CN109187963B - Preparation and use methods of electrochemical immunosensor for aflatoxin B1 detection - Google Patents
Preparation and use methods of electrochemical immunosensor for aflatoxin B1 detection Download PDFInfo
- Publication number
- CN109187963B CN109187963B CN201810951371.6A CN201810951371A CN109187963B CN 109187963 B CN109187963 B CN 109187963B CN 201810951371 A CN201810951371 A CN 201810951371A CN 109187963 B CN109187963 B CN 109187963B
- Authority
- CN
- China
- Prior art keywords
- glassy carbon
- carbon electrode
- solution
- electrode
- aflatoxin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56961—Plant cells or fungi
Abstract
The invention discloses a preparation and use method of an electrochemical immunosensor for aflatoxin B1 detection, which comprises the following steps: the preparation method of the electrochemical immunosensor and the application of the electrochemical immunosensor in the detection of the aflatoxin B1 comprise the following steps: preparing a nano-gold solution, preparing an activated glassy carbon electrode, preparing a graphene oxide/thionine/nano-gold composite material, preparing a modified glassy carbon electrode, sealing a non-specific site with bovine serum albumin, and preparing a detection base solution. The aflatoxin B1 can be detected quickly, accurately and at low cost.
Description
Technical Field
The invention relates to a preparation method and a use method of an electrochemical immunosensor, in particular to a preparation method and a use method of an electrochemical immunosensor for aflatoxin B1 detection.
Background
Aflatoxins, which are secondary metabolites of aspergillus flavus and aspergillus parasiticus, widely exist in grain and oil foods, are mycotoxins which are found to be the strongest toxins polluting agricultural products in nature and are mycotoxins which have strong carcinogenicity and pathogenicity on human and animals. The common aflatoxins are mainly classified into B1, B2, G1 and G2 according to the difference of chemical structures, wherein the toxicity of the aflatoxin B1 is the largest and far exceeds the toxicity of arsenic and potassium cyanide.
At present, aflatoxin detection methods mainly comprise an enzyme-linked immunosorbent assay, a High Performance Liquid Chromatography (HPLC), a Thin Layer Chromatography (TLC) and the like. The enzyme-linked immunosorbent assay has strong specificity and low cost, but complex samples are easily interfered due to the instability of enzyme, so that the detection accuracy is not high; the HPLC method has good selectivity and high sensitivity, but often needs to adopt a complex and fussy sample purification means to improve the selectivity, the method is time-consuming, the instrument is expensive, and the method has higher requirements on the quality of operators and experimental conditions; the TLC method uses simpler equipment and reagents, but the pretreatment of the sample is complicated, time-consuming and poor in accuracy, and a large amount of toxic organic solvent is harmful to experimenters.
Based on the above situation, a method for preparing and using an electrochemical immunosensor for aflatoxin B1 detection is needed to solve the problems of the preparation and use methods of the electrochemical immunosensor.
Disclosure of Invention
The invention aims to provide a preparation and use method of an electrochemical immunosensor for aflatoxin B1 detection, and aims to solve the problems of high cost, poor effect and great harm of the preparation and use methods of common electrochemical immunosensors.
In order to achieve the aim, the invention provides a preparation method and a use method of an electrochemical immunosensor for aflatoxin B1 detection, which comprises the following steps: a preparation method of the electrochemical immunosensor;
the method specifically comprises the following steps: preparing a nanogold solution, then placing graphene oxide, thionine and the nanogold solution into a weighing bottle with a cover, placing a stirring magneton into the weighing bottle, adding the cover, stirring for 2-8h by using a magnetic stirrer to fully and uniformly mix materials in the bottle to obtain a graphene oxide/thionine/nanogold composite material, dripping the graphene oxide/thionine/nanogold composite material on the surface of an activated glassy carbon electrode within 1-3s, then placing the glassy carbon electrode in a refrigerator at 2-8 ℃ for storage for 10-14h until the surface of the glassy carbon electrode is a layer of solid film, then placing an aflatoxin B1 monoclonal antibody and the glassy carbon electrode in a micropore reaction plate I in sequence, standing for 2-8h in the refrigerator at 2-8 ℃, then placing the glassy carbon electrode in the refrigerator at 2-8 ℃ until the surface of the glassy carbon electrode is dried, placing the bovine serum albumin solution and the glassy carbon electrode in the micropore reaction plate II in sequence, standing in a refrigerator at the temperature of 2-8 ℃ for 1-5min, taking out the glassy carbon electrode, and airing in the refrigerator at the temperature of 2-8 ℃ to obtain a modified glassy carbon electrode, namely the electrochemical immunosensor;
in the preparation process of the graphene oxide/thionine/nano-gold composite material: the concentration of the graphene oxide is 0.5-1.5wt%, the dosage of the graphene oxide is 1 volume part of A1, the dosage of the thionine is 1 weight part of B1, and the dosage of the nano gold solution is 5-12 volume parts of A1; the correlation between 1 part by volume of A1 and 1 part by weight of B1 is as follows: 1 ml for 1-5 mg;
in the preparation process of the electrochemical immunosensor: the graphene oxide/thionine/nanogold composite material is prepared from 1 part by volume of A2, the aflatoxin B1 monoclonal antibody is prepared from 100-120 parts by volume of A2, and the bovine serum albumin solution is prepared from 100-120 parts by volume of A2.
