WO2021155735A1 - 一种乳酸脱氢酶电极及其制备方法和应用 - Google Patents

一种乳酸脱氢酶电极及其制备方法和应用 Download PDF

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WO2021155735A1
WO2021155735A1 PCT/CN2021/071161 CN2021071161W WO2021155735A1 WO 2021155735 A1 WO2021155735 A1 WO 2021155735A1 CN 2021071161 W CN2021071161 W CN 2021071161W WO 2021155735 A1 WO2021155735 A1 WO 2021155735A1
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electrode
nad
coenzyme
chitosan
lactate dehydrogenase
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French (fr)
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郑岚
马润隆
马耀宏
孟庆军
王丙莲
刘庆艾
杨艳
蔡雷
公维丽
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山东省科学院生物研究所
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
    • G01N27/3272—Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
    • Y02E60/30—Hydrogen technology
    • Y02E60/50—Fuel cells

Definitions

  • the invention belongs to the technical field of detection and analysis, and specifically relates to a lactate dehydrogenase electrode and a preparation method and application thereof.
  • Oxidoreductases are the most abundant type of enzymes in nature. Among the six types of enzymes, 30% to 35% are oxidoreductases.
  • the oxidase-dependent biosensor is a mature development product, but it also has some shortcomings. For example, the oxidase needs to consume oxygen during its action and is susceptible to O 2 interference; the oxidase reaction products are mostly H 2 O 2 , H 2 O 2 Accumulation will affect sensor detection.
  • Dehydrogenases are more abundant in nature, and nearly 400 kinds of dehydrogenases have been reported in nature. The dehydrogenase biosensor relies on the coenzyme to complete the reaction, and the coenzyme can directly participate in the reaction without O 2 interference, and the reaction is more sensitive and rapid.
  • dehydrogenase biosensors have become a research hotspot and play an important role in medical detection and food fermentation control.
  • M.Gamellaa et al. monitored the malate-lactic acid fermentation process in the winemaking process by preparing an electrochemical biosensor for malate dehydrogenase.
  • Vinay Narwal et al. used a gold electrode modified by preparing lactate dehydrogenase nanoparticles to determine lactate.
  • Liang Yunyu and others developed a glutamate dehydrogenase voltammetric sensor for sensitive ammonium ion determination.
  • NAD + nicotinyl coenzyme
  • NADH nicotinyl coenzyme
  • the catalysis of most dehydrogenases requires the participation of nicotinyl coenzyme [NAD + , NADH], which binds to the enzyme in the enzymatic reaction and directly participates in the reaction as an oxidant or a reducing agent.
  • NAD + is in the oxidized state
  • NADH is in the reduced state
  • the oxidized coenzyme and the reduced coenzyme are converted into each other through the redox reaction of hydrogenation or dehydrogenation.
  • NAD + is not encased inside by enzyme molecules when it performs the function of electron transfer.
  • the traditional dehydrogenase electrode needs to add free NAD + to the reaction system to achieve the reaction between the dehydrogenase and the substrate.
  • free NAD + cannot be reused, and free NAD + needs to be added repeatedly for each measurement.
  • NAD + is expensive, usually much more expensive than the product obtained from the enzymatic reaction, and its price needs to be about $100/g.
  • the addition of free NAD + also has the disadvantages of time-consuming and complicated operation, which is not conducive to the convenient use and popularization of dehydrogenase electrodes. It is necessary to immobilize the coenzyme NAD + and regenerate it for recycling.
  • the immobilization of coenzyme NAD + on the electrode surface and effective regeneration technology are two key technologies for constructing dehydrogenase electrodes/sensors.
  • the dehydrogenase electrode NAD + has only a few hundred molecular weights, and it is difficult to fix it with traditional methods. It is difficult to embed it in a semi-permeable membrane. If the coenzyme is combined with a polymer carrier to make it polymerized, it can be solved. This problem.
  • Another key issue to realize the recycling of dehydrogenase electrodes is the regeneration of NAD+.
  • NADH has a high oxidation overpotential, too slow electron transfer rate, and unsatisfactory kinetic characteristics.
  • NAD + Redox regeneration based on electronic mediators NAD + is an effective regeneration method that can reduce the oxidation potential of NADH, increase the electron transfer rate on the electrode surface, and realize the use of dehydrogenase electrodes. NAD + regeneration.
  • the present invention provides a coenzyme factor complex, an enzyme electrode, an enzyme sensor, and a preparation method and application thereof.
  • the present invention first chemically modifies the small molecule NAD + inactive parts of the coenzyme factor by chemical means; chemically connects the chemically modified NAD + with the chitosan carrier to obtain the NAD + -chitosan complex (coenzyme). Factor complex).
  • the enzyme electrode is prepared, and the steps are as follows: modify carbon nanotubes on the surface of the substrate electrode, use carbon nanotubes as the substrate material for NADH detection; electrodeposit ABTS on the electrode, and use ABTS as the electronic mediator to achieve NAD + In-situ regeneration; dropwise addition of prepared NAD + -chitosan complex to realize the immobilization of small molecule NAD + ; connect dehydrogenase to chitosan carrier through glutaraldehyde cross-linking to obtain dehydrogenase electrode.
  • a variety of dehydrogenase electrodes/biosensors can be prepared to detect malic acid, glucose, lactic acid and other substrates. Therefore, the technical method of immobilization and in-situ regeneration of NAD + and electrode preparation technology of the present invention can be widely used in the preparation process of dehydrogenase electrode/sensor, to realize the recycling of dehydrogenase electrode, and to construct dehydrogenase. Electrode/biosensing provides an effective technical approach.
  • a coenzyme factor complex is provided, which is obtained by compounding a coenzyme factor and a load material.
  • the compounding means that the coenzyme factor and the carrier are attached, combined, integrated or linked to each other. Therefore, they are not physically separate components, but instead can be compounded together as a single component (covalently or ionic bonded complex).
  • the coenzyme factor can be a natural coenzyme or an artificial coenzyme.
  • "Coenzyme” or “redox cofactor” refers to act as an enzymatic transfer of redox equivalents (e.g., hydrogen ions (H -)) receptor molecules, and the enzymatic oxidation of transferring reducing equivalents from the substrate ( For example, target analyte) to enzyme to coenzyme transfer.
  • redox equivalent refers to a concept commonly used in redox chemistry, which is well known to those skilled in the art.
  • coenzyme-dependent enzyme ie, target analyte
  • coenzyme-dependent enzyme ie, target analyte
  • electrons transferred from the substrate of the coenzyme-dependent enzyme ie, target analyte
  • coenzymes include, but are not limited to, NAD, NADP, PQQ, thio-NAD, thio-NADP, and the like.
  • the coenzyme is an artificial coenzyme.
  • artificial coenzymes include, but are not limited to, artificial NAD(P)/NAD(P)H compounds, which are natural NAD/NADH or chemical derivatives of natural NADP/NADPH.
  • the artificial coenzyme can chemically modify the inactive part of the natural coenzyme, so that the coenzyme can carry a chemical group for immobilization without affecting the active function of the coenzyme factor, thereby realizing the immobilization of the coenzyme.
  • the amino group on NAD adenine can be modified to obtain an artificial coenzyme NAD + compound.
  • the loading material may be a polymer carrier.
  • loading materials include, but are not limited to, chitosan, agarose, sodium alginate, polyethylene glycol, etc.; preferably chitosan, which is a kind of The high-molecular water-soluble polysaccharide with free amino groups has good film-forming properties and can be used as a carrier of coenzyme factors; more preferably, the chitosan is a medium viscosity chitosan (200-400 mPa.s).
  • cofactor complex of the present invention may be a NAD + - chitosan complex
  • the NAD + - Chitosan Complex by an amino group on the adenine NAD + NAD + can be modified to obtain an artificial coenzyme, Then it is prepared by covalently linking with chitosan, which actually realizes the immobilization of NAD +.
  • the preparation method of the NAD + -chitosan complex includes:
  • the heating reaction is preferably water bath heating, and the water bath reaction conditions are at 60 to 80°C for 0.5 to 3 hours, preferably at 70°C for 1 hour;
  • the pH is adjusted to be strongly alkaline, preferably 10-11
  • the heating reaction is preferably water bath heating
  • the water bath reaction conditions are reaction at 60-80°C for 0.5-3h, preferably 70°C water bath reaction for 1h;
  • the heating reaction is preferably water bath heating, and the water bath reaction conditions are at 60-80°C for 0.5 to 3 hours, preferably at 70°C for 1 hour;
  • the heating reaction is preferably water bath heating, and the water bath reaction conditions are at 60-80° C. for 0.5 to 3 hours, preferably at 70° C. for 1 h.
  • the second aspect of the present invention provides the application of the above-mentioned coenzyme factor complex in an enzyme electrode and/or preparation of an enzyme electrode.
