CN102680683A - Periodontal bacteria impedance immunosensor based on conductive polymer - Google Patents
Periodontal bacteria impedance immunosensor based on conductive polymer Download PDFInfo
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- CN102680683A CN102680683A CN2012101738589A CN201210173858A CN102680683A CN 102680683 A CN102680683 A CN 102680683A CN 2012101738589 A CN2012101738589 A CN 2012101738589A CN 201210173858 A CN201210173858 A CN 201210173858A CN 102680683 A CN102680683 A CN 102680683A
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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
Technical field
The present invention relates to a kind of detection by quantitative 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 technology is simple, cost is low, has 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 type of healthy Chronic Non-Communicable Diseases of harm humans by the World Health Organization (WHO); Serious harm oral cavity and whole body health; Be the important risk factor of systemic diseases such as angiocardiopathy, diabetes, research recently finds that especially periodontitis can significantly increase the incidence of disease of some cancer.At present; The incidence of disease of China's adult group periodontosis is up to more than 80%; Above middle age for sending out the crowd well; The increase of aging population has further increased the weight of the medical burden of patient and sanitation system, and therefore in time monitoring and prevent and treat periodontitis has become public health system and health problem public and that pay close attention to and be badly in need of solving.
Periodontitis is because of complicacy, and bibliographical information inherent cause, immune factor etc. are all in close relations with the onset of periodontitis, but the bacterium factor is the initiating agent that causes periodontitis all the time, is the essential factor that causes periodontal to destroy.The number change of suffering from the local periodontal dominant bacteria of tooth is to estimate 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; The patient to the curative effect difference of drug therapy then to a certain extent the side reflected the clinical value of specific aim medication, in time understand the dominant bacteria field planting situation of periodontal affected part, clinical medicine supplemental treatment periodontitis is also had great importance.
But periodontal bacteria detects a part that does not become the periodontal routine inspection at present, and very big reason is to relatively lag behind because be suitable for the technology and the means development of the other Bacteria Detection of clinical chair at present.The means that tradition is used for the periodontal bacteria detection by quantitative mainly are microbe growth and quantitative PCR technique, have defectives such as testing conditions is harsh, sense cycle is long, cost is high, the other detection use of clinical chair inconvenience.Although we are in the recent period based on the electrical properties of bacterial cell; The binding immunoassay magnetic separation technique; Set up based on the periodontal bacteria galvanochemistry of liquid electrode micro flow chip and surveyed the bacterium method; Realized the quick unmarked electrical impedance quantitative test of the main pathogenic bacteria porphyromonas gingivalis of periodontal, but the sensitivity of detection system, stability, portability, anti-interference, throwing property need still further to improve.
Along with the progress of micro-nano processing, new material and new unit technology, detect opportunity and the approach that provides new simultaneously and rapidly for realizing the many bacterial classifications of the other periodontal of chair based on the research and development of the high-performance biology sensor of conductive polymer micro-nano rice structure.Do not see the electrical properties that utilizes the periodontal bacteria cell at present both at home and abroad as yet, based on the biology sensor report that periodontal bacteria directly quantizes that is used for of conducting polymer.
Summary of the invention
The objective of the invention is to the limitation on the other detection technique of present periodontal bacteria chair; Provide a kind of highly sensitive, sensing range is wide, the response time is short, strong interference immunity; And technology is simple, cost is low; But be easy to prepared in batches and transform, have the periodontal bacteria impedance immunosensor of conducting polymer modified electrode to the disposal type end product.
A kind of periodontal bacteria impedance immunosensor provided by the invention comprises the work microelectrode of being located on the solid phase substrate, and with said solid phase substrate covalent bonding micro flow chip passage together, said micro flow chip passage is located on the said work microelectrode; The two ends of said micro flow chip passage are respectively equipped with injection port and outlet; Said work microelectrode is connected with electrochemical workstation through lead;
Deposit conducting polymer on the surface of said work microelectrode, be modified with the periodontal bacteria specific antibody on the said conducting polymer.
In the above-mentioned impedance immunosensor, said solid phase substrate can be monox substrate, glass or flexible substrates.
In the above-mentioned impedance immunosensor, said work microelectrode can be gold electrode or platinum electrode.
In the above-mentioned impedance immunosensor, said work microelectrode can be interdigital electrode, and said interdigital electrode can comprise 10 pairs ~ 15 pairs branch electrodes, and the interval between the said branch electrodes can be 20 μ m ~ 50 μ m; The width of said branch electrodes can be 50 μ m ~ 100 μ m.
