CN103698383A - Preparation method and application of electrochemical immunosensor for detecting diethylstilbestrol - Google Patents
Preparation method and application of electrochemical immunosensor for detecting diethylstilbestrol Download PDFInfo
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- CN103698383A CN103698383A CN201310696498.5A CN201310696498A CN103698383A CN 103698383 A CN103698383 A CN 103698383A CN 201310696498 A CN201310696498 A CN 201310696498A CN 103698383 A CN103698383 A CN 103698383A
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Abstract
The invention provides an electrochemical immunosensor for detecting diethylstilbestrol. According to the immunosensor, by an electrode surface modifying technique, compounds for preparing carbon nanotube-ionic liquid-chitosan (MWCNTs-IL-CS) are modified to the surface of an electrode, then composited gold nanoparticles (AuNPs) are dropwise added to the surface of the modified electrode to simultaneously modify complete antigens DES-BSA of the diethylstilbestrol, antibody compounds (PRGO-Au-Ab-HRP) and diethylstilbestrol standard substances at the surface of the electrode, and the DES-BSA and the standard substances are simultaneously and competitively bound with binding sites of antibodies on an antibody compound so as to change the concentration of the standard substance; before and after hydrogen peroxide (H2O2) is added to a detection base solution, a working curve is drawn according to the relation between the signal variation and the concentration of the standard substances. The electrochemical immunosensor can be used for detecting the diethylstilbestrol in foods, is strong in selectivity and high in sensitivity, is simple and rapid to operate and is suitable for detection on the diethylstilbestrol in foods.
Description
Technical field
The invention belongs to nano material and food additives, a kind of electrochemical immunosensor that detects diethylstilbestrol is provided, can be for the detection of diethylstilbestrol in food.
Background technology
Diethylstilbestrol (DES) is a kind of synthetic nonsteroidal hormone, is widely used in clinical medicine and as animal growth promoting agent.Recently, much research shows that diethylstilbestrol is a kind of potential carcinogen, and in the U.S. and many European Union member countries, diethylstilbestrol is prohibited for promoting the aspects such as growth of animal.Therefore, set up a kind of quick, sensitive, simple detection method is necessary, at present the method for the detection diethylstilbestrol of report mainly comprises that gas chromatography-mass spectrometry, reflective of chemistry and pressurization capillary electric chromatogram are illegal etc., these methods often need complicated treatment step, and complicated operation together, detection time is longer.
Summary of the invention
This research has realized a kind of method of utilizing immune response combined with electrochemical to detect the diethylstilbestrol in food, has the advantages such as highly sensitive, simple to operate and with low cost.By modifying one deck nano-complex in gold electrode surfaces, be used for the electric signal of amplification sensor, then complete antigen of diethylstilbestrol is being modified to electrode surface, standard items competition by itself and diethylstilbestrol is in conjunction with the antibody complex being labeled, and the enzyme being combined in like this on the antibody complex of electrode surface just can catalytic substrate generation electric signal.Utilize the relation between change in electric and diethylstilbestrol standard items concentration, drawing curve, so just can reach the object that detects diethylstilbestrol content.
Summary of the invention
One of content of the present invention has been to provide a kind of preparation method who detects the electrochemical immunosensor of diethylstilbestrol in food.
Two of content of the present invention is by the immunosensor building, for detection of the diethylstilbestrol in food.
Technical scheme of the present invention, comprises the following steps:
1, a preparation method who detects the electrochemical immunosensor of diethylstilbestrol in food, is characterized in that, comprises the following steps;
(1) preparation of carbon nano-tube-ionic liquid-chitosan nano compound (MWCNTs-IL-CS) and golden nanometer particle:
The shitosan of 50 mg is dissolved in 1% the acetic acid of 20 ml, obtains shitosan and is dissolved in, and a certain amount of carbon nano-tube and particle are dissolved in the chitosan solution obtaining by ultrasonic, obtain MWCNTs-IL-CS.
Get 1 ml, 1% HAuCl
44H
2o is dissolved in 99 ml water, adds a certain amount of sodium citrate solution, and 100 ℃ of 1 h that reflux, obtain AuNPs nano particle.