  • an enzyme electrode comprising:
  • the base electrode, and the base material supported by the base electrode are The base electrode, and the base material supported by the base electrode.
  • the base electrode includes, but is not limited to: a glassy carbon (GCE) electrode, a gold electrode, a graphite electrode, and a carbon paste electrode, preferably GCE.
  • GCE has good mechanical stability, light stability and high conductivity.
  • the base material is a carbon material
  • the carbon material includes, but is not limited to: activated carbon, graphene, carbon nanofibers, carbon nanospheres, glassy carbon, carbon aerogels, carbon nanotubes (CNTs), preferably carbon nanotubes,
  • CNTs carbon nanotubes
  • the carbon nanotubes have large specific surface area and small internal resistance and are porous, can significantly improve the analytical performance of the chemically modified electrode, and at the same time have good electrical conductivity and chemical stability.
  • the carbon nanotubes include not only multi-walled and single-walled carbon nanotubes, but also functionalized carbon nanotubes modified by amination and carboxylation.
  • the enzyme electrode includes a GCE electrode, and CNTs supported on the GCE electrode.
  • the loading can be carried out by drip coating.
  • the enzyme electrode includes:
  • a substrate electrode wherein a substrate material is carried on the substrate electrode, a medium is deposited on the surface of the substrate material, and the surface of the medium is coated with a coenzyme factor complex and an enzyme.
  • the base electrode includes, but is not limited to: a glassy carbon (GCE) electrode, a gold electrode, a graphite electrode, and a carbon paste electrode, preferably GCE.
  • GCE has good mechanical stability, light stability and high conductivity.
  • the base material is a carbon material
  • the carbon material includes, but is not limited to: activated carbon, graphene, carbon nanofibers, carbon nanospheres, glassy carbon, carbon aerogels, carbon nanotubes (CNTs), preferably carbon nanotubes,
  • CNTs carbon nanotubes
  • the carbon nanotubes have large specific surface area and small internal resistance and are porous, can significantly improve the analytical performance of the chemically modified electrode, and at the same time have good electrical conductivity and chemical stability.
  • the carbon nanotubes include not only multi-walled and single-walled carbon nanotubes, but also functionalized carbon nanotubes modified by amination and carboxylation.
  • “medium” refers to a compound that increases the reactivity of a reduced coenzyme obtained by reacting with an analyte and transfers electrons to an electrode system or a suitable optical indicator/optical indicator system.
  • the mediator can be any chemical substance (usually electrochemically active), which can participate in a reaction scheme involving analytes, coenzyme-dependent enzymes, coenzymes and their reaction products to produce detectable electrochemically active reaction products.
  • the participation of the mediator in the reaction involves interaction with any one of the analyte, coenzyme-dependent enzyme, coenzyme, or a species that is a reaction product of one of these (for example, the coenzyme reaction is in a different oxidation state) The change in its oxidation state.
  • the medium can also be stable in its oxidized form, can optionally exhibit reversible redox electrochemistry, can exhibit good solubility in aqueous solutions, and can react quickly to produce electrochemically active reaction products.
  • Examples of the medium include, but are not limited to, 2,2'-diazobis(3-ethylbenzothiazolin-6-sulfonic acid) diammonium salt (ABTS), azo compounds or azo precursors, Benzoquinone, Prussian blue, nitrosoaniline or precursors based on nitrosoaniline, thiazine or thiazine derivatives, transition metal complexes such as potassium ferricyanide, ruthenium complexes such as hexylamine ruthenium chloride, Osmium derivatives, quinone or quinone derivatives, phenazine or phenazine-based precursors, and combinations of phenazine derivatives and hexaammine ruthenium chloride, and their derivatives.
  • ABTS 2,2'-diazobis(3-ethylbenzothiazolin-6-sulfonic acid) diammonium salt
  • ABTS 2,2'-diazobis(
  • the mediator when the coenzyme is NAD/NADH, the mediator may be 2,2'-diazobis(3-ethylbenzothiazolin-6-sulfonic acid) diammonium salt (ABTS).
  • ABTS 2,2'-diazobis(3-ethylbenzothiazolin-6-sulfonic acid) diammonium salt
  • carbon nanotubes and ABTS + are modified on the surface of the GCE electrode, the carbon nanotubes can realize the catalytic oxidation of NADH, and ABTS + can increase electron transfer.
  • ABTS + and NADH undergo redox, the 4-position of the pyridine ring in NADH reacts with ABTS + radical cations, and the hydrogen ion obtained by ABTS + is converted into ABTS, and NADH is oxidized to NAD + , realizing NAD + in-situ regeneration.
  • ABTS loses electrons and produces ABTS + at the positive electrode of the electrode, and enters the next cycle.
  • the "enzyme” specifically refers to a coenzyme-dependent enzyme
  • the "coenzyme-dependent enzyme” refers to an enzyme that requires an organic or inorganic cofactor called a coenzyme for catalytic activity.
  • the coenzyme-dependent enzyme may be a dehydrogenase.
  • dehydrogenase refers to a protein or polypeptide capable of catalyzing the oxidation of a substrate by transferring a hydride ion (H ⁇ ), which is a redox equivalent, to an acceptor molecule.
  • dehydrogenases include, but are not limited to, glucose dehydrogenase, alcohol dehydrogenase, glycerol dehydrogenase, lactate dehydrogenase, L-amino acid dehydrogenase, malate dehydrogenase or sorbitol dehydrogenase Etc., especially NAD(P)/NAD(P)H-dependent dehydrogenase.
  • the fourth aspect of the present invention provides a preparation method of the above-mentioned enzyme electrode.
  • the preparation method of the enzyme electrode is not particularly limited, but the enzyme electrode can be applied on a surface of a base electrode or in the form of a membrane by using the following method It is coated to form: for example, drop coating, electrodeposition, sputtering, electron beam, thermal deposition, spin coating, screen printing, inkjet printing, doctor blade or gravure printing.
  • the preparation method includes:
  • step 1)
  • the base electrode includes, but is not limited to: a glassy carbon (GCE) electrode, a gold electrode, a graphite electrode, and a carbon paste electrode, preferably GCE.
  • GCE has good mechanical stability, light stability and high conductivity.
  • the base material is a carbon material
  • the carbon material includes, but is not limited to: activated carbon, graphene, carbon nanofibers, carbon nanospheres, glassy carbon, carbon aerogels, carbon nanotubes (CNTs), preferably carbon nanotubes.
  • Carbon nanotubes have a large specific surface area and a small internal resistance of porosity, which can significantly improve the analytical performance of chemically modified electrodes, and at the same time have good electrical conductivity and chemical stability.
  • the carbon nanotubes include not only multi-walled and single-walled carbon nanotubes, but also functionalized carbon nanotubes modified by amination and carboxylation.
  • step 2)
  • Mediator refers to a compound that increases the reactivity of a reduced coenzyme obtained by reacting with an analyte and transfers electrons to an electrode system or a suitable optical indicator/optical indicator system.
  • the mediator can be any chemical substance (usually electrochemically active), which can participate in a reaction scheme involving analytes, coenzyme-dependent enzymes, coenzymes and their reaction products to produce detectable electrochemically active reaction products.
  • the participation of the mediator in the reaction involves interaction with any one of the analyte, coenzyme-dependent enzyme, coenzyme, or a species that is a reaction product of one of these (for example, the coenzyme reaction is in a different oxidation state) The change in its oxidation state (for example, reduction).
  • a variety of media exhibit suitable electrochemical behavior.
  • the medium can also be stable in its oxidized form, can optionally exhibit reversible redox electrochemistry, can exhibit good solubility in aqueous solutions, and can react quickly to produce electrochemically active reaction products.
  • Examples of the medium include, but are not limited to, ABTS, azo compounds or azo precursors, benzoquinone, Prussian blue, nitrosoaniline or precursors based on nitrosoaniline, thiazine or thiazine derivatives, Transition metal complexes such as potassium ferricyanide, ruthenium complexes such as hexylamine ruthenium chloride, osmium derivatives, quinone or quinone derivatives, phenazine or phenazine-based precursors, and phenazine derivatives and chlorinated The combination of ruthenium hexaammine and their derivatives.
  • the mediator when the coenzyme factor is NAD/NADH, the mediator can be 2,2'-diazobis(3-ethylbenzothiazolin-6-sulfonic acid) diammonium salt (ABTS).
  • ABTS 2,2'-diazobis(3-ethylbenzothiazolin-6-sulfonic acid) diammonium salt
  • step 3
  • the coenzyme factor complex is obtained by the complex of the coenzyme factor and the load material.
  • the compounding means that the coenzyme factor and the carrier are attached, combined, integrated or linked to each other. Therefore, they are not physically separate components, but instead can be compounded together as a single component (covalently or ionic bonded complex).
  • the coenzyme factor can be a natural coenzyme or an artificial coenzyme.
