WO2007136386A2

WO2007136386A2 - Droplet-based on-chip sample preparation for mass spectrometry

Info

Publication number: WO2007136386A2
Application number: PCT/US2006/021699
Authority: WO
Inventors: Aaron R. Wheeler; Robin L. Garrell; Chang-Jin Kim; Hyejin Moon
Original assignee: The Regents Of The University Of California
Priority date: 2005-06-06
Filing date: 2006-06-06
Publication date: 2007-11-29
Also published as: WO2007136386A3

Abstract

The present invention relates to droplet-based on-chip sample preparation for mass spectrometry. Specifically, sample processing is performed in matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) using one or more droplet-based microfluidic devices to manipulate droplets containing samples and reagents and place them to specified locations on an array.

Description

DROPLET-BASED ON-CHIP SAMPLE PREPARATION FOR MASS SPECTROMETRY

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Grant No. NCC 2- 1364 awarded by NASA, and Grant No. 1 F32 EB03696-01 awarded by the NIH. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the fields of microfluidics, lab-on-a-chip, and micro total analysis system (μ-TAS), especially droplet-based microfluidics or digital microfluidics.

2. Description of the Related Art

The push to sequence the human genome brought an unprecedented level of attention to the field of genomics. In recent years, attention has been turned to the field of proteomics. First conceptualized in the mid-1990s, proteomics has undergone a meteoric rise in popularity, with more than 2000 papers published in the field in 2003. Proteomics, like genomics, requires methods and instruments capable of collecting, storing, cataloguing, and analyzing vast amounts of information. The technological challenges for proteomics may be even greater than those for genomics, given that an organism has a single genome but may express hundreds of different proteomes, depending on environmental and developmental cues. The development of new methods and instrumentation with the capacity for rapid, high-throughput data collection is crucial for continued progress.

Current standard methods in proteomics rely on the pairing of two technologies: analytical separations (e.g., two-dimensional gel electrophoresis, 2DGE) and mass spectrometry (MS) detection. One mode of mass spectrometry, matrix -assisted laser desorption/ionization (MALDI) coupled with time-of-flight (TOF) analyzers, has become popular for high-throughput proteomics applications. In MALDI, which was introduced in the late 1980s, a protein sample is cocrystallized with an organic matrix. When the crystal is irradiated with energy of an appropriate wavelength, the sample is simultaneously desorbed and ionized.

The sample array geometry of most Matrix- Assisted Laser Desorption/Ionization-Mass Spectrometry (MALDI-MS) systems makes it appealing for high-throughput proteomics applications. However, typical proteomics analyses require many steps; a crucial step is mixing the sample with matrix. Repetitive pipetting of reagents onto MALDI targets is time-consuming and can lead to sample loss, dilution, and contamination. High-end commercial instruments utilize robotically controlled deposition, but such instruments are expensive and require careful maintenance. Other methods for high-throughput deposition of sample and matrix include using lithographically patterned targets, microfabricated picoliter droplet delivery devices, or micro fluidic channels. Of these methods, only patterned targets, which facilitate easier spot deposition but do not eliminate pipetting, have gained widespread use. Sample processing for MALDI-MS applications is typically accomplished manually (by pipetting) or in some cases may be partially accomplished by droplet dispensing robots. MALDI-MS is used for many applications, including analysis of proteins, nucleic acids, and synthetic polymers.

What is needed, however, are methods and devices that streamline the processing required for MALDI-MS applications. The present invention satisfies these needs. SUMMARY OF THE INVENTION

The present invention relates to droplet-based on-chip sample preparation for mass spectrometry. Specifically, sample processing is performed in matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) using one or more droplet-based microfluidic devices to dispense, transport, merge, mix, cut, and deliver droplets containing samples and reagents to specified locations on an array. As part of the sample processing, reactions may take place in one or more droplets. Substances in droplets may be concentrated by solvent evaporation from the droplets. Droplets may also be used to deposit materials on the device surface by precipitation or evaporation. Droplets may be used to dissolve one or more components from a solid sample on the device, and may be used to transport the dissolved substances. Droplets may also be used to remove solids or particulates from the surface and transport them elsewhere on the device. Samples or substances may be desorbed directly from the device for analysis by mass spectrometry. The mass spectrometry analysis may include peptide mass fingerprinting and database identification.

