant is in about scope of 0.001 to about 10%w/w.

469. the droplet microdrive of claim 455, the amount of wherein said surfactant is in about scope of 0.001 to about 1%w/w.

470. the droplet microdrive of claim 455, the amount of wherein said surfactant is in about scope of 0.001 to about 0.1%w/w.

471. the droplet microdrive of claim 455, wherein said fill fluid comprise that silicone oil and described surfactant are Triton X-15, the amount of described Triton X-15 about 0.001 to the scope of about 10% w/w.

472. the droplet microdrive of claim 455, wherein said fill fluid comprise that silicone oil and described surfactant are Triton X-15, the amount of described Triton X-15 about 0.001 to the scope of about 1% w/w.

473. the droplet microdrive of claim 455, wherein said fill fluid comprise that silicone oil and described surfactant are Triton X-15, the amount of described Triton X-15 about 0.001 to the scope of about 0.1% w/w.

474. a droplet microdrive, it comprises base material, and described base material comprises:

(a) be used to spray or distribute the electrode of droplet operation; With

(b) electromagnet, itself and droplet are fully approaching to impel all basically pearls to be retained in the droplet in described division or batch operation process.

475. the droplet microdrive of claim 474, wherein said operation is a splitting operation.

476. the droplet microdrive of claim 474, wherein said operation is batch operation.

477. droplet microdrive, it comprises base material, described base material comprises the electrode that is used to carry out the droplet operation, wherein the described base material of at least a portion comprises the electrode that is used to carry out the droplet operation and lacks second base material, can carry out the droplet operation on the described base material of substantially parallel base material lacking like this.

478. a droplet microdrive, it comprises one or more electrode that is used to carry out the droplet operation, and wherein one or more described electrode comprises opening, is used to transmit light to the droplet that is positioned on the described electrode or the transmission light from described droplet.

479. droplet microdrive, it comprises the electrode that is used to carry out the droplet operation of one or more substantial transparent, wherein the covered thing of one or more described electrode part covers, described overcover leaves window, is used to transmit light to the droplet that is positioned on the described electrode or the transmission light from described droplet.

480. a droplet microdrive comprises opaque fill fluid and transparent droplet on it.

481. a droplet microdrive, it comprises:

(a) particle suspension; With

(b) electrode, it is set to be used for use and comprises the particulate droplet and carry out the droplet operation.

482. the droplet microdrive of claim 481, wherein said electrode are set to be used for distribute one or more to comprise one or more particulate droplet from described particle suspension.

483. the droplet microdrive of claim 481, wherein said particle comprises biological cell.

484. the droplet microdrive of claim 481, wherein said particle comprises pearl.

485. the droplet microdrive of claim 481 comprises droplet, comprises individual particle in the described droplet.

486. a droplet microdrive, it comprises droplet, comprises individual particle in the described droplet.

487. the droplet microdrive of claim 486, wherein said droplet are filled fluid and surround.

488. the method for transfer particle, it comprises providing and comprises described particulate droplet and described droplet is transferred on the droplet microdrive.

489. a system, the droplet microdrive that it comprises claim 482 also comprises transmitter, and described transmitter is set to be used to detect character and/or the signal from the droplet that distributes.

490. the system of claim 489, wherein said system are by sequencing and be configured to have transmitter, described transmitter is set to be used for based on from the character of the droplet of described distribution and/or the particulate quantity of the described droplet of signal detection.

491. the system of claim 489, wherein said system are by sequencing and be configured to have transmitter, described transmitter is set to be used for based on from the character of the droplet of described distribution and/or the particulate type of the described droplet of signal detection.

492. the system of claim 491, wherein said particle is color-coded.

493. the system of claim 491, wherein said particle is a mark.

494. the system of claim 491, wherein said particle comprises the cell of the markd antibody of mark.

495. the system of claim 491, wherein said particle comprises the cell that is marked with the antibody that fluoresces.

496. being turned to by program, the system of claim 489, wherein said system can merge with the droplet of the interactional material of described individual particle comprising the droplet of individual particle and comprising.

