CN102680683B - Periodontal bacteria impedance immunosensor based on conductive polymer - Google Patents

Periodontal bacteria impedance immunosensor based on conductive polymer Download PDF

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Publication number
CN102680683B
CN102680683B CN201210173858.9A CN201210173858A CN102680683B CN 102680683 B CN102680683 B CN 102680683B CN 201210173858 A CN201210173858 A CN 201210173858A CN 102680683 B CN102680683 B CN 102680683B
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immunosensor
microelectrode
electrode
periodontal bacteria
micro flow
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CN102680683A (en
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裴振华
全保刚
牛忠英
施生根
汤楚华
史亮
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306 HOSPITAL OF PLA
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306 HOSPITAL OF PLA
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Abstract

The invention discloses a periodontal bacteria impedance immunosensor based on a conductive polymer modified electrode. The immunosensor comprises a working microelectrode which is arranged on a solid phase substrate and a microfluidic chip channel which is covalently bonded with the solid phase substrate together, wherein the microfluidic chip channel is formed on the working microelectrode; two ends of the microfluidic chip channel are respectively provided with a sample inlet and a sample outlet; the working microelectrode is connected with an electrochemical workstation through a wire; polypyrrole is deposited on the surface of the working microelectrode; and a specific antibody is modified on the polypyrrole. The periodontal bacteria impedance immunosensor has the advantages that the sample detection is performed in a micro-nano space, a small amount of agents and samples are consumed, and the cost is reduced; and moreover, a conductive polymer film or a nanowire array is modified on the surface of the working microelectrode, the fixed quantity of antibodies in a unit area and the detection specific surface area are increased, so that the sensitivity, interference resistance and response time of the sensor are greatly improved.