In the above-mentioned impedance immunosensor, the material of said micro flow chip passage can be dimethyl silicone polymer (polydimethylsilicone, PDMS).
In the above-mentioned impedance immunosensor, said specific antibody is a periodontal bacteria thalline surface protein antibody, and the present invention can promptly can be used for the detection of various biomolecules through changing the biomolecule recognition component-specific antibody on the said conductive polymer coating.
The conductive polymer coating that deposits on the work microelectrode provided by the invention surface can obtain on said work microelectrode through the non-template electrochemical method; Periodontal bacteria specific antibody on the conductive polymer coating can be fixing on said conductive polymer coating through chemical crosslink technique.
The present invention also provides the above-mentioned application of periodontal bacteria impedance immunosensor in periodontal bacteria quantizes.
The present invention is owing to take above technical scheme, and have 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 increase unit area antibody fixed qty and detect specific surface area, thus sensitivity, anti-interference and the response time of improving sensor greatly; 3, technology is simple, through changing the biomolecule recognition component-specific antibody on the said conductive polymer coating, promptly can be used for the detection of various biomolecules; 4, required micro-nano process technology and biotechnology are ripe, but are easy to prepared in batches and transform to the disposal type end product.
Description of drawings
Fig. 1 is according to immunosensor structural representation of the present invention.
Fig. 2 is the cyclic voltammetry curve that cyclic voltammetry prepares polypyrrole according to the present invention.
Fig. 3 is the stereoscan photograph of the polypyrrole of cyclic voltammetry polymerization according to the present invention.
Each mark is following among the figure: 1 monox substrate, 2 interdigital electrodes, 3 micro flow chip passages, 4 injection ports, 5 outlets, 6 leads, 7 electrochemical workstations.
Embodiment
Below in conjunction with accompanying drawing the present invention is further specified, but the present invention is not limited to following examples.
Periodontal bacteria impedance immunosensor provided by the invention comprise the interdigital electrode 2 be located on the monox substrate 1 and with this monox substrate 1 covalent bonding micro flow chip passage 3 together; And this micro flow chip passage 3 is located on the interdigital electrode 2; This interdigital electrode 2 is for being the titanium coating of 10nm and thickness is deposited upon one for the 80nm gold electrode (this electrode is fixed on the monox substrate 1 more firmly) by thickness; It comprises 15 pairs of branch electrodes; Be spaced apart 50 μ m between the branch electrodes; Its width is 100 μ m, and this interdigital electrode 2 can adopt the photoetching technique of standard to prepare: the silicon chip that at first obtains carrying out electrode pattern through whirl coating on monox substrate 1, preceding baking, exposure and step of developing; Then carrying out the thin layer that successively prepares titanium and gold on the silicon chip of figure through hot vapour deposition method; Get final product after with acetone soln photoresist being removed at last; The material of this micro flow chip passage 3 is a dimethyl silicone polymer, and it highly is 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 through lead 6; Deposit polypyrrole layer on the surface of interdigital electrode 2, be modified with specific antibody on this polypyrrole layer.
In the above-mentioned immunosensor, the branch electrodes of forming interdigital electrode 2 can be 10 pairs ~ 15 pairs, and the spacing between the 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 the above-mentioned immunosensor can be passed through cyclic voltammetry (Cyclic Voltammetry; CV) prepare; It is that a side in the interdigital electrode 2 is interdigital that three-electrode system, working electrode are taked in experiment, interdigital as to electrode with opposite side; Contrast electrode can be the accurate contrast electrode of homemade Ag/AgCl, and (method for making is following: get one section thick filamentary silver; The oxide layer on surface is removed in polishing through physics, is positioned over acetone, ethanol and deionized water for ultrasonic then respectively and cleans each 5 minutes, and it is subsequent use that nitrogen dries up the back; Press the volume ratio mixed configuration electrolytic solution of 1:1 with concentrated hydrochloric acid and deionized water; As working electrode, platinized platinum or stainless steel substrates are to electrode with the filamentary silver that cleans up, and reaction is 10 minutes under the constant voltage of 1.4V; Obtain the accurate contrast electrode of Ag/AgCl of uniform surface, maroon at last); The bath composition that uses in the experiment is the camphorsulfonic acid of 0.04M, the pyrroles of 0.2M, the PBS of the pH=6.86 of 2.0M; The sweep limit of setting cyclic voltammetric is-0.1V ~ 1.2V (vs.Ag/AgCl) in the experiment, and sweep speed is 20mA/s, and cycle index is 5 times; The polymerization parameter that from cyclic voltammogram shown in Figure 3, can obtain this individual system is following: the oxidation peak of pyrrole monomer appears at 0.9V, and pyrroles's polymerization current density is 1mA/cm
2The oxidation of pyrrole monomer is carried out very soon, and the first lap circulation just can be seen the polypyrrole of black at electrode surface basically; Cause the electrode surface conductivity to descend owing to there has been polypyrrole to cover working electrode, so will be much smaller than first round-robin electric current 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 the above-mentioned immunosensor.At first the antibody immobile liquid (comprises 2 mg/ml antibody; 60 mM EDC, 2 mg/ml NHS) inject in the micro flow chip passage 3 through injection port 4; Hatched altogether 3 hours with the polypyrrole room temperature, use phosphate buffer (pH 7.2) flushing to remove unnecessary antibody immobile liquid then; Use bovine serum albumin solution (1M, pH 7.2) to seal non-specific site then; Use phosphate buffer (pH 7.2) flushing to remove unnecessary bovine serum albumin(BSA) at last.