Preferably, the nanogold solution is prepared by pouring 1 part by volume of a chloroauric acid solution with the mass fraction of 0.005-0.05% of A3 into a container, heating and stirring until the solution boils for 1-10min, adding 0.01-0.1 part by volume of a sodium citrate solution with the mass fraction of 0.2-5% of A3, continuing to react for 1-20min until the synthetic solution does not change color any more, stopping heating, continuing to stir, after the synthetic solution is cooled to 20-28 ℃, bottling, and storing in a refrigerator with the temperature of 2-8 ℃.
Preferably, before the graphene oxide/thionine/nanogold composite material is dropped on the glassy carbon electrode, sequentially polishing alumina powder with the particle sizes of 0.1-0.6 mu m and 0.01-0.1 mu m on chamois leather until the surface is smooth and no scratch is visible to naked eyes, washing the alumina powder remained on the glassy carbon electrode after polishing with deionized water, then sequentially putting the glassy carbon electrode into absolute ethyl alcohol and deionized water to be ultrasonically cleaned for 2-10min, putting the cleaned glassy carbon electrode into 0.03-0.5mol/L sulfuric acid solution, then scanning for 2-15 circles within the voltage range of 0-3.6V by using a cyclic voltammetry scanning method to activate the electrode, and after activation, washing the activated electrode with deionized water.
Preferably, the bovine serum albumin solution is prepared by dissolving 0.1-0.5g of bovine serum albumin in a pH6.5-7.4 phosphate buffer solution to a constant volume of 60-120mL to obtain a bovine serum albumin solution with a volume fraction of 0.1-0.4%.
Preferably, the phosphate buffer solution with pH6.5-7.4 is prepared by mixing 0.01-0.15mol/L dipotassium hydrogen phosphate and 0.01-0.15mol/L sodium dihydrogen phosphate in a ratio of 1: 0.9-0.5.
Preferably, the stirrer is a magnetic stirrer, the magnetic stirrer comprises a stirring magneton and a magnetic stirring table, the stirring magneton is arranged in the weighing bottle, and the weighing bottle is arranged on the magnetic stirring table.
Preferably, the method comprises the following steps: the use method of the electrochemical immunosensor comprises the following specific steps: preparing a phosphate buffer solution with the pH value of 6.5-7.4, adding an antigen stock solution into the phosphate buffer solution, diluting for multiple times to obtain a plurality of antigen diluent base solutions with different concentrations and reduced proportion, stirring the different base solutions for 20-70s by using a magnetic stirrer respectively to uniformly mix the different base solutions, dripping the different stirred base solutions on the surface of a glassy carbon electrode, and detecting by using a three-electrode system respectively.
Preferably, the three-electrode system comprises: the modified glassy carbon electrode is a working electrode, the platinum electrode is a counter electrode, and the saturated potassium chloride electrode is a reference electrode.
Preferably, the detection process of the electrochemical immunosensor on the aflatoxin B1 is carried out in a nitrogen atmosphere.
The invention at least comprises the following beneficial effects:
1. the composite material probe prepared by the method has the advantages of strong conductivity and adsorption capacity, simple and convenient detection method, convenience in carrying and the like.
2. The graphene oxide has a good physical adsorption effect, the thionine can enhance the conductive effect, and the nanogold can increase the specific surface area, so that the electrochemical immunosensor has high sensitivity, short response time and low detection cost, and the rapid detection of the aflatoxin B1 is realized.
3. The electrochemical immunosensor disclosed by the invention shows strong specificity, and other non-specific molecules in a sample have no influence on a detection result, and the electrochemical immunosensor is high in sensitivity and rapidness in detection.
4. The method for processing the aflatoxin corn sample is simple.
5. The method can accurately perform qualitative and quantitative detection on the aflatoxin B1, and the lowest detection limit can reach 2.394 multiplied by 10-12μ g/kg at 2.394X 10-12—3.810×10-11μ g/kg and 1.172X 10-10—0.7863×10-9μ g/kg and 2.347X 10-8—1.169×10-7The linear relationship is good in mu g/kg.
Drawings
FIG. 1 is a schematic diagram of the preparation of the electrochemical immunosensor of the present invention and the specific binding of the sensor to an antibody.
FIG. 2 is a side sectional view of the electrochemical immunosensor preparation of the present invention and the specific binding of the sensor to antibodies.
