CN111521658A - Preparation method of high-sensitivity sensor for pesticide detection, product and application thereof - Google Patents
Preparation method of high-sensitivity sensor for pesticide detection, product and application thereof Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 35
- 239000000575 pesticide Substances 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 48
- 239000002105 nanoparticle Substances 0.000 claims abstract description 40
- 239000002131 composite material Substances 0.000 claims abstract description 31
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- 229910052906 cristobalite Inorganic materials 0.000 claims abstract description 30
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 30
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 30
- 229910052682 stishovite Inorganic materials 0.000 claims abstract description 30
- 229910052905 tridymite Inorganic materials 0.000 claims abstract description 30
- 229910021397 glassy carbon Inorganic materials 0.000 claims abstract description 22
- 229920000642 polymer Polymers 0.000 claims abstract description 22
- 108010022752 Acetylcholinesterase Proteins 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 15
- 229920001661 Chitosan Polymers 0.000 claims abstract description 14
- 102000004190 Enzymes Human genes 0.000 claims abstract description 13
- 108090000790 Enzymes Proteins 0.000 claims abstract description 13
- 229940088598 enzyme Drugs 0.000 claims abstract description 13
- 229940022698 acetylcholinesterase Drugs 0.000 claims abstract description 10
- 239000000758 substrate Substances 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims description 55
- 238000003756 stirring Methods 0.000 claims description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical group O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 25
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 20
- 239000006185 dispersion Substances 0.000 claims description 20
- 238000001035 drying Methods 0.000 claims description 20
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 20
- 102100033639 Acetylcholinesterase Human genes 0.000 claims description 19
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- 229920001940 conductive polymer Polymers 0.000 claims description 18
- 238000004070 electrodeposition Methods 0.000 claims description 18
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 16
- KAESVJOAVNADME-UHFFFAOYSA-N Pyrrole Chemical compound C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 claims description 16
- 239000003987 organophosphate pesticide Substances 0.000 claims description 16
- 229920000128 polypyrrole Polymers 0.000 claims description 15
- 239000008055 phosphate buffer solution Substances 0.000 claims description 14
- 239000000178 monomer Substances 0.000 claims description 12
- 239000011259 mixed solution Substances 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- 229910052757 nitrogen Inorganic materials 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 10
- 229910001961 silver nitrate Inorganic materials 0.000 claims description 10
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 10
- 239000012279 sodium borohydride Substances 0.000 claims description 10
- 239000001509 sodium citrate Substances 0.000 claims description 10
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 10
- 239000012153 distilled water Substances 0.000 claims description 9
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 claims description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 8
- 241001481789 Rupicapra Species 0.000 claims description 8
- 239000003792 electrolyte Substances 0.000 claims description 8
- 229910021389 graphene Inorganic materials 0.000 claims description 8
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- 238000004140 cleaning Methods 0.000 claims description 6
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- 238000009210 therapy by ultrasound Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 230000007423 decrease Effects 0.000 claims description 5
- 229920000767 polyaniline Polymers 0.000 claims description 5
- 230000035945 sensitivity Effects 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 3
- 230000005764 inhibitory process Effects 0.000 claims description 3
- GKWLILHTTGWKLQ-UHFFFAOYSA-N 2,3-dihydrothieno[3,4-b][1,4]dioxine Chemical compound O1CCOC2=CSC=C21 GKWLILHTTGWKLQ-UHFFFAOYSA-N 0.000 claims description 2
- 230000003068 static effect Effects 0.000 claims description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims 1
- 239000002953 phosphate buffered saline Substances 0.000 claims 1
- 239000002086 nanomaterial Substances 0.000 abstract description 5
- 238000000835 electrochemical detection Methods 0.000 abstract description 3
- 230000003321 amplification Effects 0.000 abstract description 2
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 2
- 102000012440 Acetylcholinesterase Human genes 0.000 abstract 1
- 239000003905 agrochemical Substances 0.000 abstract 1
- 239000000447 pesticide residue Substances 0.000 description 3
- 102000003914 Cholinesterases Human genes 0.000 description 1
- 108090000322 Cholinesterases Proteins 0.000 description 1
- 206010010071 Coma Diseases 0.000 description 1
- 208000012661 Dyskinesia Diseases 0.000 description 1
- 206010033799 Paralysis Diseases 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 231100000570 acute poisoning Toxicity 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229940048961 cholinesterase Drugs 0.000 description 1
- 231100000739 chronic poisoning Toxicity 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
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- 208000030159 metabolic disease Diseases 0.000 description 1
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- 238000002203 pretreatment Methods 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
Abstract
The invention relates to a preparation method of a high-sensitivity sensor for pesticide detection, a product and application thereof, wherein the preparation method comprises the following steps: a biological enzyme layer, a nano particle layer/polymer layer and an electrode substrate, wherein the nano particles are Ag-SiO2(ii) a The electrode substrate is made of glassy carbon; the enzyme solution is acetylcholinesterase and chitosan acetic acid solution. A method for preparing the sensor for quickly, sensitively and easily detecting agricultural chemical features that the biochemical, nano material and sensing technique are combined together based on the high specificity of biological recognition and the amplification of electrochemical detection signal by nano material to construct a new composite nano materialA modified enzyme sensor.