The present invention is a method for cocrystallizing sample and matrix for MALDI-MS. The method utilizes a solution handling technique known as digital πύcrofluidics. In digital microfluidics, droplets are moved over an array of electrodes by means of Electro Wetting-On-Dielectric (EWOD), dielectrophoresis and/or other mechanisms. In EWOD, the local wettability of a surface is reversibly changed by applying potentials between electrodes buried beneath hydrophobic, dielectric layers. By applying a sequence of potentials to adjacent electrodes on an array, liquid droplets can be made to travel across the surface. Droplets may also move in response to an applied field gradient, without any apparent contact angle change, a process known as dielectrophoresis. Hereafter, we use the term to "digital microfluidics" to encompass any mechanism used to manipulate droplets on an array of electrodes. Several configurations of digital micro fluidics-based devices can be used in the present invention, including single-plate open air devices, parallel-plate devices filled with silicone oil, and parallel-plate open-air devices. Digital microfluidics-based devices are reconfigurable and can handle neutral and charged analytes, particulates, proteins, cells and microorganisms.

The present invention also discloses a method to realize digital microfluidics actuation across a two-dimensional plane (rather than simply across one or two rows of electrodes) and to use this technique to create a fully portable micro fluidic device. Digital microfluidics is well-suited to MALDI, as both techniques rely on array geometries, which stands in contrast to channel-based microfluidic devices for MALDI-MS, which require rastering or complex networks of holes to mate with MALDI-MS targets. A method in accordance with the present invention comprises performing sample processing using at least one droplet-based microfluidic device capable of manipulating droplets containing samples, placing the droplets at specified locations on an array; and moving the droplets within the array. Such a method optionally includes the droplet-based microfluidic devices being formed in an array geometry, applying a sequence of electrical signals to enable transport of droplets across the droplet-based microfluidic device surface, the sequence of electrical signals being applied to a pattern of electrodes buried beneath a dielectric layer on the droplet-based microfluidic device surface, the sequence of electrical signals dividing the droplet, and the droplets comprising a homogeneous liquid, emulsion or suspension.

The method further optionally comprises placing a droplet containing a reagent on the array, and moving the droplet containing the reagent within the array, the droplets comprising solutions in which cells or particles are suspended, the samples reacting with chemical agents in the droplets, the samples being acted on by catalysts in the droplets, the samples being concentrated by evaporating liquid from the droplet, the samples being precipitated from the droplet, the samples being crystallized by evaporating liquid from the droplet, the droplets being used to dissolve the samples, the samples being purified by selective precipitation, the droplet-based microfluidics device being used with a mass spectrometer, and a plurality of samples being processed in parallel.

A device in accordance with the present invention comprises a first plate comprising an array of first electrodes, a second plate, comprising at least a second electrode, wherein the first plate and the second plate are spaced apart such that a droplet can travel between the first plate and the second plate, a first layer of one or more materials, covering the array of first electrodes, and a second layer of one or more materials, covering the at least second electrode, wherein application of electrical signals between selective electrodes within the array of first electrodes and the at least one second electrode moves the droplet between the top plate and the bottom plate.

Such a device further optionally includes the first layer of material being Teflon or other coating material, a first droplet being moved between the first plate and the second plate, and dried between the first plate and the second plate, a second droplet being moved between the first plate and the second plate to where the first droplet was dried, the second droplet containing a reagent. The dried spot is then analyzed in situ by MALDI-MS. In a two-plate device, this entails removing the top plate.

Finally, such a device further optionally includes devices with a single plate with an array of electrodes. In this configuration, droplets are manipulated on the array by applying potentials between electrodes on a single plane, with no need for a top plate. The droplet is then analyzed in situ by MALDI-MS.

BRIEF DESCRIPTION QF THE DRAWINGS

Referring now to the drawings in which like reference numbers represent corresponding parts throughout:

FIGS. l(a)-(f) illustrate a matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) application; FIGS. 2-3 illustrate digital micro fluidics devices in accordance with the present invention;

FIGS. 4A-4B illustrate droplet movement experiment on an digital microfluidics device; FIG. 5 shows images and mass spectra of insulin cocystallized with DHB, FA, and SA;

FIGS. 6A-6C illusrate sample MALDI spectra of insulin-DHB created with different techniques in accordance with the present invention;

FIGS. 7A- 7C illustrate other proteins and peptides as analyzed with digital microfiuidics-MALDI in accordance with the present invention;

FIGS. 8A-8B illustrate residual nonspecific adsorption for various sizes of insulin spots in accordance with the present invention;

FIG. 9 illustrates a process chart showing typical steps used in practicing the invention; and FIGS. 10A-D illustrate a digital microfluidics-driven reduction of insulin into the constitutive peptides (insulin A and insulin B) followed by tryptic digestion.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT In the following description of the preferred embodiment, reference is made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention.

