CN101285792B - Mediator electrochemical enzyme electrode and method for making same - Google Patents
Mediator electrochemical enzyme electrode and method for making same Download PDFInfo
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- CN101285792B CN101285792B CN2008100616234A CN200810061623A CN101285792B CN 101285792 B CN101285792 B CN 101285792B CN 2008100616234 A CN2008100616234 A CN 2008100616234A CN 200810061623 A CN200810061623 A CN 200810061623A CN 101285792 B CN101285792 B CN 101285792B
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Abstract
The invention discloses a mediator electrochemical enzyme electrode and a method for making the same. Acrylonitrile copolymer with a functionalized mediator is prepared through the coupling reaction of acrylonitrile copolymer and an electric mediator with a reactive group in dimethylformamide. The dimethylformamide is taken as solvent, the functionalized acrylonitrile copolymer is taken as a raw material, an electrochemical electrode is taken as an accepting electrode, a copolymer fiber membrane is prepared on the surface of the electrode through an electrostatic spinning method, then a covalent method is used to fix xidoreductase on a fiber layer on the surface of the electrode to form an activated layer of fibers. In the invention, extremely high specific surface area of the electrostatic spinning fiber, the reaction group on the surface of the electrostatic spinning fiber and the fixed electric mediator ensure that the electrochemical enzyme electrode has higher enzyme-carrying efficiency, high stability and high response strength. The mediator electrochemical enzyme electrode has the advantages of simple preparation process, repeated use, large surface area of an effective enzyme membrane, strong anti-jamming ability and so on, and is suitable for batch production.
Description
Technical field
The present invention relates to a kind of mediator electrochemical enzyme electrode and preparation method thereof.
Background technology
Up to now, the development of biology sensor was gone through nearly 50 years, had attracted world wide scientist's huge concern, and had been applied to more field.The formation of biology sensor comprises two parts: molecular recognition elements and transducer.Measured matter is by diffusing into the bio-sensing rete, and behind molecular recognition, generation biochemical reaction, the transducer that the information that is produced is responded converts to and the concentration dependent electric signal of measured object.Molecular recognition elements has played key effect in the function aspects of decision biology sensor, and its contained biological components is enzyme, tissue, antibody/antigen, bacterium, yeast.Fixed enzyme electrode is owing to characteristics such as stable, controlled, low interference obtain maximum research and application.Such as, the enzyme of aqueous solution form usually can only with substrate-function once, be difficult to reclaim, but when enzyme is immobilized, can very fast from reaction mixture, separating, and can reuse; And, by the microenvironment of regulation and control immobilised enzymes, can obtain the character of a lot of demands, as high sensitivity, response fast.
Polymer film provides the good supporting body for enzyme in the immobilization of electrode surface, and the feature of polymeric multilayer film tool three-D space structure, the many potential fields that can utilize can be provided, concentration height, the galvanochemistry corresponding signal of its activating agent are big, and has higher chemistry, machinery and electrochemical stability, in addition, with respect to the method for other modified electrodes, the preparation of thin polymer film requires not harsh to the surface state of basal electrode.The tunica fibrosa of electrostatic spinning preparation can be easy to realize this sandwich construction, and because electrostatic interaction, and tunica fibrosa can be firm is attached to electrode surface.Electrostatic spinning is to flow in electrostatic field and distortion by charged Polymer Solution or melt, is solidified to form the spinning process of ultrafine fiber through solvent evaporation or melt cooling.Can on receiving screen, form nano-scale fiber (Z.-M.Huang by electrostatic spinning, Y.-Z.Zhang, M.Kotaki, S.Ramakrishna. " A review on polymer nanofibers by electrospinning andtheir applications in nanocomposties ", Compos.Sci.Technol., 2003,63:2223-2253).And, by changing electrostatic spinning apparatus, can obtain having the tunica fibrosa (W.E.Teo of required character, S.Ramakrishna. " A review on electrospinning design and nanofiber assemblies ", Nanotechnology, 2006,17, R89-R106). as enzyme immobilization carrier, electrostatic spinning fiber film has clearly advantage with respect to other materials: the first, preparation technology is very simple; The second, tunica fibrosa has high specific surface area, can have higher enzyme immobilization efficient and reduce the substrate diffusional resistance.Many researchers have attempted enzyme is fixed in (H.-F.Jia on the electrostatic spinning fiber film, G.-Y.Zhu, B.Vugrinovich. " Enzyme-carrying polymeric nanofibers prepared via electrospinning for use asunique biocatalysts ", Biotechnol.Prog.2002,18:1027-1032; T.E.Herricks, S.-H.Kim, J.-B.Kim. " Direct fabrication of enzyme-carrying polymer nanofibers byelectrospinning ", J.Mater.Chem., 2005,15:3241-3245; Y.-H.Wang, Y.-L.Hsieh. " Enzyme immobilization to ultra-fine cellulose fibers via amphiphilic polyethyleneglycol spacers ", J.Polym.Sci.Part A:Polym.Chem., 2004,42:4289-4299; L.-L.Wu, X.-Y.Yuan, J.Sheng. " Immobilization of cellulase in nanofibrous PVAmembranes by electrospinning ", J.Membr.Sci., 2005,250:167-173).Then use aspect preparing electrostatic spinning few at enzyme electrode.CN1737560A mixes enzyme with polymer solution, co-spun yarn prepares enzyme electrode then.The applicable type of polymer of but this method is limited, also must be water miscible because most of enzyme all is water miscible, used polymkeric substance.Therefore, utilize electrostatic spinning fiber as active layer in enzyme electrode preparation still tool have very important significance.