497. the system of claim 496, wherein said can being selected from: pearl, particle, particulate, nano particle, medicine, protein, peptide, antigen, toxin and antibody with the interactional material of described individual particle.

498. the system of claim 498, wherein said system are further turned to representing described particle that the existence or the amount of the material of the response of described material are measured by program.

499. the system of claim 496, wherein said particle is the T particle.

500. the system of claim 489 comprises and surpasses 50 droplets, each droplet comprises the particle of pre-determined quantity.

501. the system of claim 489 comprises and surpasses 250 droplets, each droplet comprises the particle of pre-determined quantity.

502. the system of claim 489 comprises and surpasses 1000 droplets, each droplet comprises the particle of pre-determined quantity.

503. the method for the droplet that comprises individual particle is provided, and described method comprises:

(a) provide the droplet that comprises particle suspension;

(b) droplet from (a) distributes droplet so that the droplet of distribution to be provided; With

(c) whether the droplet of determining described distribution comprises individual particle and/or required grain type.

504. the method for claim 503 also comprises repeating step 503 (b) and 503 (c), until the droplet that comprises individual particle that identifies pre-determined quantity.

505. the method for claim 503, wherein said distribution are the electrode mediations.

506. the method for claim 503, wherein said distribution are electric wetting mediations.

507. the method for claim 503, wherein said distribution are the two-dimensional electrophoresis mediations.

508. the method for claim 503, wherein the described droplet of 503 (a) is surrounded by oily medium.

509. the method for claim 508, wherein said oily medium and described droplet be unmixing basically.

510. the method for claim 503 is wherein by based on the character of the droplet of described distribution and/or the particulate quantity in the described droplet of signal detection and performing step 503 (c).

511. the method for claim 510 wherein uses transmitter to realize described detection step.

512. the method for claim 503 is wherein by based on the character of the droplet of described distribution and/or the particulate type in the described droplet of signal detection and performing step 503 (c).

513. the method for claim 503 also comprises can merging with the droplet of the interactional material of described individual particle comprising the droplet of individual particle and comprising.

514. the method for claim 513, wherein said can being selected from: pearl, particle, particulate, nano particle, medicine, protein, peptide, antigen, toxin and antibody with the interactional material of described individual particle.

515. the method for claim 513 also comprises existence or the amount of the described particle of representative to the material of the response of described material of determining.

516. the method for claim 515, wherein said determining step comprises the mensuration based on the step of droplet.

517. the method for claim 513, wherein said particle comprises the T cell.

518. the method for claim 503 also comprises and will comprise droplet and the merging of described particle suspension that a plurality of particulate distribute.

519. the method for claim 503 also comprises:

(a) division comprises the droplet of a plurality of particulate distribution to produce two or more filial generation droplets; With

(b) analyze one or more described filial generation droplet and whether have single droplet.

520. the method for claim 503 also comprises:

(a) will comprise droplet and the merging of damping fluid droplet that a plurality of particulate distribute;

(b) division comprises the droplet of a plurality of particulate distribution to produce two or more filial generation droplets; With

(c) analyze one or more described filial generation droplet and whether have single droplet.

521. the method for claim 503, the droplet that does not wherein contain the particulate distribution is transferred back described particle suspension.

522. the method for claim 503, the droplet that does not wherein contain the particulate distribution goes out of use.

523. the method for claim 503 is wherein made the sorting decision based on detected signal in the single droplet.

524. the method for claim 523, wherein said signal comprise from the fluorescence of described particle bonded molecule.

525. the method for claim 523, wherein said signal comprises radioactive particulate.

526. the method for claim 523, wherein said signal comprises sensed image.

527. the method for claim 503, the droplet that wherein contains individual particle is transferred to droplet microdrive pond.

528. the method for claim 503 is wherein carried out droplet operation containing on the droplet of individual particle.

529. the method for claim 503 is wherein analyzed the droplet that contains individual particle.

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