Technical Description

The present invention enables automated, integrated, reconfigurable, high- throughput sample processing for matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) applications. Sample processing for MALDI-MS is accomplished manually (by pipetting) or in some cases may be partially accomplished by droplet dispensing robots. The present invention relies on droplet-based microfluidics (also known as "digital microfluidics") to create, dispense, transport, merge, mix, cut, and deliver droplets containing samples and reagents to specified locations on an array. Droplet-based microfluidics is described in detail in the publications attached hereto as appendices. Creating a droplet can mean dispensing a droplet from a reservoir, ejecting a droplet from a channel, or dividing a larger droplet.

Briefly, in droplet-based microfludics, a sequence of potentials is applied to adjacent electrodes buried beneath dielectric layers. When a potential is applied across electrodes in the device, two phenomena may be observed: (1) a droplet may move towards the biased electrode, and (2) the contact angle between the droplet and device surface may decrease because of a change in the local wettability of the surface. A change in surface wettability is not required for droplet movement. Droplet-based microfluidic devices are formed in an array geometry, which makes it an attractive technology for use with MALDI-MS (which is typically used to analyze arrays of processed samples). The present invention is the first to use droplet-based microfluidics for on-chip sample preparation on an array for mass spectrometry.

The present invention makes use of technology for manipulating droplets of homogeneous liquids, as well as liquid droplets that contain suspended liquid droplets (emulsions) or suspended solids (suspensions), cells or microorganisms, by droplet- based microfluidics, which is described in detail in the publications attached hereto as appendices. A homogeneous liquid may be a pure liquid, or a liquid in which one or more components have been dissolved. Droplet manipulations may include any or all of the following functions: droplet generation or dispensing, movement, merging, dividing or cutting, joining, mixing, and concentrating or drying by evaporation. Droplets may also be manipulated so as to dissolve or suspend solids. The droplets may consist of pure liquids, solvents, solutions, or suspensions. Soluble substances or suspended particles in the droplets may be reagents, polymers or other agents such as surfactants that modify the physical properties of the droplets, samples (analytes), labeling agents (molecular or particulate) and/or catalysts, collectively called chemical agents herein. The present invention required the development of specific device designs and utilization parameters to establish feasibility of manipulating samples and reagents for MALDI-MS.

MALDI-MS is used for many applications, including analysis of proteins, nucleic acids, and synthetic polymers. It is in the former capacity, the analysis of proteins, that this technique has become an extremely important tool used in virtually every biochemistry laboratory in the world. Proteomics, the study of all proteins expressed in a given sample, is an important field in chemistry and biology, with applications for both clinical diagnostics and basic science. The capacity to streamline the processing required for proteomics and other MALDI-MS applications will be widely desired in both industry and academia.

As described above, the method has been demonstrated to be useful for the . function of mixing and crystallizing protein samples and MALDI matrices. The method has also been demonstrated to be useful for the purification step, and for the combining of reagents or catalysts, such as reducing agents, alkylating agents or enzymes, with samples to be analyzed and performing those reactions on the device. The feasibility of directly desorbing substances from the device for mass spectral analysis has also been demonstrated. The feasibility of processing two or more samples simultaneously has been demonstrated. Work is on-going to extend the range of usable reagents, integrate other processing steps, and to develop denser arrays of electrodes for high-throughput analysis.

Related Art

Standard laboratory practices for sample preparation for MALDI-MS, especially those for proteomics applications, require many tedious steps. A MALDI- MS proteomics analysis is described in FIGS. l(a)-(f). FIG. l(a) illustrates the step of 2-D gel electrophoresis, FIG. l(b) illustrates the step of excising separated protein spots, FIG. l(c) illustrates the step of processing the samples, which may include any or all of the following: sample purification, disulfide reduction, proteolytic digestion and peptide recovery, FIG. l(d) illustrates the step of crystallizing the sample with a matrix, wherein a matrix can be a substance that co-deposits with the sample, FIG. l(e) illustrates the step of collecting peptide mass fingerprints using MALDI, and FIG. l(e) illustrates the step of identifying the protein by searching one or more proteomic databases.