For intensifier electrode response with improve the antijamming capability of electrode, often adopt electron mediator based on the amperometric sensor of oxidoreducing enzyme.With respect to dissolved oxygen DO, electron mediator can make the line style scope of electrode response widen, and overpotential reduces.Simultaneously, noise, background current and undesired signal all reduce, and owing to eliminated hydrogen peroxide, make the mission life of biology sensor prolong.Applicable mediator is a lot, comprises the potassium ferricyanide, quinone, ferrocene, ferrocene derivatives and porphyrin.The ferrocene range of application is very general, this is because ferrocene is hydrophobic, when being used for aqueous systems, has avoided mediator coming off from electrode surface, and ferrocene presents excellent electrochemical reversibility, and can regulate its oxidation-reduction potential by changing its structure.In the thin polymer film modified electrode, ferrocene and derivant thereof also are widely adopted, as ferrocene aldehyde radical and shitosan side chain are reacted, then shitosan and the enzyme with this modified is co-deposited to electrode surface (A.Garcia, C.Peniche-Covas, B.Chico, B.K.Simpson, R.Villalonga. " Ferrocenebranched chitosan for the construction of a reagentless amperometric hydrogenperoxide biosensor ", Macromol.Biosci.2007,7,435-439).But because still may there be the low problem of sensor stability in coming off of fabulous water wettability of shitosan and enzyme when electrochemical analysis.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of mediator electrochemical enzyme electrode and preparation method thereof is provided
Mediator electrochemical enzyme electrode comprises basal electrode and active layer, and basal electrode is provided with active layer, it is characterized in that:
1) active layer is the nano fibrous membrane and the fixing oxidoreducing enzyme of electron mediator functionalization acrylonitrile copolymer;
2) nano fibrous membrane of active layer is prepared by electrospinning process;
3) in the acrylonitrile copolymer, comonomer is acrylic acid, methacrylic acid, maleic anhydride, itaconic acid or glycidyl methacrylate, it is 5～20% that comonomer accounts for the acrylonitrile copolymer mole percentage, and the viscosity average molecular weigh of acrylonitrile copolymer is 30,000～100,000; The massfraction that electron mediator accounts for acrylonitrile copolymer is 2.5%～15.8%;
4) electron mediator comprises ferrocene, porphyrin, iron porphyrin or the zinc protoporphyrin that contains reactive group, and reactive group comprises amino or hydroxyl;
5) oxidoreducing enzyme is hydrogen peroxidase, peroxidase, cytochrome, glucose oxidase, cholesterol oxidase, alcohol dehydrogenase or laccase.
