CN110596218A - Preparation method of enzyme sensor for detecting organophosphorus pesticide, product and application thereof - Google Patents
Preparation method of enzyme sensor for detecting organophosphorus pesticide, product and application thereof Download PDFInfo
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Abstract
The invention provides a preparation method of an enzyme sensor for detecting organophosphorus pesticide, a product and an application thereof, wherein the enzyme sensor comprises an electrode substrate and a biological identification unit, wherein the biological identification unit is a biological enzyme layer prepared from acetylcholinesterase (AChE) and chitosan acetic acid solution; the electrode substrate is a glassy carbon electrode made of glassy carbon materials, and Ag-Fe is adopted3O4Nanoparticles and conductive polymers. The linear range of the sensor prepared by the invention for detecting the existing organophosphorus pesticide is 3.0 multiplied by 10‑13~6.0×10‑9mol/L, the detection limit of the sensor to the prior organophosphorus pesticide is 1.0 multiplied by 10‑13mol/L. The sensor is based on the high specificity of biological recognition and the amplification effect of the nano material on electrochemical detection signals, combines biochemistry, the nano material and sensing technology, and constructs a novel enzyme sensor modified by the composite nano material.
Description
Technical Field
The invention relates to a chemical sensor, in particular to a preparation method of an enzyme sensor for detecting organophosphorus pesticide, a product and application thereof.
Background
The organophosphorus pesticide mainly refers to phosphate, phosphorothioate and phosphoramidate organophosphorus compounds, and has the characteristics of high insecticidal efficiency, small phytotoxicity to plants, low persistence in the environment and the like, so that the organophosphorus pesticide becomes the most commonly used pesticide in China and is widely applied to various crops, particularly vegetables and fruits. However, the residual pesticide enters the human body along with the food chain, can inhibit the activity of cholinesterase in the human body, causes the metabolic disorder of nerve conduction media, and causes various acute and chronic poisoning conditions, such as dyskinesia, coma, paralysis and even death. Therefore, the monitoring and the detection of the organophosphorus pesticide are timely, accurate and sensitive, and the urgent problem to be solved urgently is formed.
The traditional pesticide residue detection method mainly comprises the following steps: gas chromatography-mass spectrometry, high performance liquid chromatography, spectrometry, immunoassay, chemiluminescence, etc. Although the method can accurately detect the residual quantity of the pesticide, the method has the defects of expensive equipment, complicated sample pretreatment method, long analysis period and the like.
The electrochemical detection technology is widely used for detecting samples due to the advantages of high sensitivity, high speed, simple and convenient operation and the like. Because the content of the pesticide residue is very low, the development of a high-sensitivity electrochemical sensor is of great significance for detecting the pesticide residue.
The basic structural unit of the enzyme biosensor is composed of a substance recognition element (immobilized enzyme membrane) and a signal converter (matrix electrode). when an enzymatic reaction occurs on the enzyme membrane, the produced electroactive substance is responded to by the matrix electrode. The matrix electrode is used for converting chemical signals into electrical signals, and can be carbon electrode (such as carbolic plate, glassy carbon electrode, and carbon shed electrode), R electrode, and corresponding modified electrode.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a preparation method of an enzyme sensor for detecting organophosphorus pesticide.
Yet another object of the present invention is to: provides an enzyme sensor product for detecting organophosphorus pesticide prepared by the method.
Yet another object of the present invention is to: provides an application of the product.