Description
Technical Field
The invention relates to a chemical sensor, in particular to a preparation method of a high-sensitivity sensor for pesticide detection, a product and application thereof.
Background
The residual pesticide enters into human body along with food chain, and can inhibit cholinesterase activity in human body, and cause metabolism disorder of nerve conduction medium, and 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.
Disclosure of Invention
In view of the above-mentioned disadvantages of the prior art, the present invention aims to provide a method for preparing a high-sensitivity sensor for detecting pesticides.
Yet another object of the present invention is to: provides a high-sensitivity sensor product for pesticide detection 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: a preparation method of a high-sensitivity sensor for pesticide detection is characterized by comprising the following steps: a biological enzyme layer, a nano particle layer/polymer layer and an electrode substrate, wherein the nano particles are Ag-SiO2(ii) a The electrode substrate is made of glassy carbon; the enzyme solution is a solution of acetylcholinesterase and chitosan acetic acid, and the specific steps are as follows:
(1) taking SiO2Ultrasonically 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-SiO2A dispersion solution of composite nanoparticles;
(2) putting the cleaned glassy carbon electrode into an M polymer monomer 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 composite modified electrode;
(3) taking 2 mu L-20 mu L of Ag-SiO2Dripping the nano particle dispersion solution on the surface of an air-dried polymer composite material modified electrode, naturally drying, uniformly mixing 1-20 mu L of AChE (100U/mL) with 0.5-5 mg/mL chitosan acetic acid solution with the same volume, dripping 1-20 mu L of the mixed solution on the modified electrode, and naturally drying to obtain the Ag-SiO2Nanoparticle/polymer modified acetylcholinesterase biosensors.
The M polymer monomer is one of pyrrole, aniline and 3, 4-ethylenedioxythiophene.
The invention provides a high-sensitivity sensor for pesticide detection, which is prepared by any one of the methods.
The invention provides an application of a high-sensitivity 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 5.0min, 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 decrease is caused by the inhibition of the enzyme activity, the degree of current decrease is proportional to the concentration of the organophosphorus pesticide in the solution, and the inhibitionSystem rate (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 detection limit of the sensor to the existing organophosphorus pesticide is 1.0 × 10-13mol/L。
The invention has the advantages that: provides a sensor for rapidly, sensitively, simply and conveniently detecting organophosphorus pesticides. 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
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
A high sensitivity sensor for pesticide detection, the sensor comprising: a biological enzyme layer, a nano particle layer/polymer layer and an electrode substrate, wherein the nano particles are Ag-SiO2(ii) a The electrode substrate is made of glassy carbon; the enzyme solution is a solution of acetylcholinesterase and chitosan acetic acid, and is prepared by the following steps:
(1) taking 3 mg of SiO with an average diameter of 20 nm2Ultrasonically dispersing the powder in 50 mL of secondary distilled water, adding 2.5 mL of 0.1mol/L sodium citrate solution, stirring for 10 min, slowly adding 8mL of 0.5mol/L sodium borohydride and 0.25 mL of 0.1mol/L silver nitrate while stirring, and stirring for 1 h to obtain the Ag-SiO2A dispersion solution of composite nanoparticles;
(2) 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 1:13Adding the cleaned glassy carbon electrode into an aqueous solution containing 0.1M pyrrole monomer and 0.5 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 0.5 mA cm/cm-2The electric quantity of the electrodeposition is 1.2 coulombs; then, the obtained polypyrrole film is cleaned and placed in a three-electrode system for electrochemical reduction, the applied voltage is 1.1V, the electrolyte is PBS buffer solution, and after reaction, the polypyrrole film is repeatedly washed by water to obtain a conductive polymer composite material modified electrode;
(3) taking 8 mu L of Ag-SiO2Dripping the nanoparticle dispersion solution on the surface of the dried polymer composite modified electrode, naturally drying, uniformly mixing 2 mu L of AChE (100U/mL) with 1mg/mL chitosan acetic acid solution with the same volume, dripping 8 mu L of the mixed solution on the electrode modified with the nanoparticles/conductive polymer, and naturally drying to obtain the Ag-SiO2Nanoparticle/polymer modified acetylcholinesterase biosensors.