The steps of FIGS. l(c) and l(d) typically require between several hours to days to accomplish, involving tens-hundreds of pipetting steps. Expensive, high-end MALDI-MS instruments are equipped with robots that can accomplish the step of FIG. l(d), but the processes in the step of FIG. l(c) remain a bottleneck in virtually all MALDI-MS proteomics methods. In the present invention, droplet-based microfluidics is used to integrate and automate the steps of FIGS. l(c) and l(d). The steps of FIGS. l(a) and l(b) may also be incorporated by integrating micro fluidic separation columns onto a single high-throughput proteomics analysis platform. This would distill a process that requires many hours or days of laboratory time into an automated, high-throughput, programmable, and reconfigurable method that will require only minutes of laboratory time.

Experimental Approaches and Results

Stock solutions of analytes, including bovine insulin (100 μM), bovine insulin chain B (40 μM), horse heart cytochrome c (14.5 μM), and horse skeletal myoglobin (59 μM), were prepared in deionized (DI) water or with 0.2% trifluoroacetic acid (TFA). Stock solutions were kept frozen; working solutions were diluted and used within 1 day. Working solutions of matrixes, including 2,5-dihydroxybenzoic acid (DHB), ferulic acid (FA), and sinapinic acid (SA), were prepared in DI water containing TFA and acetonitrile and were used within 1 day. Various working concentrations of analytes and matrixes were evaluated for the feasibility of moving droplets by digital microfluidics. The following concentrations were used to obtain the results presented here: insulin (1.75 μM, 0.025% TFA), insulin chain B (2 μM, 0.025% TFA), cytochrome c (1.85 μM, 0.025% TFA), myoglobin (1.45 μM, 0.0125% TFA), DHB (10 mg/mL, 0.05% TFA, with 5% acetonitrile), FA (3 mg/mL, 0.0375% TFA, with 15% acetonitrile), and SA (10 mg/mL, 0.1% TFA, with 33% acetonitrile).

Teflon- AF 1600 resin was purchased from DuPont (Wilmington, DE). Working solutions of 6% (w/v) were formed in Fluorinert FC-40 solvent; solutions were used as made or diluted (v/v with FC-40).

Fabrication and Use of Digital Micro fluidic Devices

FIGS. 2-3 illustrate a digital microfluidic device in accordance with the present invention. As shown in FIG. 2, device 200 is formed each device was formed from a bottom plate 202 having individually addressable electrodes 204-212 and a top plate 214 with one contiguous electrode 216. Droplet 218 is shown between bottom plate 202 and top plate 214.

Bottom plate 202 is formed from quartz wafers coated with a 3500- A layer of phosphorus-doped polysilicon. Polysilicon electrodes 204-212 are patterned using standard photolithographic techniques and reactive ion etching. A typical formation of the electrodes 204-212 is to grow a thermal oxide (1500 A) on the polysilicon of bottom plate 202 in an oxidation furnace. Holes through the oxide to the electrical contacts were formed with photolithography and wet etching with buffered hydrofluoric acid. The devices were then primed with hexamethyldisilazane vapor and spin-coated (2000 rpm, 60 s) with 5% Teflon-AF layer 220. The devices were postbaked on a hot plate (160 ⁰C, 10 min) and in a furnace (330 ⁰C, 30 min) to form a uniform 7500- A layer 220 of Teflon-AF. Layer 220 cam be made from any polymer to achieve the desired surface hydrophobicity or hydrophilicity, or wettability. Additional layers 221 of dielectric or other material can be used to control the electron flow between electrodes 204-212 and any droplets that are placed on layer 220.

The top plate 214 is a glass piece with the electrode 216 being formed from indium-tin oxide (ITO). A 150 A layer 222 of Teflon- AF was spin-coated (0.5%, processed as above) onto the ITO-coated top plate 214. Layer 222 can be made from any polymer to achieve the desired surface hydrophobicity or hydrophilicity, and does not have to be the same material as layer 220. The two plates 202 and 214 are joined with spacers 224 (at approximately 300 μm apart), which can be formed from three pieces of double-sided tape or other materials. Other spacings between plates 202 and 214 are possible within the scope of the present invention.