The purpose of the invention is realized by the following scheme: the enzyme sensor comprises an electrode substrate and a biological recognition unit, wherein the biological recognition unit is a biological enzyme prepared from acetylcholinesterase (AChE) and chitosan acetic acid solutionA layer; the electrode substrate is a glassy carbon electrode made of glassy carbon materials, and Ag-Fe is adopted3O4Nanoparticle and conductive polymer modification, comprising the following steps:
1) modification of the electrode substrate:
taking Fe3O4Ultrasonically dispersing the powder in secondary distilled water, adding sodium citrate solution, slowly adding sodium borohydride and silver nitrate while stirring, and stirring for a certain time to obtain Ag-Fe3O4A dispersion solution of composite nanoparticles;
putting the cleaned glassy carbon electrode into an M polymer monomer and graphene oxide solution, introducing nitrogen for 30 minutes, performing constant-current electrodeposition by adopting a three-electrode system, and performing electrochemical reduction on the obtained modified electrode to obtain a polymer/graphene oxide composite modified electrode;
2 mul ~ 20 mul Ag-Fe was taken3O4Dripping the dispersion solution of the composite nanoparticles on the surface of the dried polymer/graphene oxide composite material modified electrode, and naturally drying to obtain the modified electrode;
2) preparation of biological enzyme layer
Uniformly mixing 1 mu L of ~ 20 mu L of AChE (100U/mL) with equal volume of 0.5 mg/mL ~ 5 mg/mL chitosan acetic acid solution to obtain a biological enzyme mixed solution;
3) preparation of acetylcholine enzyme biosensor
Dripping 1 μ L ~ 20 μ L mixed solution of biological enzyme on the modified electrode, and naturally drying to obtain Ag-Fe3O4Nanoparticle/polymer modified acetylcholinesterase biosensors.
Wherein, the M polymer monomer is one of pyrrole, aniline and 3, 4-ethylenedioxythiophene.
The invention provides an enzyme sensor for detecting organophosphorus pesticide, which is prepared by any one of the methods.
The invention provides an application of an enzyme sensor in organophosphorus pesticide detection.
The invention carries out detection by the following steps:
the acetylcholinesterase catalyzes a substrate, namely thioacetylcholine (ATCH), to hydrolyze, so that products, namely thiocholine and acetic acid are generated, the thiocholine is easily oxidized under a specific potential to generate current, and the magnitude of a current signal is in a linear relation with the concentration of the thiocholine.
Recording the i-t response and the working curve of the sensor to different concentrations of ATCH under the condition of +0.6V by using a CHI832 type electrochemical system;
adding ATCH with known concentration into PBS after sensor signal is stabilized at +0.6V working potential, stirring for 5.0 min, and recording generated steady-state current I in static solution after current is stabilized1(ii) a Adding pesticide with a certain concentration into another ATCH solution under the same conditions, stirring, standing for several minutes, and measuring the steady-state current I of various organophosphorus pesticides with different concentrations2. The current decreases due to the inhibition of the enzyme activity, the degree of current decrease is proportional to the concentration of organophosphorus pesticide in the solution, and the inhibition ratio (I)1-I2)/I1Is linear with the logarithm of the concentration of organophosphorus, so that the concentration of organophosphorus pesticide can be detected by measuring the decrease in enzyme activity.
The method can realize the detection of the concentration of the organophosphorus pesticide by simple steps, and the linear range of the sensor for detecting the existing organophosphorus pesticide is 3.0 multiplied by 10-13 ~6.0×10-9mol/L, the detection limit of the sensor to the prior organophosphorus pesticide is 1.0 multiplied by 10-13 mol/L。
The sensor is based on the high specificity of biological recognition and the amplification effect of the nano material on electrochemical detection signals, combines biochemistry, the nano material and sensing technology, and constructs a novel enzyme sensor modified by the composite nano material.
Drawings
FIG. 1 is an i-t operating curve for thioacetylcholine at operating potential +0.6V for the sensor prepared in example 1.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The embodiments are implemented on the premise of the technical scheme of the invention, and detailed implementation modes and specific operation processes are given, but the protection scope of the invention is not limited by the following embodiments.
