CN111189897A - Biosensor for detecting organophosphorus pesticide and preparation and application thereof - Google Patents
Biosensor for detecting organophosphorus pesticide and preparation and application thereof Download PDFInfo
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- 239000003987 organophosphate pesticide Substances 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title abstract description 10
- 229920001661 Chitosan Polymers 0.000 claims abstract description 101
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims abstract description 81
- 239000004408 titanium dioxide Substances 0.000 claims abstract description 43
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims abstract description 38
- 108010022752 Acetylcholinesterase Proteins 0.000 claims abstract description 34
- 229940022698 acetylcholinesterase Drugs 0.000 claims abstract description 34
- 239000002131 composite material Substances 0.000 claims abstract description 32
- 229910021389 graphene Inorganic materials 0.000 claims abstract description 28
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 27
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 27
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 22
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- 239000000758 substrate Substances 0.000 claims abstract description 21
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- 239000007864 aqueous solution Substances 0.000 claims description 37
- 238000001514 detection method Methods 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 21
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 18
- 229910052709 silver Inorganic materials 0.000 claims description 16
- 239000004332 silver Substances 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 238000009713 electroplating Methods 0.000 claims description 13
- 229910021397 glassy carbon Inorganic materials 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 11
- 239000008367 deionised water Substances 0.000 claims description 10
- 229910021641 deionized water Inorganic materials 0.000 claims description 10
- 229960000583 acetic acid Drugs 0.000 claims description 9
- 239000012362 glacial acetic acid Substances 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 9
- 238000012986 modification Methods 0.000 claims description 8
- 230000004048 modification Effects 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 239000011248 coating agent Substances 0.000 claims description 5
- 238000000576 coating method Methods 0.000 claims description 5
- GNFTZDOKVXKIBK-UHFFFAOYSA-N 3-(2-methoxyethoxy)benzohydrazide Chemical compound COCCOC1=CC=CC(C(=O)NN)=C1 GNFTZDOKVXKIBK-UHFFFAOYSA-N 0.000 claims description 4
- YHWCPXVTRSHPNY-UHFFFAOYSA-N butan-1-olate;titanium(4+) Chemical compound [Ti+4].CCCC[O-].CCCC[O-].CCCC[O-].CCCC[O-] YHWCPXVTRSHPNY-UHFFFAOYSA-N 0.000 claims description 4
- 239000000575 pesticide Substances 0.000 claims description 4
- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 claims description 3
- 238000007306 functionalization reaction Methods 0.000 claims description 3
- 238000004381 surface treatment Methods 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 2
- 238000005498 polishing Methods 0.000 claims description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 15
- 230000005518 electrochemistry Effects 0.000 abstract description 2
- 102100033639 Acetylcholinesterase Human genes 0.000 description 27
- 238000012360 testing method Methods 0.000 description 19
- 238000001903 differential pulse voltammetry Methods 0.000 description 12
- 238000002474 experimental method Methods 0.000 description 12
- 238000002791 soaking Methods 0.000 description 11
- 239000003153 chemical reaction reagent Substances 0.000 description 10
- GFFIJCYHQYHUHB-UHFFFAOYSA-N 2-acetylsulfanylethyl(trimethyl)azanium Chemical compound CC(=O)SCC[N+](C)(C)C GFFIJCYHQYHUHB-UHFFFAOYSA-N 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- 229940088598 enzyme Drugs 0.000 description 9
- 230000003647 oxidation Effects 0.000 description 7
- 238000007254 oxidation reaction Methods 0.000 description 7
- 230000003197 catalytic effect Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 239000008055 phosphate buffer solution Substances 0.000 description 6
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 description 4
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 4
- 229960004373 acetylcholine Drugs 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 229910052719 titanium Inorganic materials 0.000 description 4
- 239000010936 titanium Substances 0.000 description 4
- OGIDPMRJRNCKJF-UHFFFAOYSA-N titanium oxide Inorganic materials [Ti]=O OGIDPMRJRNCKJF-UHFFFAOYSA-N 0.000 description 4
- 235000013311 vegetables Nutrition 0.000 description 4
- 241000277305 Electrophorus electricus Species 0.000 description 3
