CN103822949A - Preparation method and application of electrochemical immunosensor for detecting escherichia coli - Google Patents
Preparation method and application of electrochemical immunosensor for detecting escherichia coli Download PDFInfo
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- CN103822949A CN103822949A CN201410015673.4A CN201410015673A CN103822949A CN 103822949 A CN103822949 A CN 103822949A CN 201410015673 A CN201410015673 A CN 201410015673A CN 103822949 A CN103822949 A CN 103822949A
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Abstract
The invention discloses a method for detecting escherichia coli by using an electrochemical biosensor. The method organically combines a nanotechnology, a biotechnology and an electrochemical sensing analysis technology, and comprises the following steps: firstly, preparing a compound of graphene, gold nanoparticles and EDOT and modifying the compound on an electrode; after optimizing conditions, carrying out subsequent detection under the optimal conditions; modifying an antigen 1 on the electrode, then inactivating escherichia coli with different concentrations at the temperature of 100 DEG C and modifying the inactivated escherichia coli on the electrode; finally, modifying an HRP-marked antigen 2 on the electrode. The linear corresponding relation is found between current detected by the sensor and the concentration of the escherichia coli, and accordingly the detection of the escherichia coli is implemented. The electrochemical biosensor prepared with the method is proved to have the advantages of high selectivity, high sensitivity, simplicity and quickness in operation and the like.
Description
Technical field
The invention belongs to nano material and food harmful bacteria detection field, provide a kind of detection colibacillary electrochemical immunosensor, can be used for colibacillary detection in food.
Background technology
Escherichia coli are the one in the food-borne pathogens that toxicity is the strongest.It often occupy in people and other homeothermal animal enteron aisles, is distributed widely in occurring in nature, is usually used to as important indicator bacteria in environment water monitoring and food safety detection.Infect enteropathogenic E．Coli and can cause the diseases such as diarrhoea, hemorrhagic colitis, septicemia, kidney failure and meningitis.It is reported, the whole world causes at least 6.5 hundred million people's morbidities because of Infective Escherichia coli every year, and causes approximately 800,000 5 years old following death of child, in the pathogeny spectrum the many regional Escherichia coli of China diarrhoea patient, also accounts for first place.Thus, we should set up a kind of colibacillary method of simple, quick, accurate, sensitive detection.Colibacillary traditional detection method mainly contains dilution plate counting method, multitube fermentation method, filter membrane method etc., although these method results are more accurate, often complex operation, length consuming time, can not meet the demand of fast detecting.
This research organically combines nanometer technology, biotechnology and electrochemical sensing assays technology three, prepared electro-chemistry immunity biology sensor based on Graphene, golden nanometer particle, EDOT with fast detecting Escherichia coli, that this sensor has is highly sensitive, analysis speed is fast, the feature such as simple to operate and with low cost.By modify successively the compound, antigen, the antibody that strengthen electrochemical signals on glass-carbon electrode, then carrying out condition optimizing, be chosen under optimal conditions and build electrochemical sensor, for detection of containing colibacillary sample solution, utilize Escherichia coli in sample solution to be combined the curent change that front and back cause with sensor and analyze colibacillary content in testing sample solution.
Summary of the invention
One of content of the present invention is to have built colibacillary electrochemical immunosensor in a kind of detection food.
Two of content of the present invention is to have built a kind of electrochemical immunosensor, can be used for detecting the Escherichia coli in food.
Technical scheme of the present invention, comprises the following steps:
1, a preparation method who detects the Escherichia coli electrochemical immunosensor in food, is characterized in that:
(1) preparation of Graphene and nm of gold nano-particle compound (GR-AuNPs):
Prepare graphite oxide (GO); The GO that takes 100 mg is dissolved in 100 mL water, ultrasonic 1h mixes it, obtain the brownish black liquid of homogeneous, substep adds 5 mL polyvinylpyrrolidones and 40.0 μ L ammoniacal liquor, stir a few minutes, in the dispersion liquid obtaining, dropwise add 3.5 μ L hydrazine hydrates, water-bath 2 h, obtain the Graphene that polyvinylpyrrolidone is protected; The Graphene of 2.5 mg polyvinylpyrrolidone protections and the 30 mmol/L chlorauric acid solutions of 140 μ L are distributed in 10 mL distilled water, then dropwise add the NaBH of 0.25 mL0.2 mol/L
4solution, stirs 30 min, and eccentric cleaning obtains GR-AuNPs nano-complex 5 times above, dry, is stored in refrigerator;
(2) prepare 3,4-ethylene dioxythiophene, Graphene and nm of gold nano-particle compound (GR-EDOT-AuNPs)
Get a certain amount of GR-AuNPs nano-complex in ultrapure water, stir 30 min it is mixed, add EDOT and constantly stir, utilize cyclic voltammetry to be scanned up to 0.6 V by-0.2 and carry out electropolymerization, can prepare GR-EDOT-AuNPs nano-complex.