Description of the drawings: a-graphene oxide/thionine/nanogold composite material, B-graphene oxide/thionine/nanogold composite material is dripped on the surface of a glassy carbon electrode to form a film, a C-aflatoxin B1 monoclonal antibody is adsorbed on the surface of the film of the glassy carbon electrode, D-bovine serum albumin seals non-specific sites adsorbed on the surface of the electrode, an E-aflatoxin B1 monoclonal antibody is specifically combined with aflatoxin B1 antigen, a-glassy carbon electrode, B-graphene oxide/thionine/nanogold composite material is dripped on the surface of the glassy carbon electrode to form a film, a C-aflatoxin B1 monoclonal antibody is adsorbed on the surface of the film of the glassy carbon electrode, D-bovine serum specific sites adsorbed on the surface of the closed electrode, and E-aflatoxin B1 monoclonal antibody is specifically combined with aflatoxin B1 antigen.
Detailed Description
The technical content of the invention is further explained by the following figures and embodiments:
example 1
1. The electrochemical immunosensor is prepared by the following steps:
preparing a nanogold solution, and then placing 1 volume part of graphene oxide with the concentration of 1.01wt% of A1, 1 weight part of B1 thionine and 8.333 volume parts of A1 nanogold solution into a weighing bottle with a cover, wherein the relationship between 1 volume part of A1 and 1 weight part of B1 is as follows: 1 ml corresponds to 1.25 mg, a stirring magneton is placed in a weighing bottle, the weighing bottle is covered and then placed on a magnetic stirring table to be stirred for about 4 hours to fully and uniformly mix materials in the bottle to obtain a graphene oxide/thionine/nanogold composite material, the graphene oxide/thionine/nanogold composite material 2S with the volume of 1 part A2 is dripped on the surface of an activated glassy carbon electrode, then the glassy carbon electrode is placed in a refrigerator with the temperature of 4 ℃ to be stored for 12 hours until the surface of the glassy carbon electrode presents a layer of solid film, then a 111.111 volume part of aflatoxin B1 monoclonal antibody of A2 and the glassy carbon electrode are sequentially placed in a micropore reaction plate I and then placed in the refrigerator with the temperature of 4 ℃ for 4 hours, then the glassy carbon electrode is placed in the refrigerator with the temperature of 4 ℃ until the surface of the glassy carbon electrode is dried, and then a 111.111 volume part of bovine serum protein solution of A2 and the glassy carbon electrode are sequentially placed in a micropore reaction plate II, standing in a refrigerator at 4 ℃ for 2min, taking out the glassy carbon electrode, and airing in the refrigerator at 4 ℃ to obtain a modified glassy carbon electrode, namely the electrochemical immunosensor;
1) and preparing a nano gold solution. Pouring 1 part by volume of a chloroauric acid solution with the mass fraction of A3 being 0.01% into a container, heating and stirring until the solution boils and lasts for 2min, adding 0.04 part by volume of a sodium citrate solution with the mass fraction of A3 being 1%, continuing to react for 10min until the synthetic liquid does not change color any more, stopping heating, continuing stirring, after the synthetic liquid is cooled to 25 ℃, bottling, and storing in a refrigerator at 4 ℃; the color change of the nano gold solution in the synthesis process is as follows: colorless → light gray → dark red → magenta → wine red → lucent red; the prepared nano gold solution is transparent and clear in color, good in transparency and almost free of impurity particles, and can be used.
2) Preparing an activated glassy carbon electrode. Sequentially polishing a glassy carbon electrode on chamois by using alumina powder with the particle size of 0.3 mu m and 0.05 mu m until the surface is smooth and no scratch is visible to naked eyes, washing the residual alumina powder on the polished glassy carbon electrode with deionized water, then respectively ultrasonically cleaning the glassy carbon electrode in absolute ethyl alcohol and deionized water for 5min, putting the cleaned glassy carbon electrode into 0.1mol/L sulfuric acid solution, then scanning for 10 circles within the voltage range of 0-1.8V by using a cyclic voltammetry scanning method to activate the electrode, and after activation is finished, washing the activated electrode with deionized water.
3) Bovine serum albumin solution was prepared. Dissolving 0.2g of bovine serum albumin by using a PH7.0 phosphate buffer solution to a constant volume of 100mL to obtain a bovine serum albumin solution with a volume fraction of 0.2%, and storing at 4 ℃.
4) A phosphate buffer solution at pH7.0 was prepared. 0.05mol/L dipotassium hydrogenphosphate and 0.05mol/L sodium dihydrogenphosphate were mixed at a ratio of 1:0.7 to obtain a phosphate buffer solution having a pH of 7.0.