Example 1
An enzyme sensor for detecting organophosphorus pesticide comprises an electrode substrate and a biological recognition unit, wherein the biological recognition unit is a biological enzyme layer prepared from acetylcholinesterase (AChE) and chitosan acetic acid solution; the electrode substrate is a glassy carbon electrode made of glassy carbon materials, and Ag-Fe is adopted3O4Modifying nano particles, and preparing the following steps:
1) modification of the electrode substrate:
taking 3 mg of Fe with the average diameter of 20 nm3O4Ultrasonically dispersing the powder in 50 mL of secondary distilled water, adding 2.5 mL of 0.1mol/L sodium citrate solution with concentration, stirring for 10 min, slowly adding 8mL of 0.5mol/L sodium borohydride with concentration and 0.25 mL of 0.1mol/L silver nitrate with concentration while stirring, and stirring for 1 h to obtain silver-doped Ag-Fe3O4A dispersion solution of composite nanoparticles;
using 0.5 μm diameter Al for the cleaned bare glassy carbon electrode2O3Grinding and polishing on chamois leather to obtain mirror surface, sequentially adding acetone and HNO at volume ratio of 1:13Carrying out ultrasonic treatment on NaOH (1mol/L) and secondary water, then putting the electrode into a solution containing 0.1M pyrrole monomer and 0.5 mg/ml graphene oxide, introducing nitrogen for 30 minutes, and then carrying out constant current electrodeposition by adopting a three-electrode system, wherein the applied current is 0.5 mA cm-2The electric quantity of the electrodeposition is 1.2 coulombs; cleaning the obtained polypyrrole film modified electrode, placing the cleaned polypyrrole film modified electrode in a three-electrode system for electrochemical reduction, applying a voltage of 1.1V, using a PBS (phosphate buffer solution) as an electrolyte, and repeatedly cleaning the polypyrrole film modified electrode with water after reaction to obtain a conductive polymer modified electrode, namely a polypyrrole/graphene oxide composite modified electrode;
taking 8 mu L of Ag-Fe3O4Dripping the dispersion solution of the composite nanoparticles on the surface of the dried polymer/graphene oxide composite material modified electrode, and naturally drying to obtain the modified electrode;
2) preparation of biological enzyme layer
Uniformly mixing 2 mu L of AChE with the concentration of 100U/mL and chitosan acetic acid solution with the equal volume concentration of 1mg/mL to obtain a biological enzyme mixed solution;
3) preparation of acetylcholine enzyme biosensor
Dripping 8 μ L of the mixed solution of the biological enzyme on an electrode modified with the nano particles/conductive polymer, and naturally drying to obtain Ag-Fe3O4Nanoparticle/polymer modified acetylcholinesterase biosensors.
FIG. 1 is a graph showing the i-t response curves of the resulting sensors at a working potential of +0.6V for various concentrations of thioacetylcholine.
Example 2
An enzyme sensor for detecting organophosphorus pesticide, similar to example 1, is prepared by the following steps:
1) modification of the electrode substrate:
taking 3 mg of Fe with the average diameter of 30 nm3O4Ultrasonically dispersing the powder in 35 mL of secondary distilled water, adding 1.88 mL of sodium citrate solution with the concentration of 0.1mol/L, stirring for 10 min, then slowly adding 5mL of sodium borohydride with the concentration of 0.5mol/L and 0.35 mL of silver nitrate with the concentration of 0.1mol/L while stirring, and stirring for 2 h to obtain silver-doped Ag-Fe3O4A dispersion solution of composite nanoparticles;
using 0.5 μm diameter Al for bare glassy carbon electrode2O3Grinding and polishing on chamois leather to obtain mirror surface, sequentially adding acetone and HNO at volume ratio of 1:13Performing ultrasonic treatment on NaOH (1mol/L) and secondary water, then putting the electrode into an aqueous solution containing 0.2M pyrrole monomer and 1.0 mg/ml graphene oxide, introducing nitrogen for 30 minutes, and performing constant-current electrodeposition by adopting a three-electrode system with the applied current of 2 mA cm-2The electric quantity of the electrodeposition is 2.0 coulombs; then, cleaning the obtained polypyrrole film modified electrode, placing the cleaned polypyrrole film modified electrode in a three-electrode system for electrochemical reduction, applying a voltage of 1.2V, using a PBS (phosphate buffer solution) as an electrolyte, and repeatedly cleaning the polypyrrole film modified electrode with water after reaction to obtain a conductive polymer modified electrode, namely a polypyrrole/graphene oxide composite modified electrode;
taking 13 mu L of Ag-Fe3O4Drop coating of composite nanoparticle dispersion solution on air-dried polypyrrole/oxideModifying the surface of the electrode by using the graphene composite material, and naturally airing to obtain the electrode modified with the nano particles/conductive polymer;
2) preparation of biological enzyme layer
Uniformly mixing 10 mu L of AChE with the concentration of 100U/mL and chitosan acetic acid solution with the equal volume concentration of 1mg/mL to obtain a biological enzyme mixed solution;
3) preparation of acetylcholine enzyme biosensor
Dripping 10 μ L of the mixed solution of the biological enzyme on an electrode modified with the nano particles/conductive polymer, and naturally drying to obtain Ag-Fe3O4Nanoparticle/polymer modified acetylcholinesterase biosensors. The sensor is used for detecting organophosphorus pesticide.