The one of preparation as claimed in claim 1 detects colibacillary electrochemical immunosensor, as follows for colibacillary detecting step:
(1), take Ag/AgCl as contrast electrode, take Pt electrode as to electrode, the electrode of modifying compound and antibody, determinand is working electrode, is connected to electrochemical workstation, at HQ and H
2o
2in solution, scan, detect electrochemical signals corresponding to variable concentrations Escherichia coli standard solution, drawing curve;
(2) testing sample solution is substituted to Escherichia coli standard solution, detect according to the method for drafting of working curve.
Advantage of the present invention and feature are:
(1) the present invention has prepared the compound good conductivity of Graphene, golden nanometer particle, EDOT, becomes the excellent material that builds sensor and selects;
(2) the electrochemical immunosensor that prepared by the present invention is highly sensitive, detection speed is fast;
(3) it is strong, highly sensitive that the electrochemica biological sensor that prepared by the present invention has selectivity, and the advantage such as fast simple to operate, can be used for Site Detection.
Embodiment
Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.In addition, should also be understood that those skilled in the art can make various changes or modification to the present invention after having read the content that the present invention lectures, these equivalent form of values fall within the application's appended claims limited range equally.
Embodiment 1
A preparation method who detects colibacillary electrochemical sensor, step is as follows:
(1) Escherichia coli are cultivated 12 h at pure bacterium liquid at 37 ℃ of LB nutrient culture media.The quantity of viable bacteria is determined by colony counting method.
(2) drip the glass-carbon electrode surface that 6 μ L EDOT-GR-AuNPs uniform solution were processed in alcohol, ambient temperature overnight is dried;
(3) antibody 1 is modified in electrode surface, got 10
4the Escherichia coli solution of cfu/mL, bacterium liquid boils after 15 min deactivations in boiling water, makes electrode overnight incubation in this bacterium liquid;
(4) by two anti-modifications in electrode surface of HRP mark;
(5) preparing respectively pH is 6,6.5,7.4,8.0, and the phosphate buffer of 8.5 0.05 mol/L, adds appropriate HQ and H
2o
2and it is stirred;
(6) take Ag/AgCl as contrast electrode, take Pt electrode as to electrode, with very working electrode of (4) gained sensor electrical, connect electrochemical workstation scanning, in the solution of (5) gained, scan, obtain the condition optimizing of best PH.
Embodiment 2
A preparation method who detects colibacillary electrochemical sensor, step is as follows:
(1) Escherichia coli are cultivated 12 h at pure bacterium liquid at 37 ℃ of LB nutrient culture media.The quantity of viable bacteria is determined by colony counting method.
(2) drip the glass-carbon electrode surface that 6 μ L EDOT-GR-AuNPs uniform solution were processed in alcohol, ambient temperature overnight is dried;
(3) antibody 1 is modified in electrode surface, got 10
4the Escherichia coli solution of cfu/mL, bacterium liquid boils after 15 min deactivations in boiling water, makes electrode overnight incubation in this bacterium liquid;
(4) by two anti-modifications in electrode surface of HRP mark;
(5) phosphate buffer of 0.05 mol/L that preparation pH is 7.4, adds 0,0.5,1.0,1.5 of appropriate HQ and different amounts, the H of 2.0mmol
2o
2and it is stirred;
(6) take Ag/AgCl as contrast electrode, take Pt electrode as to electrode, with very working electrode of (4) gained sensor electrical, connect electrochemical workstation scanning, in the solution of (5) gained, scan, obtain best H
2o
2the condition optimizing of concentration.
Embodiment 3
A preparation method who detects colibacillary electrochemical sensor, step is as follows:
(1) Escherichia coli are cultivated 12 h at pure bacterium liquid at 37 ℃ of LB nutrient culture media.The quantity of viable bacteria is determined by colony counting method.
(2) drip the glass-carbon electrode surface that 6 μ L EDOT-GR-AuNPs uniform solution were processed in alcohol, ambient temperature overnight is dried;
(3) antibody 1 is modified in electrode surface, got 10
4the Escherichia coli solution of cfu/mL, bacterium liquid boils after 15 min deactivations in boiling water, makes electrode overnight incubation in this bacterium liquid;
(4) by two anti-modifications in electrode surface of HRP mark;
(5) phosphate buffer of 0.05 mol/L that preparation pH is 7.4, adds appropriate H
2o
2and the HQ of 5.0,7.0,9.0,11.0,13.0mmol it is stirred;
(6) take Ag/AgCl as contrast electrode, take Pt electrode as to electrode, with very working electrode of (4) gained sensor electrical, connect electrochemical workstation scanning, in the solution of (5) gained, scan, obtain the condition optimizing of best HQ concentration.