- 229910021607 Silver chloride Inorganic materials 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000002484 cyclic voltammetry Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000002848 electrochemical method Methods 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101100384355 Mus musculus Ctnnbip1 gene Proteins 0.000 description 1
- YTAHJIFKAKIKAV-XNMGPUDCSA-N [(1R)-3-morpholin-4-yl-1-phenylpropyl] N-[(3S)-2-oxo-5-phenyl-1,3-dihydro-1,4-benzodiazepin-3-yl]carbamate Chemical compound O=C1[C@H](N=C(C2=C(N1)C=CC=C2)C1=CC=CC=C1)NC(O[C@H](CCN1CCOCC1)C1=CC=CC=C1)=O YTAHJIFKAKIKAV-XNMGPUDCSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000003421 catalytic decomposition reaction Methods 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- GTKRFUAGOKINCA-UHFFFAOYSA-M chlorosilver;silver Chemical compound [Ag].[Ag]Cl GTKRFUAGOKINCA-UHFFFAOYSA-M 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- OEBRKCOSUFCWJD-UHFFFAOYSA-N dichlorvos Chemical compound COP(=O)(OC)OC=C(Cl)Cl OEBRKCOSUFCWJD-UHFFFAOYSA-N 0.000 description 1
- ZOMNIUBKTOKEHS-UHFFFAOYSA-L dimercury dichloride Chemical class Cl[Hg][Hg]Cl ZOMNIUBKTOKEHS-UHFFFAOYSA-L 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000002070 nanowire Substances 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 210000000225 synapse Anatomy 0.000 description 1
- 238000004506 ultrasonic cleaning Methods 0.000 description 1
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- 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
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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
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
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- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
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- Pathology (AREA)
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Abstract
The invention relates to the field of electrochemistry, in particular to a biosensor for detecting organophosphorus pesticide and preparation and application thereof. The biosensor comprises a working electrode, a reference electrode and a counter electrode, wherein the working electrode is obtained by sequentially modifying a silver nanowire, graphene, a titanium dioxide/chitosan composite material and a chitosan film on the surface of a substrate electrode and then fixing a mixture of acetylcholinesterase and bovine serum albumin. Has good sensitivity, specificity, repeatability and stability.
Description
Technical Field
The invention relates to the field of electrochemistry, in particular to a biosensor for detecting organophosphorus pesticide and preparation and application thereof.
Background
Pesticides, particularly Organophosphorus Pesticides (OPs), have been widely used for the past decades to protect crops from insect attack. However, organophosphorus pesticides can inhibit enzymes in neurosynaptic-acetylcholinesterase (AChE), leading to accumulation of acetylcholine in humans, overstimulation of its receptors in synapses and eventual damage to the nervous system, causing harm to humans and animals. Therefore, there is an increasing need for efficient, simple and rapid detection of Ops in various samples.
Among the currently applied methods for determining the level of OPs, the electrochemical method has the advantages of high reliability, simple instrument operation and use method, high speed of obtaining results, high sensitivity and capability of determining complex samples. However, the biosensor reported at present has the main technical characteristics that the surface of a glassy carbon electrode is coated with a chitosan solution, and acetylcholinesterase is added after the glassy carbon electrode is placed and dried in the air, so that the biosensor has the defects of low sensitivity, small detection range, poor stability and the like. The sensor has poor sensitivity and stability because the sensor can not well amplify the electric signal of the acetylcholinesterase for hydrolyzing the acetylthiocholine, can not well fix the biological enzyme and keep the catalytic activity of the enzyme for a long time.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a biosensor for detecting organophosphorus pesticide, and its preparation and application.
In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions: the invention provides a biosensor for detecting organophosphorus pesticide, which comprises a working electrode, a reference electrode and a counter electrode, wherein the working electrode is obtained by modifying silver nanowires, graphene, a titanium dioxide/chitosan composite material and chitosan on the surface of a substrate electrode in sequence and then fixing a mixture of acetylcholinesterase and bovine serum albumin.