Example 3
An enzyme sensor for detecting organophosphorus pesticide, similar to example 1, is prepared by the following steps:
1) modification of the electrode substrate:
taking 5 mg of Fe with the average diameter of 80 nm3O4Ultrasonically dispersing the powder in 50 mL of secondary distilled water, adding 4 mL of sodium citrate solution with the concentration of 0.5mol/L, stirring for 10 min, slowly adding 10mL of sodium borohydride with the concentration of 0.5mol/L and 0.5 mL of silver nitrate with the concentration of 0.1mol/L while stirring, and stirring for 5 h to obtain silver-doped Ag-Fe3O4A dispersion solution of composite nanoparticles;
using 0.5 μm diameter Al for bare glassy carbon electrode2O3Grinding and polishing on chamois leather to obtain mirror surface, sequentially adding acetone and HNO at volume ratio of 1:13Performing ultrasonic treatment on NaOH (1mol/L) and secondary water, putting the cleaned glassy carbon electrode into a solution containing 0.05M aniline monomer and 1.0 mg/ml graphene oxide, introducing nitrogen for 30 minutes, and performing constant current electrodeposition by adopting a three-electrode system, wherein the applied current is 1 mA cm-2The electric quantity of the electrodeposition is 1.6 coulombs; washing the obtained polyaniline membrane electrode, placing the polyaniline membrane electrode in a three-electrode system for electrochemical reduction, applying a voltage of 1.0V, using a PBS (phosphate buffer solution) as an electrolyte, repeatedly washing with water after reaction, and drying in the air to obtain a conductive polymer modified electrode, namely the poly-polymer modified electrodeModifying the electrode by using an aniline/graphene oxide composite material;
5 mu L of Ag-Fe is taken3O4Dripping the dispersion solution of the composite nanoparticles on the surface of the electrode modified by the dried polyaniline/graphene oxide composite material, and naturally drying to obtain the electrode modified by the nanoparticles/conductive polymer;
2) preparation of biological enzyme layer
Uniformly mixing 2 mu L of AChE (100U/mL) with 1mg/mL chitosan acetic acid solution with the same volume to obtain a biological enzyme mixed solution;
3) preparation of acetylcholine enzyme biosensor
Dripping 2 μ L of the mixed solution of the biological enzyme on the modified electrode, and naturally drying to obtain Ag-Fe3O4Nanoparticle/conducting polymer modified acetylcholinesterase biosensor.
Example 4
An enzyme sensor for detecting organophosphorus pesticide, similar to example 1, is prepared by the following steps:
1) modification of the electrode substrate:
5 mg of Fe with an average diameter of 200 nm were taken3O4Ultrasonically dispersing the powder in 50 mL of secondary distilled water, adding 4 mL of 1mol/L sodium citrate solution, stirring for 10 min, slowly adding 3mL of 0.2mol/L sodium borohydride and 0.25 mL of 0.3mol/L silver nitrate while stirring, and stirring for 8 h to obtain silver-doped Ag-Fe3O4A dispersion solution of composite nanoparticles;
using 0.5 μm diameter Al for bare glassy carbon electrode2O3Grinding and polishing on chamois leather to obtain mirror surface, sequentially adding acetone and HNO at volume ratio of 1:13Carrying out ultrasonic treatment on NaOH (1mol/L) and secondary water, then putting the cleaned glassy carbon electrode into a solution containing 0.2M pyrrole monomer and 1.0 mg/ml graphene oxide, introducing nitrogen for 30 minutes, and then carrying out constant current electrodeposition by adopting a three-electrode system, wherein the applied current is 2 mA cm-2The electric quantity of the electrodeposition is 2.0 coulombs; then, the obtained polypyrrole film modified electrode is cleaned and placed in a three-electrode system for electrochemical reduction, the applied voltage is 1.2V, and electricity is generatedThe electrolyte is PBS buffer solution, and after reaction, the electrolyte is repeatedly washed by water to obtain a conductive polymer modified electrode, namely the polypyrrole/graphene oxide composite material modified electrode is obtained;
5 mu L of Ag-Fe is taken3O4Dripping the dispersion solution of the composite nanoparticles on the surface of the electrode modified by the air-dried polypyrrole/graphene oxide composite material, and naturally drying to obtain the electrode modified with the nanoparticles/conductive polymer;
2) preparation of biological enzyme layer
Uniformly mixing 2 mu L of AChE with the concentration of 100U/mL and chitosan acetic acid solution with the equal volume concentration of 1mg/mL to obtain a biological enzyme mixed solution;
3) preparation of acetylcholine enzyme biosensor
Dripping 2 μ L of the mixed solution of the biological enzyme on an electrode modified with the nano particles/conductive polymer, and naturally drying to obtain Ag-Fe3O4And (3) obtaining the organophosphorus pesticide detection sensor by using the nanoparticle/conductive polymer modified acetylcholine enzyme biosensor.