2. The electrochemical immunosensor is used for detecting aflatoxin B1 and specifically comprises the following steps:
preparing phosphate buffer solution with pH of 7.0, adding antigen stock solution into the phosphate buffer solution, and sequentially diluting with gradient concentration to obtain 10-4、10-5、10-6、10-7、10-8、10-9Mu g/L of antigen dilution liquid base liquid, stirring the base liquid for 46s by a magnetic stirrer respectively to uniformly mix the base liquid, dripping different stirred base liquid on the surface of a glassy carbon electrode, and adopting a three-electrode system to carry out detection respectively, wherein during detection, an antigen binding cluster on an aflatoxin B1 monoclonal antibody on the glassy carbon electrode and an antigenic determinant on an aflatoxin B1 antigenic molecule are mutually attracted, so that the aflatoxin B1 monoclonal antibody is specifically bound with the aflatoxin B1 antigen, electron transfer is carried out, a weak electric signal is generated, signal amplification is realized through thionine, and finally, a cyclic voltammetry image is presented in an electrochemical workstation.
1) The three-electrode system includes: the modified glassy carbon electrode is a working electrode, the platinum electrode is a counter electrode, and the saturated potassium chloride electrode is a reference electrode.
2) The detection process of the electrochemical immunosensor on the aflatoxin B1 is carried out in a nitrogen atmosphere.
3) The method for processing the aflatoxin corn sample comprises the following steps: 0.5g of ground corn is weighed and mixed with 25mL of methanol-water mixed solution (methanol: deionized water = 7: 3), the mixture is put into a centrifuge after being uniformly mixed, and the centrifuge is used for 5min at the speed of 4000r/min, and supernatant is taken for standby.
Example 2
1. The electrochemical immunosensor is prepared by the following steps:
preparing a nano-gold solution, and then placing 1 volume part of graphene oxide with the concentration of 0.5wt% of A1, 1 weight part of thionine B1 and 5 volume parts of a1 nano-gold solution into a weighing bottle with a cover, wherein the relation between 1 volume part of A1 and 1 weight part of B1 is as follows: 1 ml corresponds to 1 mg, a stirring magneton is placed in a weighing bottle, the weighing bottle is covered and then placed on a magnetic stirring table to be stirred for about 4 hours to fully and uniformly mix materials in the bottle to obtain a graphene oxide/thionine/nanogold composite material, the graphene oxide/thionine/nanogold composite material 1S is dripped on the surface of an activated glassy carbon electrode in 1 volume part of A2, then the glassy carbon electrode is placed in a refrigerator at 2 ℃ to be stored for 10 hours until the surface of the glassy carbon electrode presents a layer of solid film, then 100 volume parts of A2 aflatoxin B1 monoclonal antibody and the glassy carbon electrode are sequentially placed in a first micropore reaction plate, the first micropore reaction plate is placed in the refrigerator at 2 ℃ for 2 hours, then the glassy carbon electrode is placed in the refrigerator at 2 ℃ until the surface of the glassy carbon electrode is dried, then 100 volume parts of A2 bovine serum protein solution and the glassy carbon electrode are sequentially placed in a second micropore reaction plate, standing in a refrigerator at 2 ℃ for 1min, taking out the glassy carbon electrode, and airing in the refrigerator at 2 ℃ to obtain a modified glassy carbon electrode, namely the electrochemical immunosensor;
1) and preparing a nano gold solution. Pouring 1 part by volume of a chloroauric acid solution with the mass fraction of A3 of 0.005% into a container, heating and stirring until the solution boils and lasts for 1min, adding 0.01 part by volume of a sodium citrate solution with the mass fraction of A3 of 0.2%, continuously reacting for 1min until the synthetic liquid does not change color any more, stopping heating, continuously stirring, after the synthetic liquid is cooled to 20 ℃, bottling, and storing in a refrigerator at 2 ℃; the color change of the nano gold solution in the synthesis process is as follows: colorless → light gray → dark red → magenta → wine red → lucent red; the prepared nano gold solution is transparent and clear in color, good in transparency and almost free of impurity particles, and can be used.
2) Preparing an activated glassy carbon electrode. Sequentially polishing a glassy carbon electrode on chamois by using alumina powder with the particle size of 0.1 mu m and 0.01 mu m until the surface is smooth and no scratch is visible to naked eyes, washing the residual alumina powder on the polished glassy carbon electrode with deionized water, then respectively ultrasonically cleaning the glassy carbon electrode in absolute ethyl alcohol and deionized water for 2min, putting the cleaned glassy carbon electrode in 0.03mol/L sulfuric acid solution, then scanning for 2 circles within the voltage range of 1.8-2.4V by using a cyclic voltammetry scanning method to activate the electrode, and after activation, washing the activated electrode with deionized water.
3) Bovine serum albumin solution was prepared. Dissolving 0.1g of bovine serum albumin by using a PH6.5 phosphate buffer solution to a constant volume of 60mL to obtain a bovine serum albumin solution with a volume fraction of 0.1%, and storing at 2 ℃.
4) A phosphate buffer solution at pH6.5 was prepared. 0.01mol/L dipotassium hydrogenphosphate and 0.01mol/L sodium dihydrogenphosphate were mixed at a ratio of 1:0.9 to obtain a phosphate buffer solution having a pH of 6.5.