Furthermore, the mass of the silver nanowire is 0.0355 mu g-0.071 mu g per square millimeter of the working electrode. Alternatively, 0.071 μ g.
Furthermore, the mass of the graphene on each square millimeter of the working electrode is 0.007 to 0.014 mu g. Alternatively, 0.014. mu.g.
Further, the titanium dioxide/chitosan composite material is prepared by the following method and then is modified on the surface of the silver nanowire-graphene modified electrode:
a. uniformly mixing tetra-n-butyl titanate, glacial acetic acid, deionized water and absolute ethyl alcohol to obtain titanium dioxide sol gel;
b. dissolving chitosan in water to obtain a chitosan water solution;
c. and mixing the chitosan aqueous solution and the titanium dioxide sol-gel to prepare the titanium dioxide/chitosan composite material.
Preferably, in the step a, the molar ratio of tetrabutyl titanate, glacial acetic acid, deionized water and absolute ethyl alcohol is 1: 1: 4: 50.
preferably, in the step b, the concentration of the chitosan is 0.002g/ml based on the total amount of the chitosan aqueous solution.
Preferably, in the step c, the mixing volume ratio of the titanium dioxide sol gel used for preparing the titanium dioxide/chitosan composite material to the chitosan aqueous solution is 99: 1-95: 5. comprises 99: 1-97: 3. 97 (b): 3-95: 5. specifically, it may be 99: 1. 98: 2. 97 (b): 3. 96: 4. 95: 5. preferably, 99: 1.
further, the chitosan film is obtained by electrifying and electroplating the working electrode on the surface of the electrode in a chitosan aqueous solution.
Preferably, the plating of the chitosan film is performed in a chitosan aqueous solution having a chitosan volume fraction of 0.2%, wherein the chitosan aqueous solution is the total amount.
Preferably, the electroplating condition is that the electroplating voltage is-2.5V and the time is 10 s-25 s. Including 10 s-15 s, 15 s-20 s, 20 s-25 s. For example, 10s, 11s, 12s, 13s, 14s, 15s, 16s, 17s, 18s, 19s, 20s, 21s, 22s, 23s, 24s, 25 s; preferably, it is 20 s.
Further, the mixture of acetylcholinesterase and bovine serum albumin is prepared by the following method and then modified on the surface of the electrode modified by the silver nanowire-graphene-titanium dioxide/chitosan composite material-chitosan film:
dissolving acetylcholinesterase in aqueous solution of bovine serum albumin to obtain mixture of acetylcholinesterase and bovine serum albumin.
Preferably, the concentration ratio of the acetylcholinesterase to the bovine serum albumin is 3 mg/ml-5 mg/ml: 10 mg/ml. For example, it may be 3 mg/ml: 10mg/ml, 4 mg/ml: 10mg/ml, 5 mg/ml: 10 mg/ml. More preferably, it is 5 mg/ml: 10 mg/ml.
Further, the substrate electrode is selected from a glassy carbon electrode.
Preferably, the diameter of the base electrode is 3 mm.
Furthermore, the reference electrode, the counter electrode and the working electrode in the biosensor form a three-electrode system.
Preferably, the reference electrode is selected from any one of a saturated calomel electrode or a silver-silver chloride electrode (Ag/AgCl); more preferably, the reference electrode is a silver/silver chloride (Ag/AgCl) electrode.
Preferably, the counter electrode is a platinum wire electrode.
The second aspect of the invention provides a preparation method of a working electrode of a biosensor, which comprises the steps of sequentially modifying a silver nanowire, graphene, a titanium dioxide/chitosan composite material and a chitosan film on the surface of a substrate electrode, and then fixing a mixture of acetylcholinesterase and bovine serum albumin.
Further, the preparation method of the working electrode comprises the following steps:
(1) preparing a titanium dioxide/chitosan composite material:
d. uniformly mixing tetra-n-butyl titanate, glacial acetic acid, deionized water and absolute ethyl alcohol to obtain titanium dioxide sol gel;
e. dissolving chitosan in water to obtain a chitosan water solution;
f. and mixing the chitosan aqueous solution and the titanium dioxide sol-gel to prepare the titanium dioxide/chitosan composite material.