(2) preparation of antibody complex:
10 mg DA and 1 mg GO are dissolved in the PBS that 10 ml pH are 8.5, and ultrasonic 2 min form dispersion uniformly molten.60 ℃ are stirred 24 h, obtain PRGO, are again dissolved in 10 ml water; 500 μ l HAuCl
44H
2o (10 mg/mL) adds as in solution obtained above, stirs after 2 h, then adds a certain amount of sodium citrate solution, stirs 2 h, eccentric cleaning, then be dissolved in 10 ml water; Finally, 100 μ l, the HRP of 1 mg/mL and 100 μ l, the Ab of 5 μ g/mL joins in the above-mentioned solution of 2 ml, and 4 ℃ are stirred a night, by PBS damping fluid eccentric cleaning, are dissolved in the PBS that 2 ml contain 1%BSA standby.
(3) detect the preparation of diethylstilbestrol immunosensor:
Drip the MWCNTs-IL-CS of 10 μ l in the gold electrode surfaces of handling well, dry under room temperature; Drip again the AuNPs of 20 μ l at the electrode surface of modifying, dry under room temperature; Drip a certain amount of complete antigen of diethylstilbestrol DES-BSA on modified electrode surface, dry under room temperature, each step is modified rear with PBS buffer solution cleaning electrode.
A kind of electrochemical immunosensor that detects diethylstilbestrol of preparing as mentioned above, as follows for detection of the step of diethylstilbestrol:
(1) take Ag/AgCl electrode as contrast electrode, platinum electrode is to electrode, drips diethylstilbestrol standard items and antibody complex simultaneously and as working electrode, connect electrochemical workstation on modified electrode surface, is containing H
2o
2in liquid at the bottom of the PBS of HQ, by cyclic voltammetry, detect, scanning voltage is 0.6--0.6 V, detects and adds H
2o
2the variation of front and back electrochemical signals and the relation between diethylstilbestrol standard items concentration, drawing curve;
(2) determinand is replaced to diethylstilbestrol standard items, according to the method for working curve, detect.
Described a kind of electrochemical immunosensor that detects diethylstilbestrol, material therefor Jun chemical reagents corporation buys.
Advantage and feature that the present invention embodies are:
(1) the MWCNTs-IL-CS compound conductive capability that the present invention adopts is strong, and good biocompatibility becomes the excellent material that builds electrochemical immunosensor;
(2) the transducer sensitivity that prepared by the present invention is high, detection speed is fast, simple to operate;
(3) the present invention detects the method for diethylstilbestrol, simple to operate, quick, is convenient to actual detection.
Embodiment
Below in conjunction with embodiment, further set forth 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 tells about, these equivalent form of values fall within claims limited range described in the application equally.
Embodiment 1
A preparation method who detects the electrochemical immunosensor of diethylstilbestrol, step is as follows:
The MWCNTs-IL-CS of (1) 10 μ l is in the gold electrode surfaces of handling well, dry under room temperature;
(2) AuNPs that drips 20 μ l is at the electrode surface of modifying, dry under room temperature;
(3) drip a certain amount of complete antigen of diethylstilbestrol DES-BSA on modified electrode surface, dry under room temperature, each step is modified rear with PBS buffer solution cleaning electrode.
(4) take Ag/AgCl electrode as contrast electrode, platinum electrode is to electrode, drips diethylstilbestrol standard items and antibody complex simultaneously and as working electrode, connect electrochemical workstation on (3) modified electrode surface, is containing H
2o
2in liquid at the bottom of the PBS of HQ, by cyclic voltammetry, detect.
Embodiment 2
A preparation method who detects the electrochemical immunosensor of diethylstilbestrol, step is as follows:
The MWCNTs-IL-CS of (1) 20 μ l is in the gold electrode surfaces of handling well, dry under room temperature;
(2) AuNPs that drips 20 μ l is at the electrode surface of modifying, dry under room temperature;
(3) drip a certain amount of complete antigen of diethylstilbestrol DES-BSA on modified electrode surface, dry under room temperature, each step is modified rear with PBS buffer solution cleaning electrode.
(4) take Ag/AgCl electrode as contrast electrode, platinum electrode is to electrode, drips diethylstilbestrol standard items and antibody complex simultaneously and as working electrode, connect electrochemical workstation on (3) modified electrode surface, is containing H
2o
2in liquid at the bottom of the PBS of HQ, by cyclic voltammetry, detect the variation of electrochemical signals.
Embodiment 3
The MWCNTs-IL-CS of (1) 20 μ l is in the gold electrode surfaces of handling well, dry under room temperature;
(2) AuNPs that drips 10 μ l is at the electrode surface of modifying, dry under room temperature;
(3) drip a certain amount of complete antigen of diethylstilbestrol DES-BSA on modified electrode surface, dry under room temperature, each step is modified rear with PBS buffer solution cleaning electrode.