The i-t response working curve of the product as an organophosphorus pesticide detection sensor to different concentrations of thioacetylcholine at the working potential of +0.6V is shown in figure 1.
Example 2
A high-sensitivity sensor for pesticide detection is prepared by the following steps similar to the example 1:
(1) taking 3 mg of SiO with an average diameter of 30 nm2Ultrasonically dispersing the powder in 35mL of secondary distilled water, adding 1.88 mL of 0.1mol/L sodium citrate solution, stirring for 10 min, slowly adding 5mL of 0.5mol/L sodium borohydride and 0.35 mL of 0.1mol/L silver nitrate while stirring, and stirring for 2 hours to obtain the Ag-SiO2A dispersion solution of composite nanoparticles;
(2) 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; placing the cleaned glassy carbon electrodeAdding into 0.2M pyrrole monomer and 1.0 mg/ml graphene oxide water solution, introducing nitrogen for 30 min, and performing constant current electrodeposition with a three-electrode system with applied current of 2 mA cm-2The electric quantity of the electrodeposition is 2.0 coulombs; cleaning the obtained polypyrrole film, placing the polypyrrole film 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 with water after reaction to obtain a conductive polymer composite material modified electrode;
(3) taking 13 mu L of Ag-SiO2And (3) dripping the nanoparticle dispersion solution on the surface of the dried polymer composite modified electrode, naturally drying, uniformly mixing 10 mu L of AChE (100U/mL) with 1mg/mL chitosan acetic acid solution with the same volume, dripping 10 mu L of the mixed solution on the electrode modified with the nanoparticles/conductive polymer, and naturally drying to obtain the organophosphorus pesticide detection sensor.
Example 3
A high-sensitivity sensor for pesticide detection is prepared by the following steps similar to the example 1:
(1) 5 mg of SiO with an average diameter of 80 nm are taken2Ultrasonically dispersing the powder in 50 mL of secondary distilled water, adding 4 mL0.5 mol/L sodium citrate solution, stirring for 10 min, slowly adding 10mL of 0.5mol/L sodium borohydride and 0.5 mL of 0.1mol/L silver nitrate while stirring, and stirring for 5 hours to obtain the Ag-SiO2A dispersion solution of composite nanoparticles;
(2) 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; putting the cleaned glassy carbon electrode into an aqueous solution containing aniline monomer with the content of 0.05M and graphene oxide with the content of 1.0 mg/ml, introducing nitrogen for 30 minutes, and then 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 film, placing the polyaniline film in a three-electrode system for electrochemical reduction, applying a voltage of 1.0V and using PBS as an electrolyte, reacting, and repeatedly washing with water to obtain a conductive polymer compoundSynthesizing a material modified electrode;
(3) taking 5 mu L of Ag-SiO2And (3) dripping the nanoparticle dispersion solution on the surface of the dried conductive polymer composite modified electrode, naturally drying, uniformly mixing 2 mu L of AChE (100U/mL) with 1mg/mL chitosan acetic acid solution with the same volume, dripping 2 mu L of the mixed solution on the electrode modified with the nanoparticles/conductive polymer, and naturally drying to obtain the organophosphorus pesticide detection sensor.
