CN101140256B - Preparation method of photofixation horse radish peroxidase electric pole and uses thereof - Google Patents
Preparation method of photofixation horse radish peroxidase electric pole and uses thereof Download PDFInfo
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- CN101140256B CN101140256B CN2007101333755A CN200710133375A CN101140256B CN 101140256 B CN101140256 B CN 101140256B CN 2007101333755 A CN2007101333755 A CN 2007101333755A CN 200710133375 A CN200710133375 A CN 200710133375A CN 101140256 B CN101140256 B CN 101140256B
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- horse radish
- radish peroxidase
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- electric pole
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Abstract
The present invention provides a photo-fixation horse radish peroxidase preparation method and an application method. 0.5 to 2 portions by weight of polyacrylic ester pre-polymer are dissolved into 100 portions by weight of organic solvent and then mixed with horse radish peroxidase solution to prepare mixed solution. The mixed solution is dipped on an electrode substrate surface, which is illuminated for 0.1h to 3h by visible lights at normal temperature to polymerize the pre-polymer and obtain a photo-fixation horse radish peroxidase electrode. The present invention utilizes characteristicsand excellent biological compatibility of polyacrylic esters to prepare the photo-fixation horse radish peroxidase electrode. The photo-fixation horse radish peroxidase electrode prepared by the present invention has the advantages of wide applicable scope and biological catalytic activation for H<SUB>2</SUB>O<SUB>2</SUB>, organic peroxide and oxygen in aqueous phase, aqueous organic phase and organic phase, quick response, excellent stability and repeatability, long service life, high accuracy, good precision and wide measuring range.
Description
Technical field
The present invention relates to a kind of light fixed enzyme electrode, is a kind of with preparation method and the application thereof of polyacrylate as the photofixation horse radish peroxidase electric pole of bonding agent specifically.
Background technology
The biology sensor that the twentieth century middle and later periods grows up is a cross discipline that is interpenetrated and grown up by multiple subjects such as biology, analytical chemistry, physics, medical science, and it has single-minded, sensitive, advantage fast and accurately.Obtained a series of in recent ten years at the valuable biology sensor in aspects such as clinical detection, biochemical analysis.It is enzyme electrode that maximum biology sensors is used in research the earliest.Enzyme is fixed on the enzyme electrode of making on the surface of electrochemical electrode, and it utilizes enzyme in biochemical reaction special catalytic action can take place, and the chemical substance that consumes in the course of reaction or produce is transformed into electric signal with converter notes.
In numerous enzyme electrodes, the superoxide enzyme modified electrode occupies an important position, and wherein (E.C.1.11.1.7) application of modified electrode is in the majority for horseradishperoxidase, HRP with horseradish peroxidase.Horseradish peroxidase is at room temperature very stable, and low price, makes easily, is morning, widely used a kind of enzyme preparation of commercialization.At present existing several different methods is fixed on horseradish peroxidase on the different electrodes, adopts different analytical approachs to detect hydrogen peroxide, phenolic compound, aminated compounds and horseradish peroxidase inhibitor etc.The problem that exists is that prepared horseradish peroxidase electrode stability is relatively poor, and the obscission of enzyme in use easily takes place.
In the preparation of bioelectrochemistry enzyme sensor, the technology of most critical comprises: how (1) is fixed on electrode surface with enzyme.(2) catalytic activity of being maintained fixed enzyme how.(3) enzyme modified electrode can use in organic phase.The method of using has absorption method, investment, and self-assembly method etc., wherein absorption method causes the loss of enzyme easily, and electrode life is short, and the amount of self-assembly method immobilized enzyme is less, and required time of manufacturing process is longer.So the method for immobilised enzymes mainly is an investment.Use investment, require immobilization material can form stable film, have biological affinity, can keep the characteristics such as catalytic activity of enzyme.Used gel mainly contains polyacrylamide, Polyvinylchloride, methylcellulose etc. in traditional investment, the enzyme electrode less stable of these material preparations, and long-term the use comes off from electrode easily; Reversibility is relatively poor under acid-base condition, is unfavorable for repeatedly using.
Summary of the invention
The object of the present invention is to provide and a kind ofly have advantages of higher stability, response is fast and favorable reproducibility, can under acid-base condition, has stronger reversibility, the photofixation horse radish peroxidase electric pole that can be used for the safety detection, environment monitoring, production run control etc. of industries such as chemical industry, medicine, environment is that the preparation biology sensor is sought new immobilization material.
Another object of the present invention is to provide the preparation method of above-mentioned photofixation horse radish peroxidase electric pole.
The present invention also aims to provide the application of above-mentioned photofixation horse radish peroxidase electric pole.
Design philosophy of the present invention is that the thin polymer film that the light curing agent polyacrylate forms in the photopolymerization process has stronger viscosity and advantages of higher stability.Utilize this characteristic of polyacrylate and good bioaffinity, the preparation photofixation horse radish peroxidase electric pole.