FIG. 3 illustrates a top view of plate 202 in a typical digital microfluidics pattern, which comprises sixteen 1-mm² electrodes 204-212 (having a typical 4-μm gap between electrodes) where each electrode 204-212 is connected to an electrical contact pad 226. Aqueous droplets 218 (0.5 μL) are sandwiched between the two plates 202 and 214 and are moved by applying ac potentials (1 kHz, 75 Vrms) between the electrode 216 in the top plate 214 and successive electrodes 204-212 in the bottom plate 202. Once the solution composition had been optimized (described below), droplet 216 movement is facile and fast. Droplet 216 movement can be monitored and recorded by a CCD camera mated to an imaging lens positioned over the top of the device 200.

Mass Spectrometry

After droplet movement, digital microfluidics devices 200 were stored in a chamber under house vacuum; 0.5-μL droplets dried in 1 -2 min. Matrix and sample cocrystals were imaged by light microscopy. Typically, several spots were deposited on each digital microfluidics device 200. When deposition was complete, the bottom plate of the digital microfluidics device 200 was affixed with double-sided tape into a 1-mm-deep milled-out groove on a standard stainless steel MALDI target. A mass spectrometer is then used to collect MALDI-MS data.

Typically, 500 shots were collected per spectrum, with the laser power adjusted for different matrixes. Data were normalized to the protein analyte peak; some data were baseline subtracted, smoothed with a running average of 15 points, or both. All MALDI data were replicated at least two times. To compare the new technique with conventional MALDI, five identical spots were prepared (insulin deposited first, followed by DHB) on an digital microfluidics device and on a stainless steel MALDI target. The spectra were evaluated for rms noise, signal-to- noise (S/N), and resolution.

Results and Discussion

Suitability of Solvent for digital micro fluidics-MALDI

Many potential applications of digital microfluidics require the use of organic solvents. For example, acetonitrile is often used to increase the solubility of matrixes for MALDI-MS. Some matrixes, such as DHB, require little or no acetonitrile, while other matrixes, such as SA, require significant concentrations of acetonitrile. The feasibility of using device 200 to actuate a wide range of liquids, both solvents and solutions, has been demonstrated. The liquids include solvents having a wide range of polarities, and include ionic liquids, chlorinated solvents, alcohols, carboxylic acids, aldehydes, amides, sulfoxides, ethers, heterocycles and nitriles, but are not limited to these listed liquids.

Digital Microfluidics-Driven Droplet Movement FIGS. 4A-4B illustrate droplet movement experiment on an digital microfluidics device.

As shown in FIG. 4a, a droplet 400 of insulin was moved to a designated electrode 402 as shown in FIG. 4a. The droplet 400 was then allowed to dry on electrode 402. Similarly, as shown in FIG. 4B, a droplet 404 of FA was moved to the electrode 402 on top of the droplet 400 of insulin. Droplet 404 was then allowed to dry. dried spot (FIG. 4b); and (4) the droplet was allowed to dry. Droplets were routinely driven on and between each line of electrodes on each device.

Mass Spectrometry

FIGS. 5A-5D show images and mass spectra of insulin cocystallized with DHB, FA, and SA.

MALDI-MS was used to analyze spots of protein and matrix prepared by digital microfluidics.

The appearance of crystals on digital microfluidics devices (FIGS. 5 A-C) was similar to those formed on a standard stainless steel target (FIG. 5D). Likewise, the digital microfluidics-MALDI spectra of insulin exhibit the expected strong signal at 5.7 kDa, with similar peak shape as for spectra collected from a standard target. The small peaks at higher mass were usually observed and were likely caused by the matrix forming a photochemically induced adduct with the peptide. DHB spot 500 (FIG. 5A) and FA spot 502 (FIG. 5B) spots were prepared as shown in FIG. 4. An insulin droplet was moved and dried, followed by a droplet of matrix DHB, which is known as a "universal matrix" used for peptides, proteins, nucleotides, and synthetic polymers, was found to work quite well with digital microfluidics.