Description

A kind of periodontal bacteria impedance immunosensor based on conducting polymer
Technical field
The present invention relates to a kind of quantitative testing device of periodontal bacteria, be specifically related to a kind of highly sensitive, sensing range is wide, the response time is short, strong interference immunity, and technique is simple, cost is low, there is the periodontal bacteria impedance immunosensor of conducting polymer modified electrode.
Background technology
Periodontitis is worldwide frequently-occurring disease, common disease, classified as the third-largest class Chronic Non-Communicable Diseases of harm humans health by the World Health Organization (WHO), serious harm oral cavity and whole body health, be the important risk factor of the systemic disease such as angiocardiopathy, diabetes, research recently finds that periodontitis can significantly increase the incidence of disease of some cancer especially.At present, the incidence of disease of China's adult group periodontosis is up to more than 80%, above middle age for sending out well crowd, the increase of aging population has further increased the weight of the medical burden of patient and sanitation system, and therefore monitoring in time and prevent and treat periodontitis has become public health system and health problem public and concern and urgent need solution.
Periodontitis is because of complexity, and bibliographical information inherent cause, immune factor etc. are all in close relations with the onset of periodontitis, but bacterium factors is the initiating agent that causes periodontitis all the time, is the essential factor that causes Periodontoclasia.The number change of suffering from the local periodontal dominant bacteria of tooth is to evaluate the important indicator of the state of an illness.In addition, at present clinical in the medicine supplemental treatment process of periodontitis, the routine administration of Different Individual is basic identical, patient to the curative effect difference of drug therapy to a certain extent side reflected the clinical value of specific aim medication, the dominant bacteria field planting situation of understanding in time periodontal affected part, also has great importance to clinical medicine supplemental treatment periodontitis.
But periodontal bacteria detects a part that does not become periodontal routine inspection at present, very large reason is to relatively lag behind because be suitable at present technology and the means development of the other Bacteria Detection of clinical chair.The means that tradition quantitatively detects for periodontal bacteria are mainly microbe growth and quantitative PCR technique, have testing conditions harshness, sense cycle is long, cost is high, the other defects such as using inconvenience that detects of clinical chair.Although we are the electrical properties based on bacterial cell in the recent period, binding immunoassay magnetic separation technique, the periodontal bacteria galvanochemistry of having set up based on liquid electrode micro flow chip is surveyed bacterium method, realized the quick unmarked electrical impedance quantitative test of periodontal Main Pathogenic Bacteria porphyromonas gingivalis, but the sensitivity of detection system, stability, portability, anti-interference, throwing property still require further improvement.
Along with the progress of micro-nano processing, new material and new unit technology, the research and development of the high-performance biology sensor based on conductive polymer micro-nano rice structure detect for realizing the many bacterial classifications of the other periodontal of chair the opportunity and the approach that provide new simultaneously and rapidly.There is not yet at present the electrical properties that utilizes periodontal bacteria cell both at home and abroad, the biology sensor directly quantizing for the periodontal bacteria report based on conducting polymer.
Summary of the invention
The object of the invention is for the limitation in the other detection technique of current periodontal bacteria chair, provide a kind of highly sensitive, sensing range is wide, the response time is short, strong interference immunity, and technique is simple, cost is low, be easy in batches preparation and to what can disposal type end product transform, there is the periodontal bacteria impedance immunosensor of conducting polymer modified electrode.
A kind of periodontal bacteria impedance immunosensor provided by the invention, comprises the work microelectrode of being located in solid phase substrate, the micro flow chip passage together with described solid phase substrate covalent bonding, and described micro flow chip passage is located on described work microelectrode; The two ends of described micro flow chip passage are respectively equipped with injection port and outlet; Described work microelectrode is connected with electrochemical workstation by wire;
On the surface of described work microelectrode, deposit conducting polymer, on described conducting polymer, be modified with periodontal bacteria specific antibody.
In above-mentioned impedance immunosensor, described solid phase substrate can be monox substrate, glass or flexible substrates.
In above-mentioned impedance immunosensor, described work microelectrode can be gold electrode or platinum electrode.
In above-mentioned impedance immunosensor, described work microelectrode can be interdigital electrode, and described interdigital electrode can comprise 10 pairs ~ 15 pairs branch electrodes, and the interval between described branch electrodes can be 20 μ m ~ 50 μ m; The width of described branch electrodes can be 50 μ m ~ 100 μ m.
In above-mentioned impedance immunosensor, the material of described micro flow chip passage can be dimethyl silicone polymer (polydimethylsilicone, PDMS).
In above-mentioned impedance immunosensor, described specific antibody is periodontal bacteria thalline surface protein antibody, and the present invention can, by changing the biomolecule recognition component-specific antibody on described conductive polymer coating, can be used for the detection of various biomolecules.
The conductive polymer coating depositing on work microelectrode provided by the invention surface can obtain by non-template electrochemical method on described work microelectrode; Periodontal bacteria specific antibody on conductive polymer coating, can be fixing on described conductive polymer coating by chemical crosslink technique.
The present invention also provides the application in periodontal bacteria quantizes of above-mentioned periodontal bacteria impedance immunosensor.