(4) take Ag/AgCl electrode as contrast electrode, platinum electrode is to electrode, drips diethylstilbestrol standard items and antibody complex simultaneously and as working electrode, connect electrochemical workstation on (3) modified electrode surface, is containing H
2o
2in liquid at the bottom of the PBS of HQ, by cyclic voltammetry, detect the variation of electrochemical signals.
Embodiment 4
The MWCNTs-IL-CS of (1) 10 μ l is in the gold electrode surfaces of handling well, dry under room temperature;
(2) AuNPs that drips 20 μ l is at the electrode surface of modifying, dry under room temperature;
(3) drip a certain amount of complete antigen of diethylstilbestrol DES-BSA on modified electrode surface, dry under room temperature, each step is modified rear with PBS buffer solution cleaning electrode.
(4) take Ag/AgCl electrode as contrast electrode, platinum electrode is to electrode, drips diethylstilbestrol standard items and antibody complex simultaneously and as working electrode, connect electrochemical workstation on (3) modified electrode surface, is containing H
2o
2in liquid at the bottom of the PBS of HQ, by cyclic voltammetry, detect.Scanning voltage is 0.6--0.6 V, detects and adds H
2o
2the variation of front and back electrochemical signals and the relation between diethylstilbestrol standard items concentration, drawing curve;
Embodiment 5
The MWCNTs-IL-CS of (1) 10 μ l is in the gold electrode surfaces of handling well, dry under room temperature;
(2) AuNPs that drips 20 μ l is at the electrode surface of modifying, dry under room temperature;
(3) drip a certain amount of complete antigen of diethylstilbestrol DES-BSA on modified electrode surface, dry under room temperature, each step is modified rear with PBS buffer solution cleaning electrode.
(4) take Ag/AgCl electrode as contrast electrode, platinum electrode is to electrode, drips diethylstilbestrol standard items and antibody complex simultaneously and as working electrode, connect electrochemical workstation on (3) modified electrode surface, is containing H
2o
2in liquid at the bottom of the PBS of HQ, by cyclic voltammetry, detect.Scanning voltage is 0.6--0.6 V, detects and adds H
2o
2the variation of front and back electrochemical signals and the relation between diethylstilbestrol standard items concentration, drawing curve;
(5) determinand is replaced to diethylstilbestrol standard items, according to the method for working curve, detect.
(6) adopt electrochemical immunosensor to detect the milk sample 1 of buying from market, average detected 6 times, does not detect and contains diethylstilbestrol, belongs to qualified samples; Mark-on recovery test, its relative standard deviation is 4.5%.
Embodiment 6
The MWCNTs-IL-CS of (1) 10 μ l is in the gold electrode surfaces of handling well, dry under room temperature;
(2) AuNPs that drips 20 μ l is at the electrode surface of modifying, dry under room temperature;
(3) drip a certain amount of complete antigen of diethylstilbestrol DES-BSA on modified electrode surface, dry under room temperature, each step is modified rear with PBS buffer solution cleaning electrode.
(4) take Ag/AgCl electrode as contrast electrode, platinum electrode is to electrode, drips diethylstilbestrol standard items and antibody complex simultaneously and as working electrode, connect electrochemical workstation on (3) modified electrode surface, is containing H
2o
2in liquid at the bottom of the PBS of HQ, by cyclic voltammetry, detect.Scanning voltage is 0.6--0.6 V, detects and adds H
2o
2the variation of front and back electrochemical signals and the relation between diethylstilbestrol standard items concentration, drawing curve;
(5) determinand is replaced to diethylstilbestrol standard items, according to the method for working curve, detect.
(6) adopt electrochemical immunosensor to detect the milk sample 2 of buying from market, average detected 6 times, does not detect and contains diethylstilbestrol, belongs to qualified samples; Mark-on recovery test, its relative standard deviation is 5.3%.