2. The electrochemical immunosensor is used for detecting aflatoxin B1 and specifically comprises the following steps:
preparing phosphate buffer solution with pH of 6.5, adding antigen stock solution into the phosphate buffer solution, and sequentially diluting with gradient concentration to obtain 10-4、10-5、10-6、10-7、10-8、10-9Mu g/L of antigen dilution liquid base liquid, stirring the base liquid for 20s by a magnetic stirrer respectively to uniformly mix the base liquid, dripping different stirred base liquid on the surface of a glassy carbon electrode, and adopting a three-electrode system to detect respectively, wherein during detection, an antigen binding cluster on an aflatoxin B1 monoclonal antibody on the glassy carbon electrode and an antigenic determinant on an aflatoxin B1 antigenic molecule are mutually attracted, so that the aflatoxin B1 monoclonal antibody is specifically bound with the aflatoxin B1 antigen, electron transfer is generated, a weak electric signal is generated, signal amplification is realized through thionine, and finally, a cyclic voltammetry image is presented at an electrochemical workstation.
1) The three-electrode system includes: the modified glassy carbon electrode is a working electrode, the platinum electrode is a counter electrode, and the saturated potassium chloride electrode is a reference electrode.
2) The detection process of the electrochemical immunosensor on the aflatoxin B1 is carried out in a nitrogen atmosphere.
3) The method for processing the aflatoxin corn sample comprises the following steps: 0.5g of ground corn is weighed and mixed with 25mL of methanol-water mixed solution (methanol: deionized water = 7: 3), the mixture is put into a centrifuge after being uniformly mixed, and the centrifuge is used for 5min at the speed of 4000r/min, and supernatant is taken for standby.
Example 3
1. The electrochemical immunosensor is prepared by the following steps:
preparing a nanogold solution, and then placing 1 volume part of graphene oxide with the concentration of 1.5wt% of A1, 1 weight part of thionine B1 and 12 volume parts of a1 nanogold solution into a weighing bottle with a cover, wherein the relationship between 1 volume part of A1 and 1 weight part of B1 is as follows: 1 ml corresponds to 5 mg, a stirring magneton is placed in a weighing bottle, the weighing bottle is covered and then placed on a magnetic stirring table to be stirred for about 4 hours to fully and uniformly mix materials in the bottle to obtain a graphene oxide/thionine/nanogold composite material, the graphene oxide/thionine/nanogold composite material 3S is dripped on the surface of an activated glassy carbon electrode in 1 volume part of A2, then the glassy carbon electrode is placed in a refrigerator at 8 ℃ to be stored for 14 hours until the surface of the glassy carbon electrode presents a layer of solid film, then 120 volume parts of aflatoxin B1 monoclonal antibody of A2 and the glassy carbon electrode are sequentially placed in a first micropore reaction plate, the first micropore reaction plate is placed in the refrigerator at 8 ℃ for 8 hours, then the glassy carbon electrode is placed in the refrigerator at 8 ℃ until the surface of the glassy carbon electrode is dried, and then 120 volume parts of A2 bovine serum protein solution and the glassy carbon electrode are sequentially placed in a second micropore reaction plate, standing in a refrigerator at 8 ℃ for 5min, taking out the glassy carbon electrode, and airing in the refrigerator at 8 ℃ to obtain a modified glassy carbon electrode, namely the electrochemical immunosensor;
1) and preparing a nano gold solution. Pouring 1 part by volume of a chloroauric acid solution with the mass fraction of A3 being 0.05% into a container, heating and stirring until the solution boils and lasts for 10min, adding 0.1 part by volume of a sodium citrate solution with the mass fraction of A3 being 5%, continuing to react for 20min until the synthetic liquid does not change color any more, stopping heating, continuing to stir, after the synthetic liquid is cooled to 28 ℃, bottling, and storing in a refrigerator with the temperature of 8 ℃; the color change of the nano gold solution in the synthesis process is as follows: colorless → light gray → dark red → magenta → wine red → lucent red; the prepared nano gold solution is transparent and clear in color, good in transparency and almost free of impurity particles, and can be used.
2) Preparing an activated glassy carbon electrode. Sequentially polishing a glassy carbon electrode on chamois by using alumina powder with the particle size of 0.6 mu m and 0.1 mu m until the surface is smooth and no scratch is visible to naked eyes, washing the residual alumina powder on the polished glassy carbon electrode with deionized water, then respectively ultrasonically cleaning the glassy carbon electrode in absolute ethyl alcohol and deionized water for 10min, putting the cleaned glassy carbon electrode in 0.5mol/L sulfuric acid solution, then scanning for 15 circles within the voltage range of 2.4-3.6V by using a cyclic voltammetry scanning method to activate the electrode, and after activation, washing the activated electrode with deionized water.
3) Bovine serum albumin solution was prepared. Dissolving 0.5g of bovine serum albumin by using a PH7.4 phosphate buffer solution to a constant volume of 120mL to obtain a bovine serum albumin solution with a volume fraction of 0.4%, and storing at 8 ℃.