The present invention, owing to taking above technical scheme, has the following advantages: 1, pattern detection is carried out in micro-nano space, consumes trace reagent and sample, has reduced cost; 2, work microelectrode finishing conductive polymer membrane or nano-wire array, increases unit area antibody fixed qty and detects specific surface area, thus sensitivity, anti-interference and the response time of greatly improving sensor; 3, technique is simple, by changing the biomolecule recognition component-specific antibody on described conductive polymer coating, can be used for the detection of various biomolecules; 4, required micro-nano process technology and biotechnology maturation, is easy in batches preparation and to transforming by disposal type end product.
Brief description of the drawings
Fig. 1 is according to immunosensor structural representation of the present invention.
Fig. 2 is the cyclic voltammetry curve that cyclic voltammetry is prepared polypyrrole according to the present invention.
Fig. 3 is the stereoscan photograph of the polypyrrole of cyclic voltammetry polymerization according to the present invention.
In figure, each mark is as follows: 1 monox substrate, 2 interdigital electrodes, 3 micro flow chip passages, 4 injection ports, 5 outlets, 6 wires, 7 electrochemical workstations.
Embodiment
Below in conjunction with accompanying drawing, the present invention will be further described, but the present invention is not limited to following examples.
Periodontal bacteria impedance immunosensor provided by the invention comprises the interdigital electrode 2 be located on monox substrate 1 and micro flow chip passage 3 together with this monox substrate 1 covalent bonding, and this micro flow chip passage 3 is located on interdigital electrode 2, titanium coating and thickness that this interdigital electrode 2 is is 10nm by thickness are the electrode (can make this electrode be fixed on more firmly on monox substrate 1) that 80nm gold is deposited upon one, it comprises 15 pairs of branch electrodes, between branch electrodes, be spaced apart 50 μ m, its width is 100 μ m, this interdigital electrode 2 can adopt the photoetching technique of standard to be prepared: first by whirl coating on monox substrate 1, front baking, exposure and the step of developing obtain carrying out the silicon chip of electrode pattern, then on the silicon chip of carrying out figure, successively prepare titanium and golden thin layer by hot vapour deposition method, after finally photoresist being removed with acetone soln, the material of this micro flow chip passage 3 is dimethyl silicone polymer, and it is highly 50 μ m, the two ends of this micro flow chip passage 3 are respectively equipped with injection port 4 and outlet 5, interdigital electrode 2 is connected with electrochemical workstation 7 by wire 6, on the surface of interdigital electrode 2, deposit polypyrrole layer, in this polypyrrole layer, be modified with specific antibody.
In above-mentioned immunosensor, the branch electrodes of composition interdigital electrode 2 can be 10 pairs ~ 15 pairs, and the spacing between branch electrodes can be adjusted in the scope between 20 μ m ~ 50 μ m, and its width can be adjusted in the scope between 50 μ m ~ 100 μ m.
Polypyrrole layer in above-mentioned immunosensor can be passed through cyclic voltammetry (Cyclic Voltammetry, CV) be prepared, three-electrode system is taked in experiment, working electrode is that the side in interdigital electrode 2 is interdigital, interdigital as to electrode using opposite side, contrast electrode can be the accurate contrast electrode of homemade Ag/AgCl, and (method for making is as follows: get one section of thick filamentary silver, through physics, surperficial oxide layer is removed in polishing, then be positioned over respectively acetone, ethanol and deionized water for ultrasonic and clean each 5 minutes, nitrogen dries up rear for subsequent use; Press the volume ratio mixed configuration electrolytic solution of 1:1 with concentrated hydrochloric acid and deionized water, using the filamentary silver cleaning up as working electrode, platinized platinum or stainless steel substrates are to electrode, under the constant voltage of 1.4V, react 10 minutes, finally obtain the accurate contrast electrode of Ag/AgCl of uniform surface, maroon), the bath composition using in experiment is the camphorsulfonic acid of 0.04M, the pyrroles of 0.2M, the phosphate buffered solution of the pH=6.86 of 2.0M; The sweep limit of setting cyclic voltammetric in experiment is-0.1V ~ 1.2V(vs.Ag/AgCl), sweep speed is 20mA/s, cycle index is 5 times; The polymerization parameter that can obtain this individual system from the cyclic voltammogram shown in Fig. 3 is as follows: the oxidation peak of pyrrole monomer appears at 0.9V, and pyrroles's polymerization current density is 1mA/cm 2; The oxidation of pyrrole monomer is carried out very soon, and first lap circulation just can be seen at electrode surface the polypyrrole of black substantially; Cause electrode surface conductivity to decline owing to there being polypyrrole to cover working electrode, so will be much smaller than the electric current of first circulation at the electric current at second circulation time oxidizing potential place of scanning.
Fixing EDC (1-(3-dimethylamino-propyl)-3-ethyl carbodiimide salt)-NHS (N monohydroxy succinimide) the chemical crosslink technique sessile antibody that adopts of antibody in above-mentioned immunosensor.First antibody immobile liquid (comprises 2 mg/ml antibody, 60 mM EDC, 2 mg/ml NHS) in injection port 4 injects micro flow chip passage 3, hatch altogether 3 hours with polypyrrole room temperature, then use phosphate buffer (pH 7.2) to rinse and remove unnecessary antibody immobile liquid; Then use bovine serum albumin solution (1M, pH 7.2) to seal non-specific site; Finally use phosphate buffer (pH 7.2) to rinse and remove unnecessary bovine serum albumin(BSA).
Detect as example taking Periodontal Pathogens porphyromonas gingivalis below, utilize above-mentioned impedance immunosensor to carry out Bacteria Detection and comprise the following steps:
1. in micro flow chip passage 3, inject sample to be detected, sample solution is in injection port 4 injects micro flow chip passage 3, and incubated at room 30 minutes, is fully caught the target bacteria in sample by the specific antibody of interdigital electrode 2 finishinges;
2. deionized water is injected in micro flow chip passage 3, object is to rinse to remove the unnecessary sample of not being combined with antibody;
3. connect interdigital electrode 2 and electrochemical workstation 7 by wire 6, by applying the alternating voltage of a characteristic frequency alternating current and the phase differential that register system feeds back to interdigital electrode 2, thereby the resistance value of the system of acquisition, read the impedance data showing in electrochemical workstation 7, the resistance value of the porphyromonas gingivalis solution that record is measured;
4. according to the porphyromonas gingivalis reference culture concentration-impedance standard curve obtaining, extrapolate the concentration of actual clinical sample Porphyromonas gingivalis.

Claims (5)

1. the periodontal bacteria impedance immunosensor based on conducting polymer, it is characterized in that: described immunosensor comprises the work microelectrode of being located in solid phase substrate, micro flow chip passage together with described solid phase substrate covalent bonding, described micro flow chip passage is located on described work microelectrode; The two ends of described micro flow chip passage are respectively equipped with injection port and outlet; Described work microelectrode is connected with electrochemical workstation by wire;
On the surface of described work microelectrode, deposit polypyrrole, on described polypyrrole, be modified with specific antibody;
Described work microelectrode is interdigital electrode;
Described interdigital electrode comprises 10 pairs~15 pairs branch electrodes, between described branch electrodes, is spaced apart 20 μ m~50 μ m; The width of described branch electrodes is 50 μ m~100 μ m.
2. immunosensor according to claim 1, is characterized in that: described solid phase substrate is monox substrate, glass or flexible substrates.
3. immunosensor according to claim 1 and 2, is characterized in that: described work microelectrode is gold electrode or platinum electrode.
4. immunosensor according to claim 1, is characterized in that: the material of described micro flow chip passage is dimethyl silicone polymer.
5. immunosensor according to claim 1, is characterized in that: described specific antibody is periodontal bacteria thalline surface protein antibody.
CN201210173858.9A 2012-05-30 2012-05-30 Periodontal bacteria impedance immunosensor based on conductive polymer Expired - Fee Related CN102680683B (en)

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CN103558389A (en) * 2013-11-06 2014-02-05 彭新凯 Immunoassay sensor based on micro-spacing array electrode and production method as well as method for detecting colon bacillus O157:H7 in food
CN104849322B (en) * 2015-04-22 2017-10-24 中国农业大学 A kind of impedance biosensor and bio-impedance determination method
CN108474802A (en) * 2015-12-21 2018-08-31 黄荣堂 Detection device
EP3918316A4 (en) * 2019-01-31 2022-10-05 Femtodx Measurement techniques for semiconductor nanowire-based sensors and related methods
CN110632138A (en) * 2019-11-01 2019-12-31 江南大学 Interdigital electrode chip
CN112986553A (en) * 2019-12-14 2021-06-18 南京岚煜生物科技有限公司 Preparation method of immunity electrode
CN111351931A (en) * 2020-02-23 2020-06-30 南京岚煜生物科技有限公司 Preparation method of immuno-electrode for detecting novel coronavirus

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101400993A (en) * 2005-11-10 2009-04-01 阿普尔拉股份有限公司 Microfluidic systems including porous polymer electrodes
CN101517403A (en) * 2006-09-14 2009-08-26 新加坡科技研究局 Electrochemical sensor with interdigitated microelectrodes and conductive polymer
WO2011156088A1 (en) * 2010-06-09 2011-12-15 Massachusetts Institute Of Technology Conducting polymer with actively switchable absorbency
EP2423162A1 (en) * 2010-08-24 2012-02-29 Leibniz-Institut für Polymerforschung Dresden e.V. Fabrication of carbon nano- or microtubes by using a self-rolling process
CN102445472A (en) * 2010-10-14 2012-05-09 北京华凯瑞微流控芯片科技有限责任公司 Microfluidic chip-based sensor and preparation method thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8206664B2 (en) * 2010-07-06 2012-06-26 Xerox Corporation Methods of producing multi-layered microfluidic devices

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101400993A (en) * 2005-11-10 2009-04-01 阿普尔拉股份有限公司 Microfluidic systems including porous polymer electrodes
CN101517403A (en) * 2006-09-14 2009-08-26 新加坡科技研究局 Electrochemical sensor with interdigitated microelectrodes and conductive polymer
WO2011156088A1 (en) * 2010-06-09 2011-12-15 Massachusetts Institute Of Technology Conducting polymer with actively switchable absorbency
EP2423162A1 (en) * 2010-08-24 2012-02-29 Leibniz-Institut für Polymerforschung Dresden e.V. Fabrication of carbon nano- or microtubes by using a self-rolling process
CN102445472A (en) * 2010-10-14 2012-05-09 北京华凯瑞微流控芯片科技有限责任公司 Microfluidic chip-based sensor and preparation method thereof

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