The preparation method of mediator electrochemical enzyme electrode comprises the steps:
1) earlier acrylonitrile copolymer and the electron mediator that contains amino or hydroxyl are dissolved in dimethyl formamide, the mass ratio of acrylonitrile copolymer and electron mediator is 1/1～5/1, add N then, N '-dicyclohexylcarbodiimide, its concentration in dimethyl formamide is 5～15 mg/ml, and reaction is 12～48 hours in the time of 15 ℃～45 ℃.With product absolute ethyl alcohol precipitating, washing 60 ℃～70 ℃ following vacuum drying 24～48 hours, obtains the acrylonitrile copolymer that contains acrylic acid, methacrylic acid or furoate clothing of electron mediator functionalization;
2) acrylonitrile copolymer that contains acrylic acid, methacrylic acid or furoate clothing with the electron mediator functionalization is dissolved in the dimethyl formamide, is mixed with massfraction and is 3%～10% copolymer solution; With platinum electrode, gold electrode or glass-carbon electrode is dash receiver, when voltage is that 10～20 kilovolts, rate of extrusion are 0.5～3.0 milliliter/hour, collect tunica fibrosa when receiving range is 10～20 centimetres, spinning time remaining 2～10 hours obtains depositing the electrode of nano fibrous membrane;
3) electrode surface that will deposit nano fibrous membrane immerses in the buffer solution of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide, wherein the concentration of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride is 5～20 mg/ml, the concentration of N-hydroxy-succinamide is 3～12 mg/ml, 4 ℃～25 ℃ activation 2～10 hours of vibrating down, then with buffer solution washing 3～5 times; The electrode surface of activation is immersed the buffer solution that contains 1～10 mg/ml oxidoreducing enzyme, 4 ℃～25 ℃ following oscillating reactionss after 3～24 hours, obtain containing the electrode of the fiber surface immobilized enzyme of acrylic acid, methacrylic acid or itaconic acid, preserve stand-by down for 4 ℃.
The preparation method of mediator electrochemical enzyme electrode comprises the steps:
1) earlier acrylonitrile copolymer is dissolved in dimethyl formamide, adding the electron mediator that contains amino or hydroxyl then reacted 12～48 hours in the time of 15 ℃～45 ℃, the mass ratio of acrylonitrile copolymer and electron mediator is 1/1～5/1, with product absolute ethyl alcohol precipitating, washing, 60 ℃～70 ℃ following vacuum drying 24～48 hours, obtain the acrylonitrile copolymer that contains maleic anhydride or methacrylic acid shrink base glycerol ester of electron mediator functionalization;
2) acrylonitrile copolymer that contains maleic anhydride or methacrylic acid shrink base glycerol ester with the electron mediator functionalization is dissolved in the dimethyl formamide, is mixed with massfraction and is 3%～10% copolymer solution; With platinum electrode, gold electrode or glass-carbon electrode is dash receiver, when voltage is that 10～20 kilovolts, rate of extrusion are that 0.5～3.0 milliliter/hour, receiving range are 10～20 centimetres and collect tunica fibrosas, spinning time remaining 2～10 hours obtains depositing the electrode of nano fibrous membrane;
3) electrode surface that will deposit nano fibrous membrane immerses the buffer solution contain 1～10 mg/ml oxidoreducing enzyme, 4 ℃～25 ℃ following oscillating reactionss after 3～24 hours, obtain containing the electrode of the fiber surface immobilized enzyme of maleic anhydride or glycidyl methacrylate, preserve stand-by down for 4 ℃.
Described oxidoreducing enzyme is hydrogen peroxidase, peroxidase, cytochrome, glucose oxidase, cholesterol oxidase, alcohol dehydrogenase or laccase.
Described electron mediator is ferrocene, porphyrin, iron porphyrin or the zinc protoporphyrin that contains reactive group, and reactive group comprises amino or hydroxyl.
The beneficial effect that the present invention compared with prior art has:
1) selected polymkeric substance obtains easily, and contains reactive group;
2) the reaction efficiency height of electron mediator and multipolymer, and the product detachment process is easy;
3) the enzyme electrode preparation method is simple, and the fibrage of electrode surface contains reactive group, is convenient to enzyme immobilization;
What 4) high specific surface area of fiber composite layer and high porosity made electrode carries enzyme efficient height, and the substrate diffusional resistance is little; And the introducing of electron mediator obviously reduced the detectability of electrode, accelerated response speed, improved electrode response, but widened the linear response range of check and analysis things, increased the antijamming capability of electrode;
5) immobilization of enzyme and electron mediator has improved the response stability of electrode greatly, thereby has reduced the cost of manufacture of electrode and improved the ubiquity that electrode uses.
Embodiment:
Following embodiment is done more detailed description to the present invention, but described embodiment is not construed as limiting the invention.