Claims (2)
1. an electrochemical immunosensor that detects diethylstilbestrol, is characterized in that, comprises the following steps:
(1) preparation of carbon nano-tube-ionic liquid-chitosan nano compound (MWCNTs-IL-CS) and golden nanometer particle:
The shitosan of 50 mg is dissolved in 1% the acetic acid of 20 ml, obtains shitosan and is dissolved in, and a certain amount of carbon nano-tube and particle are dissolved in the chitosan solution obtaining by ultrasonic, obtain MWCNTs-IL-CS; Get 1 ml, 1% HAuCl
44H
2o is dissolved in 99 ml water, adds a certain amount of sodium citrate solution, and 100 ℃ of 1 h that reflux, obtain AuNPs nano particle;
(2) preparation of antibody complex:
10 mg DA and 1 mg GO are dissolved in the PBS that 10 ml pH are 8.5, and ultrasonic 2 min form dispersion uniformly molten; 60 ℃ are stirred 24 h, obtain PRGO, are again dissolved in 10 ml water; 500 μ l HAuCl
44H
2o (10 mg/mL) adds as in solution obtained above, stirs after 2 h, then adds a certain amount of sodium citrate solution, stirs 2 h, eccentric cleaning, then be dissolved in 10 ml water; Finally, 100 μ l, the HRP of 1 mg/mL and 100 μ l, the Ab of 5 μ g/mL joins in the above-mentioned solution of 2 ml, and 4 ℃ are stirred a night, by PBS damping fluid eccentric cleaning, are dissolved in the PBS that 2 ml contain 1%BSA standby;
(3) detect the preparation of diethylstilbestrol immunosensor:
Drip the MWCNTs-IL-Cs of 10 μ l in the gold electrode surfaces of handling well, dry under room temperature; Drip again the AuNPs of 20 μ l at the electrode surface of modifying, dry under room temperature; Drip a certain amount of complete antigen of diethylstilbestrol DES-BSA on modified electrode surface, dry under room temperature, each step is modified rear with PBS buffer solution curve electrode again.
2. a kind of electrochemical immunosensor that detects diethylstilbestrol of preparation as claimed in claim 1, step for detection of diethylstilbestrol is as follows: (1) take Ag/AgCl electrode as contrast electrode, platinum electrode is to electrode, on modified electrode surface, drip diethylstilbestrol standard items and antibody complex and as working electrode simultaneously, connect electrochemical workstation, containing H
2o
2in liquid at the bottom of the PBS of HQ, by cyclic voltammetry, detect, scanning voltage is 0.6--0.6 V, detects and adds H
2o
2the variation of front and back electrochemical signals and the relation between diethylstilbestrol standard items concentration, drawing curve;
(2) determinand is replaced to diethylstilbestrol standard items, according to the method for working curve, detect.
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Cited By (4)
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| CN104849449A (en) * | 2015-05-13 | 2015-08-19 | 东南大学 | Application of enzyme-labeled antibody-gold nanoprobe in catalyzed coloration and dark-field imaging of diaminobenzidine |
| CN105758849A (en) * | 2016-03-16 | 2016-07-13 | 济南大学 | Application and preparation method of dual-functional diethylstilbestrol non-enzymatic sensor constructed on basis of two-dimensional composite material |
| CN110455893A (en) * | 2019-07-23 | 2019-11-15 | 惠州市钰芯电子材料有限公司 | A kind of ionic liquid of Sensitive Detection dopamine/MCNT modified electrode and the preparation method and application thereof |
| CN113671003A (en) * | 2021-07-26 | 2021-11-19 | 仲恺农业工程学院 | Nano-material ionic liquid composite modified electrode, preparation method thereof and application of nano-material ionic liquid composite modified electrode in diethylstilbestrol determination |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104849449A (en) * | 2015-05-13 | 2015-08-19 | 东南大学 | Application of enzyme-labeled antibody-gold nanoprobe in catalyzed coloration and dark-field imaging of diaminobenzidine |
| CN105758849A (en) * | 2016-03-16 | 2016-07-13 | 济南大学 | Application and preparation method of dual-functional diethylstilbestrol non-enzymatic sensor constructed on basis of two-dimensional composite material |
| CN105758849B (en) * | 2016-03-16 | 2018-03-27 | 济南大学 | A kind of preparation method and application of the difunctional stilbestrol without enzyme sensor based on Two-dimensional Composites structure |
| CN110455893A (en) * | 2019-07-23 | 2019-11-15 | 惠州市钰芯电子材料有限公司 | A kind of ionic liquid of Sensitive Detection dopamine/MCNT modified electrode and the preparation method and application thereof |
| CN113671003A (en) * | 2021-07-26 | 2021-11-19 | 仲恺农业工程学院 | Nano-material ionic liquid composite modified electrode, preparation method thereof and application of nano-material ionic liquid composite modified electrode in diethylstilbestrol determination |
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Application publication date: 20140402 |