According to technical scheme provided by the invention, the preparation method of photofixation horse radish peroxidase electric pole is as follows: the performed polymer of 1～5 part of polyacrylate (PEA) is dissolved in 100 parts of organic solvents at normal temperatures, evenly mixes with horseradish peroxidase solution equal-volume; This mixed solution dripped be coated in electrode surface, radiation of visible light makes its dry solidification under the room temperature, and the time of radiation of visible light dry solidification is 0.1～3h, obtains photofixation horse radish peroxidase electric pole, and unit is a mass parts.
Described organic solvent is the mixed solution of acetone and normal butyl alcohol, and its volume ratio is 1: 3～3: 1.
The manufacturing process of the performed polymer of polyacrylate is: take by weighing 20～40 parts of methyl methacrylates, and 30～50 parts of butyl acrylates, 1～3 part of vinyl acetate, 0.5～2 part of methacrylic acid fills N
2Gas shiled; add 1～3 part of benzoyl peroxide; slowly be warming up to 70～95 ℃, behind the reaction 2h, detect viscosity; when viscosity reaches 120mPas; cooling adds 0.5～3 part of ethylene glycol dimethacrylate and 5～15 parts of dioctyl phthalates, 0.5～5 part of benzoin isobutyl ether; make the performed polymer of polyacrylate, unit is a weight portion.
Utilize polyacrylate that horseradish peroxidase is fixed on glass-carbon electrode and graphite electrode surface, form stable horseradish peroxidase-polyacrylate modified electrode; In polyacrylate, horseradish peroxidase directly and between the electrode transmits electronics.
In the polyacrylate performed polymer, add an amount of acetone,, be beneficial to the exposed of hydrophobic active center, promote the electron transport between horseradish peroxidase and the electrode with the orientation of control horseradish peroxidase in polyacrylate.
Modified electrode of the present invention comprises following application:
(1) horseradish peroxidase in the electrode can electrical catalyze reduction O in aqueous solution
2, H
2O
2With organic peroxide etc.Stable photofixation horse radish peroxidase electric pole can be used for H
2O
2Quantitative measurement with organic peroxide.
(2) photofixation horse radish peroxidase electric pole also shows Direct Electrochemistry and catalytic activity in the mixed solution of hydrophilic organic solvents such as ethanol and DMF and water, shows that organic solvent does not influence the conformation and the catalysis thereof of enzyme significantly.
(3) photofixation horse radish peroxidase electric pole can catalytic reduction oxygen, be expected to become the positive pole of biological fuel cell.
Because the polyacrylate of the present invention's preparation has stronger viscosity, advantages of higher stability and good bioaffinity, therefore the enzyme electrode of preparation has good stable and reappearance, long service life, and the accuracy height, precision is good, and measurement range is wide.In water, organic water phase and organic phase, energy catalytic reduction hydrogen peroxide, organic peroxide and oxygen etc.
Because horseradish peroxidase is grouped together in the polyacrylate, so enzyme is difficult for leaking, and polyacrylate provides adapt circumstance for wherein enzyme simultaneously, can keep the original conformation and the catalytic activity of enzyme, so this enzyme has advantages of higher stability.
Description of drawings
Fig. 1 is the cyclic voltammogram of horseradish peroxidase modified electrode of the present invention.
Fig. 2 is the cyclic voltammogram that the horseradish peroxidase modified electrode of the embodiment of the invention 1 detects hydrogen peroxide.
Fig. 3 is the cyclic voltammogram that the horseradish peroxidase modified electrode of the embodiment of the invention 2 detects organic peroxide.
Embodiment
Among Fig. 1, horizontal ordinate is with respect to the current potential of saturated calomel electrode (SCE) (E), and unit is V, and the ordinate electric current is (I), and unit is a microampere (μ A).
Among Fig. 2, the concentration of hydrogen peroxide (μ M) is respectively: (a) 0, (b) 15.6, (c) 30.2, and (d) the 44.6. horizontal ordinate is the current potential (E) with respect to saturated calomel electrode (SCE), and unit is V, and the ordinate electric current is (I), and unit is a microampere (μ A).
Among Fig. 3, the concentration of organic peroxide (μ M) is respectively: (a) 0, (b) 15.6, (c) 30.2, and (d) the 44.6. horizontal ordinate is the current potential (E) with respect to saturated calomel electrode (SCE), and unit is V, and the ordinate electric current is (I), and unit is a microampere (μ A).