Claims (8)
1. A preparation method of an enzyme sensor for detecting organophosphorus pesticide, the enzyme sensor comprises an electrode substrate and a biological recognition unit, and is characterized in that the biological recognition unit is a biological enzyme layer prepared from acetylcholinesterase (AChE) and chitosan acetate solution; the electrode substrate is a glassy carbon electrode made of glassy carbon materials, and Ag-Fe is adopted3O4Nanoparticle and conductive polymer modification, comprising the following steps:
1) modification of the electrode substrate:
taking Fe3O4Ultrasonically dispersing the powder in secondary distilled water, adding sodium citrate solution, slowly adding sodium borohydride and silver nitrate while stirring, and stirring for a certain time to obtain Ag-Fe3O4A dispersion solution of composite nanoparticles;
putting the cleaned glassy carbon electrode into an M polymer monomer and graphene oxide solution, introducing nitrogen for 30 minutes, performing constant-current electrodeposition by adopting a three-electrode system, and performing electrochemical reduction on the obtained modified electrode to obtain a polymer/graphene oxide composite modified electrode;
2 mul ~ 20 mul Ag-Fe was taken3O4Dripping the dispersion solution of the composite nanoparticles on the surface of the dried polymer/graphene oxide composite material modified electrode, and naturally drying to obtain the modified electrode;
2) preparation of biological enzyme layer
Uniformly mixing 1 mu L of ~ 20 mu L of AChE (100U/mL) with equal volume of 0.5 mg/mL ~ 5 mg/mL chitosan acetic acid solution to obtain a biological enzyme mixed solution;
3) preparation of acetylcholine enzyme biosensor
Dripping 1 μ L ~ 20 μ L mixed solution of biological enzyme on the modified electrode, and naturally drying to obtain Ag-Fe3O4Nanoparticle/polymer modified acetylcholinesterase biosensors.
2. The method for preparing the enzyme sensor for detecting the organophosphorus pesticide according to claim 1, wherein the M polymer monomer is one of pyrrole, aniline, and 3, 4-ethylenedioxythiophene.
3. The method for preparing the enzyme sensor for detecting organophosphorus pesticide according to claim 1 or 2, characterized by comprising the steps of:
1) modification of the electrode substrate:
taking 3 mg of Fe with the average diameter of 20 nm3O4Ultrasonically dispersing the powder in 50 mL of secondary distilled water, adding 2.5 mL of 0.1mol/L sodium citrate solution with concentration, stirring for 10 min, slowly adding 8mL of 0.5mol/L sodium borohydride with concentration and 0.25 mL of 0.1mol/L silver nitrate with concentration while stirring, and stirring for 1 h to obtain silver-doped Ag-Fe3O4A dispersion solution of composite nanoparticles;
using 0.5 μm diameter Al for the cleaned bare glassy carbon electrode2O3Grinding and polishing on chamois leather to obtain mirror surface, sequentially adding acetone and HNO at volume ratio of 1:13NaOH (1mol/L) and secondary water, then the electrode was put in a solution containing 0.1M pyrrole monomer and 0.5 mg/ml of oxygenIntroducing nitrogen into the graphene solution for 30 minutes, and then performing constant-current electrodeposition by adopting a three-electrode system, wherein the applied current is 0.5 mA cm-2The electric quantity of the electrodeposition is 1.2 coulombs; cleaning the obtained polypyrrole film modified electrode, placing the cleaned polypyrrole film modified electrode in a three-electrode system for electrochemical reduction, applying a voltage of 1.1V, using a PBS (phosphate buffer solution) as an electrolyte, and repeatedly cleaning the polypyrrole film modified electrode with water after reaction to obtain a conductive polymer modified electrode, namely a polypyrrole/graphene oxide composite modified electrode;
taking 8 mu L of Ag-Fe3O4Dripping the dispersion solution of the composite nanoparticles on the surface of the dried polymer/graphene oxide composite material modified electrode, and naturally drying to obtain the modified electrode;
2) preparation of biological enzyme layer
Uniformly mixing 2 mu L of AChE with the concentration of 100U/mL and chitosan acetic acid solution with the equal volume concentration of 1mg/mL to obtain a biological enzyme mixed solution;
3) preparation of acetylcholine enzyme biosensor
Dripping 8 μ L of the mixed solution of the biological enzyme on an electrode modified with the nano particles/conductive polymer, and naturally drying to obtain Ag-Fe3O4Nanoparticle/polymer modified acetylcholinesterase biosensors.