Embodiment 1
1) earlier with acrylonitrile copolymer (viscosity average molecular weigh 100,000, the comonomer mole percentage is 5%) and amino ferrocene in dimethyl formamide, dissolve, the mass ratio of acrylonitrile copolymer and electron mediator is 1/1 to add N then, N '-dicyclohexylcarbodiimide, its concentration in dimethyl formamide is 5 mg/ml, and reaction is 12 hours in the time of 15 ℃.With product absolute ethyl alcohol precipitating, washing 60 ℃ of following vacuum drying 24 hours, obtains containing of ferrocene functionalization of acrylic acid acrylonitrile copolymer;
2) with containing of ferrocene functionalization acrylic acid acrylonitrile copolymer be dissolved in the dimethyl formamide, be mixed with massfraction and be 3% copolymer solution; With the platinum electrode is dash receiver, and when voltage is that 10 kilovolts, rate of extrusion are 0.5 milliliter/hour, collect tunica fibrosa when receiving range is 10 centimetres, spinning time remaining 2 hours obtains depositing the electrode of nano fibrous membrane;
3) electrode surface that will deposit nano fibrous membrane immerses in the buffer solution of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide, wherein the concentration of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride is 5 mg/ml, the concentration of N-hydroxy-succinamide is 3 mg/ml, 25 ℃ of activation 2 hours of vibrating down, then with buffer solution washing 3 times; The electrode surface immersion of activation is contained the catalatic buffer solution of 1 mg/ml, after 3 hours, obtain containing the electrode of acrylic acid fiber surface immobilized enzyme, preserve stand-by down for 4 ℃ 25 ℃ of following oscillating reactionss.
Embodiment 2
1) earlier with acrylonitrile copolymer (viscosity average molecular weigh 30,000, the comonomer mole percentage is 20%) and contain amino porphyrin and in dimethyl formamide, dissolve, the mass ratio of acrylonitrile copolymer and electron mediator is 5/1, add N then, N '-dicyclohexylcarbodiimide, its concentration in dimethyl formamide is 15 mg/ml, and reaction is 48 hours in the time of 45 ℃.With product absolute ethyl alcohol precipitating, washing 70 ℃ of following vacuum drying 48 hours, obtains the acrylonitrile copolymer that contains methacrylic acid of porphyrin functionalization;
2) acrylonitrile copolymer that contains methacrylic acid with the porphyrin functionalization is dissolved in the dimethyl formamide, is mixed with massfraction and is 3%～10% copolymer solution; With the gold electrode is dash receiver, and when voltage is that 20 kilovolts, rate of extrusion are 3.0 milliliters/hour, collect tunica fibrosa when receiving range is 20 centimetres, spinning time remaining 10 hours obtains depositing the electrode of nano fibrous membrane;
3) electrode surface that will deposit nano fibrous membrane immerses in the buffer solution of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide, wherein the concentration of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride is 20 mg/ml, the concentration of N-hydroxy-succinamide is 12 mg/ml, 4 ℃ of activation 10 hours of vibrating down, then with buffer solution washing 5 times; The electrode surface of activation is immersed the buffer solution that contains 10 mg/ml glucose oxidases, after 24 hours, obtain containing the electrode of methacrylic acid fiber surface immobilized enzyme, preserve stand-by down for 4 ℃ 4 ℃ of following oscillating reactionss.
Embodiment 3
1) earlier with acrylonitrile copolymer (viscosity average molecular weigh 30,000, the comonomer mole percentage is 20%) and contain amino iron porphyrin and in dimethyl formamide, dissolve, the mass ratio of acrylonitrile copolymer and electron mediator is 1/1, add N then, N '-dicyclohexylcarbodiimide, its concentration in dimethyl formamide is 15 mg/ml, and reaction is 48 hours in the time of 45 ℃.With product absolute ethyl alcohol precipitating, washing 70 ℃ of following vacuum drying 48 hours, obtains the acrylonitrile copolymer that contains methacrylic acid of iron porphyrin functionalization;
2) acrylonitrile copolymer that contains methacrylic acid with the iron porphyrin functionalization is dissolved in the dimethyl formamide, is mixed with massfraction and is 5% copolymer solution; With the glass-carbon electrode is dash receiver, and when voltage is that 20 kilovolts, rate of extrusion are 3.0 milliliters/hour, collect tunica fibrosa when receiving range is 20 centimetres, spinning time remaining 10 hours obtains depositing the electrode of nano fibrous membrane;
3) electrode surface that will deposit nano fibrous membrane immerses in the buffer solution of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide, wherein the concentration of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride is 20 mg/ml, the concentration of N-hydroxy-succinamide is 12 mg/ml, 4 ℃ of activation 10 hours of vibrating down, then with buffer solution washing 5 times; The electrode surface of activation is immersed the buffer solution that contains 10 mg/ml glucose oxidases, after 24 hours, obtain containing the electrode of itaconic acid fiber surface immobilized enzyme, preserve stand-by down for 4 ℃ 4 ℃ of following oscillating reactionss.
Embodiment 4
1) earlier with acrylonitrile copolymer (viscosity average molecular weigh 30,000, the comonomer mole percentage is 20%) and contain amino zinc protoporphyrin and in dimethyl formamide, dissolve, acrylonitrile copolymer is 1/1 with the mass ratio that contains amino zinc protoporphyrin, add N then, N '-dicyclohexylcarbodiimide, its concentration in dimethyl formamide is 15 mg/ml, and reaction is 48 hours in the time of 45 ℃.With product absolute ethyl alcohol precipitating, washing 70 ℃ of following vacuum drying 48 hours, obtains the acrylonitrile copolymer that contains methacrylic acid of zinc protoporphyrin functionalization;
2) acrylonitrile copolymer that contains methacrylic acid with the zinc protoporphyrin functionalization is dissolved in the dimethyl formamide, is mixed with massfraction and is 5% copolymer solution; With the platinum electrode is dash receiver, and when voltage is that 20 kilovolts, rate of extrusion are 3.0 milliliters/hour, collect tunica fibrosa when receiving range is 20 centimetres, spinning time remaining 10 hours obtains depositing the electrode of nano fibrous membrane;
3) electrode surface that will deposit nano fibrous membrane immerses in the buffer solution of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide, wherein the concentration of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride is 20 mg/ml, the concentration of N-hydroxy-succinamide is 12 mg/ml, 4 ℃ of activation 10 hours of vibrating down, then with buffer solution washing 5 times; The electrode surface of activation is immersed the buffer solution that contains 10 mg/ml glucose oxidases, after 24 hours, obtain containing the electrode of itaconic acid fiber surface immobilized enzyme, preserve stand-by down for 4 ℃ 4 ℃ of following oscillating reactionss.
Embodiment 5
Replace glucose oxidase with peroxidase, all the other are identical with embodiment 3.
Embodiment 6
Replace glucose oxidase with cytochrome, all the other are identical with embodiment 3.
Embodiment 7
Replace glucose oxidase with cholesterol oxidase, all the other are identical with embodiment 3.
Embodiment 8
Replace glucose oxidase with alcohol dehydrogenase, all the other are identical with embodiment 3.
Embodiment 9
Replace glucose oxidase with laccase, all the other are identical with embodiment 3.
Embodiment 10
1) earlier with acrylonitrile copolymer (viscosity average molecular weigh 100,000, the comonomer mole percentage is 5%) in dimethyl formamide, dissolve, the mass ratio of the ferrocene of acrylonitrile copolymer and hydroxyl is 5/1, adding the ferrocene that contains hydroxyl then reacted 12 hours in the time of 15 ℃, the mass ratio of the ferrocene of acrylonitrile copolymer and hydroxyl is 5/1, with product absolute ethyl alcohol precipitating, washing, 60 ℃ of following vacuum drying 24 hours, obtain the acrylonitrile copolymer that contains maleic anhydride of ferrocene functionalization;
2) acrylonitrile copolymer that contains maleic anhydride with the ferrocene functionalization is dissolved in the dimethyl formamide, is mixed with massfraction and is 3% copolymer solution; With the platinum electrode is dash receiver, and when voltage is that 10 kilovolts, rate of extrusion are that 0.5 milliliter/hour, receiving range are 10 centimetres and collect tunica fibrosas, spinning time remaining 2 hours obtains depositing the electrode of nano fibrous membrane;
3) electrode surface that will deposit nano fibrous membrane immerses and to contain the catalatic buffer solution of 1 mg/ml, 25 ℃ of following oscillating reactionss after 3 hours, obtains containing the electrode of the fiber surface immobilized enzyme of maleic anhydride, preserves stand-by down for 4 ℃.
Embodiment 11
1) earlier with acrylonitrile copolymer (viscosity average molecular weigh 30,000, the comonomer mole percentage is 20%) in dimethyl formamide, dissolve, adding the porphyrin that contains hydroxyl then reacted 48 hours in the time of 45 ℃, acrylonitrile copolymer is 1/1 with the mass ratio that contains the porphyrin of hydroxyl, with product absolute ethyl alcohol precipitating, washing 70 ℃ of following vacuum drying 48 hours, obtains the acrylonitrile copolymer that contains glycidyl methacrylate of porphyrin functionalization;
2) acrylonitrile copolymer that contains methacrylic acid shrink base glycerol ester with the porphyrin functionalization is dissolved in the dimethyl formamide, is mixed with massfraction and is 10% copolymer solution; With the gold electrode is dash receiver, and when voltage is that 20 kilovolts, rate of extrusion are that 3.0 milliliters/hour, receiving range are 20 centimetres and collect tunica fibrosas, spinning time remaining 10 hours obtains depositing the electrode of nano fibrous membrane;
3) electrode surface that will deposit nano fibrous membrane immerses the buffer solution contain 10 mg/ml glucose oxidases, 4 ℃ of following oscillating reactionss after 24 hours, obtain containing the electrode of the fiber surface immobilized enzyme of glycidyl methacrylate, preserve stand-by down for 4 ℃.
Embodiment 12
1) earlier with acrylonitrile copolymer (viscosity average molecular weigh 30,000, the comonomer mole percentage is 20%) in dimethyl formamide, dissolve, adding the iron porphyrin that contains hydroxyl then reacted 48 hours in the time of 45 ℃, the mass ratio of the iron porphyrin of acrylonitrile copolymer and hydroxyl is 1/1, with product absolute ethyl alcohol precipitating, washing 70 ℃ of following vacuum drying 48 hours, obtains the acrylonitrile copolymer that contains glycidyl methacrylate of iron porphyrin functionalization;
2) acrylonitrile copolymer that contains methacrylic acid shrink base glycerol ester with the iron porphyrin functionalization is dissolved in the dimethyl formamide, is mixed with massfraction and is 10% copolymer solution; With the glass-carbon electrode is dash receiver, and when voltage is that 20 kilovolts, rate of extrusion are that 3.0 milliliters/hour, receiving range are 20 centimetres and collect tunica fibrosas, spinning time remaining 10 hours obtains depositing the electrode of nano fibrous membrane;
3) electrode surface that will deposit nano fibrous membrane immerses the buffer solution contain 10 mg/ml peroxidase, 4 ℃ of following oscillating reactionss after 24 hours, obtain containing the electrode of the fiber surface immobilized enzyme of glycidyl methacrylate, preserve stand-by down for 4 ℃.
Embodiment 13
1) earlier with acrylonitrile copolymer (viscosity average molecular weigh 30,000, the comonomer mole percentage is 20%) in dimethyl formamide, dissolve, adding the zinc protoporphyrin that contains amino then reacted 48 hours in the time of 45 ℃, acrylonitrile copolymer is 1/1 with the mass ratio that contains amino zinc protoporphyrin, with product absolute ethyl alcohol precipitating, washing 70 ℃ of following vacuum drying 48 hours, obtains the acrylonitrile copolymer that contains glycidyl methacrylate of zinc protoporphyrin functionalization;
2) acrylonitrile copolymer that contains methacrylic acid shrink base glycerol ester with the zinc protoporphyrin functionalization is dissolved in the dimethyl formamide, is mixed with massfraction and is 10% copolymer solution; With the glass-carbon electrode is dash receiver, and when voltage is that 20 kilovolts, rate of extrusion are that 3.0 milliliters/hour, receiving range are 20 centimetres and collect tunica fibrosas, spinning time remaining 10 hours obtains depositing the electrode of nano fibrous membrane;
3) electrode surface that will deposit nano fibrous membrane immerses the buffer solution contain 10 mg/ml glucose oxidases, 4 ℃ of following oscillating reactionss after 24 hours, obtain containing the electrode of the fiber surface immobilized enzyme of glycidyl methacrylate, preserve stand-by down for 4 ℃.
Embodiment 14
Replace glucose oxidase with peroxidase, all the other are identical with example 11.
Embodiment 15
Replace glucose oxidase with cytochrome, all the other are identical with example 11.
Embodiment 16
Replace glucose oxidase with cholesterol oxidase, all the other are identical with example 11.
Embodiment 17
Replace glucose oxidase with alcohol dehydrogenase, all the other are identical with example 11.
Embodiment 18
Replace glucose oxidase with laccase, all the other are identical with example 11.
The glucose oxidase electrode parameter of part examples preparation of the present invention sees Table 1.
The glucose oxidase electrode parameter of table 1 preparation
| Routine number | Comonomer | Electron mediator | The comonomer mole fraction | Electron mediator/copolymer quality ratio | The glucose linear detection range (mM/liter) | Lowest detectable limit (mM/liter) | Response time (second) | Sensitivity (microampere rises/mM) | Maximum current (microampere) |
| 2 | Methacrylic acid | The hydroxyl porphyrin | 20％ | 0.152 | 0～13 | 0.1279 | 2.5 | 0.25548 | 4.05 |
| 3 | Methacrylic acid | The iron carbonyl porphyrin | 20％ | 0.152 | 0～17 | 0.1079 | 1.7 | 0.19511 | 4.32 |
| 4 | Itaconic acid | The hydroxyl zinc protoporphyrin | 20％ | 0.152 | 0～11 | 0.1447 | 2.9 | 0.17784 | 3.99 |
| 11 | Glycidyl methacrylate | The hydroxyl porphyrin | 20％ | 0.152 | 0～16 | 0.09564 | 3.5 | 0.15354 | 4.21 |
| 12 | Glycidyl methacrylate | The iron carbonyl porphyrin | 20％ | 0.152 | 0～18 | 0.0824 | 3.0 | 0.22212 | 4.11 |
| 13 | Glycidyl methacrylate | The hydroxyl zinc protoporphyrin | 20％ | 0.152 | 0～19 | 0.0795 | 2.7 | 0.19785 | 4.21 |
Claims (5)
1. a mediator electrochemical enzyme electrode comprises basal electrode and active layer, and basal electrode is provided with active layer, it is characterized in that:
1) active layer is the nano fibrous membrane and the fixing oxidoreducing enzyme of electron mediator functionalization acrylonitrile copolymer;
2) nano fibrous membrane of active layer is prepared by electrospinning process;
3) in the acrylonitrile copolymer, comonomer is acrylic acid, methacrylic acid, maleic anhydride, itaconic acid or glycidyl methacrylate, it is 5～20% that comonomer accounts for the acrylonitrile copolymer mole percentage, and the viscosity average molecular weigh of acrylonitrile copolymer is 30,000～100,000; The massfraction that electron mediator accounts for acrylonitrile copolymer is 2.5%～15.8%;
4) electron mediator comprises ferrocene, porphyrin, iron porphyrin or the zinc protoporphyrin that contains reactive group, and reactive group comprises amino or hydroxyl;
5) oxidoreducing enzyme is hydrogen peroxidase, peroxidase, cytochrome, glucose oxidase, cholesterol oxidase, alcohol dehydrogenase or laccase.
2. the preparation method of a mediator electrochemical enzyme electrode as claimed in claim 1 is characterized in that comprising the steps:
1) earlier acrylonitrile copolymer and the electron mediator that contains amino or hydroxyl are dissolved in dimethyl formamide, the mass ratio of acrylonitrile copolymer and electron mediator is 1/1～5/1, add N then, N '-dicyclohexylcarbodiimide, its concentration in dimethyl formamide is 5～15 mg/ml, reaction is 12～48 hours in the time of 15 ℃～45 ℃, with product absolute ethyl alcohol precipitating, washing, 60 ℃～70 ℃ following vacuum drying 24～48 hours, obtain the acrylonitrile copolymer that contains acrylic acid, methacrylic acid or furoate clothing of electron mediator functionalization;
2) acrylonitrile copolymer that contains acrylic acid, methacrylic acid or furoate clothing with the electron mediator functionalization is dissolved in the dimethyl formamide, is mixed with massfraction and is 3%～10% copolymer solution; With platinum electrode, gold electrode or glass-carbon electrode is dash receiver, when voltage is that 10～20 kilovolts, rate of extrusion are 0.5～3.0 milliliter/hour, collect tunica fibrosa when receiving range is 10～20 centimetres, spinning time remaining 2～10 hours obtains depositing the electrode of nano fibrous membrane;
3) electrode surface that will deposit nano fibrous membrane immerses in the buffer solution of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide, wherein the concentration of 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride is 5～20 mg/ml, the concentration of N-hydroxy-succinamide is 3～12 mg/ml, 4 ℃～25 ℃ activation 2～10 hours of vibrating down, then with buffer solution washing 3～5 times; The electrode surface of activation is immersed the buffer solution that contains 1～10 mg/ml oxidoreducing enzyme, 4 ℃～25 ℃ following oscillating reactionss after 3～24 hours, obtain containing the electrode of the fiber surface immobilized enzyme of acrylic acid, methacrylic acid or itaconic acid, preserve stand-by down for 4 ℃.
3. the preparation method of a mediator electrochemical enzyme electrode as claimed in claim 1 is characterized in that comprising the steps:
1) earlier acrylonitrile copolymer is dissolved in dimethyl formamide, adding the electron mediator that contains amino or hydroxyl then reacted 12～48 hours in the time of 15 ℃～45 ℃, the mass ratio of acrylonitrile copolymer and electron mediator is 1/1～5/1, with product absolute ethyl alcohol precipitating, washing, 60 ℃～70 ℃ following vacuum drying 24～48 hours, obtain the acrylonitrile copolymer that contains maleic anhydride or methacrylic acid shrink base glycerol ester of electron mediator functionalization;
2) acrylonitrile copolymer that contains maleic anhydride or methacrylic acid shrink base glycerol ester with the electron mediator functionalization is dissolved in the dimethyl formamide, is mixed with massfraction and is 3%～10% copolymer solution; With platinum electrode, gold electrode or glass-carbon electrode is dash receiver, when voltage is that 10～20 kilovolts, rate of extrusion are that 0.5～3.0 milliliter/hour, receiving range are 10～20 centimetres and collect tunica fibrosas, spinning time remaining 2～10 hours obtains depositing the electrode of nano fibrous membrane;
3) electrode surface that will deposit nano fibrous membrane immerses the buffer solution contain 1～10 mg/ml oxidoreducing enzyme, 4 ℃～25 ℃ following oscillating reactionss after 3～24 hours, obtain containing the electrode of the fiber surface immobilized enzyme of maleic anhydride or glycidyl methacrylate, preserve stand-by down for 4 ℃.
4. as the preparation method of claim 2 or 3 described a kind of mediator electrochemical enzyme electrodes, it is characterized in that described oxidoreducing enzyme is hydrogen peroxidase, peroxidase, cytochrome, glucose oxidase, cholesterol oxidase, alcohol dehydrogenase or laccase.
5. as the preparation method of claim 2 or 3 described a kind of mediator electrochemical enzyme electrodes, it is characterized in that described electron mediator is ferrocene, porphyrin, iron porphyrin or the zinc protoporphyrin that contains reactive group, reactive group comprises amino or hydroxyl.
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| CN101915797B (en) * | 2010-07-27 | 2011-09-28 | 北京师范大学 | Method for preparing electrostatic spinning immobilized laccase electrode |
| CN107817284B (en) * | 2017-10-24 | 2019-08-02 | 中国地质大学（北京） | The preparation method of enzyme electrode is imitated in Electrospun immobilization for disinfection by-products detection |
| CN108448141B (en) * | 2018-01-19 | 2020-07-21 | 天津大学 | Flexible skin energy supply device |
| CN109942879B (en) * | 2019-02-28 | 2021-07-27 | 东南大学 | Poly (methacrylic acid-itaconic acid) modified film and preparation method and application thereof |
| CN110642461B (en) * | 2019-09-23 | 2021-11-02 | 西南科技大学 | Alkali-activated leather-immobilized bacillus pumilus/H2O2Method for purifying printing and dyeing wastewater |
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| CN1737560A (en) * | 2005-09-02 | 2006-02-22 | 天津大学 | Static electricity spinning prepared fixed enzymic electrode and method thereof |
| CN1850983A (en) * | 2006-02-20 | 2006-10-25 | 扬州大学 | Method for preparing biosensor using porous membrane immobilized enzyme |
| CN1837266A (en) * | 2006-03-22 | 2006-09-27 | 浙江大学 | Phospholipid modified acrylon nano fiber composite film and its preparation and immobilized enzyme thereof |
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