Embodiment 1
Take by weighing the 20g methyl methacrylate, the 30g butyl acrylate, the 1g vinyl acetate, the 0.5g methacrylic acid fills N
2Gas shiled adds the 1g benzoyl peroxide, slowly is warming up to 95 ℃; behind the reaction 2h, detect viscosity, when viscosity reaches 120mPas; cooling adds 0.5g ethylene glycol dimethacrylate and 5g dioctyl phthalate, and the 0.5g benzoin isobutyl ether is made the PEA performed polymer.Above-mentioned PEA performed polymer with 0.1g, under the lucifuge condition be 1: 3 normal butyl alcohol with 10g massfraction ratio: the acetone organic solvent is mixed with solution, be that 5% horseradish peroxidase aqueous solution equal-volume mixes with massfraction again, stir and make mixed solution.Get 10 μ l mixed solutions and drip and be coated in the glass-carbon electrode surface, make polymkeric substance performed polymer generation polymerization, obtain photo-immobilization horseradish peroxidase modified electrode at 25 ℃ of following radiation of visible light 0.1h.This electrode has excellent performance, response time fast (30 seconds), electrode life long (200 days), accuracy height (＜5%), precision good (＜5 ‰) is in water, organic water phase and organic phase, can detect hydrogen peroxide, organic peroxide etc., measurement range is wide.
Embodiment 2
Take by weighing the 40g methyl methacrylate, the 50g butyl acrylate, the 3g vinyl acetate, the 2g methacrylic acid fills N
2Gas shiled adds the 3g benzoyl peroxide, slowly is warming up to 70 ℃, behind the reaction 2h, detects viscosity, and when viscosity reached 120mPas, cooling added 3g ethylene glycol dimethacrylate and 15g dioctyl phthalate, and the 5g benzoin isobutyl ether is made the PEA performed polymer.Above-mentioned PEA performed polymer with 0.5g, under the lucifuge condition be 3: 1 normal butyl alcohol with 10g massfraction ratio: the acetone organic solvent is mixed with solution, be that 10% horseradish peroxidase aqueous solution equal-volume mixes with massfraction again, stir and make mixed solution.Get 10 μ l mixed solutions and drip and be coated in graphite electrode surface, make polymkeric substance performed polymer generation polymerization, obtain photo-immobilization horseradish peroxidase modified electrode at 20 ℃ of following radiation of visible light 3h.This electrode has excellent performance, response time fast (30 seconds), long service life (30 days), accuracy height (＜5%), precision good (＜5 ‰) is in water, organic water phase and organic phase, can detect hydrogen peroxide and organic peroxide etc., measurement range is wide.
Embodiment 3
Take by weighing the 25g methyl methacrylate, the 35g butyl acrylate, the 2g vinyl acetate, the 1g methacrylic acid fills N
2Gas shiled adds the 2g benzoyl peroxide, slowly is warming up to 80 ℃, behind the reaction 2h, detects viscosity, and when viscosity reached 120mPas, cooling added 2g ethylene glycol dimethacrylate and 10g dioctyl phthalate, and the 2g benzoin isobutyl ether is made the PEA performed polymer.Above-mentioned PEA performed polymer with 0.3g, under the lucifuge condition be 1: 1 normal butyl alcohol with 10g massfraction ratio: the acetone organic solvent is mixed with solution, be that 10% horseradish peroxidase aqueous solution equal-volume mixes with massfraction again, stir and make mixed solution.Get 10 μ l mixed solutions and drip and be coated in graphite electrode surface, make polymkeric substance performed polymer generation polymerization, obtain photo-immobilization horseradish peroxidase modified electrode at 20 ℃ of following radiation of visible light 1h.This electrode has excellent performance, response time fast (30 seconds), long service life (30 days), accuracy height (＜5%), precision good (＜5 ‰) is in water, organic water phase and organic phase, can detect hydrogen peroxide and organic peroxide etc., measurement range is wide.
Embodiment 4
Take by weighing the 30g methyl methacrylate, the 40g butyl acrylate, the 2g vinyl acetate, the 1.5g methacrylic acid fills N
2Gas shiled adds the 2g benzoyl peroxide, slowly is warming up to 85 ℃, behind the reaction 2h, detects viscosity, and when viscosity reached 120mPas, cooling added 1g ethylene glycol dimethacrylate and 15g dioctyl phthalate, and the 2g benzoin isobutyl ether is made the PEA performed polymer.Above-mentioned PEA performed polymer with 0.4g, under the lucifuge condition be 1: 2 normal butyl alcohol with 10g massfraction ratio: the acetone organic solvent is mixed with solution, be that 10% horseradish peroxidase aqueous solution equal-volume mixes with massfraction again, stir and make mixed solution.Get 10 μ l mixed solutions and drip and be coated in graphite electrode surface, make polymkeric substance performed polymer generation polymerization, obtain photo-immobilization horseradish peroxidase modified electrode at 20 ℃ of following radiation of visible light 0.3h.This electrode has excellent performance, response time fast (30 seconds), long service life (30 days), accuracy height (＜5%), precision good (＜5 ‰) is in water, organic water phase and organic phase, can detect hydrogen peroxide and organic peroxide etc., measurement range is wide.