  • a "coenzyme” or “redox cofactor” refers to the enzymatic oxidation of the transfer function as reducing equivalents (e.g., hydrogen ions (H -)) receptor molecules, and the enzymatic transfer The redox equivalent is transferred from the substrate (eg, target analyte) to the enzyme to the coenzyme.
  • reducing equivalents e.g., hydrogen ions (H -)
  • Redox equivalent refers to a concept commonly used in redox chemistry, which is well known to those skilled in the art.
  • coenzyme-dependent enzyme ie, target analyte
  • coenzyme-dependent enzyme ie, target analyte
  • electrons transferred from the substrate of the coenzyme-dependent enzyme ie, target analyte
  • coenzymes include, but are not limited to, NAD, NADP, PQQ, thio-NAD, thio-NADP, and the like.
  • the coenzyme is an artificial coenzyme.
  • artificial coenzymes include, but are not limited to, artificial NAD(P)/NAD(P)H compounds, which are natural NAD/NADH or chemical derivatives of natural NADP/NADPH.
  • the artificial coenzyme can chemically modify the inactive part of the natural coenzyme, so that the coenzyme can carry a chemical group for immobilization without affecting the active function of the coenzyme factor, thereby realizing the immobilization of the coenzyme.
  • the amino group on NAD adenine can be modified to obtain an artificial coenzyme NAD compound.
  • the supporting material may be a polymer carrier.
  • the supporting material include, but are not limited to, chitosan, agarose, sodium alginate, polyethylene glycol, etc.; preferably chitosan, chitosan Sugar is a high molecular weight water-soluble polysaccharide with free amino groups, which has good film-forming properties and can be used as a carrier for coenzyme factors; more preferably, the chitosan is medium-viscosity chitosan (200-400 mPa.s).
  • the coenzyme factor complex of the present invention may be a NAD + -chitosan complex, the NAD + -chitosan complex, and the NAD + -chitosan complex can pass through the NAD + adenine
  • the amino group on the upper part is modified to obtain an artificial NAD + coenzyme, which is then covalently linked with chitosan to prepare it, thereby actually realizing the immobilization of NAD +.
  • step 4
  • the "enzyme” specifically refers to a coenzyme-dependent enzyme
  • the "coenzyme-dependent enzyme” refers to an enzyme that requires an organic or inorganic cofactor called a coenzyme for catalytic activity.
  • the coenzyme-dependent enzyme may be a dehydrogenase.
  • dehydrogenase refers to a protein or polypeptide capable of catalyzing the oxidation of a substrate by transferring a hydride ion (H ⁇ ), which is a redox equivalent, to an acceptor molecule.
  • dehydrogenases include, but are not limited to, glucose dehydrogenase, alcohol dehydrogenase, glycerol dehydrogenase, lactate dehydrogenase, L-amino acid dehydrogenase, malate dehydrogenase or sorbitol dehydrogenase Etc., especially NAD(P)/NAD(P)H-dependent dehydrogenase.
  • step 4 loading the enzyme on the material prepared in step 3) can be achieved by using glutaraldehyde to cross-link the enzyme.
  • the loading material of the coenzyme factor complex in step 3) is chitosan
  • glutaraldehyde through the action of glutaraldehyde, the free amino group of chitosan is covalently linked to a free aldehyde on glutaraldehyde to synthesize a Schiff base structure
  • Another free aldehyde of glutaraldehyde is connected with the enzyme to realize the immobilization of the enzyme.
  • a method for regenerating a coenzyme factor comprising:
  • step 1)
  • the base electrode includes, but is not limited to: a glassy carbon (GCE) electrode, a gold electrode, a graphite electrode, and a carbon paste electrode, preferably GCE.
  • GCE has good mechanical stability, light stability and high conductivity.
  • the base material is a carbon material
  • the carbon material includes, but is not limited to: activated carbon, graphene, carbon nanofibers, carbon nanospheres, glassy carbon, carbon aerogels, carbon nanotubes (CNTs), preferably carbon nanotubes,
  • CNTs carbon nanotubes
  • the carbon nanotubes have large specific surface area and small internal resistance and are porous, can significantly improve the analytical performance of the chemically modified electrode, and at the same time have good electrical conductivity and chemical stability.
  • the carbon nanotubes include not only multi-walled and single-walled carbon nanotubes, but also functionalized carbon nanotubes modified by amination and carboxylation.
  • step 2)
  • Mediator refers to a compound that increases the reactivity of a reduced coenzyme obtained by reacting with an analyte and transfers electrons to an electrode system or a suitable optical indicator/optical indicator system.
  • the mediator can be any chemical substance (usually electrochemically active), which can participate in a reaction scheme involving analytes, coenzyme-dependent enzymes, coenzymes and their reaction products to produce detectable electrochemically active reaction products.
  • the participation of the mediator in the reaction involves interaction with any one of the analyte, coenzyme-dependent enzyme, coenzyme, or a species that is a reaction product of one of these (for example, the coenzyme reaction is in a different oxidation state) The change in its oxidation state (for example, reduction).
  • a variety of media exhibit suitable electrochemical behavior.
  • the medium can also be stable in its oxidized form, can optionally exhibit reversible redox electrochemistry, can exhibit good solubility in aqueous solutions, and can react quickly to produce electrochemically active reaction products.
  • Examples of the medium include, but are not limited to, ABTS, azo compounds or azo precursors, benzoquinone, Prussian blue, nitrosoaniline or precursors based on nitrosoaniline, thiazine or thiazine derivatives, Transition metal complexes such as potassium ferricyanide, ruthenium complexes such as hexylamine ruthenium chloride, osmium derivatives, quinone or quinone derivatives, phenazine or phenazine-based precursors, and phenazine derivatives and chlorinated The combination of ruthenium hexaammine and their derivatives.
  • the mediator when the coenzyme factor is NAD, the mediator may be 2,2'-diazobis(3-ethylbenzothiazolin-6-sulfonic acid) diammonium salt (ABTS).
  • ABTS 2,2'-diazobis(3-ethylbenzothiazolin-6-sulfonic acid) diammonium salt
  • carbon nanotubes and ABTS + are modified on the surface of the GCE electrode, the carbon nanotubes can realize the catalytic oxidation of NADH, and ABTS + can increase electron transfer.
  • ABTS + and NADH undergo redox, the 4-position of the pyridine ring in NADH reacts with ABTS + radical cations, and the hydrogen ion obtained by ABTS + is converted into ABTS, and NADH is oxidized to NAD + , realizing NAD + in-situ regeneration.
  • ABTS loses electrons and produces ABTS + at the positive electrode of the electrode, and enters the next cycle.
  • the sixth aspect of the present invention provides the application of the above-mentioned coenzyme factor regeneration method in enzyme electrodes and/or enzyme sensors.
  • the seventh aspect of the present invention provides the application of the above-mentioned coenzyme factor complex and/or enzyme electrode in preparing an enzyme sensor.
  • an enzyme sensor which comprises at least two electrodes, and the electrodes at least comprise the above-mentioned coenzyme factor complex and/or the above-mentioned enzyme electrode.
  • the enzyme sensor of the invention has higher detection sensitivity, higher detection repeatability and long-term storage stability.
  • the enzyme sensor includes two or three electrodes.
  • the enzyme sensor is a two-electrode or three-electrode enzyme sensor.
  • the electrodes are a working electrode and a counter electrode; wherein, the working electrode is the aforementioned coenzyme factor complex and/or the aforementioned enzyme electrode.
  • the electrodes are a working electrode, a counter electrode, and a reference electrode; wherein, the working electrode is the aforementioned coenzyme factor complex and/or The above enzyme electrode.
  • the counter electrode is a platinum electrode; the reference electrode is an Ag/AgCl electrode.
  • a method for electrochemically measuring the concentration or presence of a target analyte to be tested comprising: combining the enzyme electrode and/or the enzyme sensor with the target analyte to be tested. Contact with the liquid sample, measure the response current intensity of the target analyte to be tested, and analyze the concentration or presence of the target analyte.
  • the target analytes include, but are not limited to, amino acids, glucose, ethanol, glycerol, lactic acid, malic acid, pyruvate, sorbitol, triglycerides, and uric acid.
  • a malate dehydrogenase electrode comprising: a GCE electrode on which carbon nanotubes are loaded, and ABTS is deposited on the surface of the carbon nanotubes , The surface of the ABTS is loaded with chitosan-NAD + complex and malate dehydrogenase.
  • the preparation method of the malate dehydrogenase electrode includes:
  • the preparation method of the malate dehydrogenase electrode includes:
  • the carbon nanotube dispersion (0.1%) was dripped onto the working surface of the GCE electrode and dried.
  • the electrode is CNTs/GCE.
  • the CNTs/GCE was immersed in the ABTS deposition stock solution, and cyclic voltammetry (CV) scans were performed at a scan rate of 50mV/s in the potential range of -200 ⁇ 600mV at different times.