(3) Modification and functionalization of the working electrode:
g. surface treatment of a substrate electrode: polishing the surface of the substrate electrode to make the surface smooth;
h. modifying silver nanowires and graphene: sequentially coating the silver nanowire solution and the graphene solution on the surface of the substrate electrode treated in the step g, and airing to obtain a silver nanowire-graphene modified electrode;
i. modifying the titanium dioxide/chitosan composite material: coating the titanium dioxide/chitosan composite material prepared in the step (1) on the silver nanowire-graphene modified electrode, airing, and electroplating a chitosan film to obtain the silver nanowire-graphene-titanium dioxide/chitosan composite material-chitosan modified electrode;
j. immobilization of a mixture of acetylcholinesterase and bovine serum albumin: and adding a mixture of acetylcholinesterase and bovine serum albumin onto the silver nanowire-graphene-titanium dioxide/chitosan composite material-chitosan modified electrode, and airing to obtain the working electrode.
Further, the chitosan has a formula (C)6H11NO4)NThe molecular weight of the monoliths is 161.2.
Preferably, in the step d, the molar ratio of tetrabutyl titanate, glacial acetic acid, deionized water and absolute ethyl alcohol is 1: 1: 4: 50;
preferably, in the step e, the concentration of the chitosan is 0.002g/ml based on the total amount of the chitosan aqueous solution.
Preferably, in the step f, the mixing volume ratio of the titanium dioxide sol gel used for preparing the titanium dioxide/chitosan composite material to the chitosan aqueous solution is 99: 1-95: 5. comprises 99: 1-97: 3. 97 (b): 3-95: 5, specifically, it may be 99: 1. 98: 2. 97 (b): 3. 96: 4. 95: 5. preferably, 99: 1.
preferably, in step g, the substrate electrode is selected from a glassy carbon electrode. Further, the diameter of the base electrode is 3 mm.
Preferably, in step g, the base electrode may be polished with alumina powder.
Preferably, in the step h, the concentration of the silver nanowires is 1mg/ml to 2mg/ml based on the total amount of the silver nanowire aqueous solution. Including 1mg/ml to 1.5mg/ml, 1.5mg/ml to 2 mg/ml. Specifically, 1mg/ml, 1.2mg/ml, 1.4mg/ml, 1.5mg/ml, 1.6mg/ml, 1.8mg/ml and 2mg/ml may be mentioned. Preferably, it is 2 mg/ml. The concentration of the graphene is 0.2 mg/ml-0.4 mg/ml based on the total amount of the graphene aqueous solution. Comprises 0.2 mg/ml-0.3 mg/ml, 0.3 mg/ml-0.4 mg/ml. Preferably, it is 0.4 mg/ml.
Preferably, in step i, the electroplating of the chitosan membrane is performed in a chitosan aqueous solution with a chitosan volume fraction of 0.2%, wherein the chitosan aqueous solution is taken as a reference.
Preferably, in the step i, the electroplating condition is that the electroplating voltage is-2.5V and the time is 10 s-25 s. Including 10 s-15 s, 15 s-20 s, 20 s-25 s. For example, 10s, 11s, 12s, 13s, 14s, 15s, 16s, 17s, 18s, 19s, 20s, 21s, 22s, 23s, 24s, 25s may be used. Preferably, it is 20 s.
Preferably, in step j, the concentration ratio of the acetylcholinesterase to the bovine serum albumin based on the total amount of the mixture of acetylcholinesterase and bovine serum albumin is: 3 mg/ml-5 mg/ml: 10 mg/ml. For example, it may be 3 mg/ml: 10mg/ml, 4 mg/ml: 10mg/ml, 5 mg/ml: 10 mg/ml. Preferably, 5 mg/ml: 10 mg/ml.
The invention provides a biosensor for detecting organophosphorus pesticide, which is prepared by the method.