4. The method for preparing the enzyme sensor for detecting organophosphorus pesticide according to claim 1 or 2, characterized by comprising the steps of:
1) modification of the electrode substrate:
taking 3 mg of Fe with the average diameter of 30 nm3O4Ultrasonically dispersing the powder in 35 mL of secondary distilled water, adding 1.88 mL of sodium citrate solution with the concentration of 0.1mol/L, stirring for 10 min, then slowly adding 5mL of sodium borohydride with the concentration of 0.5mol/L and 0.35 mL of silver nitrate with the concentration of 0.1mol/L while stirring, and stirring for 2 h to obtain silver-doped Ag-Fe3O4A dispersion solution of composite nanoparticles;
using 0.5 μm diameter Al for bare glassy carbon electrode2O3Pasting on chamois leather, grinding and polishing to obtain mirror surface, sequentially adding acetone and HN with volume ratio of 1:1O3Performing ultrasonic treatment on NaOH (1mol/L) and secondary water, then putting the electrode into an aqueous solution containing 0.2M pyrrole monomer and 1.0 mg/ml graphene oxide, introducing nitrogen for 30 minutes, and performing constant-current electrodeposition by adopting a three-electrode system with the applied current of 2 mA cm-2The electric quantity of the electrodeposition is 2.0 coulombs; then, cleaning the obtained polypyrrole film modified electrode, placing the cleaned polypyrrole film modified electrode in a three-electrode system for electrochemical reduction, applying a voltage of 1.2V, using a PBS (phosphate buffer solution) as an electrolyte, and repeatedly cleaning the polypyrrole film modified electrode with water after reaction to obtain a conductive polymer modified electrode, namely a polypyrrole/graphene oxide composite modified electrode;
taking 13 mu L of Ag-Fe3O4Dripping the dispersion solution of the composite nanoparticles on the surface of the electrode modified by the air-dried polypyrrole/graphene oxide composite material, and naturally drying to obtain the electrode modified with the nanoparticles/conductive polymer;
2) preparation of biological enzyme layer
Uniformly mixing 10 mu L of AChE with the concentration of 100U/mL and chitosan acetic acid solution with the equal volume concentration of 1mg/mL to obtain a biological enzyme mixed solution;
3) preparation of acetylcholine enzyme biosensor
Dripping 10 μ L of the mixed solution of the biological enzyme on an electrode modified with the nano particles/conductive polymer, and naturally drying to obtain Ag-Fe3O4Nanoparticle/polymer modified acetylcholinesterase biosensors. The sensor is used for detecting organophosphorus pesticide.