Example 4
A high-sensitivity sensor for pesticide detection is prepared by the following steps similar to the example 1:
(1) 5 mg of SiO with an average diameter of 200 nm are taken2Ultrasonically 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 mL0.3mol/L silver nitrate while stirring, and stirring for 8 hours to obtain the Ag-SiO2A dispersion solution of composite nanoparticles;
(2) 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; putting the cleaned glassy carbon electrode into an aqueous solution containing 0.2M pyrrole monomer and 1.0 mg/ml graphene oxide, introducing nitrogen for 30 minutes, and then performing 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; cleaning the obtained polypyrrole film, placing the polypyrrole film 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 with water after reaction to obtain a conductive polymer composite material modified electrode;
(3) taking 5 mu L of Ag-SiO2Dripping the nano particle dispersion solution on the surface of an air-dried polymer composite material modified electrode, naturally drying, uniformly mixing 2 mu L of AChE (100U/mL) with 1mg/mL chitosan acetic acid solution with the same volume, dripping 2 mu L of the mixed solution on the electrode modified with the nano particles/conductive polymer, and naturally drying to obtain the organophosphorus pesticide detection sensor。
Claims (9)
1. A preparation method of a high-sensitivity sensor for pesticide detection is characterized in that the sensor comprises the following steps: a biological enzyme layer, a nano particle layer/polymer layer and an electrode substrate, wherein the nano particles are Ag-SiO2(ii) a The electrode substrate is made of glassy carbon; the enzyme solution is a solution of acetylcholinesterase and chitosan acetic acid, and comprises the following preparation steps:
(1) taking SiO2Ultrasonically dispersing the powder in secondary distilled water, adding sodium citrate solution, slowly adding sodium borohydride and silver nitrate while stirring, and fully stirring to obtain Ag-SiO2A dispersion solution of composite nanoparticles;
(2) putting the cleaned glassy carbon electrode into an M polymer monomer 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 composite modified electrode;
(3) taking 2 mu L-20 mu L of Ag-SiO2Dripping the nano particle dispersion solution on the surface of an air-dried polymer composite material modified electrode, naturally drying, uniformly mixing 1-20 mu L of AChE (100U/mL) with 0.5-5 mg/mL chitosan acetic acid solution with the same volume, dripping 1-20 mu L of the mixed solution on the modified electrode, and naturally drying to obtain the Ag-SiO2Nanoparticle/polymer modified acetylcholinesterase biosensors.
2. The method for preparing a high-sensitivity sensor for pesticide detection according to claim 1, wherein the M polymer monomer is one of pyrrole, aniline, and 3, 4-ethylenedioxythiophene.
3. The method for preparing a high-sensitivity sensor for pesticide detection according to claim 1 or 2, characterized by comprising the steps of:
(1) taking 3 mg of SiO with an average diameter of 20 nm2The powder is dispersed in 50 mL of redistilled water by ultrasonic wave, and 2 is added5 mL0.1mol/L sodium citrate solution, stirring for 10 min, slowly adding 8mL of 0.5mol/L sodium borohydride and 0.25 mL of 0.1mol/L silver nitrate while stirring, and stirring for 1 h to obtain the Ag-SiO2A dispersion solution of composite nanoparticles;
(2) 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 1:13Adding the cleaned glassy carbon electrode into an aqueous solution containing 0.1M pyrrole monomer and 0.5 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 0.5 mA cm/cm-2The electric quantity of the electrodeposition is 1.2 coulombs; then, the obtained polypyrrole film is cleaned and placed in a three-electrode system for electrochemical reduction, the applied voltage is 1.1V, the electrolyte is PBS buffer solution, and after reaction, the polypyrrole film is repeatedly washed by water to obtain a conductive polymer composite material modified electrode;
(3) taking 8 mu L of Ag-SiO2Dripping the nanoparticle dispersion solution on the surface of the dried polymer composite modified electrode, naturally drying, uniformly mixing 2 mu L of AChE (100U/mL) with 1mg/mL chitosan acetic acid solution with the same volume, dripping 8 mu L of the mixed solution on the electrode modified with the nanoparticles/conductive polymer, and naturally drying to obtain the Ag-SiO2Nanoparticle/polymer modified acetylcholinesterase biosensors.
The i-t response working curve of the product as an organophosphorus pesticide detection sensor to different concentrations of thioacetylcholine at the working potential of +0.6V is shown in figure 1.