Embodiment 4
A preparation method who detects colibacillary electrochemical sensor, step is as follows:
(1) Escherichia coli are cultivated 12 h at pure bacterium liquid at 37 ℃ of LB nutrient culture media.The quantity of viable bacteria is determined by colony counting method.
(2) drip the glass-carbon electrode surface that 6 μ L EDOT-GR-AuNPs uniform solution were processed in alcohol, ambient temperature overnight is dried;
(3) antibody 1 is modified in electrode surface, configured the Escherichia coli solution of variable concentrations take PH as 7.4 PBS damping fluid, bacterium liquid boils after 15 min deactivations in boiling water, makes electrode overnight incubation in this bacterium liquid;
(4) by two anti-modifications in electrode surface of HRP mark;
(5) prepare respectively the phosphate buffer that pH is 7.4 0.05 mol/L, add appropriate HQ and H
2o
2and it is stirred;
(6) take Ag/AgCl as contrast electrode, take Pt electrode as to electrode, with very working electrode of (4) gained sensor electrical, connect electrochemical workstation scanning, in the solution of (5) gained, scan, detect the change in electric of electrochemical immunosensor and the relation of e. coli concentration, drawing curve.
Embodiment 5
A preparation method who detects colibacillary electrochemical sensor, step is as follows:
(1) drip the glass-carbon electrode surface that 6 μ L EDOT-GR-AuNPs uniform solution were processed in alcohol, ambient temperature overnight is dried;
(2) antibody 1 is modified in electrode surface, replaced Escherichia coli standard solution with milk sample 1, make electrode overnight incubation in this sample;
(3) by two anti-modifications in electrode surface of HRP mark;
(4) prepare respectively the phosphate buffer that pH is 7.4mol/L, add appropriate HQ and H
2o
2and it is stirred;
(5) take Ag/AgCl as contrast electrode, take Pt electrode as to electrode, with very working electrode of (3) gained sensor electrical, connect electrochemical workstation scanning, in the solution of (4) gained, scan, detect the electric signal of electrochemical immunosensor, Parallel testing 10 times, utilize working curve to obtain its colibacillary concentration, contrast with national standard, this milk is qualified samples.
Embodiment 6
A preparation method who detects colibacillary electrochemical sensor, step is as follows:
(1) drip the glass-carbon electrode surface that 6 μ L EDOT-GR-AuNPs uniform solution were processed in alcohol, ambient temperature overnight is dried;
(2) antibody 1 is modified in electrode surface, replaced Escherichia coli standard solution with milk sample 2, make electrode overnight incubation in this sample;
(3) by two anti-modifications in electrode surface of HRP mark;
(4) prepare respectively the phosphate buffer that pH is 7.4mol/L, add appropriate HQ and H
2o
2and it is stirred;
(5) take Ag/AgCl as contrast electrode, take Pt electrode as to electrode, with very working electrode of (3) gained sensor electrical, connect electrochemical workstation scanning, in the solution of (4) gained, scan, detect the electric signal of electrochemical immunosensor, Parallel testing 10 times, contrasts with national standard, belongs to qualified samples, recovery testu, its relative deviation is 3.6%.
Claims (2)
1. a preparation method who detects the Escherichia coli electrochemical immunosensor in food, is characterized in that:
(1) preparation of Graphene and nm of gold nano-particle compound (GR-AuNPs):
Prepare graphite oxide (GO); The GO that takes 100 mg is dissolved in 100 mL water, ultrasonic 1h mixes it, obtain the brownish black liquid of homogeneous, substep adds 5 mL polyvinylpyrrolidones and 40.0 μ L ammoniacal liquor, stir a few minutes, in the dispersion liquid obtaining, dropwise add 3.5 μ L hydrazine hydrates, after water-bath 2 h, obtain the Graphene of polyvinylpyrrolidone protection; Take the Graphene of 2.5 mg polyvinylpyrrolidone protections and the 30 mmol/L chlorauric acid solutions of 140 μ L and be distributed in 10 mL distilled water, then dropwise add the NaBH of 0.25 mL 0.2 mol/L
4solution, stirs 30 min, and eccentric cleaning obtains GR-AuNPs nano-complex 5 times above, dry, is stored in refrigerator;
(2) prepare 3,4-ethylene dioxythiophene, Graphene and nm of gold nano-particle compound (GR-EDOT-AuNPs)
Get a certain amount of GR-AuNPs nano-complex in ultrapure water, stir 30 min it is mixed, add EDOT and constantly stir, utilize cyclic voltammetry to be scanned up to 0.6 V by-0.2 and carry out electropolymerization, can prepare GR-EDOT-AuNPs nano-complex.