5. The method for preparing the enzyme sensor for detecting organophosphorus pesticide according to claim 1 or 2, characterized by comprising the steps of:
1) modification of the electrode substrate:
taking 5 mg of Fe with the average diameter of 80 nm3O4Ultrasonically dispersing the powder in 50 mL of secondary distilled water, adding 4 mL of sodium citrate solution with the concentration of 0.5mol/L, stirring for 10 min, slowly adding 10mL of sodium borohydride with the concentration of 0.5mol/L and 0.5 mL of silver nitrate with the concentration of 0.1mol/L while stirring, and stirring for 5 h to obtain silver-doped Ag-Fe3O4Composite nanoparticlesA dispersion solution of seeds;
using 0.5 μm diameter Al for bare glassy carbon electrode2O3Grinding and polishing on chamois leather to obtain mirror surface, sequentially adding acetone and HNO at volume ratio of 1:13Performing ultrasonic treatment on NaOH (1mol/L) and secondary water, putting the cleaned glassy carbon electrode into a solution containing 0.05M aniline monomer and 1.0 mg/ml graphene oxide, introducing nitrogen for 30 minutes, and performing constant current electrodeposition by adopting a three-electrode system, wherein the applied current is 1 mA cm-2The electric quantity of the electrodeposition is 1.6 coulombs; cleaning the obtained polyaniline membrane electrode, placing the polyaniline membrane electrode in a three-electrode system for electrochemical reduction, applying a voltage of 1.0V, wherein an electrolyte is a PBS (phosphate buffer solution), repeatedly washing with water after reaction, and drying in the air to obtain a conductive polymer modified electrode, namely the polyaniline/graphene oxide composite modified electrode;
5 mu L of Ag-Fe is taken3O4Dripping the dispersion solution of the composite nanoparticles on the surface of the electrode modified by the dried polyaniline/graphene oxide composite material, and naturally drying to obtain the electrode modified by the nanoparticles/conductive polymer;
2) preparation of biological enzyme layer
Uniformly mixing 2 mu L of AChE (100U/mL) with 1mg/mL chitosan acetic acid solution with the same volume to obtain a biological enzyme mixed solution;
3) preparation of acetylcholine enzyme biosensor
Dripping 2 μ L of the mixed solution of the biological enzyme on the modified electrode, and naturally drying to obtain Ag-Fe3O4Nanoparticle/conducting polymer modified acetylcholinesterase biosensor.
6. The method for preparing the enzyme sensor for detecting organophosphorus pesticide according to claim 1 or 2, characterized by comprising the steps of:
1) modification of the electrode substrate:
5 mg of Fe with an average diameter of 200 nm were taken3O4Ultrasonically dispersing the powder in 50 mL of secondary distilled water, adding 4 mL of 1mol/L sodium citrate solution, stirring for 10 min, and slowly adding 3mL of 0.2mol/L borohydride while stirringDissolving sodium and 0.25 mL of silver nitrate with the concentration of 0.3mol/L, and stirring for 8 hours to obtain Ag-Fe doped with silver3O4A dispersion solution of composite nanoparticles;
using 0.5 μm diameter Al for bare glassy carbon electrode2O3Grinding and polishing on chamois leather to obtain mirror surface, sequentially adding acetone and HNO at volume ratio of 1:13Carrying out ultrasonic treatment on NaOH (1mol/L) and secondary water, then putting the cleaned glassy carbon electrode into a solution containing 0.2M pyrrole monomer and 1.0 mg/ml graphene oxide, introducing nitrogen for 30 minutes, and then carrying out constant current electrodeposition by adopting a three-electrode system, wherein the applied current is 2 mA cm-2The electric quantity of the electrodeposition is 2.0 coulombs; then, cleaning the obtained polypyrrole film modified electrode, placing the cleaned polypyrrole film modified electrode in a three-electrode system for electrochemical reduction, applying a voltage of 1.2V, using a PBS (phosphate buffer solution) as an electrolyte, and repeatedly washing the polypyrrole film modified electrode with water after reaction to obtain a conductive polymer modified electrode, namely the polypyrrole/graphene oxide composite modified electrode;
5 mu L of Ag-Fe is taken3O4Dripping the dispersion solution of the composite nanoparticles on the surface of the electrode modified by the air-dried polypyrrole/graphene oxide composite material, and naturally drying to obtain the electrode modified with the nanoparticles/conductive polymer;
2) preparation of biological enzyme layer
Uniformly mixing 2 mu L of AChE with the concentration of 100U/mL and chitosan acetic acid solution with the equal volume concentration of 1mg/mL to obtain a biological enzyme mixed solution;
3) preparation of acetylcholine enzyme biosensor
Dripping 2 μ L of the mixed solution of the biological enzyme on an electrode modified with the nano particles/conductive polymer, and naturally drying to obtain Ag-Fe3O4And (3) obtaining the organophosphorus pesticide detection sensor by using the nanoparticle/conductive polymer modified acetylcholine enzyme biosensor.