4. The method for preparing a high-sensitivity sensor for pesticide detection according to claim 1 or 2, characterized by comprising the steps of:
(1) taking 3 mg of SiO with an average diameter of 30 nm2Ultrasonically dispersing the powder in 35mL of secondary distilled water, adding 1.88 mL of 0.1mol/L sodium citrate solution, stirring for 10 min, slowly adding 5mL of 0.5mol/L sodium borohydride and 0.35 mL of 0.1mol/L silver nitrate while stirring, and stirring for 2 hours to obtain the final productTo Ag-SiO2A dispersion solution of composite nanoparticles;
(2) 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; putting the cleaned glassy carbon electrode into an aqueous solution containing 0.2M pyrrole monomer and 1.0 mg/ml graphene oxide, introducing nitrogen for 30 minutes, and then performing 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; cleaning the obtained polypyrrole film, placing the polypyrrole film 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 with water after reaction to obtain a conductive polymer composite material modified electrode;
(3) taking 13 mu L of Ag-SiO2And (3) dripping the nanoparticle dispersion solution on the surface of the dried polymer composite modified electrode, naturally drying, uniformly mixing 10 mu L of AChE (100U/mL) with 1mg/mL chitosan acetic acid solution with the same volume, dripping 10 mu L of the mixed solution on the electrode modified with the nanoparticles/conductive polymer, and naturally drying to obtain the organophosphorus pesticide detection sensor.
5. The method for preparing a high-sensitivity sensor for pesticide detection according to claim 1 or 2, characterized by comprising the steps of:
(1) 5 mg of SiO with an average diameter of 80 nm are taken2Ultrasonically dispersing the powder in 50 mL of secondary distilled water, adding 4 mL of 0.5mol/L sodium citrate solution, stirring for 10 min, slowly adding 10mL of 0.5mol/L sodium borohydride and 0.5 mL0.1mol/L silver nitrate while stirring, and stirring for 5 hours to obtain the Ag-SiO2A dispersion solution of composite nanoparticles;
(2) 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; the cleaned glassy carbon electrode was placed in a chamber containing 0.05M aniline monomer and 1.0 mg/ml of oxygenIntroducing nitrogen into graphene aqueous solution for 30 minutes, and then 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 film, placing the polyaniline film in a three-electrode system for electrochemical reduction, applying a voltage of 1.0V, wherein an electrolyte is a PBS (phosphate buffered saline) buffer solution, and repeatedly washing the polyaniline film with water after reaction to obtain a conductive polymer composite material modified electrode;
(3) taking 5 mu L of Ag-SiO2And (3) dripping the nanoparticle dispersion solution on the surface of the dried conductive polymer composite modified electrode, naturally drying, uniformly mixing 2 mu L of AChE (100U/mL) with 1mg/mL chitosan acetic acid solution with the same volume, dripping 2 mu L of the mixed solution on the electrode modified with the nanoparticles/conductive polymer, and naturally drying to obtain the organophosphorus pesticide detection sensor.
6. The method for preparing a high-sensitivity sensor for pesticide detection according to claim 1 or 2, characterized by comprising the steps of:
(1) 5 mg of SiO with an average diameter of 200 nm are taken2Ultrasonically 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 mL0.3mol/L silver nitrate while stirring, and stirring for 8 hours to obtain the Ag-SiO2A dispersion solution of composite nanoparticles;
(2) 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; putting the cleaned glassy carbon electrode into an aqueous solution containing 0.2M pyrrole monomer and 1.0 mg/ml graphene oxide, introducing nitrogen for 30 minutes, and then performing 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; cleaning the obtained polypyrrole film, placing the polypyrrole film in a three-electrode system for electrochemical reduction, applying a voltage of 1.2V, using a PBS (phosphate buffer solution) as an electrolyte, reacting, and repeatedly cleaning with water to obtain the conductive polymer composite material modified electrode；
(3) Taking 5 mu L of Ag-SiO2And (3) dripping the nanoparticle dispersion solution on the surface of the dried polymer composite modified electrode, naturally drying, uniformly mixing 2 mu L of AChE (100U/mL) with 1mg/mL chitosan acetic acid solution with the same volume, dripping 2 mu L of the mixed solution on the electrode modified with the nanoparticles/conductive polymer, and naturally drying to obtain the organophosphorus pesticide detection sensor.
7. A high sensitivity sensor for detection of pesticides, characterized by being prepared according to the method of any one of claims 1 to 6.
8. Use of the high sensitivity sensor of claim 7 for the detection of organophosphorus pesticides.
9. The use of the high sensitivity sensor of claim 8 for the detection of organophosphorus pesticides, characterized in that the detection is carried out by the steps of:
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;
under the working potential of +0.6V, ATCH with known concentration is added into PBS after the sensor signal is stabilized, stirring is carried out for 5.0min, and the generated steady-state current I is recorded in a static solution after the 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(ii) a 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)/I1Linearly related to the logarithm of the concentration of organophosphorus, and thus can be determined by measuring the enzymeThe reduction of activity is used to determine the concentration of organophosphorus pesticide.
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