4) A phosphate buffer solution at pH7.4 was prepared. 0.15mol/L dipotassium hydrogen phosphate and 0.15mol/L sodium dihydrogen phosphate were mixed in a ratio of 1:0.5 to obtain a phosphate buffer solution having a pH of 7.4.
2. The electrochemical immunosensor is used for detecting aflatoxin B1 and specifically comprises the following steps:
preparing phosphate buffer solution with pH of 7.4, adding antigen stock solution into the phosphate buffer solution, and sequentially diluting with gradient concentration to obtain 10-4、10-5、10-6、10-7、10-8、10-9Mu g/L of antigen dilution liquid base liquid, stirring the base liquid for 70s by using a magnetic stirrer respectively to uniformly mix the base liquid, dripping different stirred base liquid on the surface of a glassy carbon electrode, and adopting a three-electrode system to carry out detection respectively, wherein during detection, an antigen binding cluster on an aflatoxin B1 monoclonal antibody on the glassy carbon electrode and an antigenic determinant on an aflatoxin B1 antigenic molecule are mutually attracted, so that the aflatoxin B1 monoclonal antibody is specifically bound with the aflatoxin B1 antigen, electron transfer is carried out, a weak electric signal is generated, signal amplification is realized through thionine, and finally, a cyclic voltammetry image is presented in an electrochemical workstation.
1) The three-electrode system includes: the modified glassy carbon electrode is a working electrode, the platinum electrode is a counter electrode, and the saturated potassium chloride electrode is a reference electrode.
2) The detection process of the electrochemical immunosensor on the aflatoxin B1 is carried out in a nitrogen atmosphere.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principles of the present invention are intended to be included within the scope of the present invention.
Claims (9)
1. A preparation method of an electrochemical immunosensor for aflatoxin B1 detection is characterized by comprising the following steps:
preparing a nanogold solution, then placing graphene oxide, thionine and the nanogold solution into a weighing bottle with a cover, stirring for 2-8h by using a stirrer to fully and uniformly mix materials in the bottle to obtain a graphene oxide/thionine/nanogold composite material, dripping the graphene oxide/thionine/nanogold composite material on the surface of an activated glassy carbon electrode within 1-3s, then placing the glassy carbon electrode into a refrigerator at 2-8 ℃ for storage for 10-14h until the surface of the glassy carbon electrode presents a layer of solid film, then placing an aflatoxin B1 monoclonal antibody and the glassy carbon electrode into a microporous reaction plate I in sequence, standing the microporous reaction plate I for 2-8h in the refrigerator at 2-8 ℃, then placing the glassy carbon electrode into the refrigerator at 2-8 ℃ until the surface of the glassy carbon electrode is dried, then placing a bovine serum albumin solution and the glassy carbon electrode into a microporous reaction plate II in sequence, standing in a refrigerator at 2-8 ℃ for 1-5min, taking out the glassy carbon electrode, and airing in the refrigerator at 2-8 ℃ to obtain a modified glassy carbon electrode, namely the electrochemical immunosensor;
in the preparation process of the graphene oxide/thionine/nano-gold composite material: the concentration of the graphene oxide is 0.5-1.5wt%, the dosage of the graphene oxide is 1 volume part of A1, the dosage of the thionine is 1 weight part of B1, and the dosage of the nano gold solution is 5-12 volume parts of A1; the correlation between 1 part by volume of A1 and 1 part by weight of B1 is as follows: 1 ml for 1-5 mg; in the preparation process of the electrochemical immunosensor: the graphene oxide/thionine/nanogold composite material is prepared from 1 part by volume of A2, the aflatoxin B1 monoclonal antibody is prepared from 100-120 parts by volume of A2, and the bovine serum albumin solution is prepared from 100-120 parts by volume of A2.
2. The method for preparing the electrochemical immunosensor for aflatoxin B1 detection according to claim 1, wherein the nanogold solution is prepared by pouring 1 part by volume of A3 solution with a mass fraction of 0.005-0.05% of chloroauric acid into a container, heating and stirring until the solution boils and lasts for 1-10min, adding 0.01-0.1 part by volume of A3 solution with a mass fraction of 0.2-5% of sodium citrate, continuing to react for 1-20min until the synthetic solution does not change color, stopping heating, continuing to stir, after the synthetic solution is cooled to 20-28 ℃, bottling and storing in a refrigerator with a temperature of 2-8 ℃.
3. The method for preparing the electrochemical immunosensor for aflatoxin B1 detection according to claim 1, it is characterized in that before the graphene oxide/thionine/nanogold composite material is dropped on the glassy carbon electrode, the glassy carbon electrode is sequentially polished on chamois leather by alumina powder with the grain diameters of 0.1-0.6 mu m and 0.01-0.1 mu m until the surface is smooth and no visible scratch is generated, the alumina powder remained on the glassy carbon electrode after polishing is washed clean by deionized water, then putting the glassy carbon electrode into absolute ethyl alcohol and deionized water in sequence, ultrasonically cleaning for 2-10min, putting the cleaned glassy carbon electrode into 0.03-0.5mol/L sulfuric acid solution, and then scanning for 2-15 circles within the voltage range of 0-3.6V by using a cyclic voltammetry scanning method to activate the electrode, and washing the activated electrode clean by using deionized water after activation.
