CN102608190A - Preparation method of acetylcholinesterase sensor used for detecting pesticide residue - Google Patents
Preparation method of acetylcholinesterase sensor used for detecting pesticide residue Download PDFInfo
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- CN102608190A CN102608190A CN201210088856XA CN201210088856A CN102608190A CN 102608190 A CN102608190 A CN 102608190A CN 201210088856X A CN201210088856X A CN 201210088856XA CN 201210088856 A CN201210088856 A CN 201210088856A CN 102608190 A CN102608190 A CN 102608190A
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Abstract
The invention relates to a preparation method of an acetylcholinesterase sensor used for detecting pesticide residue and is characterized in that: carboxymethyl chitosan-nano-Au complex is prepared, a glass carbon electrode is subjected to cleaning, activation and characterization, the carboxymethyl chitosan-nano-Au complex is dispensed, EDC-NHS mixture is dispensed, acetylcholinesterase is dispensed, a working curve of the acetylcholinesterase sensor is established, the performances of the acetylcholinesterase sensor such as repeatability, accuracy, stability and regenerative ability are detected, and a real sample is detected by the acetylcholinesterase sensor. Compared with chitosan, the carboxymethyl chitosan is more soluble in water, the biocompatibility is better, so that the carboxymethyl chitosan-nano-Au complex obtained by the in-situ reduction method is more stable and is more suitable for the fixation of biomolecule acetylcholinesterase. The EDC-NHS mixture can activate the carboxyl in the nano complex to form an intermediate body, so that the acetylcholinesterase can be fixed on the electrode more stably. According to the acetylcholinesterase sensor prepared by the method, the range of pesticide detection is wider, the limit of detection is lower, the sensitivity is high, the stability is good, the regenerative ability is high, and good recovery rate and repeatability on the analysis of the real sample are obtained. The preparation method of the acetylcholinesterase sensor used for detecting pesticide residue has good application prospect in the quick detection of organophosphorus and carbamic acid ester pesticide residue.
Description
Technical field
The invention provides a kind of preparation method who detects the acetylcholinesterase sensor of residues of pesticides, belong to the biosensor technology field.
Background technology
China is a large agricultural country, and agricultural has very important effect to national economy and people's lives, and agricultural chemicals is prevention and elimination of disease and pests in the agricultural production in modern times, ensures agricultural foison, guarantees the important means of production of staple food supply.A large amount of uses of agricultural chemicals, when improving crop yield, the negative effect that its residues of pesticides exceed standard also displays gradually.So-called residues of pesticides are meant the general name that remains in trace pesticide substance, toxic metabolite, degradation product and impurity in biosome, agricultural byproducts and the environment after agricultural chemicals uses.Generally not volatile and in plant, in the soil, be difficult for decomposing, and residual quantity is higher after the fat-soluble strong applications of pesticide.Along with China's living standards of the people improve constantly; The quality security problem of agricultural product more and more receives publicity; Especially the agricultural chemicals residue problem has become the focus of public attention in the vegetables, and the whole nation is annual all to have the edible acute poisoning incident that is caused by the agricultural product of pollution by pesticides of causes up to a hundred, has a strong impact on the healthy of consumers in general; Therefore the food-safety problem that is exceeded standard and caused by residues of pesticides more and more receives the national governments and the public's attention.
The agricultural chemicals overwhelming majority of using now is organophosphorus and carbamate compound.Have about the method for Detecting Pesticide and technology multiple, be the conventional sense method or modern age method for quick all have certain defective.Both at home and abroad the conventional sense method to residues of pesticides mainly is the physico-chemical analysis method, and utilize instrument such as gas chromatograph, liquid chromatograph, gas chromatograph-mass spectrometer, the LC-MS appearance etc. promptly said usually carry out the analyzing and testing of persticide residue.The advantage of these methods be can be qualitative, quantitative the detection persticide residue, also be the state specified standards detection method.These methods need be carried out complicated preprocessing process such as extract and separate to sample, need lot of manpower and material resources, and detection time, length and instrument cost an arm and a leg; Need the professional and technical personnel to safeguard; The detection cost is high, is inappropriate for on-the-spot the detection, can not satisfy miscellaneous agricultural product and detect demand.The fast determining method of China's residues of pesticides is that enzyme suppresses test paper method and enzyme suppresses AAS (agricultural residual fast detecting appearance), can realize the field quick detection of organophosphorus pesticide and carbamate chemicals for agriculture, has good practical values.Quick measuring card is the change color through the visual inspection card, and the vegetable sample that therefore generally can only be used for severe overweight carries out observational measurement.The application that enzyme suppresses AAS is also relatively more extensive, and the residual tacheometer of domestic existing Multiple Pesticides all is based on this principle.Beam split light ratio juris is based on that the variation of absorbance detects; But pigments a large amount of in the vegetable and fruit can cause very big influence to AAS; Cause the inaccurate of testing result; And said method exists the recovery low, and false retrieval, omission ratio are higher, poor repeatability, be difficult to satisfy the shortcomings such as requirement of low-residual and detection by quantitative.
The purpose of invention is to provide a kind of can overcome above-mentioned defective and simple to operate, quick and precisely, highly sensitive, the preparation method of the current mode acetylcholinesterase sensor of the detection residues of pesticides of good reproducibility.
Its technical scheme is: a kind of preparation method who detects the acetylcholinesterase sensor of residues of pesticides; It is characterized in that: the preparation of CMC-nano-Au composite; The group shape process of current mode acetylcholinesterase sensor is coated with CMC-nano-Au composite for dripping; Drip and to be coated with N-hydroxy-succinamide (NHS), droplet be coated with the acetylcholine esterase with 1-ethyl-3-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC) potpourri carries out activation.
Described a kind of preparation method who detects the acetylcholinesterase sensor of residues of pesticides; It is characterized in that: the cleaning of glass-carbon electrode (d=3mm); The structure at acetylcholinesterase sensor sensing interface and process characterize; The foundation of acetylcholinesterase working sensor curve, the detection of acetylcholinesterase sensor performance, the acetylcholinesterase sensor is to the detection of actual sample.
Described a kind of preparation method who detects the acetylcholinesterase sensor of residues of pesticides; It is characterized in that: the working curve of prepared current mode acetylcholinesterase sensor is: the concentration of chlopyrifos pesticides when 0.1 to, 20 μ g/L, I%=1.3497c+14.301; When the concentration of chlopyrifos pesticides is 20 to, 150 μ g/L, I%=0.3686c+33.135.The acetylcholinesterase sensor performance detects and comprises accuracy, stability and power of regeneration, and the acetylcholinesterase sensor is to the mensuration of the vegetable sample recovery.
Its preparation principle is: because organophosphorus pesticide has higher specificity with acetylcholinesterase and combines, acetylcholinesterase therefore commonly used is as the molecular recognition elements that detects organophosphorus pesticide.The acetylcholinesterase biology sensor is that acetylcholinesterase is fixed on electrode surface, and the substrate for enzymatic activity acetylcholine hydrolyzation generates choline and acetate.Agricultural chemicals is structurally similar a bit with the substrate acetylcholine, and it can effectively combine with the activated centre of acetylcholine, the activity of acetylcholine esterase inhibition.Choline is a kind of electroactive material; Can under certain potentials oxidation reaction take place; Can measure the concentration of residues of pesticides through the size of thiocholine oxidation peak in the volt-ampere scanning process; The variation of enzymatic reaction current signal obtains the inhibiting rate of agricultural chemicals to enzyme when relatively having or not agricultural chemicals, and this inhibiting rate becomes corresponding relation with the concentration of agricultural chemicals, just can record the content of organophosphorus or carbamate chemicals for agriculture.
The present invention adopts CMC-nano-Au composite; The EDC-NHS potpourri is modified glass-carbon electrode; Compare with traditional method, because CMC is more soluble in water than shitosan, and biocompatibility is better; Therefore more stable through the resulting CMC-nano-Au composite of local reduction way, and be more suitable for the fixing of biomolecule.The EDC-NHS potpourri can the activation nano-complex carboxyl form a kind of intermedium, make more stable being fixed on the electrode of acetylcholinesterase.To pass through the glass-carbon electrode of above-mentioned steps modified, process enzyme sensor after, the precision that detects agricultural chemicals is higher, wider, realizes small-sized, convenient, as to be applicable to on-the-spot detection purpose.Before the current mode acetylcholinesterase biology sensor that adopts the present invention to process can be gathered, go on the market at vegetables and fruits; Carry out the fast measuring of residues of pesticides; Directly whether residues of pesticides are exceeded standard and detect; The fruits and vegetables of avoiding containing remains of pesticide because of food cause poisoning, and the technical support of residue detection is provided for agricultural product security production and consumption.
For reaching above purpose, take following technical scheme to realize: a kind of preparation method who detects the acetylcholinesterase sensor of residues of pesticides is characterized in that: the cleaning of glass-carbon electrode before the preparation of (1) current mode acetylcholinesterase sensor; Activation and performance test; If the spike potential difference in the test loop volt-ampere curve is below 80 mV, and as far as possible near 64 mV, said glass-carbon electrode can use; Otherwise to clean again, up to meeting the requirements.(2) on the glass-carbon electrode that cleans up, drip and be coated with CMC-nano-Au composite, after drying naturally under the room temperature, drip and be coated with N-hydroxy-succinamide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC) potpourri carries out activation; Behind 1 h; Rinse well with PBS, drip and to be coated with the acetylcholine esterase, 2 h post-flush are clean; Current mode acetylcholinesterase sensor preparation finishes, and it is subsequent use to put in the refrigerator 4 ℃ of preservations.
For reaching above purpose; Take following technical scheme to realize: a kind of preparation method who detects the acetylcholinesterase sensor of residues of pesticides; It is characterized in that: a series of chlopyrifos titer of (1) configuration, in order to working curve, sensing range and the detectability of the current mode acetylcholinesterase biology sensor that draws above-mentioned preparation; (2) electrode after the agricultural chemicals inhibition is immersed pralidoxime 10 min, record the regenerability of its acetylcholinesterase biology sensor; (3) to the degree of accuracy of acetylcholinesterase biology sensor, but stability is estimated with reuse; (4) through detecting the recovery that actual vegetable sample draws this sensor.
The present invention at first prepares CMC, and in the water that can be dissolved in neutrality of this shitosan, its biocompatibility is better; Adopt local reduction way to prepare CMC-nano-Au composite, the preparation method is simple, and the compound particle diameter is little; Size is even, good dispersion degree.The use of CMC-nano-Au composite has effectively increased the current-responsive of acetylcholinesterase biology sensor, has further increased the precision that detects.The carboxyl of EDC in can the activation nano-complex forms a kind of intermedium, can with enzyme in the amino reaction, form acid amides and combine, enzyme is fixed on the electrode, but this intermedium is unstable, need NHS stable.The EDC-NHS potpourri makes the fixing more stable of enzyme, has improved the stability of acetylcholinesterase biology sensor greatly.
The preparation technology of said acetylcholinesterase biology sensor is following: (1) is dripped 5 μ L CMC-nm of gold nano-complexes and is coated on the good glass-carbon electrode of pre-service; After the drying, rinse out the compound that does not combine with phosphate buffer under the room temperature; (2) get 5 μ L EDC-NHS drips of solution and be coated in the electrode surface that nano-complex is modified, leave standstill 1 h under the normal temperature, surperficial with the PBS damping fluid flushing of PH=7.5 then; (3) after the above-mentioned electrode of making dries; Get 5 μ L acetylcholinesterase drips of solution and be coated on the electrode, 2 h under the normal temperature, surperficial with the phosphate buffer flushing of PH=7.5 then; The acetylcholinesterase biology sensor completes, and is kept under 4 ℃ of conditions subsequent use.
Embodiment
Embodiment: (1) Preparation of Carboxymethylchitosan: with 20 g shitosans, 200 mL, 3.5 M NaOH add in the flask, and-18 ° of C stir 24 h.Then 30 g chloroacetic acids are dissolved in the 40 mL isopropyl alcohols, slowly join together in the flask, reaction 7 h under 50 ° of C; The sediment that reaction is obtained carries out suction filtration, and with ethanol/water (3:1) solution washing, obtains the CMC sodium salt; It is immersed in 75% ethanol, add 32% HCl, react 30 min; And filter, last, dry 12 h obtain the CMC solid under 60 ° of C.(2) preparation of CMC-nano-Au composite: 20 mL, 1% CMCs (W/V) WS adds 200 μ L, 1% HAuCl in stirring
4(W/V) WS.Behind 10 min, slowly add the freshly prepared 0.4 M KBH of 100 μ L
4The rapid pulverize of solution is red, and the generation of nm of gold is described.Continue to stir 3 h, obtain CMC-nano-Au composite.(3) cleaning of glass-carbon electrode: before said glass-carbon electrode is modified, at first immerse " piranha " solution (H of heat
2SO
4: 30% H
2O
2=soaking 15 min in 3:1), water cleans up, and next uses the Al of 0.3 μ m, 30 nm
2O
3Slurry is polished to minute surface on chamois leather, the polishing back moves in the ultrasonic water bath and cleans with deionized water flush away surface contaminants, and each 5 min repeat secondary, use the HNO of 6 mol/L then successively
3, absolute ethyl alcohol and deionized water ultrasonic cleaning, nitrogen environment is dry down; (4) activation of glass-carbon electrode: thoroughly after the washing, electrode is at 0.5 mol/L H
2SO
4Use the cyclic voltammetry activation in the solution, sweep limit 1.0～-1.0V, scan repeatedly till reaching stable cyclic voltammogram.The test of the glass-carbon electrode that (5) pre-service is good: containing 2 * 10 of 0.1 mol/L KCl at last
-3Mol/L K
3Fe (CN)
6Carry out cyclic voltammetry scan in the solution, to test the performance of said glass-carbon electrode, sweep velocity 50 mV/s; Sweep limit is-0.1V～0.6V, when the spike potential difference in the said cyclic voltammetry curve below 80 mV, and as far as possible near 64 mV; Resulting glass-carbon electrode can use; Otherwise to return again in the step (1), handle said glass-carbon electrode, up to meeting the requirements; (6) 5 μ L CMC-nano-Au composites are dripped be coated on the good glass-carbon electrode of pre-service, dry 2 h under the room temperature rinse out the compound that does not combine with phosphate buffer, and nitrogen dries up; (7) get 5 μ L EDC-NHS mixed solutions and drip the electrode surface that is coated in the nano-complex modification, leave standstill 1 h under the normal temperature, with the PBS damping fluid flushing surface of PH=7.5, nitrogen dries up then; (8) get 5 μ L acetylcholinesterase drips of solution and be coated on the electrode, 2 h under the normal temperature, with the PBS damping fluid flushing surface of PH=7.5, nitrogen dries up then, and the acetylcholinesterase biology sensor completes, and is kept under 4 ℃ of conditions subsequent use.(9) the above-mentioned acetylcholinesterase sensor for preparing is carried out the cyclic voltammetric test with 50 mV/s sweep velocitys in the PBS of the pH 7.5 of the chlorinated thio acetylcholine (ATCl) that contains 1 mM, potential window is 0.0V ~ 1.2V.(10) the chlopyrifos standard solution of configuration 0.05 μ g/L-200 μ g/L; When agricultural chemicals is measured; Above-mentioned acetylcholinesterase sensor is immersed in 10 min in the chlopyrifos standard solution of variable concentrations; In the PBS that contains 1 mM ATCl, carry out cyclic voltammetry scan then, inhibiting rate I can be tried to achieve by following formula:
I(%)=(i
P,?control?i
P,?exp)/i
P,?control×100%
I wherein
P, controlAnd i
P, expBe respectively modified electrode through the agricultural chemicals inhibition with through after the agricultural chemicals inhibition; Peak current in ATCl solution; Pesticide concentration and inhibiting rate are certain linear, make working curve diagram, obtain linear relationship and detectability between pesticide concentration and the inhibiting rate.(11) accuracy of acetylcholinesterase biology sensor is studied through deviation experiment in organizing.Same electrode carries out replication acquisition group within variance coefficient six times; Repeat the different electrode pair normal concentration evaluation of pesticides of six preparations and obtain the between-group variation coefficient.When electrode not the time spent be kept in 4 ℃ of refrigerators, the variation of 7 days and the response of 30 days after-currents obtains the stability of this sensor.(12) after agricultural chemicals is suppressed sensor be immersed in 10 min in the pralidoxime iodide solution of 4.0 mM, rinse well with PBS, in the PBS of 1 mM ATCl, carry out cyclic voltammetry scan, to detect its power of regeneration; (13) thoroughly clean up vegetables and with washed with de-ionized water 3 times, certain density agricultural chemicals in the sprinkling, place 24 h after, do solvent supersonic with pH 7.5 PBSs and handle 30 min (changing solvent three times), to detect the recovery of actual sample.
The detection method of this kind acetylcholinesterase biology sensor residues of pesticides is simple to operate; The scope that detects pesticide concentration is wide; Detection time is short, and is highly sensitive, good stability; Power of regeneration good and to actual sample analysis has the recovery and reappearance preferably, meet China's Fast Determination of Pesticide Residue technical development with the internationalization requirement.
Claims (3)
1. preparation method who detects the acetylcholinesterase sensor of residues of pesticides; It is characterized in that: the group shape process of current mode acetylcholinesterase sensor is coated with CMC-nano-Au composite for dripping; Drip and to be coated with the EDC-NHS potpourri, drip and be coated with the acetylcholine esterase.
2. a kind of preparation method who detects the acetylcholinesterase sensor of residues of pesticides as claimed in claim 1; It is characterized in that: the structure at acetylcholinesterase sensor sensing interface and process characterize; The foundation of acetylcholinesterase working sensor curve; The detection of acetylcholinesterase sensor performance, the acetylcholinesterase sensor is to the detection of actual sample.
3. a kind of preparation method who detects the acetylcholinesterase sensor of residues of pesticides as claimed in claim 1; It is characterized in that: the working curve of prepared current mode acetylcholinesterase sensor is: the concentration of chlopyrifos pesticides when 0.1 to, 20 μ g/L, I%=1.3497c+14.301; When the concentration of chlopyrifos pesticides is 20 to, 150 μ g/L, I%=0.3686c+33.135, the acetylcholinesterase sensor performance detects and comprises selectivity, stability and power of regeneration, and the acetylcholinesterase sensor is to the mensuration of the vegetable sample recovery.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104458844A (en) * | 2014-12-09 | 2015-03-25 | 江南大学 | Preparation method of nanogold with controllable size on presence of dioxide carbon switchable surfactant, and applications of nanogold in electrochemical sensor |
| CN106053568A (en) * | 2016-08-12 | 2016-10-26 | 浙江大学 | Composite material modified electrode for measurement of vitamin C concentration and application thereof |
| CN107389916A (en) * | 2017-06-26 | 2017-11-24 | 安徽安龙基因医学检验所有限公司 | A kind of preparation method of CD133 immunomagnetic beadses |
| CN110794013A (en) * | 2019-11-15 | 2020-02-14 | 山东省农业科学院农业质量标准与检测技术研究所 | Detect aflatoxin's electrochemical sensor |
| CN112782132A (en) * | 2019-11-07 | 2021-05-11 | 华南理工大学 | Method for detecting organic phosphate flame retardant based on acetylcholinesterase-gold nanoclusters |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104458844A (en) * | 2014-12-09 | 2015-03-25 | 江南大学 | Preparation method of nanogold with controllable size on presence of dioxide carbon switchable surfactant, and applications of nanogold in electrochemical sensor |
| CN106053568A (en) * | 2016-08-12 | 2016-10-26 | 浙江大学 | Composite material modified electrode for measurement of vitamin C concentration and application thereof |
| CN107389916A (en) * | 2017-06-26 | 2017-11-24 | 安徽安龙基因医学检验所有限公司 | A kind of preparation method of CD133 immunomagnetic beadses |
| CN112782132A (en) * | 2019-11-07 | 2021-05-11 | 华南理工大学 | Method for detecting organic phosphate flame retardant based on acetylcholinesterase-gold nanoclusters |
| CN112782132B (en) * | 2019-11-07 | 2022-05-24 | 华南理工大学 | Method for detecting organic phosphate flame retardant based on acetylcholinesterase-gold nanoclusters |
| CN110794013A (en) * | 2019-11-15 | 2020-02-14 | 山东省农业科学院农业质量标准与检测技术研究所 | Detect aflatoxin's electrochemical sensor |
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Application publication date: 20120725 |