2. the one of preparation as claimed in claim 1 detects colibacillary electrochemical immunosensor, as follows for colibacillary detecting step:
(1), take Ag/AgCl as contrast electrode, take Pt electrode as to electrode, the electrode of modifying compound and antibody, determinand is working electrode, is connected to electrochemical workstation, at HQ and H
2o
2in solution, scan, detect electrochemical signals corresponding to variable concentrations Escherichia coli standard solution, drawing curve;
(2) testing sample solution is substituted to Escherichia coli standard solution, detect according to the method for drafting of working curve.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104198558A (en) * | 2014-09-05 | 2014-12-10 | 天津工业大学 | Preparation method of novel escherichia coli electrochemical sensor |
| CN104407132A (en) * | 2014-10-24 | 2015-03-11 | 济南大学 | Electrochemical sensor for detection of Escherichia coli and preparation method thereof |
| CN104483489A (en) * | 2014-12-03 | 2015-04-01 | 东南大学 | Electronic mediator type enzymatic electrochemical immunodetection method of p53 protein |
| CN104764774A (en) * | 2015-01-22 | 2015-07-08 | 济南大学 | Biosensor for detecting escherichia coli and preparation method thereof |
| CN106442920A (en) * | 2016-11-11 | 2017-02-22 | 大连海事大学 | Ship ballast water drainage real-time monitoring device and method |
| CN108776161A (en) * | 2018-04-11 | 2018-11-09 | 红河学院 | AFB1Electrochemical immunosensor and preparation method thereof and its be used for AFB1Detection |
| CN111157599A (en) * | 2020-01-06 | 2020-05-15 | 杭州电子科技大学 | Electrochemical immunosensor for detecting escherichia coli and preparation method and application thereof |
| CN112525965A (en) * | 2020-10-15 | 2021-03-19 | 南京农业大学 | Escherichia coli electrochemical detection method based on bacteria-mediated azido alkyne cycloaddition and atom transfer radical polymerization |
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| CN103454331A (en) * | 2013-09-06 | 2013-12-18 | 南京理工大学 | Per-oxidized poly(3,4-ethylenedioxythiophene-graphite oxide (PEDOT/GO) modified electrode and electrochemical detection method thereof to pesticide imidacloprid |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104198558A (en) * | 2014-09-05 | 2014-12-10 | 天津工业大学 | Preparation method of novel escherichia coli electrochemical sensor |
| CN104198558B (en) * | 2014-09-05 | 2017-05-17 | 天津工业大学 | Preparation method of novel escherichia coli electrochemical sensor |
| CN104407132A (en) * | 2014-10-24 | 2015-03-11 | 济南大学 | Electrochemical sensor for detection of Escherichia coli and preparation method thereof |
| CN104407132B (en) * | 2014-10-24 | 2016-05-25 | 济南大学 | A kind of colibacillary electrochemical sensor and preparation method thereof that detects |
| CN104483489A (en) * | 2014-12-03 | 2015-04-01 | 东南大学 | Electronic mediator type enzymatic electrochemical immunodetection method of p53 protein |
| CN104764774A (en) * | 2015-01-22 | 2015-07-08 | 济南大学 | Biosensor for detecting escherichia coli and preparation method thereof |
| CN104764774B (en) * | 2015-01-22 | 2018-01-30 | 济南大学 | A kind of biology sensor for detecting Escherichia coli and preparation method thereof |
| CN106442920A (en) * | 2016-11-11 | 2017-02-22 | 大连海事大学 | Ship ballast water drainage real-time monitoring device and method |
| CN108776161A (en) * | 2018-04-11 | 2018-11-09 | 红河学院 | AFB1Electrochemical immunosensor and preparation method thereof and its be used for AFB1Detection |
| CN108776161B (en) * | 2018-04-11 | 2020-10-09 | 红河学院 | AFB1Electrochemical immunosensor, preparation method thereof and application thereof in AFB (active carbon B)1Detection of (2) |
| CN111157599A (en) * | 2020-01-06 | 2020-05-15 | 杭州电子科技大学 | Electrochemical immunosensor for detecting escherichia coli and preparation method and application thereof |
| CN112525965A (en) * | 2020-10-15 | 2021-03-19 | 南京农业大学 | Escherichia coli electrochemical detection method based on bacteria-mediated azido alkyne cycloaddition and atom transfer radical polymerization |
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Application publication date: 20140528 |