4. The method for preparing an electrochemical immunosensor for aflatoxin B1 detection according to claim 1, wherein the bovine serum albumin solution is prepared by dissolving 0.1-0.5g of bovine serum albumin in pH6.5-7.4 phosphate buffer solution to a volume of 60-120mL, and the volume fraction of the bovine serum albumin solution is 0.1-0.4%.
5. The method for preparing an electrochemical immunosensor for aflatoxin B1 detection according to claim 4, wherein the phosphate buffer solution with pH of 6.5-7.4 is prepared by mixing 0.01-0.15mol/L dipotassium hydrogen phosphate and 0.01-0.15mol/L sodium dihydrogen phosphate in a ratio of 1: 0.9-0.5.
6. The method for preparing the electrochemical immunosensor for aflatoxin B1 detection according to claim 1, wherein the stirrer is a magnetic stirrer, the magnetic stirrer comprises a stirring magneton and a magnetic stirring table, the stirring magneton is placed in a weighing bottle, and the weighing bottle is placed on the magnetic stirring table.
7. The use method of the electrochemical immunosensor prepared by the preparation method according to claim 1, is characterized by comprising the following steps: preparing a phosphate buffer solution with the pH value of 6.5-7.4, adding an antigen stock solution into the phosphate buffer solution, diluting for multiple times to obtain a plurality of antigen diluent base solutions with different concentrations and reduced proportion, stirring the different base solutions for 20-70s by using a magnetic stirrer respectively to uniformly mix the different base solutions, dripping the different stirred base solutions on the surface of a glassy carbon electrode, and detecting by using a three-electrode system respectively.
8. The method of using the electrochemical immunosensor of claim 7, wherein the three-electrode system comprises: the modified glassy carbon electrode is a working electrode, the platinum electrode is a counter electrode, and the saturated potassium chloride electrode is a reference electrode.
9. The use method of the electrochemical immunosensor according to claim 7, wherein the detection process of the electrochemical immunosensor for aflatoxin B1 is performed in a nitrogen atmosphere.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810951371.6A CN109187963B (en) | 2018-08-21 | 2018-08-21 | Preparation and use methods of electrochemical immunosensor for aflatoxin B1 detection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810951371.6A CN109187963B (en) | 2018-08-21 | 2018-08-21 | Preparation and use methods of electrochemical immunosensor for aflatoxin B1 detection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109187963A CN109187963A (en) | 2019-01-11 |
| CN109187963B true CN109187963B (en) | 2021-11-05 |
Family
ID=64918630
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810951371.6A Active CN109187963B (en) | 2018-08-21 | 2018-08-21 | Preparation and use methods of electrochemical immunosensor for aflatoxin B1 detection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109187963B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109342526B (en) * | 2018-11-26 | 2020-12-11 | 东南大学 | Method for detecting aflatoxin B1 by electrochemical aptamer sensor |
| CN109709188B (en) * | 2019-02-27 | 2020-08-11 | 山东理工大学 | Preparation method and application of sandwich type immunosensor marked by nitrogen-sulfur double-doped graphene oxide |
| CN110208343B (en) * | 2019-05-10 | 2021-04-20 | 江苏大学 | Preparation method of ratiometric electrochemical biosensor for detecting aflatoxin B1 |
| CN111982989A (en) * | 2020-08-06 | 2020-11-24 | 成都师范学院 | SiO (silicon dioxide)2Preparation method and application of-MWCNTs (multi-wall carbon nanotubes) enzymatic glucose electrochemical sensor |
| CN112098490A (en) * | 2020-08-21 | 2020-12-18 | 华南理工大学 | Electrochemical biosensor for detecting aflatoxin B1 and preparation and application thereof |
| CN112179962B (en) * | 2020-09-29 | 2023-06-16 | 陕西科技大学 | Detection method of aflatoxin based on iron ion probe-nano gold/glassy carbon electrode modified electrode |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013157917A2 (en) * | 2012-04-20 | 2013-10-24 | Institut Penyelidikan Dan Kemajuan Pertanian Malaysia (Mardi) | A biosensor and preparation method thereof |
| CN105651840A (en) * | 2016-04-01 | 2016-06-08 | 商丘师范学院 | Mimic electrochemical immunosensor for detecting beta-amyloid protein oligomers and preparation method thereof |
| CN106093390A (en) * | 2016-06-01 | 2016-11-09 | 山东理工大学 | A kind of PtCu@g C3n4the preparation method and application of the electrochemical immunosensor of/rGO mark |
| CN106324057A (en) * | 2016-08-01 | 2017-01-11 | 红河学院 | Method for detecting aflatoxin through immunosensor |