SA spots 504 (FIG. 5C) were deposited manually followed by digital microfluidics- driven movement and drying of an insulin droplet. This result demonstrates that if matrixes that are not water soluble are desirable, the technique of pre-coating a high- throughput target with matrix could be used for digital microfluidics-MALDI devices. For a comparison of digital microfluidics-MALDI to conventional MALDI, five spots 506 were prepared by depositing insulin and then DHB on an digital microfluidics device and on a stainless steel target (FIG. 5D). The two kinds of spectra had similar S/N ( 43.1 and 68.4 for conventional MALDI and digital microfluidics-MALDI, respectively). 103, 136 Conventional MALDI had slightly better resolution (62 for conventional MALDI and 88 for digital microfluidics- MALDI) (508, 275), but digital microfluidics-MALDI had lower noise (10. for conventional MALDI and 6 for digital microfluidics-MALDI) (60, 29). No attempt was made to optimize digital microfluidics-MALDI for spectral properties; even so, the digital niicrofluidics devices proved to be an effective alternative to conventional targets for MALDI-MS. It should be noted that another form of digital microfluidics, in which droplets are suspended in silicone oil,30-33 would probably not be suitable for this technique for two reasons. First, suspending media such as volatile or nonvolatile hydrocarbons or silicone oils may also interfere with protein co-crystallization with the matrix. Second, MALDI signals are severely degraded by the presence of nonvolatile liquids.

Cocrystallization of sample and matrix for MALDI-MS is process-driven, with many recipes and variants to choose from. We have replicated three common cocrystallization recipes using digital microfluidics, including the following: (1) the "sample first" technique, for which sample is deposited first, followed by matrix, (2) the "dried drop" technique, for which sample and matrix are mixed and dried together, and (3) the "sandwich" technique, for which a layer of sample is deposited between two layers of matrix.

FIGS. 6A-6C illusrate sample MALDI spectra of insulin-DHB created with different techniques in accordance with the present invention.

Spots formed with the sample first technique, as shown in FIG. 6A, were prepared as depicted in FIG. 4. Spots formed with the dried drop technique, as shown in FIG. 6B, were prepared by using digital microfluidics to merge a droplet of insulin and DHB and allowing the combined droplet to dry. Prior to drying, the droplet was moved back and forth between electrodes several times, which has been shown to increase mixing efficiency. Spots formed with the sandwich technique, as shown in FIG. 6C, were prepared by using digital microfluidics to deposit a droplet of DHB, insulin, and then DHB again. The signal-to-noise ratios 600-604 were similar for spectra formed by each technique, with the sample first and sandwich techniques giving a slightly narrower analyte peak. The compatibility of digital microfluidics- MALDI-MS with common matrix/sample preparation recipes demonstrates that digital microfluidics should be useful for many applications of MALDIMS.

In addition to insulin (FIGS. 5 and 6), several other proteins and peptides were analyzed with digital microfluidics-MALDI, as shown in FIGS. 7A-7C. Samples with a wide range of molecular weights were probed, including insulin chain B (3495), cytochrome c (12 400), and myoglobin (16 900).

One concern for methodologies using digital microfluidics for biochemical applications is the potential for molecules such as peptides and proteins to nonspecifically adsorb on the hydrophobic Teflon- AF surface. As shown in FIGS. 8A-8B, MALDI-MS proved to be a convenient tool to probe this phenomenon. Two sets of spectra are shown, formed from droplets containing 1.75 or 0.175 μM insulin (FIGS. 8a and 8b, respectively). For each concentration, a droplet of insulin was moved across a series of electrodes to a designated point and dried. DHB droplets were then moved and deposited some to the dried spot of insulin (main panels of FIGS. 8A and 8B) to produce spectra 800 and 802; and other DHB droplets were moved to an electrode over which the insulin had traveled (inset panels) to produce spectra 804 and 806. Each inset spectrum was scaled to the parent ion peak of the main panel spectrum. Although MALDI-MS is typically not a quantitative technique, the spectra qualitatively suggest that the level of surface fouling at high concentrations of insulin is low, and the amount of fouling at low concentrations is below the detection limit of the technique.

Alternative Embodiments and Uses Additional uses of the invention could include a tool for drug discovery or biosensing. Specifically, the present invention could be used to purify samples, or perform separations or extractions.

With regard to additional embodiments, the present invention may be a fully integrated component of a mass spectrometer, as well as a standalone component. In addition, the present invention works as a sample preparation device that processes samples in parallel rather than sequentially. The present invention also allows processing of each sample in an individualized way. Differences in sample processing may include the number of steps, the type of steps and reagents used for each step, the concentrations of samples and reagents, and the time allowed for each step.

With regard to equivalents, note that the surfaces are not limited to hydrophobic surfaces, and the surfaces can be any dielectric material. The present invention also includes the movement of any type of liquid. FIGS. 10A-D illustrate a digital microfluidics-driven reduction of insulin into the constitutive peptides (insulin A and insulin B) followed by tryptic digestion. A 0.5 μL droplet of insulin (5 μM in 3:1 acetonitrile:DI water) was merged with a 0.6 μL droplet containing the reducing reagent, TCEP (500 μg/mL in DI water). The combined droplet was actively mixed (~5 min, RT) and then merged with a 1 μL droplet containing trypsin (0.5 μM in DI water). The combined droplet was again actively mixed (-10 min, RT) and then dried. Matrix was added, and a MALDI mass spectrum was collected, which is shown in FIG. 1OB.

In other experiments, cytochrome c and ubiquitin were digested by merging 0.5 μL droplets of analyte (2 μM in 3:1 acetonitrile:DI water) and trypsin (0.5 μM in DI water), and actively mixing (~10 min, RT); the combined droplet was then dried and exposed to matrix. MALDI mass spectra were collected (FIGS. 1OC and 10D). Asterisks denote peptides identified by the proteomic database search engine, Mascot. For each of these spectra, the Mowse scores (61 and 95 respectively) identifications were significant (p < 0.05). Sequence coverages were 40 and 51%. Although the analytes in Fig. 10A- D were not completely digested, these preliminary data demonstrate that digital micro fluidics can be used for rapid processing of proteomic samples.

FIG. 1OA is a video sequence depicting reduction and digestion of a droplet of insulin, and FIG. 1OB is a figure of the spectrum of a processed insulin spot. FIGS. 1OC and 1OD show tryptic digests of cytochrome c and ubiquitin (processed by digital microfluidics). Each reaction represented was allowed to react for no more than 30 min, at room temperature.

Process Chart

FIG. 9 illustrates a process chart showing typical steps used in practicing the invention.

Box 900 illustrates performing sample processing using at least one droplet- based micro fluidic device capable of manipulating droplets containing samples; Box 902 illustrates placing the droplets at specified locations on an array within the droplet-based microfluidic device.

Box 904 illustrates moving the droplets within the array.

Conclusion

This concludes the description including the preferred embodiments of the present invention. The foregoing description of the preferred embodiment of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching.

This work demonstrates that digital microfluidics is compatible with matrixes, samples, concentrations, and recipes typically used for MALDI-MS analyses. The results indicate that digital microfluidics-MALDI-MS has the potential to be useful for high-throughput proteomics analyses. The proteomics strategy employing 2DGE and MS utilizes the measurement of proteolyic peptide fragments as the means for protein identification. We have also integrated proteolytic digestion as a sample processing step. For these experiments, droplets containing trypsin or other proteolytic enzyme and protein analyte are to be merged, incubated at room temperature, and dried, followed by deposition of a third droplet containing matrix. The present invention can also be extended to devices with dense arrays of electrodes, which will be a useful tool for automating sample preparation for high-throughput proteomics analyses.

A method in accordance with the present invention comprises performing sample processing using at least one droplet-based microfluidic device capable of manipulating droplets containing samples, placing the droplets at specified locations on an array, and moving the droplets within the array.

Such a method optionally includes the droplet-based microfluidic devices being formed in an array geometry, applying a sequence of electrical signals to enable transport of droplets across the droplet-based microfluidic device surface, the sequence of electrical signals being applied to a pattern of electrodes buried beneath a dielectric layer on the droplet-based microfluidic device surface, the sequence of electrical signals dividing the droplet, the droplets comprising a homogeneous liquid, and the sample having particles or cells suspended in the droplet.

The method further optionally comprises placing a droplet containing a reagent on the array, and moving the droplet containing the reagent within the array, the droplets comprising solutions in which cells are suspended, the samples being concentrated by evaporating liquid from the droplet, the samples being crystallized by evaporating liquid from the droplet, the droplets being used to dissolve the samples, the samples being purified by selective precipitation, the droplet-based microfluidics device being integrated with a mass spectrometer, and a plurality of samples being processed in parallel.

A device in accordance with the present invention comprises a first plate comprising an array of first electrodes, a second plate, comprising at least a second electrode, wherein the first plate and the second plate are spaced apart such that a droplet can travel between the first plate and the second plate, a first layer of material, covering the array of first electrodes, and a second layer of material, covering the at least second electrode, wherein application of electrical signals between selective electrodes within the array of first electrodes and the at least one second electrode moves the droplet between the top plate and the bottom plate.

Such a device further optionally includes the first layer of material being Teflon or other coating materials, a first droplet being moved between the first plate and the second plate, and dried between the first plate and the second plate, a second droplet being moved between the first plate and the second plate to where the first droplet was dried, the second droplet containing a reagent, and the droplet being analyzed by a mass spectrometer. For two-plate devices, the top plate must be removed prior to analysis by mass spectrometry, for one-plate devices (i.e., with no top), the device can be inserted into the spectrometer immediately after processing. It is intended that the scope of the invention be limited not by this detailed description, but rather by the claims appended hereto. The above specification, examples and data provide a complete description of the manufacture and use of the apparatus and method of the invention. Since many embodiments of the invention can be made without departing from the scope of the invention, the invention resides in the claims hereinafter appended and the equivalents thereto.

Claims

WHAT IS CLAIMED IS:

1. A method of performing sample preparation for mass spectrometry, comprising: performing sample processing using at least one droplet-based microfluidic device capable of manipulating droplets containing samples; placing the droplets at specified locations on a surface of the droplet-based microfluidic device; and moving the droplets on the surface.

2. The method of claim 1, wherein the droplet-based microfluidic devices are formed in an array geometry.

3. The method of claim 1, further comprising applying a sequence of electrical signals to enable transport of droplets across the surface of the droplet-based microfluidic device.

4. The method of claim 3, wherein the sequence of electrical signals is applied to a pattern of electrodes buried beneath a dielectric layer on the droplet-based microfluidic device surface.

5. The method of claim 3, wherein the sequence of electrical signals creates the droplet.

6. The method of claim 1, wherein the droplet comprises a homogeneous liquids.

7. The method of claim 1, further comprising: placing a droplet containing a chemical agent on a surface of the droplet-based microfluidic device; and moving the droplet containing the chemical agent on the surface.

8. The method of claim 1, wherein at least some liquid is removed from the droplet by evaporation.

9. The method of claim 1, wherein the samples are precipitated from the droplet.

10. The method of claim 9, wherein a droplet is used to dissolve the dried samples.

11. The method of claim 10, wherein the samples are purified by selective dissolution.

12. The method of claim 1, further comprising: placing droplets containing a matrix solution on a surface of the droplet-based microfluidic device; and moving the droplets containing the matrix solution on the surface.

13. The method of claim 1 , wherein the droplet-based microfiuidics device is used with a mass spectrometer.

14. The method of claim 1, wherein a plurality of samples are processed in parallel.

15. A microfluidic device for processing droplets containing samples to be analyzed using mass spectrometry, comprising: a first plate comprising an array of first electrodes; a second plate, comprising at least a second electrode, wherein the first plate and the second plate are spaced apart such that a droplet can travel between the first plate and the second plate; a first layer of material, covering the array of first electrodes; and a second layer of material, covering the at least second electrode, wherein application of electrical signals between selected electrodes within the array of first electrodes and the at least one second electrode moves the droplet between the top plate and the bottom plate.

16. The microfluidic device of claim 15, further comprising an additional layer deposited on at least one of the first layer and the second layer wherein the additional layer modifies the surface wettability.

17. The microfluidic device of claim 15, wherein a first droplet is moved between the first plate and the second plate, and dried between the first plate and the second plate.

18. The microfluidic device of claim 15, wherein a second droplet is moved between the first plate and the second plate to where the first droplet was dried.

19. The microfluidic device of claim 16, wherein the droplet is analyzed by a mass spectrometer while the droplet is on the first plate.

20. A microfluidic device for processing droplets containing samples to be analyzed using mass spectrometry, comprising: a plate comprising a plurality of electrodes; and a layer of material, covering the plurality of electrodes, wherein application of electrical signals between selected electrodes within the plurality of electrodes moves a droplet on the plate.