Detecting with periodontal pathogenic bacteria porphyromonas gingivalis below is example, utilizes above-mentioned impedance immunosensor to carry out Bacteria Detection and may further comprise the steps:
1. in micro flow chip passage 3, inject sample to be detected, sample solution injects in the micro flow chip passage 3 through injection port 4, and incubated at room 30 minutes makes that the target bacteria in the sample is fully caught by the specific antibody of interdigital electrode 2 finishinges;
2. deionized water is injected in the micro flow chip passage 3, purpose is removed not the unnecessary sample with antibodies for flushing;
3. connect interdigital electrode 2 and electrochemical workstation 7 through lead 6; Through applying the alternating voltage of a CF and alternating current and the phase differential that register system feeds back to for interdigital electrode 2; Thereby the resistance value of the system of acquisition; Read the impedance data that shows in the 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 that obtains, extrapolate the concentration of porphyromonas gingivalis in the actual clinical sample.
Claims (7)
1. periodontal bacteria impedance immunosensor based on conducting polymer; It is characterized in that: said immunosensor comprises work microelectrode and the said solid phase substrate covalent bonding micro flow chip passage of being located on the solid phase substrate together, and said micro flow chip passage is located on the said work microelectrode; The two ends of said micro flow chip passage are respectively equipped with injection port and outlet; Said work microelectrode is connected with electrochemical workstation through lead;
Deposit polypyrrole on the surface of said work microelectrode, be modified with specific antibody on the said polypyrrole.
2. immunosensor according to claim 1 is characterized in that: said solid phase substrate is monox substrate, glass or flexible substrates.
3. immunosensor according to claim 1 and 2 is characterized in that: said work microelectrode is gold electrode or platinum electrode.
4. according to arbitrary described immunosensor among the claim 1-3, it is characterized in that: said work microelectrode is an interdigital electrode.
5. immunosensor according to claim 4 is characterized in that: said interdigital electrode comprises 10 pairs ~ 15 pairs branch electrodes, is spaced apart 20 μ m ~ 50 μ m between the said branch electrodes; The width of said branch electrodes is 50 μ m ~ 100 μ m.
6. according to the described immunosensor of claim 1-5, it is characterized in that: the material of said micro flow chip passage is a dimethyl silicone polymer.
7. according to arbitrary described immunosensor among the claim 1-6, it is characterized in that: said specific antibody is a periodontal bacteria thalline surface protein antibody.
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Cited By (7)
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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 |
| CN104849322A (en) * | 2015-04-22 | 2015-08-19 | 中国农业大学 | Impedance biosensor and bio-impedance detection analysis method |
| CN108474802A (en) * | 2015-12-21 | 2018-08-31 | 黄荣堂 | Detection device |
| CN110632138A (en) * | 2019-11-01 | 2019-12-31 | 江南大学 | Interdigital electrode chip |
| 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 |
| US20200256828A1 (en) * | 2019-01-31 | 2020-08-13 | FemtoDx | Fluid flow conductance measurement devices and related methods |
| WO2021115491A1 (en) * | 2019-12-14 | 2021-06-17 | 南京岚煜生物科技有限公司 | Preparation method for immunoelectrode |
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| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
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| US20200256828A1 (en) * | 2019-01-31 | 2020-08-13 | FemtoDx | Fluid flow conductance measurement devices and related methods |
| CN110632138A (en) * | 2019-11-01 | 2019-12-31 | 江南大学 | Interdigital electrode chip |
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| 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 |
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