| CN107422013A (en) * | 2017-06-29 | 2017-12-01 | 东南大学 | A kind of immunity biosensor for determining aflatoxin B1 and preparation method and application |
| CN108318553A (en) * | 2018-02-06 | 2018-07-24 | 红河学院 | AFB1Electrochemical immunosensor and preparation method thereof and its be used for aflatoxins B1Detection |
-
2018
- 2018-08-21 CN CN201810951371.6A patent/CN109187963B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013157917A2 (en) * | 2012-04-20 | 2013-10-24 | Institut Penyelidikan Dan Kemajuan Pertanian Malaysia (Mardi) | A biosensor and preparation method thereof |
| CN105651840A (en) * | 2016-04-01 | 2016-06-08 | 商丘师范学院 | Mimic electrochemical immunosensor for detecting beta-amyloid protein oligomers and preparation method thereof |
| CN106093390A (en) * | 2016-06-01 | 2016-11-09 | 山东理工大学 | A kind of PtCu@g C3n4the preparation method and application of the electrochemical immunosensor of/rGO mark |
| CN106324057A (en) * | 2016-08-01 | 2017-01-11 | 红河学院 | Method for detecting aflatoxin through immunosensor |
| CN107422013A (en) * | 2017-06-29 | 2017-12-01 | 东南大学 | A kind of immunity biosensor for determining aflatoxin B1 and preparation method and application |
| CN108318553A (en) * | 2018-02-06 | 2018-07-24 | 红河学院 | AFB1Electrochemical immunosensor and preparation method thereof and its be used for aflatoxins B1Detection |
Non-Patent Citations (1)
| Title |
|---|
| 氧化石墨烯-硫堇及纳米金修饰玻碳电极电流型葡萄糖生物传感器的研究;李岩 等;《福州大学学报(自然科学版)》;20111014;第39卷(第5期);第781-785页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109187963A (en) | 2019-01-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109187963B (en) | Preparation and use methods of electrochemical immunosensor for aflatoxin B1 detection | |
| Fu et al. | Flow-injection chemiluminescent immunoassay for α-fetoprotein based on epoxysilane modified glass microbeads | |
| Leng et al. | Gold nanoparticle as an electrochemical label for inherently crosstalk-free multiplexed immunoassay on a disposable chip | |
| Wu et al. | Development of a competitive immunochromatographic assay for the sensitive detection of amantadine in chicken muscle | |
| CN102507953B (en) | Preparation method of electrochemistry immunosensor for determining alpha fetoprotein | |
| Dou et al. | A disposable electrochemical immunosensor arrays using 4-channel screen-printed carbon electrode for simultaneous detection of Escherichia coli O157: H7 and Enterobacter sakazakii | |
| CN103235145B (en) | Preparation method and application of two-channel environmental estrogen immunosensor | |
| WO2013157917A2 (en) | A biosensor and preparation method thereof | |
| CN110296979B (en) | Electrochemiluminescence method for detecting bisphenol A | |
| CN105067690B (en) | A kind of preparation method of the estradiol electrochemical immunosensor built based on molybdenum bisuphide composite | |
| CN108593920B (en) | Immunosensor for detecting zearalenone and preparation method thereof | |
| Chen et al. | A copper monosulfide-nanoparticle-based fluorescent probe for the sensitive and specific detection of ochratoxin A | |
| CN110907511A (en) | Gold-curcumin nanoparticle quenched CdS hybrid TiO2Electrochemical luminescence sensor for detecting insulin by nanobelt | |
| CN110441295B (en) | Ferritin-based packaging Ir (ppy)3Preparation method of biosensor | |
| CN109187506B (en) | Preparation method and application of electrochemiluminescence sensor based on molecularly imprinted polymer-ferroferric oxide | |
| Manetta | Aflatoxins: Their measure and analysis | |
| CN110441528B (en) | Mo based on core-shell structure2Construction of C @ C nanosphere cardiac troponin I immunosensor | |
| Zhang et al. | Rapid determination of aflatoxin B1 by an automated immunomagnetic bead purification sample pretreatment method combined with high‐performance liquid chromatography | |
| CN110988067A (en) | Electrochemical luminescence method for detecting diethylstilbestrol | |
| Hu et al. | Polydopamine-mediated quantity-based magnetic relaxation sensing for the rapid and sensitive detection of chloramphenicol in fish samples | |
| CN100405063C (en) | Fabrication method and application for citrinin immune chromatography detection test paper | |
| CN104655848B (en) | Enzyme-linked immunosorbent assay (ELISA) detection kit for detecting ractopamine as well as preparation method and application of detection kit | |
| CN111398394B (en) | Preparation method of electrochemical sensor for detecting content of chloramphenicol | |
| CN105158453A (en) | Preparation method of label-free electrochemical immunosensor for nonyl phenol detection | |
| CN110988339A (en) | Time-resolved immune quantitative test strip for detecting aflatoxin M1 in milk |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |