CN103926280A - Preparation method for electrochemical enzyme sensor - Google Patents
Preparation method for electrochemical enzyme sensor Download PDFInfo
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- CN103926280A CN103926280A CN201410175555.XA CN201410175555A CN103926280A CN 103926280 A CN103926280 A CN 103926280A CN 201410175555 A CN201410175555 A CN 201410175555A CN 103926280 A CN103926280 A CN 103926280A
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- polystyrene
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Abstract
The invention discloses a preparation method for an electrochemical enzyme sensor, relates to the technical field of food detection, medicaments, environmental analysis and industrial control, in particular to the technical field of the application of polystyrene/graphene composite nanoparticles, and is prepared by immobilizing biological enzyme through the polystyrene/graphene composite nanoparticles. The electrochemical enzyme sensor is easy to prepare, high in reproducibility and stable in performance, has the characteristics of short time, easiness in operation, high specificity, high sensitivity and the like when the electrochemical enzyme sensor is used for the electrochemical detection of biomolecules, and can be used for the quantitative detection of the biomolecules.
Description
Technical field
The present invention relates to the technical field of food inspection, medicine, environmental analysis and Industry Control, also relate to the applied technical field that is particularly related to polystyrene/graphene composite nanoparticle.
Background technology
Biology sensor is comprised of biological identification element and two parts of signal converter part conventionally.Biology sensor can be authorized mechanism or mode changeover signal classification according to biologic specificity.According to the difference of biological chemistry acceptor, biology sensor can be divided into enzyme sensor, microbiological sensor, immunosensor, tissus sensor and cell sensor etc.Biology enzyme sensor is to using enzyme as sensitive membrane, and then a series of biochemical reactions that determinand occurred on sensitive membrane surface by transducer are converted to electric signal output record by chemical signal.Because enzyme has selectivity and the catalytic performance of height, thereby realize the accurate detection to object.Having simple to operately, pollute the advantages such as few, is more satisfactory biology sensor.
But there is the shortcomings such as electrode fabrication process is complicated, easily contaminated electrode, interference measurement, biocompatibility be poor in existing biology enzyme sensor in use mediator.
Graphene has excellent electricity, calorifics and mechanical property, and it can be widely used in ultracapacitor, battery, energy storage device, nano composite material and biology sensor etc.In recent years, because nanometer technology progressively enters field of biosensors, and cause breakthrough progress.
In order to bring into play fully the advantage of Graphene in function and performance, overcome the weakness that it is easily assembled, graphene film is wrapped on even, the regular polymer microsphere of form and forms macromolecule/graphene composite particle.Adopt Graphene and polystyrene to carry out blend and prepare graphene-based polystyrene composite nanoparticle, this composite nanoparticle specific surface area greatly, good conductivity, biocompatibility advantages of higher.
Biomolecule is fixed on the interface of nano-scale, and its constant density can improve several orders of magnitude, is conducive to widen the range of linearity of detection.Nano material is combined and is made composite nano materials with macromolecular material, this composite nanoparticle has that specific surface area is large, good conductivity, biocompatibility advantages of higher have increased bioactivator fixed amount, surfactivity site, good reaction microenvironment is provided, accelerated electron transfer process, shortened the response time of signal, made sensitivity, sensing range, the repeatability of biology sensor obtain obvious enhancing.
Summary of the invention
The object of the invention is to overcome the defect of above-mentioned biomacromolecule detection method, set up a kind of preparation method who makes full use of the electrochemical enzymatic sensor of polystyrene/graphene composite nanoparticle advantage.
With the immobilized biology enzyme of polystyrene/graphene composite nanoparticle, make electrochemical enzymatic sensor.
Particularly: first polystyrene/graphene composite nanoparticle is scattered in chitosan solution, then mixes with biology enzyme, form mixed solution; Then mixed solution is dripped and be applied to clean glass-carbon electrode surface, then electrode is placed under cryogenic conditions and is dried, make electrochemical enzymatic sensor.
The present invention is with the immobilized biology enzyme of polystyrene/graphene composite nanoparticle, make electrochemical enzymatic sensor, built the biology enzyme electrochemical enzymatic sensor of a high-sensitive robotization, the electrochemical enzymatic sensor making is placed in to detection system, with electrochemical workstation, the glass-carbon electrode of process pre-treatment is as working electrode, and saturated calomel electrode is as auxiliary electrode, platinum plate electrode, as to electrode, adopts this three-electrode system to detect its current signal.Polystyrene/graphene composite nanoparticle can be given full play to the advantage of Graphene satisfactory electrical conductivity, overcomes the weakness that it is easily assembled.Polystyrene/graphene composite nanoparticle has very large specific surface area, for promoting the direct electron transfer between biomolecule and electrode surface that a favourable microenvironment is provided.The method utilizes polystyrene/graphene composite nanoparticle to be combined with biology enzyme, the electrochemical enzymatic sensor making can be glucose sensor, acetylcholine sensor and ethanol sensor etc., utilize biology enzyme to there is selectivity and the catalytic performance of height, thereby realize the accurate detection to object, prepared electrochemical enzymatic sensor has highly sensitive, detectability is low, the advantages such as repeatability and stability are better, be more satisfactory biology sensor, can be applied in fields such as food inspection, medicine, environmental analysis and Industry Control.
The present invention is first mixed to form biology enzyme and water the aqueous solution of biology enzyme, and then the aqueous solution of biology enzyme is mixed to the polystyrene/graphene-shitosan that obtains mixing-biology enzyme mixed solution with the chitosan solution that is dispersed with polystyrene/graphene composite nanoparticle.
In addition, in the aqueous solution of described biology enzyme, the massfraction of biology enzyme is identical with the massfraction that is dispersed in the polystyrene/graphene composite nanoparticle in chitosan solution.This mixing ratio is not only conducive to the transmission of electronics, and is conducive to chitosan solution film forming.
The massfraction of the polystyrene/graphene composite nanoparticle in chitosan solution is 2.0 mg/mL.This concentration is conducive to the transfer of electronics most, the too large or too little transmission that is all unfavorable for electronics of concentration.
In order to obtain clean glass-carbon electrode, before dripping painting, it by glass-carbon electrode, with meta particle diameter, is first the alumina powder polishing of 0.05 μ m, with deionized water rinsing, fall residual alumina powder again, then put into diluted nitric acid aqueous solution ultrasonic cleaning, finally use successively ethanol and redistilled water cleaning electrode surface.The material of the lip-deep oxidisability of clearing electrode or reductibility, the electrode surface that obtains having reappeared.If do not carried out this operation, can affect experimental result, or reduce the credibility of experimental result, even obtain wrong conclusion.
The nitric acid that described diluted nitric acid aqueous solution is 1:1 by volume ratio and water mix and form.This mixing ratio is conducive to the dissolving of electrode surface oxidisability or reducing substances, thereby removes residual impurity.
This electrochemical enzymatic sensor, specifically has following advantage:
(1) polystyrene/graphene composite nanoparticle can be given full play to the advantage of Graphene satisfactory electrical conductivity, overcome the weakness that it is easily assembled, have that specific surface area is large, good biocompatibility, high adsorption capacity, surfactivity advantages of higher, for fixing of biomolecule, can increase fixing molecular amounts, thus intensified response signal.
(2) polystyrene/graphene composite nanoparticle has good electric conductivity, is used for immobilized biology enzyme, and the sensor making is for promoting the direct electron transfer between enzyme and electrode surface that a favourable microenvironment is provided.Have linear relationship good, detectability is low, stability and the advantage such as reproducible.
(3) due to enzyme, there is selectivity and the catalytic performance of height, thereby realize the accurate detection to object.Having simple to operately, pollute the advantages such as few, is more satisfactory biology sensor.
Accompanying drawing explanation
Fig. 1 is the preparation of glucose oxidase electrochemica biological sensor and principle of work schematic diagram.
Fig. 2 is the chronoa mperometric plot graph of a relation of glucose oxidase electrochemica biological sensor to glucose assays.
Fig. 3 is that glucose oxidase electrochemica biological sensor adds the I-T curve relation figure after glucose in phosphate buffer solution.
Embodiment
Electrochemical enzymatic sensor can be glucose sensor, acetylcholine sensor and ethanol sensor etc.
Dilute nitric acid preparing aqueous solution: with nitric acid and deionized water, take the ratio that volume ratio is 1:1 and mix, stand-by.
One, embodiment 1:
1, prepare glucose oxidase electrochemica biological sensor.
(1) by glass-carbon electrode, with meta particle diameter, be the alumina powder polishing of 0.05 μ m, with deionized water, rinse out residual alumina powder, put into diluted nitric acid aqueous solution ultrasonic cleaning, finally use successively ethanol and redistilled water cleaning electrode surface.
(2) get polystyrene/graphene composite nanoparticle 2.0 mg, be scattered in 1 mL chitosan solution, be made into the chitosan solution of the polystyrene/graphene of 2.0 mg/mL, then mix with isocyatic glucose carbohydrate oxidase, form the mixed solution of polystyrene/graphene composite nanoparticle and glucose oxidase.
(3) mixed solution of getting polystyrene/graphene composite nanoparticle and glucose oxidase drips and is applied to pre-service electrode surface, and electrode is positioned in refrigerator under the cryogenic conditions of 4 ℃ to dry 12 hours, makes glucose oxidase sensor.
2, application:
The glucolase sensor making is placed in to detection system, with electrochemical workstation, adopts three-electrode system, detect its current signal.
Three-electrode system be the glass-carbon electrode of usining through pre-treatment as working electrode, using saturated calomel electrode as auxiliary electrode, using platinum plate electrode as to electrode.
As shown in Figure 1, the analysans in sample diffuses to the electrode surface of having modified polystyrene/graphene composite nanoparticle-glucose oxidase, reaction below electrode surface occurs:
GOD(FAD)+2e
+2H
+ GOD(FADH
2) (1)
GOD(FADH
2)+O
2→GOD(FAD)+H
2O
2 (2)
Glucose+GOD (FAD)+O
2→Gluconolactone+GOD (FADH
2) (3)
Via electrode, chemical signal is converted to electric signal output record, from drawing testing concentration.
As shown in Figure 2 be the chronoa mperometric plot of glucose oxidase electrochemica biological sensor to glucose assays, refer under saturated air conditions, in 0.1MpH 7.0 phosphate buffer solutions, continue to stir, drip continuously the current-responsive curve that glucose solution obtains.Modified electrode reaches the steady current of 90 % in 9 s, and this response is linear the increasing of increase with glucose solubility, by calculating linear response range, is 1.0 * 10
4m – 3.2 * 10
3m, sensitivity is 5.067 mAM
1cm
2, detect and be limited to 2.89 * 10
6m(S/N=3).
Be illustrated in figure 3 the electrode of glucose oxidase electrochemica biological sensor in pH 7.0 phosphate buffer solutions, add the I-T curve after 1 mM glucose.When current potential is-0.5 V, studied the stability of glucose oxidase electrochemica biological sensor to glucose assays, under continuous stirring condition, in pH 7.0 phosphate buffer solutions, add after 1 mM glucose, electric current sharply rises, stable through 2000 s after-currents, illustrate that preparing glucose oxidase electrochemica biological sensor has good stability.
By glucose oxidase electrochemica biological sensor, to the current-responsive of 0.1 mM glucose, can investigate the precision that sensor is measured.The parallel mensuration of carrying out 5 times, relative standard deviation is 0.69%, repeats to make 3 glucose sensors under optimal condition, adds respectively 0.1 mM glucose assays 5 times, relative standard deviation is 0.69%, shows that this sensor has good reappearance.
Above electro-chemical test shows, prepared biologic enzyme electrode has glucose electrochemical response ability, the wider range of linearity, low detection line, higher selectivity and good repeatability and stability fast.
Two, embodiment 2:
1, prepare acetylcholinesteraseelectrochemistry electrochemistry biosensor.
(1) get polystyrene/graphene composite nanoparticle 2.0 mg, be scattered in 1 mL chitosan solution, be made into the polystyrene/graphene solution of 2.0 mg/mL, ultrasonic dispersion, with this solution and gold size, be mixed with polystyrene/graphene-gold size solution of 1:1, ultrasonic dispersion, then mixes with isocyatic acetylcholinesterase.
(2) glass-carbon electrode used is the alumina powder polishing of 0.05 μ m with meta particle diameter, with deionized water, rinses out residual alumina powder, puts into dilute nitric acid solution ultrasonic cleaning, finally uses successively ethanol and redistilled water cleaning electrode surface.
(3) mixed solution of getting polystyrene/graphene compound substance and acetylcholinesterase drips and is applied to pre-service electrode surface, and electrode is positioned in refrigerator under the cryogenic conditions of 4 ℃ to dry 12 hours, makes acetylcholinesterase sensor.
2, application:
The acetylcholinesterase sensor making is placed in to detection system, with electrochemical workstation, adopts three-electrode system, detect its current signal.
By the prepared electrochemical sensor of the same proof of the identical test with upper example, there is higher sensitivity, stability and repeatability, low detectability and the fast-response energy to acetylcholinesterase preferably.
Three, embodiment 3:
1, prepare alcohol oxidase electrochemica biological sensor:
(1) by glass-carbon electrode, with meta particle diameter, be the alumina powder polishing of 0.05 μ m, with deionized water, rinse out residual alumina powder, put into diluted nitric acid aqueous solution ultrasonic cleaning, finally use successively ethanol and redistilled water cleaning electrode surface.
(2) get polystyrene/graphene composite nanoparticle 2.0 mg, be scattered in 1 mL chitosan solution, be made into the chitosan solution of the polystyrene/graphene of approximately 2.0 mg/mL, then mix with isocyatic alcohol oxidase, form the mixed solution of polystyrene/graphene composite nanoparticle and alcohol oxidase.
(3) mixed solution of getting polystyrene/graphene composite nanoparticle and alcohol oxidase drips and is applied to pre-service electrode surface, and electrode is positioned in refrigerator under the cryogenic conditions of 4 ℃ to dry 12 hours, makes oxidation of ethanol enzyme sensor.
2, application:
The alcohol oxidase electrochemica biological sensor making is placed in to detection system, with electrochemical workstation, adopts three-electrode system, detect its current signal.
Lead to into the same test with example 1 and also can prove that prepared sensor detects fast, precision is high, and antijamming capability is good.This sensor material therefor is simple and easy to get simultaneously, and method for making is simple, with low cost, can detect exactly ethanol, is therefore expected to be applied in quick alcohol detection fields such as food.
Claims (7)
1. a preparation method for electrochemical enzymatic sensor, is characterized in that, with the immobilized biology enzyme of polystyrene/graphene composite nanoparticle, making electrochemical enzymatic sensor.
2. preparation method according to claim 1, is characterized in that first polystyrene/graphene composite nanoparticle being scattered in chitosan solution, then mixes with biology enzyme, forms mixed solution; Then mixed solution is dripped and be applied to clean glass-carbon electrode surface, then electrode is placed under cryogenic conditions and is dried, make electrochemical enzymatic sensor.
3. preparation method according to claim 2, is characterized in that first biology enzyme and water being mixed to form to the aqueous solution of biology enzyme, and then the aqueous solution of biology enzyme is mixed with the chitosan solution that is dispersed with polystyrene/graphene composite nanoparticle.
4. preparation method according to claim 3, is characterized in that the massfraction of biology enzyme in the aqueous solution of described biology enzyme is identical with the massfraction that is dispersed in the polystyrene/graphene composite nanoparticle in chitosan solution.
5. preparation method according to claim 4, the massfraction that it is characterized in that the polystyrene/graphene composite nanoparticle in chitosan solution is 2.0 mg/mL.
6. preparation method according to claim 2, it is characterized in that before dripping painting, it by glass-carbon electrode, with meta particle diameter, is first the alumina powder polishing of 0.05 μ m, with deionized water rinsing, fall residual alumina powder again, then put into diluted nitric acid aqueous solution ultrasonic cleaning, finally use successively ethanol and redistilled water cleaning electrode surface, obtain clean glass-carbon electrode.
7. preparation method according to claim 6, is characterized in that nitric acid and water mixing that described diluted nitric acid aqueous solution is 1:1 by volume ratio form.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104828953A (en) * | 2015-04-14 | 2015-08-12 | 浙江海洋学院 | Water treatment agent and preparation method thereof |
| CN112903784A (en) * | 2021-02-04 | 2021-06-04 | 西北农林科技大学 | Enzyme ink, preparation method and biosensor |
| WO2022110959A1 (en) * | 2020-11-27 | 2022-06-02 | 山东省科学院生物研究所 | Electrode system for quickly detecting ethanol, and ethanol detecting method using electrode system |
Citations (4)
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|---|---|---|---|---|
| CN102344700A (en) * | 2011-07-07 | 2012-02-08 | 南京大学 | Preparation method of polystyrene/graphene composite particle |
| CN102636540A (en) * | 2012-04-19 | 2012-08-15 | 湖南大学 | Glucose detection sensor, and preparation and application methods thereof |
| CN102636537A (en) * | 2012-04-19 | 2012-08-15 | 湖南大学 | Enzyme-free methyl parathion detection sensor, and preparation and application methods thereof |
| CN103278541A (en) * | 2013-04-19 | 2013-09-04 | 宁波大学 | Electrochemical biosensor for detecting bisphenol-A, and preparation method and application thereof |
-
2014
- 2014-04-29 CN CN201410175555.XA patent/CN103926280A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102344700A (en) * | 2011-07-07 | 2012-02-08 | 南京大学 | Preparation method of polystyrene/graphene composite particle |
| CN102636540A (en) * | 2012-04-19 | 2012-08-15 | 湖南大学 | Glucose detection sensor, and preparation and application methods thereof |
| CN102636537A (en) * | 2012-04-19 | 2012-08-15 | 湖南大学 | Enzyme-free methyl parathion detection sensor, and preparation and application methods thereof |
| CN103278541A (en) * | 2013-04-19 | 2013-09-04 | 宁波大学 | Electrochemical biosensor for detecting bisphenol-A, and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
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| TAO QIAN等: ""Gold nanoparticles coated polystyrene/reduced graphite oxide microspheres with improved dispersibility and electrical conductivity for dopamine detection"", 《COLLOIDS ANDSURFACESB:BIOINTERFACES》, 17 August 2013 (2013-08-17), pages 310 - 314 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104828953A (en) * | 2015-04-14 | 2015-08-12 | 浙江海洋学院 | Water treatment agent and preparation method thereof |
| WO2022110959A1 (en) * | 2020-11-27 | 2022-06-02 | 山东省科学院生物研究所 | Electrode system for quickly detecting ethanol, and ethanol detecting method using electrode system |
| CN112903784A (en) * | 2021-02-04 | 2021-06-04 | 西北农林科技大学 | Enzyme ink, preparation method and biosensor |
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