A fourth aspect of the invention provides the use of a biosensor as described in the first or third aspects of the invention for detecting an organophosphorus pesticide.
In a fifth aspect of the present invention, there is provided a method for detecting an organophosphorus pesticide in a sample by using the biosensor according to the first or third aspect of the present invention.
According to the biosensor provided by the first aspect of the invention, other substrates can be detected by replacing acetylcholinesterase with other biological enzymes, and the structure of the biosensor is not required to be modified.
Compared with the prior art, the invention has the beneficial effects that:
(1) two nanowire materials of graphene and silver nanowires are introduced, so that sensing signals are effectively amplified, and the sensitivity of the sensor is improved.
(2) The titanium oxide-chitosan mixed material can provide good support for the fixation of the biological enzyme and can well maintain the activity of the enzyme. The enzyme immobilization efficiency is improved, and the sensitivity and stability of the sensor are further improved.
(3) All the materials used are materials with good biocompatibility, do not affect the activity of the enzyme, and can keep the activity of the enzyme stable for a long time.
The biosensor provided by the invention has good sensitivity, specificity, repeatability and stability.
Drawings
FIG. 1: the invention relates to a preparation flow chart of a working electrode of a biosensor.
FIG. 2: the stability detection result chart of the biosensor is provided.
FIG. 3: the sensitivity detection result graph of the biosensor is shown.
FIG. 4: the DPV detection result chart in the practical application of the biosensor in the embodiment 1 of the invention. Wherein the curve a is the test result of the electrode without soaking the vegetable leaf sample, and the curve b is the test result after soaking treatment.
FIG. 5: the DPV detection result chart of the biosensor in class 1 of the embodiment 4 of the invention in practical application. Wherein the curve a is the test result of the electrode without soaking the vegetable leaf sample, and the curve b is the test result after soaking treatment.
FIG. 6: the DPV detection result chart of the biosensor in category 2 in embodiment 4 of the invention. Wherein the curve a is the test result of the electrode without soaking the vegetable leaf sample, and the curve b is the test result after soaking treatment.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
It should be understood that the processing equipment or devices not specifically mentioned in the following examples are conventional in the art; all pressure values and ranges refer to absolute pressures.
Furthermore, it is to be understood that one or more method steps mentioned in the present invention does not exclude that other method steps may also be present before or after the combined steps or that other method steps may also be inserted between these explicitly mentioned steps, unless otherwise indicated; it is also to be understood that a combined connection between one or more devices/apparatus as referred to in the present application does not exclude that further devices/apparatus may be present before or after the combined device/apparatus or that further devices/apparatus may be interposed between two devices/apparatus explicitly referred to, unless otherwise indicated. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.
EXAMPLE 1 preparation of biosensor
1. Reagent and apparatus
1.1 reagents
Acetylcholinesterase (from electric eel), acetylthiocholine from sigma, chitosan (high viscosity, >400mpa.s), bovine serum albumin from shanghai alatin Biotech, Inc., and reagents (analytically pure) used in other experiments were all from national drug group chemical reagents, Inc.
2. Preparation of working electrode
The specific steps are shown in figure 1:
2.1 preparing a titanium dioxide/chitosan composite material: tetrabutyl titanate, glacial acetic acid, deionized water and absolute ethyl alcohol according to a molar ratio of 1: 1: 4: mixing uniformly according to the proportion of 50 to obtain titanium oxide sol gel; dissolving 1g of chitosan in 100ml of glacial acetic acid aqueous solution with the glacial acetic acid volume fraction of 1%, and continuously stirring to obtain clear and transparent chitosan aqueous solution, wherein the obtained chitosan solution is aqueous solution with the mass fraction of 1%; diluting the chitosan aqueous solution to 0.002% by using deionized water, and mixing 1 volume of the chitosan aqueous solution with 99 volumes of titanium oxide sol gel to obtain the titanium oxide-chitosan titanium dioxide/chitosan composite material.
2.2 modification and functionalization of the working electrode:
2.2.1 glassy carbon electrode surface treatment: the Glassy Carbon Electrode (GCE) had a diameter of 3mm and was polished to a mirror surface with 0.3 μm and 0.05 μm alumina powder, respectively, before use, followed by ultrasonic cleaning with deionized water. Finally, the mixture is washed clean by deionized water and dried by nitrogen at room temperature.
2.2.2 modification of silver nanowires, graphene: 4 microliter of silver nanowire aqueous solution with the concentration of 2mg/ml and 4 microliter of graphene aqueous solution with the concentration of 0.4mg/ml are sequentially and carefully dripped on the surface of the cleaned glassy carbon electrode. And then, drying the modified glassy carbon electrode in vacuum at room temperature (not overnight) to obtain the silver nanowire-graphene modified electrode.
2.2.3 modification of titanium dioxide/Chitosan composite: carefully coating a prepared titanium dioxide/chitosan composite material (4 mu L) on the surfaces of the silver nanowire and graphene modified electrodes, and then airing at room temperature (no night) to form the surfaces of the silver nanowire, graphene and titanium dioxide/chitosan composite material modified electrodes; then placing the electrode into a chitosan aqueous solution with the chitosan concentration of 0.2%, electroplating for 20 seconds under the voltage of-2.5V, and forming a chitosan film on the electrode to obtain a silver nanowire-graphene-titanium dioxide/chitosan composite material-chitosan modified electrode;
2.2.4 immobilization of acetylcholinesterase and bovine serum albumin mixture: the concentration ratio of acetylcholinesterase to bovine serum albumin added to the silver nanowire-graphene-titanium dioxide/chitosan composite material-chitosan modified electrode is as follows: 5 mg/ml: and (3) drying the mixture of 10mg/ml acetylcholinesterase and bovine serum albumin in a wet environment overnight (here, overnight), thus obtaining the working electrode.
Example 2 biosensor Performance analysis
1. Stability test
1.1 reagents
Acetylcholinesterase (from electric eel), acetylthiocholine from sigma, chitosan (high viscosity, >400mpa.s), bovine serum albumin from shanghai alatin Biotech, Inc., and reagents (analytically pure) used in other experiments were all from national drug group chemical reagents, Inc.
1.2 detection Instrument
The MCHI660E electrochemical workstation was purchased from shanghai chenhua instruments ltd.
1.3 electrochemical measurement method
The electrochemical experiments used Differential Pulse Voltammetry (DPV). In the DPV detection, the base solution is 1mM ATCL (acetylthiocholine)/1 xPBS (pH7.0) buffer solution, the detection voltage is 0.2V to 1.0V, the pulse period is 0.02S, the amplitude is 0.05V, the pulse width is 0.005S, and the standing time is 10 min. All experiments were performed at room temperature (25 degrees celsius).
1.4 detection principle
Acetylcholinesterase can catalyze the decomposition of acetylthiocholine, and the product thiocholine can undergo irreversible oxidation. The products of the catalytic decomposition process can be characterized by differential pulse voltammetry, and a distinct oxidation peak is generated near 0.6V. The stability of the same electrode can be shown in that the measurement results of a plurality of times at different times are consistent.
1.5 Experimental procedures
The fabricated electrodes were first cycled in PBS buffer using cyclic voltammetry until stable test results appeared, and then DPV measurements were performed using 1mM ATCL (solvent is also PBS buffer). The incubation time for the DPV test was 10 minutes, and the test environment was a room temperature environment. And (3) testing three groups of data each time, soaking the electrode in PBS buffer solution after the test is finished, and storing the electrode in an environment at 4 ℃. Subsequently, the electrodes were removed every 4 days and when their temperature returned to room temperature, three sets of DPVs were tested.
1.6 analysis of results
As shown in fig. 2, each curve has a distinct oxidation peak near 0.6V, indicating that the electrode has catalyzed the substrate, and the test results are nearly identical at different times, indicating that the catalysis levels at the electrodes are nearly identical at different times. Therefore, when the sensor is stored in an environment at 4 ℃ for one month, the catalytic capacity of the sensor to acetylcholine does not change obviously.
2. Sensitivity test
2.1 reagents
Acetylcholinesterase (from electric eel), acetylthiocholine from sigma, chitosan (high viscosity, >400mpa.s), bovine serum albumin from shanghai alatin Biotech, Inc., and reagents (analytically pure) used in other experiments were all from national drug group chemical reagents, Inc.
2.2 detection Instrument
CHI660E electrochemical workstation was purchased from Shanghai Chenghua instruments, Inc.
2.3 principle of detection
Double reciprocal model according to the Mie equation
To characterize the catalytic performance of the sensor on the substrate acetylcholine. Where Icat is the oxidation peak-to-peak current of DPV, CATClIs the substrate concentration, KmIs the mie constant. Calculating K from a linear equation fitted to the datam。
2.4 Experimental procedures
Continuously and circularly testing the manufactured electrode in a PBS (phosphate buffer solution) by using CV (cyclic voltammetry) until a stable test result appears; then DPV testing in substrate substrates of different concentrations, three times per concentration; and (3) selecting the oxidation peak current of each group of curves and the corresponding substrate concentration data of each group of curves according to the test result, fitting a 2.3 Mie's double reciprocal equation, and calculating the numerical value of Km.
2.5 analysis of results
As shown in FIG. 3, from the data obtained in the figure, the oxidation peak-to-peak current of each curve and the corresponding substrate concentration were reciprocal, fitting of a linear equation was performed, and K was calculated according to the equation in FIG. 2.3mHas a value of 0.315mM, KmSmall value and high sensitivity.
EXAMPLE 3 use of the sensor for actual sample detection
Firstly, sample pretreatment: cleaning and drying purchased fresh vegetable leaves, cutting and grinding, taking grinding liquid for centrifugation, taking supernate and diluting the supernate by ten times with PBS buffer solution for later use.
(II) detection: adding a small amount of DDVP in an actual sample; immersing the electrode in the sample in the embodiment 1 for incubation for 10 minutes; the electrode was removed and tested for DPV in ATCL at a concentration of 1 mM.
(III) detection principle: the organophosphorus pesticide has strong inhibition effect on acetylcholinesterase, and the catalytic performance of the inhibited acetylcholinesterase is reduced.
(IV) analyzing results: as shown in FIG. 4, the a-curve is the test result of the electrode without soaking the leaf sample, and the b-curve is the test after soaking treatment. The results show that the peak current of the oxidation peak of the curve is obviously reduced after the soaking treatment, and the catalytic performance of the electrode is obviously reduced after the soaking treatment. According to the formula Inhibit% ═ 1-Icat’/Icat 0) The inhibition ratio was 29.58% when x 100% was calculated.
Example 4
The present invention also prepares other types of biosensors with reference to example 1, and detects the stability and sensitivity thereof, followed by performing an actual sample detection experiment.
(1) The biosensor of the type 1 was tested for its stability and sensitivity in the same manner as in example 2, with a Km value of 3.625 mM.
(2) The actual sample detection experiment using the type 1 was performed in the same manner as in example 3, and the calculated inhibition ratio was 16.10%, as shown in fig. 5.
(1) The stability and sensitivity of the class 1 biosensor were measured in the same manner as in example 2, with a Km value of 1.985 mM.
(2) The actual sample detection experiment was carried out using the species 1 in the same manner as in example 3, and the results are shown in FIG. 6, and the calculated inhibition ratio was 21.15%
In conclusion, storage experiments prove that the catalytic capacity of the sensor on acetylcholine has no obvious change when the sensor is stored in an environment at 4 ℃ for one month. Sensitivity experiments prove that the sensor has higher sensitivity. In actual detection, the effect is good.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (10)
1. A biosensor for detecting organophosphorus pesticide comprises a working electrode, a reference electrode and a counter electrode, wherein the working electrode is obtained by sequentially modifying a silver nanowire, graphene, a titanium dioxide/chitosan composite material and a chitosan membrane on the surface of a substrate electrode and then fixing a mixture of acetylcholinesterase and bovine serum albumin.
2. The biosensor for detecting organophosphorus pesticide according to claim 1, wherein the mass of the silver nanowire is 0.0355 μ g to 0.071 μ g per square millimeter of the working electrode.
3. The biosensor for detecting organophosphorus pesticide according to claim 1, wherein the mass of the graphene is 0.007 μ g-0.014 μ g per square millimeter of the working electrode.
4. The biosensor for organophosphorus pesticide detection according to claim 1, wherein the base electrode is selected from glassy carbon electrodes.
5. The method for preparing the working electrode of the biosensor according to any one of claims 1 to 4, wherein the method comprises sequentially modifying the surface of the substrate electrode with the silver nanowire, the graphene, the titanium dioxide/chitosan composite material and the chitosan film, and then fixing the mixture of acetylcholinesterase and bovine serum albumin.
6. The method of claim 5, wherein the working electrode is prepared by a method comprising the steps of:
(1) preparing a titanium dioxide/chitosan composite material:
a. uniformly mixing tetra-n-butyl titanate, glacial acetic acid, deionized water and absolute ethyl alcohol to obtain titanium dioxide sol gel;
b. dissolving chitosan in water to obtain a chitosan water solution;
c. and mixing the chitosan aqueous solution and the titanium dioxide sol-gel to prepare the titanium dioxide/chitosan composite material.
(2) Modification and functionalization of the working electrode:
d. surface treatment of a substrate electrode: polishing the surface of the substrate electrode to make the surface smooth;
e. modifying silver nanowires and graphene: sequentially coating the silver nanowire aqueous solution and the graphene aqueous solution on the surface of the substrate electrode treated in the step e, and airing to obtain a silver nanowire-graphene modified electrode;
f. modifying the titanium dioxide/chitosan composite material: coating the titanium dioxide/chitosan composite material prepared in the step (1) on the silver nanowire-graphene modified electrode, airing, and electroplating a chitosan film to obtain the silver nanowire-graphene-titanium dioxide/chitosan composite material-chitosan modified electrode;
g. immobilization of a mixture of acetylcholinesterase and bovine serum albumin: and adding a mixture of acetylcholinesterase and bovine serum albumin onto the silver nanowire-graphene-titanium dioxide/chitosan composite material-chitosan modified electrode, and airing to obtain the working electrode.
7. The method of claim 6, further comprising any one or more of the following features: 1) in the step a, the molar ratio of tetrabutyl titanate, glacial acetic acid, deionized water and absolute ethyl alcohol is 1: 1: 4: 50;
2) in the step b, the concentration of the chitosan is 0.002g/ml based on the total amount of the chitosan aqueous solution;
3) in the step c, the mixing volume ratio of the titanium dioxide sol gel for preparing the titanium dioxide/chitosan composite material to the chitosan aqueous solution is 99: 1-95: 5;
4) in the step e, the concentration of the silver nanowires is 1 mg/ml-2 mg/ml by taking the total amount of the silver nanowire aqueous solution as a reference; the concentration of the graphene is 0.2 mg/ml-0.4 mg/ml based on the total amount of the graphene aqueous solution;
5) in the step f, the chitosan film is electroplated in a chitosan aqueous solution with the chitosan volume fraction of 0.2%, wherein the total amount of the chitosan aqueous solution is taken as a reference;
6) in the step f, the electroplating condition is that the electroplating voltage is minus 2.5V and the time is 10 s-25 s.
7) In the step g, the total amount of the mixture of the acetylcholinesterase and the bovine serum albumin is taken as a reference, and the concentration ratio of the acetylcholinesterase to the bovine serum albumin is as follows: 3 mg/ml-5 mg/ml: 10 mg/ml.
8. A biosensor for detecting an organophosphorus pesticide, wherein the biosensor is produced by the method according to any one of claims 5 to 7.
9. Use of the biosensor according to any one of claims 1 to 4 or the biosensor according to claim 8 for detecting organophosphorus pesticides.
10. A method for detecting an organophosphorus pesticide, which comprises detecting the organophosphorus pesticide in a sample by using the biosensor according to any one of claims 1 to 4 or the biosensor according to claim 8.
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