Claims (5)
1. the preparation method of a photofixation horse radish peroxidase electric pole is characterized in that: the performed polymer of 1～5 part of polyacrylate is dissolved in 100 parts of organic solvents at normal temperatures, evenly mixes with horseradish peroxidase solution equal-volume; This mixed solution dripped be coated in electrode surface, radiation of visible light makes its dry solidification under the room temperature, and the time of radiation of visible light dry solidification is 0.1～3h, obtains photofixation horse radish peroxidase electric pole, and unit is a mass parts;
Described organic solvent is the mixed solution of acetone and normal butyl alcohol, and its volume ratio is 1: 3～3: 1;
The manufacturing process of the performed polymer of polyacrylate is: take by weighing 20～40 parts of methyl methacrylates, and 30～50 parts of butyl acrylates, 1～3 part of vinyl acetate, 0.5～2 part of methacrylic acid fills N
2Gas shiled; add 1～3 part of benzoyl peroxide; slowly be warming up to 70～95 ℃, behind the reaction 2h, detect viscosity; when viscosity reaches 120mPas; cooling adds 0.5～3 part of ethylene glycol dimethacrylate and 5～15 parts of dioctyl phthalates, 0.5～5 part of benzoin isobutyl ether; make the performed polymer of polyacrylate, unit is a weight portion.
2. according to the preparation method of the described photofixation horse radish peroxidase electric pole of claim 1, it is characterized in that, utilize polyacrylate that horseradish peroxidase is fixed on glass-carbon electrode and graphite electrode surface, form stable horseradish peroxidase-polyacrylate modified electrode; In polyacrylate, horseradish peroxidase directly and between the electrode transmits electronics.
3. photofixation horse radish peroxidase electric pole catalytic reduction H in ethanol/water solution that utilizes claim 1 or 2 described methods to obtain
2O
2, organic peroxide, and the application of these materials of detection by quantitative.
4. photofixation horse radish peroxidase electric pole catalytic reduction H in aqueous solution that utilizes claim 1 or 2 described methods to obtain
2O
2, organic peroxide, and the application of these materials of detection by quantitative.
5. one kind is utilized photofixation horse radish peroxidase electric pole that claim 1 or 2 described methods obtain catalytic reduction oxygen in aqueous solution and in the ethanol/water solution, and the positive pole that can be used as biological fuel cell is used.
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| CN2007101333755A CN101140256B (en) | 2007-10-19 | 2007-10-19 | Preparation method of photofixation horse radish peroxidase electric pole and uses thereof |
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| CN101140256B true CN101140256B (en) | 2010-08-25 |
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| CN105353010A (en) * | 2015-10-28 | 2016-02-24 | 江南大学 | Making method of molecularly imprinted electrochemical sensing electrode for detecting propylene chorohydrin |
| CN105353022A (en) * | 2015-10-28 | 2016-02-24 | 江南大学 | Flexible electrode-based molecularly imprinted electrochemical sensor making method |
| CN107828770A (en) * | 2017-10-30 | 2018-03-23 | 北京化工大学 | It is a kind of to solidify organophosphor hydrolytic enzyme method in non-woven fabrics glazing |
| CN113215140B (en) * | 2021-06-10 | 2022-06-21 | 浙江工业大学 | Immobilization method of horseradish peroxidase |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1317574A (en) * | 2001-01-15 | 2001-10-17 | 湖南大学 | Optical immobilizing method for piezoelectric sensing medium of enzyme and microbe |
| CN1368552A (en) * | 2002-01-30 | 2002-09-11 | 武汉大学 | Visual light cross-linked composite resin method for fixing cells with xanthine oxidase activity |
| US6569651B1 (en) * | 1999-06-16 | 2003-05-27 | The United States Of America As Represented By The Secretary Of The Army | Enzymatic polymerization of anilines or phenols around a template |
| CN1809752A (en) * | 2003-03-31 | 2006-07-26 | 科学与工业研究委员会 | Method for preparing photoreactive polymers with immobilized biomolecules thereon |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6569651B1 (en) * | 1999-06-16 | 2003-05-27 | The United States Of America As Represented By The Secretary Of The Army | Enzymatic polymerization of anilines or phenols around a template |
| CN1317574A (en) * | 2001-01-15 | 2001-10-17 | 湖南大学 | Optical immobilizing method for piezoelectric sensing medium of enzyme and microbe |
| CN1368552A (en) * | 2002-01-30 | 2002-09-11 | 武汉大学 | Visual light cross-linked composite resin method for fixing cells with xanthine oxidase activity |
| CN1809752A (en) * | 2003-03-31 | 2006-07-26 | 科学与工业研究委员会 | Method for preparing photoreactive polymers with immobilized biomolecules thereon |
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