CN101551356A - Preparation of immobilization acetylcholinesterase electrochemistry biosensor - Google Patents
Preparation of immobilization acetylcholinesterase electrochemistry biosensor Download PDFInfo
- Publication number
- CN101551356A CN101551356A CNA2008102073162A CN200810207316A CN101551356A CN 101551356 A CN101551356 A CN 101551356A CN A2008102073162 A CNA2008102073162 A CN A2008102073162A CN 200810207316 A CN200810207316 A CN 200810207316A CN 101551356 A CN101551356 A CN 101551356A
- Authority
- CN
- China
- Prior art keywords
- layer
- carbon nano
- electrode
- tube
- immersed
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to a method for preparing an electrochemistry biosensor, in particular an immobilization acetylcholinesterase method and a method for preparing current type biosensor. Specifically, electrodeposition technology is used to deposit a carbon nano tube with negative charge on the surface of glassy carbon electrode; subsequently, layer-by-layer electrostatic self-assembly process is conducted in polydiene propyl dimethyl ammonium chloride solution and carbon nano tube dispersion liquid to obtain a modified electrode with high self-assembly efficiency and stable modified layer. And then, layer-by-layer self-assembly is conducted again in the polydiene propyl dimethyl ammonium chloride solution and PBS of choline esterase to obtain an immobilization acetylcholinesterase current type biosensor with high stability. The method can quickly and stably detect the concentration of parathion in remains of pesticide under normal temperature.
Description
Technical field
The present invention relates to a kind of preparation method of electrochemica biological sensor, particularly a kind of preparation method of fixedly acetylcholinesteraseelectrochemistry electrochemistry biosensor.
Background technology
Organophosphorus pesticide has irreplaceable effect in world's agricultural production.Along with the adding of WTO, the volume of trade of China and other countries' agricultural product increases year by year, and importer proposes more and more higher requirement to the quality of agricultural product.In order to improve the safe mass of agricultural products in China, enhance our international competitiveness, the Chinese government attaches great importance to the residues of pesticides problem, greatly develops the fast survey technology of agricultural chemicals, strengthens the real-time monitoring to agricultural product.The Detection ﹠ Controling of strengthening residues of pesticides in the agricultural product have become the task of top priority.
What pesticide residue analysis was commonly used in the world is chromatography, chromatograph-mass spectrometer coupling method etc., but owing to instrument complexity, costliness, complex operation, often needs processing procedure in earlier stage such as liquid-liquid extraction or leaching, is difficult to quick use.Need develop at normal temperatures fast survey technology at the current national conditions of China, the edible safety of the crops of in time guaranteeing to go on the market.
The eighties, pesticide in vegetable Fast Detection Technique some countries abroad are extensive use of.In recent years, the wholesale vegetable market of some provinces and cities of China, vegetables production base etc. also bring into use rapid method to detect, but it detects principle and all adopts photoelectric colorimetry, this quasi-instrument detection sensitivity is lower, the agricultural residual detection that has does not reach International or National lowest detectable limit standard, is unfavorable for further popularizing.
In recent years, along with developing rapidly of fields such as life science and environmental science, Application and Development based on the various biology sensors of biomaterial more and more receives publicity, and the detection that acetylcholinesteraseelectrochemistry electrochemistry biosensor is applied to organophosphorus pesticide is exactly one of them.
(acetylcholinesterase AChE) is a kind of enzyme that can selectivity catalytic substrate acetylcholine hydrolyzation, ubiquity in animal body to acetylcholinesterase.Organophosphorus pesticide is the inhibitor of AChE, can make the active obviously reduction of AChE in the body.Utilize the inhibition of organophosphorus pesticide, can measure the content of organophosphorus pesticide in the environmental sample the AChE activity.One of important method of measuring the AChE activity is an AChE biology sensor method.Wherein the principle of ampere-type AChE biology sensor is: AChE catalytic substrate acetyl thio choline (acetylthiocholine, ATCh) hydrolysis, generate thiocholine and acetate, wherein, thiocholine has electrochemical activity, can produce the redox electric current on electrode, the big I of this electric current reflects the activity of AChE indirectly under certain condition.
Carbon nano tube modified electrode has good electrochemical stability, good electrical conductivity, bigger serface and good electrocatalysis characteristic, can promote the advantages such as electron transport of electroactive material.At present, the carrier that carbon nano-tube is used as electroactive material and electroactive material has been widely used in galvanochemistry and the biology sensor, but, particularly be applied to immobilization acetylcholinesteraseelectrochemistry electrochemistry biosensor the detection research of parathion is not also appeared in the newspapers at present as yet fixing of self assembly enzyme layer by layer again after the first electro-deposition.
Summary of the invention
In order to solve organophosphorus pesticide at normal temperatures, response is quick, good stability fast survey technology, developed a kind of immobilization acetylcholinesteraseelectrochemistry electrochemistry biosensor.
The technical solution used in the present invention
This research makes electric charge on the enzyme band according to the size of isoelectric point, fix with the form interaction of Electrostatic Absorption with the carbon nano tube modified layer of oppositely charged then, made up acetylcholinesterase sensor, and be applied to the actual detected of organophosphorus pesticide parathion based on multi-walled carbon nano-tubes and diallyl dimethyl ammoniumchloride self assembly layer by layer.
At first be that the carbon nano-tube of utilizing electro-deposition techniques will have negative charge deposits to the glass-carbon electrode surface, in diallyl dimethyl ammoniumchloride solution and carbon nano-tube dispersion liquid, carry out the layer upon layer electrostatic self assembling process subsequently, obtain self assembly efficient height, the stable modified electrode of decorative layer.Then, in the PBS of diallyl dimethyl ammoniumchloride solution and cholinesterase (phosphate buffer) solution, carry out self assembly layer by layer again, obtain the higher immobilization acetylcholinesteraseelectrochemistry current mode chemical biosensor of stability.
Its concrete preparation process is as follows:
(1) after the glass-carbon electrode process is polished, cyclic voltammetry scan 10 circles carry out the electrochemical activation pre-service in the phosphate buffer of pH5.00～8.00, form two electrode system electro-deposition 2 hours with platinum electrode, form the carbon nano-tube deposition layer that has a large amount of negative charges of bright black;
(2) be 0.8molL with being immersed in ionic strength through the glass-carbon electrode after the electro-deposition in the step (1)
-11% diallyl dimethyl ammoniumchloride solution in, carry out the static self assembly of diallyl dimethyl ammoniumchloride, take out behind 10～15min and carefully wash 2min with deionized water, the sodium tetraborate solution that is immersed in pH7.0～10.0 again is 0.5～1.5gmL as the concentration of spreading agent
-1In the dispersion liquid of carbon nano-tube, carry out the static self assembly of carbon nano-tube, take out behind the 15min, carefully wash 2min with deionized water, so far, carbon nano tube modified layer is 1 layer, repeatable operation, the decorative layer number of plies up to carbon nano-tube is 5, and is last, is that the electrode of multi-walled carbon nano-tubes is dried under infrared lamp with the outermost layer that obtains;
(3) electrode after the oven dry under the infrared lamp of the gained in the step (2) being immersed in ionic strength is 0.8molL
-11% diallyl dimethyl ammoniumchloride solution in, carry out the static self assembly of diallyl dimethyl ammoniumchloride, take out behind the 15min and carefully wash 2min with deionized water, be immersed in pH again and be in the phosphate buffer solution dispersion liquid of 5.00～8.00 cholinesterase, carry out the static self assembly of carbon nano-tube, take out behind the 15min, carefully wash 2min with deionized water, so far, the cholinesterase decorative layer is 1 layer, repeatable operation, the decorative layer number of plies up to cholinesterase is 3, in order better enzyme to be fixed, continue electrode is immersed 5min in the diallyl dimethyl ammoniumchloride solution, promptly get immobilization acetylcholinesteraseelectrochemistry electrochemistry biosensor.
Beneficial effect of the present invention
Because cholinesterase is fixed on the carbon nano tube modified electrode surface effectively, has kept high enzyme activity.Therefore, immobilization acetylcholinesteraseelectrochemistry electrochemistry biosensor of the present invention can detect the parathion concentration in the remains of pesticide rapidly and stably at normal temperatures.
Description of drawings
The relation curve that Fig. 1 enzyme activity and electric current change
Fig. 2 enzyme inhibition rate and parathion concentration are born the logarithmic relationship curve
Fig. 3 enzyme inhibition rate and concentration are 10
-10-10
-4GL
-1The linear relationship of the negative logarithm of the parathion concentration in the scope
Embodiment
The invention will be further described below in conjunction with embodiment, but do not limit the present invention.
Embodiment
Below in conjunction with accompanying drawing the present invention is specified.
The preparation process of immobilization acetylcholinesteraseelectrochemistry electrochemistry biosensor is as follows:
(1) after the glass-carbon electrode process is polished, cyclic voltammetry scan 10 circles carry out the electrochemical activation pre-service in the phosphate buffer of pH7.40, form two electrode system electro-deposition 2 hours with platinum electrode, form the carbon nano-tube deposition layer that has a large amount of negative charges of bright black.
(2) be 0.8molL with being immersed in ionic strength through the glass-carbon electrode after the electro-deposition in the step (1)
-11% diallyl dimethyl ammoniumchloride solution in, carry out the static self assembly of diallyl dimethyl ammoniumchloride, take out behind the 15min and carefully wash 2min with deionized water, the sodium tetraborate solution that is immersed in pH9.18 again is 1mgmL as the concentration of spreading agent
-1In the dispersion liquid of carbon nano-tube, carry out the static self assembly of carbon nano-tube, take out behind the 15min, carefully wash 2min with deionized water, so far, carbon nano tube modified layer is 1 layer, repeatable operation, the decorative layer number of plies up to carbon nano-tube is 5, and is last, is that the electrode of multi-walled carbon nano-tubes is dried under infrared lamp with the outermost layer that obtains.
(3) electrode after the oven dry under the infrared lamp of the gained in the step (2) being immersed in ionic strength is 0.8molL
-11% diallyl dimethyl ammoniumchloride solution in, carry out the static self assembly of diallyl dimethyl ammoniumchloride, take out behind the 15min and carefully wash 2min with deionized water, be immersed in pH again and be in the phosphate buffer solution dispersion liquid of 7.40 cholinesterase, carry out the static self assembly of carbon nano-tube, take out behind the 15min, carefully wash 2min with deionized water, so far, the cholinesterase decorative layer is 1 layer, repeatable operation, the decorative layer number of plies up to cholinesterase is 3, in order better enzyme to be fixed, continue electrode is immersed 5min in the diallyl dimethyl ammoniumchloride solution, promptly get immobilization acetylcholinesteraseelectrochemistry electrochemistry biosensor.
The application of immobilization acetylcholinesteraseelectrochemistry electrochemistry biosensor
The enzyme electrode that makes is used for the detection of organophosphorus pesticide parathion: the PBS damping fluid with 6mLpH=7.40 is an electrolyte, measure its current-responsive to the acetyl chloride thiocholine down at the fixed voltage between 0.1～2.0V (vs.SCE), all tests are all carried out between 25～40 ℃.After immobilization acetylcholinesteraseelectrochemistry electrochemistry biosensor was soaked by agricultural chemicals, its current-responsive to the acetyl chloride thiocholine can change, and this variation can reflect the concentration of agricultural chemicals indirectly.
Fig. 1 immobilization acetylcholinesteraseelectrochemistry electrochemistry biosensor is as can be seen soaked by the agricultural chemicals of variable concentrations, increase along with pesticide concentration, the acetylcholinesterase enzyme activity descends thereupon in the immobilization acetylcholinesteraseelectrochemistry electrochemistry biosensor, its electric current I value to the acetyl chloride thiocholine decreases, and linear.Thus, draw the acetylcholinesterase enzyme inhibition rate and have positive correlation with residues of pesticides.
Get through linear regretional analysis:
Enzyme inhibition rate (%)=(I
0-I
1)/I
0* 100%
Wherein: I
0The immobilization acetylcholinesteraseelectrochemistry electrochemistry biosensor stable response electric current (blank electric current) that finger is not suppressed by agricultural chemicals
I
1Immobilization acetylcholinesteraseelectrochemistry electrochemistry biosensor stable response electric current after finger is suppressed by agricultural chemicals
Among Fig. 2, for the sulphur phosphorus in the organophosphorus pesticide, with the standard model stepwise dilution to 10 of 100ppm
-12GL
-1, respectively immobilization acetylcholinesteraseelectrochemistry electrochemistry biosensor is immersed pesticidal solutions 10min after, measure the acetylcholinesterase inhibiting rate.By Fig. 2 and Fig. 3 as seen, parathion concentration is 10
-10-10
-4GL
-1In the scope, the negative logarithm of the inhibiting rate of acetylcholinesterase and the sulphur phosphorus concentration in the organophosphorus pesticide is the better linearity relation, and related coefficient reaches 0.9880.Simultaneously, this immobilization acetylcholinesteraseelectrochemistry electrochemistry biosensor reaches 10 to the detectability of the sulphur phosphorus in the organophosphorus pesticide is low
-10GL
-1
Claims (2)
1. the preparation method of an immobilization acetylcholinesteraseelectrochemistry electrochemistry biosensor is characterized in that comprising the following steps:
(1) after the glass-carbon electrode process is polished, cyclic voltammetry scan 10 circles carry out the electrochemical activation pre-service in the phosphate buffer of pH5.00～8.00, form two electrode system electro-deposition 2 hours with platinum electrode, form the carbon nano-tube deposition layer that has a large amount of negative charges of bright black;
(2) be 0.8molL with being immersed in ionic strength through the glass-carbon electrode after the electro-deposition in the step (1)
-11% diallyl dimethyl ammoniumchloride solution in, carry out the static self assembly of diallyl dimethyl ammoniumchloride, take out behind 10～15min and carefully wash 2min with deionized water, the sodium tetraborate solution that is immersed in pH7.0～10.0 again is 0.5～1.5gmL as the concentration of spreading agent
-1In the dispersion liquid of carbon nano-tube, carry out the static self assembly of carbon nano-tube, take out behind the 15min, carefully wash 2min with deionized water, so far, carbon nano tube modified layer is 1 layer, repeatable operation, the decorative layer number of plies up to carbon nano-tube is 5, and is last, is that the electrode of multi-walled carbon nano-tubes is dried under infrared lamp with the outermost layer that obtains;
(3) electrode after the oven dry under the infrared lamp of the gained in the step (2) being immersed in ionic strength is 0.8molL
-11% diallyl dimethyl ammoniumchloride solution in, carry out the static self assembly of diallyl dimethyl ammoniumchloride, take out behind the 15min and carefully wash 2min with deionized water, be immersed in pH again and be in the phosphate buffer solution dispersion liquid of 5.00～8.00 cholinesterase, carry out the static self assembly of carbon nano-tube, take out behind the 15min, carefully wash 2min with deionized water, so far, the cholinesterase decorative layer is 1 layer, repeatable operation, the decorative layer number of plies up to cholinesterase is 3, in order better enzyme to be fixed, continue electrode is immersed 5min in the diallyl dimethyl ammoniumchloride solution, promptly get immobilization acetylcholinesteraseelectrochemistry electrochemistry biosensor.
2. immobilization acetylcholinesteraseelectrochemistry electrochemistry biosensor according to claim 1 is used for the detection of the residual parathion of agricultural chemicals.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008102073162A CN101551356A (en) | 2008-12-19 | 2008-12-19 | Preparation of immobilization acetylcholinesterase electrochemistry biosensor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008102073162A CN101551356A (en) | 2008-12-19 | 2008-12-19 | Preparation of immobilization acetylcholinesterase electrochemistry biosensor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101551356A true CN101551356A (en) | 2009-10-07 |
Family
ID=41155713
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2008102073162A Pending CN101551356A (en) | 2008-12-19 | 2008-12-19 | Preparation of immobilization acetylcholinesterase electrochemistry biosensor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101551356A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101923050A (en) * | 2010-07-02 | 2010-12-22 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Renewable optical biosensor for detecting organophosphorus pesticides |
| CN103115949A (en) * | 2013-01-22 | 2013-05-22 | 山东理工大学 | Preparation method of enzyme biosensor for detecting pesticide residues |
| CN104677957A (en) * | 2015-02-11 | 2015-06-03 | 安徽理工大学 | Gold-titanic niobic acid layered nanocomposite enzyme sensor and preparation method and application thereof |
| CN106645335A (en) * | 2016-09-30 | 2017-05-10 | 浙江大学 | Method for detecting glucose through one-step reduction and deposition of graphene/phenylboronic acid compound |
| CN111089889A (en) * | 2019-12-25 | 2020-05-01 | 北京农业质量标准与检测技术研究中心 | Silk-screen printing carbon nanotube sensor, preparation method thereof and pesticide detection application |
-
2008
- 2008-12-19 CN CNA2008102073162A patent/CN101551356A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101923050A (en) * | 2010-07-02 | 2010-12-22 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Renewable optical biosensor for detecting organophosphorus pesticides |
| CN101923050B (en) * | 2010-07-02 | 2012-03-14 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Renewable optical biosensor for detecting organophosphorus pesticides |
| CN103115949A (en) * | 2013-01-22 | 2013-05-22 | 山东理工大学 | Preparation method of enzyme biosensor for detecting pesticide residues |
| CN103115949B (en) * | 2013-01-22 | 2014-10-08 | 山东理工大学 | Preparation method of enzyme biosensor for detecting pesticide residues |
| CN104677957A (en) * | 2015-02-11 | 2015-06-03 | 安徽理工大学 | Gold-titanic niobic acid layered nanocomposite enzyme sensor and preparation method and application thereof |
| CN106645335A (en) * | 2016-09-30 | 2017-05-10 | 浙江大学 | Method for detecting glucose through one-step reduction and deposition of graphene/phenylboronic acid compound |
| CN106645335B (en) * | 2016-09-30 | 2019-01-01 | 浙江大学 | One step reduction deposition graphene/phenyl boric acid compound glucose detection method |
| CN111089889A (en) * | 2019-12-25 | 2020-05-01 | 北京农业质量标准与检测技术研究中心 | Silk-screen printing carbon nanotube sensor, preparation method thereof and pesticide detection application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zehani et al. | Highly sensitive electrochemical biosensor for bisphenol A detection based on a diazonium-functionalized boron-doped diamond electrode modified with a multi-walled carbon nanotube-tyrosinase hybrid film | |
| Chauhan et al. | An amperometric acetylcholinesterase sensor based on Fe3O4 nanoparticle/multi-walled carbon nanotube-modified ITO-coated glass plate for the detection of pesticides | |
| Kang et al. | Methyl parathion sensors based on gold nanoparticles and Nafion film modified glassy carbon electrodes | |
| Gong et al. | Highly sensitive visible light activated photoelectrochemical biosensing of organophosphate pesticide using biofunctional crossed bismuth oxyiodide flake arrays | |
| CN101526493B (en) | Electrochemical biosensor based on chitosan-immobilized acetylcholinesterase and application thereof | |
| Thakkar et al. | Acetylcholine esterase enzyme doped multiwalled carbon nanotubes for the detection of organophosphorus pesticide using cyclic voltammetry | |
| Kuswandi et al. | A simple visual ethanol biosensor based on alcohol oxidase immobilized onto polyaniline film for halal verification of fermented beverage samples | |
| Wang et al. | Nano-composite ZrO2/Au film electrode for voltammetric detection of parathion | |
| CN106248748B (en) | A kind of acetylcholinesterasebiosensor biosensor and its application | |
| Fan et al. | Sensitive voltammetric response of methylparathion on single-walled carbon nanotube paste coated electrodes using ionic liquid as binder | |
| Gokoglan et al. | Paper based glucose biosensor using graphene modified with a conducting polymer and gold nanoparticles | |
| Tian et al. | Fabrication of graphene quantum dots/silicon nanowires nanohybrids for photoelectrochemical detection of microcystin-LR | |
| Dhull | A Nafion/AChE-cSWCNT/MWCNT/Au-based amperometric biosensor for the determination of organophosphorous compounds | |
| CN101551356A (en) | Preparation of immobilization acetylcholinesterase electrochemistry biosensor | |
| Zezza et al. | Determination of phosphorus in biodiesel using 1: 12 phosphomolybdic modified electrode by cyclic voltammetry | |
| CN105021679A (en) | Silk-screen printing electrode sensor preparation method based on ionic liquid-multi-wall carbon nano-tube modification | |
| Hu et al. | Development of enzymatic electrochemical biosensors for organophosphorus pesticide detection | |
| Chai et al. | Carbamate insecticide sensing based on acetylcholinesterase/Prussian blue-multi-walled carbon nanotubes/screen-printed electrodes | |
| CN103743804A (en) | Organic phosphorus electrochemical biosensor based on adsorption of nano particles | |
| CN110031521A (en) | The preparation of acetylcholinesterasebiosensor biosensor and to organic phosphorus detection application | |
| Hu et al. | Acetylcholinesterase Sensor with Patterned Structure for Detecting Organophosphorus Pesticides Based on Titanium Dioxide Sol‐gel Carrier | |
| Lü et al. | Non-enzymatic electrochemical sensors based on conducting polymer hydrogels for ultrasensitive carbaryl pesticide detection | |
| Zhang et al. | A stable biosensor for organophosphorus pesticide detection based on chitosan modified graphene | |
| CN101750442B (en) | Monodispersive bimetal Au/Pt nano-particle modified electrode for detecting mercury in water and preparation method thereof | |
| CN104931554A (en) | Preparation method and application of sensor based on screen-printed carbon electrode |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20091007 |