7. An enzyme sensor for detecting organophosphorus pesticides, which is produced by the method according to any one of claims 1 to 6.
8. Use of the enzyme sensor according to claim 7 for the detection of organophosphorus pesticides.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111521658A (en) * | 2020-06-12 | 2020-08-11 | 上海纳米技术及应用国家工程研究中心有限公司 | Preparation method of high-sensitivity sensor for pesticide detection, product and application thereof |
| CN112903784A (en) * | 2021-02-04 | 2021-06-04 | 西北农林科技大学 | Enzyme ink, preparation method and biosensor |
| CN114487044A (en) * | 2022-01-20 | 2022-05-13 | 大连理工大学 | Electrochemical enzyme biosensor for detecting organophosphorus pesticide, preparation method and application |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277590A (en) * | 2011-06-13 | 2011-12-14 | 河北科技大学 | Preparation method of polypyrrole functional mediator doped with water-soluble anthraquinone or naphthoquinone compound and application thereof |
| CN102749442A (en) * | 2012-07-26 | 2012-10-24 | 济南大学 | Manufacture method of silver hybridization mesoporous ferroferric oxide antibiotic immunosensor and application thereof |
| CN102890112A (en) * | 2011-07-20 | 2013-01-23 | 上海纳米技术及应用国家工程研究中心有限公司 | Enzyme sensor for organophosphorus pesticide detection and preparation method thereof |
| CN104332639A (en) * | 2014-11-05 | 2015-02-04 | 上海纳米技术及应用国家工程研究中心有限公司 | Preparation method of co-doped conductive polymer electrode material |
| CN105223249A (en) * | 2015-10-16 | 2016-01-06 | 上海纳米技术及应用国家工程研究中心有限公司 | A kind of preparation method of the chemical sensor for thrombocytin detection |
| CN105388200A (en) * | 2015-10-16 | 2016-03-09 | 上海纳米技术及应用国家工程研究中心有限公司 | Method for preparing sensor used for organic phosphorus pesticide detection |
-
2019
- 2019-10-09 CN CN201910953571.XA patent/CN110596218A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102277590A (en) * | 2011-06-13 | 2011-12-14 | 河北科技大学 | Preparation method of polypyrrole functional mediator doped with water-soluble anthraquinone or naphthoquinone compound and application thereof |
| CN102890112A (en) * | 2011-07-20 | 2013-01-23 | 上海纳米技术及应用国家工程研究中心有限公司 | Enzyme sensor for organophosphorus pesticide detection and preparation method thereof |
| CN102749442A (en) * | 2012-07-26 | 2012-10-24 | 济南大学 | Manufacture method of silver hybridization mesoporous ferroferric oxide antibiotic immunosensor and application thereof |
| CN104332639A (en) * | 2014-11-05 | 2015-02-04 | 上海纳米技术及应用国家工程研究中心有限公司 | Preparation method of co-doped conductive polymer electrode material |
| CN105223249A (en) * | 2015-10-16 | 2016-01-06 | 上海纳米技术及应用国家工程研究中心有限公司 | A kind of preparation method of the chemical sensor for thrombocytin detection |
| CN105388200A (en) * | 2015-10-16 | 2016-03-09 | 上海纳米技术及应用国家工程研究中心有限公司 | Method for preparing sensor used for organic phosphorus pesticide detection |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111521658A (en) * | 2020-06-12 | 2020-08-11 | 上海纳米技术及应用国家工程研究中心有限公司 | Preparation method of high-sensitivity sensor for pesticide detection, product and application thereof |
| CN112903784A (en) * | 2021-02-04 | 2021-06-04 | 西北农林科技大学 | Enzyme ink, preparation method and biosensor |
| CN114487044A (en) * | 2022-01-20 | 2022-05-13 | 大连理工大学 | Electrochemical enzyme biosensor for detecting organophosphorus pesticide, preparation method and application |
| CN114487044B (en) * | 2022-01-20 | 2022-11-01 | 大连理工大学 | Preparation method and application of electrochemical enzyme biosensor for detecting organophosphorus pesticide |
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Application publication date: 20191220 |