  • the electrode was ABTS/CNTs/GCE.
  • ABTS cationic radicals deposited on the surface of the electrode can participate in the NADH oxidation process to achieve NAD + regeneration.
  • the ABTS deposition stock solution contains: 2.5 mmol/L FeCl 3 , 2.5 mmol/L K 3 Fe(CN) 6 200 mmol/L HCl, and 1 mmol/L ABTS.
  • the prepared NAD + -chitosan complex was dropped on the surface of ABTS/CNTs/GCE, dried, and the electrode was CTS-NAD + /ABTS/CNTs/GCE.
  • the preparation process of NAD + -chitosan complex includes:
  • the malate dehydrogenase sensor includes: a working electrode, a counter electrode and a reference electrode, wherein the above-mentioned malate dehydrogenase electrode is used as a working electrode; the counter electrode is a platinum electrode; and the reference electrode is an Ag/AgCl electrode .
  • a glucose dehydrogenase electrode comprising: a GCE electrode, the GCE electrode is loaded with carbon nanotubes, and ABTS is deposited on the surface of the carbon nanotubes, The surface of the ABTS is loaded with chitosan-NAD + complex and glucose dehydrogenase.
  • the preparation method of the glucose dehydrogenase electrode includes:
  • the preparation method of the glucose dehydrogenase electrode includes:
  • the carbon nanotube dispersion (0.1%) was dripped onto the working surface of the GCE electrode and dried.
  • the electrode is CNTs/GCE.
  • the CNTs/GCE was immersed in the ABTS deposition stock solution, and cyclic voltammetry (CV) scans were performed at a scan rate of 50mV/s in the potential range of -200 ⁇ 600mV at different times.
  • the electrode was ABTS/CNTs/GCE.
  • ABTS cationic radicals deposited on the surface of the electrode can participate in the NADH oxidation process to achieve NAD + regeneration.
  • the ABTS deposition stock solution contains: 2.5 mmol/L FeCl 3 , 2.5 mmol/L K 3 Fe(CN) 6 200 mmol/L HCl, and 1 mmol/L ABTS.
  • the prepared NAD + -chitosan complex was dropped on the surface of ABTS/CNTs/GCE, dried, and the electrode was CTS-NAD + /ABTS/CNTs/GCE.
  • the preparation process of NAD + -chitosan complex includes:
  • the glucose dehydrogenase sensor includes: a working electrode, a counter electrode and a reference electrode, wherein the above-mentioned glucose dehydrogenase electrode is used as a working electrode; the counter electrode is a platinum electrode; and the reference electrode is an Ag/AgCl electrode.
  • a lactate dehydrogenase electrode comprising: a GCE electrode, on which carbon nanotubes are loaded, and ABTS is deposited on the surface of the carbon nanotubes,
  • the ABTS surface includes chitosan-NAD + complex and lactate dehydrogenase.
  • the preparation method of the lactate dehydrogenase electrode includes:
  • the preparation method of the lactate dehydrogenase electrode includes:
  • the carbon nanotube dispersion (0.1%) was dripped onto the working surface of the GCE electrode and dried.
  • the electrode is CNTs/GCE.
  • the CNTs/GCE was immersed in the ABTS deposition stock solution, and cyclic voltammetry (CV) scans were performed at a scan rate of 50mV/s in the potential range of -200 ⁇ 600mV at different times.
  • the electrode was ABTS/CNTs/GCE.
  • ABTS cationic radicals deposited on the surface of the electrode can participate in the NADH oxidation process to achieve NAD + regeneration.
  • the ABTS deposition stock solution contains: 2.5 mmol/L FeCl 3 , 2.5 mmol/L K 3 Fe(CN) 6 200 mmol/L HCl, and 1 mmol/L ABTS.
  • the prepared NAD + -chitosan complex was dropped on the surface of ABTS/CNTs/GCE, dried, and the electrode was CTS-NAD + /ABTS/CNTs/GCE.
  • the preparation process of NAD + -chitosan complex includes:
  • the lactate dehydrogenase sensor includes: a working electrode, a counter electrode and a reference electrode, wherein the aforementioned lactate dehydrogenase electrode is used as a working electrode; the counter electrode is a platinum electrode; and the reference electrode is an Ag/AgCl electrode.
  • the present invention uses simple NAD + modification technology to prepare chitosan-NAD + complex (coenzyme factor complex), which is used in the preparation of dehydrogenase electrodes.
  • the complex modifies the amino group on NAD + adenine, does not affect the function of NAD + activity, and effectively improves the activity of immobilized NAD +.
  • the coenzyme factor complex is drip-coated on the electrode surface on the one hand to realize the immobilization of NAD + on the electrode surface, and on the other hand, it provides the chitosan binding site on the electrode surface for the dehydrogenase, so the coenzyme factor complex is in Coenzyme-dependent enzyme electrodes/biosensors have a wide range of applications.
  • the present invention uses ABTS as an electronic mediator, and realizes NAD + regeneration through the electron transfer of the electrode. Regeneration of NAD + in situ by electrochemical means is not only convenient and fast, but also can avoid the influence of by-products.
  • the present invention uses NAD + immobilization and in-situ regeneration technology to prepare a dehydrogenase electrode that can be reused. Moreover, compared with the traditional dehydrogenase electrode, the electrode has higher detection sensitivity, higher detection repeatability and better storage stability, so it has good practical application value.
  • Figure 1 shows the structural changes and corresponding ultraviolet absorption spectra of the chitosan-NAD + complex prepared in Example 1 of the present invention; among them, Figure 1 (I) shows the maximum ultraviolet absorption peak of NAD + at 260 nm; 1(II) is N1-carboxymethyl-NAD + exhibiting the largest UV absorption peak at 250nm; Figure 1(III) is C6-carboxymethyl NAD + exhibiting the largest UV absorption peak at 250nm and 340nm; Figure 1 (IV) shows that the chitosan-NAD + complex exhibits the largest UV absorption peak at 260 nm.
  • Figure 2 is a schematic diagram of a malate dehydrogenase electrode in Example 2 of the present invention.
  • Fig. 3 is a schematic diagram of the preparation process of malate dehydrogenase in Example 2 of the present invention and a scanning electron microscope image; among them, Fig. 3(I) is a scanning electron microscope image of a carbon nanotube modified electrode; Fig. 3(II) is an electrode after ABTS deposition Scanning electron micrograph; Figure 3 (III) is the scanning electron micrograph of the electrode after modified chitosan-NAD complex; Figure 3 (IV) is the scanning electron micrograph of the malate dehydrogenase immobilized on the electrode.
  • Figure 4 is a graph showing the repeatability results of the malate dehydrogenase electrode prepared in Example 2 of the present invention.
  • Figure 5 is a graph showing the linear range of the malate dehydrogenase electrode prepared in Example 2 of the present invention.
  • Figure 6 is a graph showing the stability results of the malate dehydrogenase electrode prepared in Example 2 of the present invention.
  • FIG. 7 is a graph showing the linear range of the glucose dehydrogenase electrode prepared in Example 3 of the present invention.
  • Figure 8 is a graph showing the repeatability results of the glucose dehydrogenase electrode prepared in Example 3 of the present invention.
  • Example 9 is a graph showing the stability results of the glucose dehydrogenase electrode prepared in Example 3 of the present invention.
  • Example 10 is a graph showing the linear range results of the lactate dehydrogenase electrode prepared in Example 4 of the present invention.
  • FIG. 11 is a graph showing the repeatability results of the lactate dehydrogenase electrode prepared in Example 4 of the present invention.
  • FIG. 12 is a diagram showing the stability results of the lactate dehydrogenase electrode prepared in Example 4 of the present invention.
  • FIG. 13 is a diagram showing the results of detecting malic acid by an unmodified ABTS dehydrogenase electrode in Example 5 of the present invention.
  • Example 14 is a graph showing the results of NADH detection using CNTs-GCE and bare electrodes in Example 6 of the present invention.
  • Example 15 is a comparison diagram of the response of the immobilized NAD + electrode to malic acid and free NAD + in Example 7 of the present invention.
  • Example 16 is a graph showing the response current intensity of malate dehydrogenase electrode obtained by using Prussian blue as the electron enzyme mediator in Example 8 of the present invention to measure malate.
  • FIG. 17 is a graph showing the response current intensity of malate dehydrogenase electrode prepared by using agarose as a load material in Example 9 of the present invention to measure malate.
  • Figure 18 shows the measurement of standard malic acid using a malate dehydrogenase electrode prepared by using other types of chitosan (high-viscosity chitosan) and standard chitosan (medium-viscosity chitosan) as loading materials in Example 10 of the present invention Comparison chart of response current intensity.
  • the present invention uses carbon nanotubes as the base material, ABTS as the electronic mediator, chitosan/chemically modified NAD + chitosan-NAD + composite film formed under the action of carbodiimide, and glutaraldehyde pair
  • the cross-linking effect of dehydrogenase produces a dehydrogenase electrode that can be reused.
  • Chitosan is a high molecular weight water-soluble polysaccharide with free amino groups. It has good film-forming properties and can be used as a carrier for NAD +. After EDC/NHS treatment, free amino groups on chitosan and free carboxyl groups on chemically modified NAD + form urea derivatives to complete immobilization of NAD + .
  • the free amino group of chitosan is covalently linked to a free aldehyde on glutaraldehyde to synthesize a Schiff base structure, and the other free aldehyde of glutaraldehyde is linked to dehydrogenase to realize the immobilization of dehydrogenase. change.
  • N1-carboxymethyl-NAD + has a blue-shifted peak, showing the largest UV absorption peak at 250nm ( Figure 1-II).
  • Sodium thiosulfate is used as a reducing agent to reduce N1-carboxymethyl-NAD+ to obtain N1-carboxymethyl-NADH which is more stable under alkaline conditions.
  • Dimroth rearrangement was performed under strong base conditions to obtain C6-carboxymethyl NADH with the amino group at the 6-position modified.
  • Formaldehyde undergoes the Cannizzaro reaction under alkaline conditions, and the rearrangement product C6-carboxymethyl NADH is oxidized to C6-carboxymethyl NAD + .
  • the compound showed the largest UV absorption peaks at 250nm and 340nm, and NADH showed the largest UV absorption peak at 340nm, indicating that NAD + and NADH derivatives co-exist during this reaction.
  • EDC/NHS EDC/NHS to treat C6-carboxymethyl NAD + and chitosan.
  • NHS treatment can enhance the stability of the carbodiimide cross-linked product.
  • the covalent bond between NAD + and chitosan carrier completes the immobilization of NAD + .
  • NAD + macromolecules show the largest UV absorption peak at 260 nm. The results show that NAD + active groups are not Affected by the fixation process.
  • the three-electrode system consists of platinum electrode as counter electrode, Ag/AgCl electrode as reference electrode, and glassy carbon electrode as working electrode.
  • the 3mm diameter glassy carbon electrode was polished to the mirror surface with a certain particle size Al 2 O 3 slurry on the polishing cloth. After each polishing, the surface dirt was washed off, and then moved into the ultrasonic water bath for cleaning, each time for 1 min. , Repeat three times, and finally use 1:1 ethanol, 1:1NHO 3 ultrasonic cleaning.
  • the electrode Immerse the electrode in the ABTS deposition stock solution (the ABTS deposition stock solution contains 2.5mM/L FeCl 3 , 2.5mM/L K 3 Fe(CN) 6 , 200mM/L HCl and 1mM/L ABTS, only for use on the day), at 50mV
  • the scan rate of /s performs cyclic voltammetry (CV) scans of different times in the potential range of -200 ⁇ 600mV. At this time, it can be seen that the redox peak value in the interface increases with the number of scan circles once and finally closes to overlap, indicating that ABTS has been deposited on the electrode.
  • CV cyclic voltammetry
  • malate dehydrogenase can catalyze the conversion of malate to oxaloacetate to generate NADH. Therefore, the content of malic acid can be quantitatively analyzed by measuring the content of NADH.
  • the carbon nanotubes and ABTS + are modified on the surface of the electrode. The carbon nanotubes can catalyze the oxidation of NADH, and ABTS + can increase electron transfer.
  • ABTS + and NADH undergo redox, the 4-position of the pyridine ring in NADH reacts with ABTS + radical cations, and the hydrogen ion obtained by ABTS + is converted into ABTS, and NADH is oxidized to NAD + , realizing NAD + in-situ regeneration.
  • ABTS loses electrons and produces ABTS + at the positive electrode of the electrode, and enters the next cycle.
  • the standard malic acid solutions with concentrations of 1, 2, 4, 6, 8, and 10 mmol/L were respectively configured, and the prepared malate dehydrogenase electrode was used to measure the response current intensity of the standard malate, and the linear range of the electrode was analyzed. Repeat the current measurement 10 times in 1, 2, 4, 6, 8, 10mmol/L malic acid solution, and analyze the repeatability of the electrode.
  • the prepared malate dehydrogenase electrode was stored at 4°C, and the electrode response was measured in a 4mmol/L malic acid solution every day to analyze the stability of the electrode.
  • Fig. 3-I shows the carbon nanotube modified electrode with obvious tube bundle distribution on the electrode surface.
  • ABTS is deposited
  • Figure 3-II after the electrochemical deposition of ABTS on carbon nanotubes, there is a clear film structure on the electrode surface.
  • Figure 3-III after modifying the chitosan-NAD complex, the membrane structure on the electrode surface was significantly thickened.
  • Figure 3-IV there are obvious protrusions on the electrode surface, indicating that malate dehydrogenase is fixed on the electrode surface.
  • the prepared malate dehydrogenase electrode was stored at 4°C, and the electrode response was measured in a 4mmol/L malic acid solution every day to analyze the stability of the electrode. As shown in Figure 6, during the 25-day test, the current intensity did not show significant fluctuations before the 20th day, indicating that the electrode remained stable within 20 days.
  • the three-electrode system consists of platinum electrode as counter electrode, Ag/AgCl electrode as reference electrode, and glassy carbon electrode as working electrode.
  • the 3mm diameter glassy carbon electrode was polished to the mirror surface with a certain particle size Al 2 O 3 slurry on the polishing cloth. After each polishing, the surface dirt was washed off, and then moved into the ultrasonic water bath for cleaning, each time for 1 min. , Repeat three times, and finally use 1:1 ethanol, 1:1NHO 3 ultrasonic cleaning.
  • the newly prepared electrode working surface was immersed in an aqueous solution containing 25% glutaraldehyde, taken out after 1 hour, and thoroughly cleaned with deionized water to introduce a certain amount of active aldehyde groups into the chitosan modified layer on the electrode surface. Then immerse the working surface of the electrode in the glucose dehydrogenase solution, take it out after 1 hour, and wash it sufficiently to take out the enzyme molecules that are not firmly bound.
  • Standard glucose solutions with concentrations of 0.55, 11, 22, 33, 44, 55 mmol/L were respectively configured, and the prepared glucose dehydrogenase electrode was used to measure the response current intensity of standard glucose, and the linear range of the electrode was analyzed.
  • the current was measured repeatedly 10 times in 1, 10, 20, 30, 40, 50mmol/L glucose solution, and the repeatability of the electrode was analyzed.
  • the prepared glucose dehydrogenase electrode was stored at 4°C, and the electrode response was measured in a 30 mmol/L glucose solution every day to analyze the stability of the electrode.
  • the prepared glucose dehydrogenase electrode was stored at 4°C, and the electrode response was measured in a 30 mmol/L glucose solution every day to analyze the stability of the electrode. As shown in Figure 9, during the 20-day test, the current intensity did not show significant fluctuations before the 15th day, indicating that the electrode remained stable within 15 days.
  • the three-electrode system consists of platinum electrode as counter electrode, Ag/AgCl electrode as reference electrode, and glassy carbon electrode as working electrode.
  • the 3mm diameter glassy carbon electrode was polished to the mirror surface with a certain particle size Al 2 O 3 slurry on the polishing cloth. After each polishing, the surface dirt was washed off, and then moved into the ultrasonic water bath for cleaning, each time for 1 min. , Repeat three times, and finally use 1:1 ethanol, 1:1NHO 3 ultrasonic cleaning.
  • the newly prepared electrode working surface was immersed in an aqueous solution containing 25% glutaraldehyde, taken out after 1 hour, and thoroughly cleaned with deionized water to introduce a certain amount of active aldehyde groups into the chitosan modified layer on the electrode surface. Then immerse the working surface of the electrode in the solution of lactate dehydrogenase, take it out after 1 hour, and wash it sufficiently to take out the enzyme molecules that are not firmly bound.
  • the standard lactic acid solutions with concentrations of 2, 4, 6, 8, and 10 mmol/L were respectively configured, and the prepared lactate dehydrogenase electrode was used to measure the response current intensity of the standard lactic acid, and the linear range of the electrode was analyzed.
  • the current was measured repeatedly 10 times in 2, 3, 4, 5, 6, 8 mmol/L lactic acid solution, and the repeatability of the electrode was analyzed.
  • the prepared lactate dehydrogenase electrode was stored at 4°C, and the electrode response was measured in a 6mmol/L lactic acid solution every day to analyze the stability of the electrode.
  • the prepared lactate dehydrogenase electrode was stored at 4°C, and the electrode response was measured in a 6mmol/L lactic acid solution every day to analyze the stability of the electrode. As shown in Figure 12, during the 28-day test, the current intensity did not fluctuate significantly before the 21st day, indicating that the electrode remained stable for 21 days.
  • the three-electrode system consists of platinum electrode as counter electrode, Ag/AgCl electrode as reference electrode, and glassy carbon electrode as working electrode.
  • the 3mm diameter glassy carbon electrode was polished to the mirror surface with a certain particle size Al 2 O 3 slurry on the polishing cloth. After each polishing, the surface dirt was washed off, and then moved into the ultrasonic water bath for cleaning, each time for 1 min. , Repeat three times, and finally use 1:1 ethanol, 1:1NHO 3 ultrasonic cleaning.
  • the three-electrode system consists of platinum electrode as counter electrode, Ag/AgCl electrode as reference electrode, and glassy carbon electrode as working electrode.
  • the 3mm diameter glassy carbon electrode was polished to the mirror surface with a certain particle size Al 2 O 3 slurry on the polishing cloth. After each polishing, the surface dirt was washed off, and then moved into the ultrasonic water bath for cleaning, each time for 1 min. , Repeat three times, and finally use 1:1 ethanol, 1:1NHO 3 ultrasonic cleaning.
  • the three-electrode system consists of platinum electrode as counter electrode, Ag/AgCl electrode as reference electrode, and glassy carbon electrode as working electrode.
  • the 3mm diameter glassy carbon electrode was polished to the mirror surface with a certain particle size Al 2 O 3 slurry on the polishing cloth. After each polishing, the surface dirt was washed off, and then moved into the ultrasonic water bath for cleaning, each time for 1 min. , Repeat three times, and finally use 1:1 ethanol, 1:1NHO 3 ultrasonic cleaning.
  • the electrode Immerse the electrode in the ABTS deposition stock solution (the ABTS deposition stock solution contains 2.5 mM/L FeCl 3 , 2.5 /L mM K 3 Fe(CN) 6 , 200 mM/L HCl and 1 mM/L ABTS, only for use on the day), at 50 mV
  • the scan rate of /s performs cyclic voltammetry (CV) scans of different times in the potential range of -200 ⁇ 600mV.
  • the redox peak value in the interface increases with the number of scan circles once and finally closes to overlap, indicating that ABTS has been deposited on the electrode.
  • the newly prepared electrode working surface was immersed in an aqueous solution containing 25% glutaraldehyde, taken out after 1 hour, and thoroughly cleaned with deionized water to introduce a certain amount of active aldehyde groups into the chitosan modified layer on the electrode surface.
  • an unmodified immobilized NAD + electrode is prepared, and the other steps are the same as the above process. Add 1 mg/mL free NAD + to a 10 mmol/L standard malic acid solution, and use the free NAD + electrode to detect the response current of the standard malic acid solution, and compare with the above-mentioned immobilized NAD + electrode detection results.
  • the electrode may occur at about 0.2V + response signal, but the signal strength is low.
  • the working signal of the immobilized NAD + electrode prepared by this patent is significantly enhanced, and the immobilized NAD + electrode is significantly better than the free NAD + electrode, indicating that the electrode preparation technology is the preparation of dehydrogenase electrodes and Ideal method for dehydrogenase biosensors.
  • the three-electrode system consists of platinum electrode as counter electrode, Ag/AgCl electrode as reference electrode, and glassy carbon electrode as working electrode.
  • the 3mm diameter glassy carbon electrode was polished to the mirror surface with a certain particle size Al 2 O 3 slurry on the polishing cloth. After each polishing, the surface dirt was washed off, and then moved into the ultrasonic water bath for cleaning, each time for 1 min. , Repeat three times, and finally use 1:1 ethanol, 1:1NHO 3 ultrasonic cleaning.
  • the three-electrode system consists of platinum electrode as counter electrode, Ag/AgCl electrode as reference electrode, and glassy carbon electrode as working electrode.
  • the 3mm diameter glassy carbon electrode was polished to the mirror surface with a certain particle size Al 2 O 3 slurry on the polishing cloth. After each polishing, the surface dirt was washed off, and then moved into the ultrasonic water bath for cleaning, each time for 1 min. , Repeat three times, and finally use 1:1 ethanol, 1:1NHO 3 ultrasonic cleaning.
  • the electrode Immerse the electrode in the ABTS deposition stock solution (the ABTS deposition stock solution contains 2.5 mM/L FeCl 3 , 2.5 mM/L K 3 Fe(CN) 6 , 200 mM/L HCl and 1 mM/LABTS, only for use on the day), at 50 mV/
  • the scan rate of s is to perform cyclic voltammetry (CV) scans of different times in the potential range of -200 ⁇ 600mV.
  • the redox peak value in the interface increases with the number of scan circles once and finally closes to overlap, indicating that ABTS has been deposited on the electrode.
  • Fully clean the working electrode and the working surface of the counter electrode with deionized water drip the agarose-NAD + compound on the working surface, and dry it again at room temperature.
  • Immerse the agarose-NAD + complex on the electrode surface in an aqueous solution containing 25% glutaraldehyde take it out after 1 hour, and rinse it with deionized water to introduce a certain amount of active aldehyde groups into the agarose modification layer on the electrode surface .
  • the agarose-NAD + complex was immersed in a malate dehydrogenase solution (240 U/mL), taken out after 1 hour, and washed sufficiently to remove the enzyme molecules that were not firmly bound.
  • a malate dehydrogenase solution 240 U/mL
  • a 10mmol/L standard malic acid solution was prepared, and the prepared malate dehydrogenase electrode was used to measure the response current intensity of the standard malate.
  • the three-electrode system consists of platinum electrode as counter electrode, Ag/AgCl electrode as reference electrode, and glassy carbon electrode as working electrode.
  • the 3mm diameter glassy carbon electrode was polished to the mirror surface with a certain particle size Al 2 O 3 slurry on the polishing cloth. After each polishing, the surface dirt was washed off, and then moved into the ultrasonic water bath for cleaning, each time for 1 min. , Repeat three times, and finally use 1:1 ethanol, 1:1NHO 3 ultrasonic cleaning.
  • the electrode Immerse the electrode in the ABTS deposition stock solution (the ABTS deposition stock solution contains 2.5 mM/L FeCl 3 , 2.5 mM/L K 3 Fe(CN) 6 200 mM/L HCl and 1 mM/L ABTS, only for use on the day), at 50 mV/
  • the scanning rate of s is in the potential range of -200 ⁇ 600mV for different times of cyclic voltammetry (CV) scanning. At this time, it can be seen that the redox peak value in the interface increases with the number of scan circles once and finally closes to overlap, indicating that ABTS has been deposited on the electrode.
  • the cyclic voltammetry curve shows ( Figure 18) that the electrical signal of other types of chitosan (high viscosity) modified electrode (1) is significantly lower than that of standard chitosan (medium viscosity) modified electrode (2), indicating that the shell
  • the type of glycan plays an important role in the electrode modification process.

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Abstract

一种乳酸脱氢酶电极及其制备方法和应用，通过化学手段将辅酶因子小分子NAD+非活性部位进行化学修饰，将化学修饰后的NAD+与壳聚糖载体共价连接得到NAD+-壳聚糖复合物，再在基底电极表面修饰碳纳米管，利用碳纳米管作为基底材料，用于NADH的检测；在电极上电沉积ABTS，使用ABTS作为电子媒介体实现NAD+原位再生；滴加NAD+-壳聚糖复合物，实现NAD+在电极表面的固定；通过戊二醛交联作用将乳酸脱氢酶酶连接到壳聚糖载体上，得到乳酸脱氢酶电极；NAD+的固定化与再生方法可与不同种类脱氢酶结合，制备多种脱氢酶电极/生物传感器，在生物传感器制备领域具有广泛的实际应用价值。

Description

一种乳酸脱氢酶电极及其制备方法和应用 技术领域
本发明属于检测分析技术领域，具体涉及一种乳酸脱氢酶电极及其制备方法和应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
氧化还原酶类是自然界品种最丰富的一类酶，在酶的六大类型中，30％～35％为氧化还原酶。氧化酶依赖性生物传感器是成熟的发展产物，但也存在一些缺点，如氧化酶在作用过程中需要消耗氧气，易受O 2的干扰；氧化酶反应产物多为H 2O 2，H 2O 2积累会对传感器检测造成影响。脱氢酶在自然界中更为丰富，现报道的自然界脱氢酶已近400种之多。脱氢酶生物传感器依托辅酶完成反应，辅酶可直接参与反应，不受O 2干扰，反应更灵敏、快速。因此，脱氢酶生物传感器成为研究热点，在医药检测、食品发酵控制等方面发挥重要作用。M.Gamellaa等人通过制备苹果酸脱氢酶电化学生物传感器监测葡萄酒酿造过程中的苹果酸-乳酸发酵过程。Vinay Narwal等用通过制备乳酸脱氢酶纳米颗粒修饰的金电极测定乳酸盐。梁云玉等人研制了谷氨酸脱氢酶伏安传感器，用于敏感的铵离子测定。
大部分脱氢酶催化作用的发挥需要烟酰型辅酶[NAD +，NADH]的参与，它在酶促反应中与酶结合，并作为氧化剂或还原剂直接参与反应。烟酰型辅酶中，NAD +为氧化态，NADH为还原态，氧化态辅酶和还原态辅酶通过加氢或脱氢的氧化还原反应相互转化。NAD +在执行电子传递功能时不被酶分子包裹于内部。因此，传统的脱氢酶电极需要通过在反应体系中添加游离NAD +实现脱氢酶与底物的反应。但是游离NAD +无法重复使用，每次测定时需要重复添加游离NAD +。NAD +的价格昂贵，通常比酶促反应所得产物要贵得多，其价格需要大约＄100/g。加入游离NAD +还存在耗时长、操作复杂等缺点，不利于脱氢酶电极的方便使用和推广。对辅酶NAD +进行固定化并再生循环使用是很有必要的。辅酶NAD +在电极表面的固定和有效再生技术是构建脱氢酶电极/传感器的两个关键技术。脱氢酶电极NAD +只有几百个分子量，传统方法很难对其进行固定，要将其包埋在半透膜中比较困难，若将辅酶与高分子载体结合，使其高分子化可以解决这一难题。实现脱氢酶电极循环使用的另一个关键问题是NAD +的再生。然而，发明人发现，NADH的氧化过电位较高，电子传递速率过慢、动力学特征不理想，会在电化学氧化过程中生成二聚体沉积在电极表面使电极钝化，从而使NADH的直接测定和NAD +的再生变得十分困难，基于电子媒介体的氧化还原再生NAD +是一种有效的再生方法，可以降低NADH氧化电位，提高电极表面电子传递速率，实现脱氢酶电极使用过程中的NAD +再生。
发明内容
针对现有技术的不足，本发明提供一种辅酶因子复合物、酶电极、酶传感器及其制备方法和应用。本发明首先通过化学手段将辅酶因子小分子NAD +非活性部位进行化学修饰；通过化学手段将化学修饰后的NAD +与壳聚糖载体共价连接，得到NAD +-壳聚糖复合物(辅酶因子复合物)。在此基础上制备酶电极，步骤如下：在基底电极表面修饰碳纳米管，利用碳纳米管作为基底材料，用于NADH的检测；在电极上电沉积ABTS，使用ABTS作为电子媒介体实现NAD +原位再生；滴加制备的NAD +-壳聚糖复合物，实现小分子NAD +的固定；通过戊二醛交联作用将脱氢酶连接到壳聚糖载体上，得到脱氢酶电极。利用该NAD +的固定化与再生方法分别与不同种类的脱氢酶结合，可以制备多种脱氢酶电极/生物传感器，实现苹果酸、葡萄糖、乳酸等多种底物的检测。因此，本发明中的固定化与原位再生NAD +的技术方法以及电极的制备技术可以广泛应用于脱氢酶电极/传感器的制备过程中，实现脱氢酶电极循环使用，为构建脱氢酶电极/生物传感提供了有效的技术途径。
为实现上述技术目的，本发明采用的技术方案如下：
本发明的第一个方面，提供一种辅酶因子复合物，所述辅酶因子复合物由辅酶因子和负载材料复合得到。
需要说明的是，所述复合是指辅酶因子与载体彼此附着、结合、整合或链接。因此，它们不是物理上分开的组分，而相反可以作为单一组分(共价或离子键合的复合物)复合在一起。
所述辅酶因子可以是天然辅酶或人工辅酶。“辅酶”或“氧化还原辅因子”是指可以充当酶促转移的氧化还原当量(例如氢负离子(H -))的受体的分子，所述酶促转移的氧化还原当量从底物(例如，目标分析物)到酶到辅酶转移。如本发明中所用的，“氧化还原当量”涉及氧化还原化学中常用的概念，其为本领域技术人员众所周知。
特别地，它涉及从辅酶依赖性酶(即，目标分析物)的底物转移到辅酶的电子或从辅酶转移到电极或指示试剂的电子。辅酶的实例包括，但不限于，NAD、NADP、PQQ、硫代-NAD、硫代-NADP等。
在一些情况下，辅酶是人工辅酶。人工辅酶的实例包括，但不限于，人工NAD(P)/NAD(P)H化合物，其是天然NAD/NADH或天然NADP/NADPH的化学衍生物。
具体的，所述人工辅酶可通过将天然辅酶的非活性部位进行化学修饰，从而在不影响辅酶因子活性功能发挥的情况下，使辅酶携带有用于固定的化学基团，进而实现辅酶的固定。
在一些情况下，可对NAD腺嘌呤上氨基进行修饰，从而获得人工辅酶NAD +化合物。
所述负载材料可以是高分子载体，负载材料的实例包括，但不限于，壳聚糖、琼脂糖、海藻酸钠、聚乙 二醇等；优选为壳聚糖，壳聚糖是一种具有游离氨基的高分子水溶性多糖，成膜性好，可作为辅酶因子的载体；更优选的，所述壳聚糖为中粘度壳聚糖(200-400mPa.s)。
在一些情况下，本发明的辅酶因子复合物可以是NAD +-壳聚糖复合物，所述NAD +-壳聚糖复合物通过对NAD +腺嘌呤上的氨基进行修饰得到人工NAD +辅酶，然后与壳聚糖共价连接制备得到，从而实际实现了NAD +的固定化。
具体的，所述NAD +-壳聚糖复合物的制备方法包括：
1)向NAD +水溶液中加入碘乙酸，加热反应得n1-羧甲基-NAD +；
2)在n1-羧甲基-NAD +水溶液中加入硫代硫酸钠溶液，调节pH至碱性，加热反应得到n1-羧甲基-NADH，同时在强碱条件下发生Dimroth重排得到n6-羧甲基-NADH；
3)加入甲醛加热反应，得c6-羧甲基NAD +。
4)调整反应体系的pH为中性后加入N-羟基琥珀酰亚胺(NHS)和(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)(EDC)，加入壳聚糖溶液，加热反应得壳聚糖-NAD +复合物。
在一些情况下，所述步骤1)中，加热反应优选为水浴加热，水浴反应条件为在60～80℃条件下反应0.5～3h，优选为70℃水浴反应1h；
在一些情况下，所述步骤2)中，pH调节至强碱性，优选为10-11，加热反应优选为水浴加热，水浴反应条件为在60～80℃条件下反应0.5～3h，优选为70℃水浴反应1h；
在一些情况下，所述步骤3)中，加热反应优选为水浴加热，水浴反应条件为在60～80℃条件下反应0.5～3h，优选为70℃水浴反应1h；
在一些情况下，所述步骤4)中，加热反应优选为水浴加热，水浴反应条件为在60～80℃条件下反应0.5～3h，优选为70℃水浴反应1h。
本发明的第二个方面，提供上述辅酶因子复合物在酶电极和/或制备酶电极中的应用。
本发明的第三个方面，提供一种酶电极，所述酶电极包括：
基底电极，以及由基底电极负载的基底材料。
所述基底电极包括，但不限于：玻碳(GCE)电极、金电极、石墨电极、碳糊电极，优选为GCE。GCE具有良好的机械稳定性、光稳定性和高导电性。
所述基底材料为碳材料，所述碳材料包括，但不限于：活性炭、石墨烯、纳米碳纤维、纳米碳球、玻璃碳、碳气凝胶、碳纳米管(CNTs)，优选碳纳米管，所述碳纳米管具有大的比表面积和较小内阻的多孔，能够显著提高化学修饰电极的分析性能，同时具有良好的导电性和化学稳定性。
所述碳纳米管既包括多壁、单壁碳纳米管，同时也包括氨基化、羧基化等修饰后的功能化碳纳米管。
在一些情况下，所述酶电极包括：GCE电极，以及负载在GCE电极上的CNTs。其中，所述负载可采用滴涂方式进行。
在一些情况下，所述酶电极包括：
基底电极，所述基底电极上负载基底材料，所述基底材料表面沉积有介质，所述介质表面包覆辅酶因子复合物和酶。
所述基底电极包括，但不限于：玻碳(GCE)电极、金电极、石墨电极、碳糊电极，优选为GCE。GCE具有良好的机械稳定性、光稳定性和高导电性。
所述基底材料为碳材料，所述碳材料包括，但不限于：活性炭、石墨烯、纳米碳纤维、纳米碳球、玻璃碳、碳气凝胶、碳纳米管(CNTs)，优选碳纳米管，所述碳纳米管具有大的比表面积和较小内阻的多孔，能够显著提高化学修饰电极的分析性能，同时具有良好的导电性和化学稳定性。
所述碳纳米管既包括多壁、单壁碳纳米管，同时也包括氨基化、羧基化等修饰后的功能化碳纳米管。
本发明中，“介质”是指增加通过与分析物反应获得的还原辅酶的反应性并将电子转移至电极系统或合适的光学指示剂/光学指示剂系统的化合物。
介质可以是任何化学物质(通常是电化学活性的)，其可以参与涉及分析物、辅酶依赖性酶、辅酶及其反应产物的反应方案，以产生可检测的电化学活性反应产物。一般，介质在反应中的参与涉及在与分析物、辅酶依赖性酶、辅酶或作为这些之一的反应产物的物类(例如，辅酶反应为不同的氧化态)中的任何一种相互作用时其氧化态的变化。介质也可以在其氧化形式中稳定，可任选地表现出可逆的氧化还原电化学，可表现出在水溶液中良好的溶解性，并且可快速反应以产生电化学活性反应产物。
所述介质的实例包括，但不限于，2,2'-联氮双(3-乙基苯并噻唑啉-6-磺酸)二铵盐(ABTS)、偶氮化合物或偶氮前体、苯醌、普鲁士蓝、亚硝基苯胺或基于亚硝基苯胺的前体、噻嗪或噻嗪衍生物、过渡金属络合物如铁氰化钾、钌络合物如己胺氯化钌、锇衍生物、醌或醌衍生物、吩嗪或基于吩嗪的前体、和吩嗪衍生物和氯化六氨合钌的组合，以及它们的衍生物。
在一些情况下，当辅酶是NAD/NADH时，介质可以是2,2'-联氮双(3-乙基苯并噻唑啉-6-磺酸)二铵盐(ABTS)。在本发明中，将碳纳米管与ABTS +修饰在GCE电极表面，碳纳米管可以实现对NADH的催化氧化， ABTS +可以增加电子传递。同时ABTS +与NADH发生氧化还原，NADH中吡啶环的4位与ABTS +自由基阳离子发生反应，ABTS +得到氢离子转变为ABTS，NADH被氧化为NAD +，实现NAD +原位再生。经过上述过程后，ABTS失去电子，在电极正极产生ABTS +，进入下一个循环。
本发明中，所述“酶”特指辅酶依赖性酶，“辅酶依赖性酶”是指需要称为辅酶的有机或无机辅因子用于催化活性的酶。
在一些情况下，辅酶依赖性酶可以是脱氢酶。如本发明中所用的，“脱氢酶”是指能够通过将作为氧化还原当量(redox equivalent)的氢负离子(H -)转移至受体分子来催化底物氧化的蛋白质或多肽。脱氢酶的实例包括，但不限于，葡萄糖脱氢酶、乙醇脱氢酶、甘油脱氢酶、乳酸脱氢酶、L-氨基酸脱氢酶、苹果酸脱氢酶或山梨糖醇脱氢酶等，尤其是NAD(P)/NAD(P)H-依赖性脱氢酶。
本发明的第四个方面，提供上述酶电极的制备方法，所述酶电极制备方法没有特别的限制，但酶电极可以通过被施用在基底电极的一个表面上或通过使用以下方法以膜的形式被涂覆来形成：例如滴涂、电沉积、溅镀、电子束、热沉积、旋涂、丝网印刷、喷墨印刷、刮刀或凹版印刷法。
在一些情况下，所述制备方法包括：
1)向基底电极表面滴涂基底材料；
2)采用电化学沉积法将介质沉积至步骤1)制备得到的负载有基底材料的基底电极上；
3)向步骤2)制备得到的材料上滴涂辅酶因子复合物；
4)向步骤3)制备得到的材料上负载酶。
步骤1)中，
在一些情况下，所述基底电极包括，但不限于：玻碳(GCE)电极、金电极、石墨电极、碳糊电极，优选为GCE。GCE具有良好的机械稳定性、光稳定性和高导电性。
基底材料为碳材料，所述碳材料包括，但不限于：活性炭、石墨烯、纳米碳纤维、纳米碳球、玻璃碳、碳气凝胶、碳纳米管(CNTs)，优选碳纳米管，所述碳纳米管具有大的比表面积和较小内阻的多孔，能够显著提高化学修饰电极的分析性能，同时具有良好的导电性和化学稳定性。
所述碳纳米管既包括多壁、单壁碳纳米管，同时也包括氨基化、羧基化等修饰后的功能化碳纳米管。
步骤2)中，
“介质”是指增加通过与分析物反应获得的还原辅酶的反应性并将电子转移至电极系统或合适的光学指示剂/光学指示剂系统的化合物。
介质可以是任何化学物质(通常是电化学活性的)，其可以参与涉及分析物、辅酶依赖性酶、辅酶及其反应产物的反应方案，以产生可检测的电化学活性反应产物。一般，介质在反应中的参与涉及在与分析物、辅酶依赖性酶、辅酶或作为这些之一的反应产物的物类(例如，辅酶反应为不同的氧化态)中的任何一种相互作用时其氧化态的变化(例如，还原)。多种介质表现出合适的电化学行为。介质也可以在其氧化形式中稳定，可任选地表现出可逆的氧化还原电化学，可表现出在水溶液中良好的溶解性，并且可快速反应以产生电化学活性反应产物。
所述介质的实例包括，但不限于，ABTS、偶氮化合物或偶氮前体、苯醌、普鲁士蓝、亚硝基苯胺或基于亚硝基苯胺的前体、噻嗪或噻嗪衍生物、过渡金属络合物如铁氰化钾、钌络合物如己胺氯化钌、锇衍生物、醌或醌衍生物、吩嗪或基于吩嗪的前体、和吩嗪衍生物和氯化六氨合钌的组合，以及它们的衍生物。
在一些情况下，当辅酶因子是NAD/NADH时，介质可以是2,2'-联氮双(3-乙基苯并噻唑啉-6-磺酸)二铵盐(ABTS)。
步骤3)中，
在一些情况下，辅酶因子复合物，所述辅酶因子复合物由辅酶因子和负载材料复合得到。
需要说明的是，所述复合是指辅酶因子与载体彼此附着、结合、整合或链接。因此，它们不是物理上分开的组分，而相反可以作为单一组分(共价或离子键合的复合物)复合在一起。
所述辅酶因子可以是天然辅酶或人工辅酶。如本发明中所用的，“辅酶”或“氧化还原辅因子”是指可以充当酶促转移的氧化还原当量(例如氢负离子(H -))的受体的分子，所述酶促转移的氧化还原当量从底物(例如，目标分析物)到酶到辅酶转移。“氧化还原当量”涉及氧化还原化学中常用的概念，其为本领域技术人员众所周知。
特别地，它涉及从辅酶依赖性酶(即，目标分析物)的底物转移到辅酶的电子或从辅酶转移到电极或指示试剂的电子。辅酶的实例包括，但不限于，NAD、NADP、PQQ、硫代-NAD、硫代-NADP等。
在一些情况下，辅酶是人工辅酶。人工辅酶的实例包括，但不限于，人工NAD(P)/NAD(P)H化合物，其是天然NAD/NADH或天然NADP/NADPH的化学衍生物。
具体的，所述人工辅酶可通过将天然辅酶的非活性部位进行化学修饰，从而在不影响辅酶因子活性功能 发挥的情况下，使辅酶携带有用于固定的化学基团，进而实现辅酶的固定。
在一些情况下，可对NAD腺嘌呤上氨基进行修饰，从而获得人工辅酶NAD化合物。
在一些情况下，所述负载材料可以是高分子载体，负载材料的实例包括，但不限于，壳聚糖、琼脂糖、海藻酸钠、聚乙二醇等；优选为壳聚糖，壳聚糖是一种具有游离氨基的高分子水溶性多糖，成膜性好，可作为辅酶因子的载体；更优选的，所述壳聚糖为中粘度壳聚糖(200-400mPa.s)。
在一些情况下，本发明的辅酶因子复合物可以是NAD +-壳聚糖复合物，所述NAD +-壳聚糖复合物，所述NAD +-壳聚糖复合物通过对NAD +腺嘌呤上的氨基进行修饰得到人工NAD +辅酶，然后与壳聚糖共价连接制备得到，从而实际实现了NAD +的固定化。
步骤4)中，
在一些情况下，所述“酶”特指辅酶依赖性酶，“辅酶依赖性酶”是指需要称为辅酶的有机或无机辅因子用于催化活性的酶。
在一些情况下，辅酶依赖性酶可以是脱氢酶。如本发明中所用的，“脱氢酶”是指能够通过将作为氧化还原当量(redox equivalent)的氢负离子(H -)转移至受体分子来催化底物氧化的蛋白质或多肽。脱氢酶的实例包括，但不限于，葡萄糖脱氢酶、乙醇脱氢酶、甘油脱氢酶、乳酸脱氢酶、L-氨基酸脱氢酶、苹果酸脱氢酶或山梨糖醇脱氢酶等，尤其是NAD(P)/NAD(P)H-依赖性脱氢酶。
需要说明的是，步骤4)中，将酶负载至步骤3)制备得到的材料上可采用戊二醛对酶的交联作用得以实现。具体的，当步骤3)中辅酶因子复合物的负载材料为壳聚糖时，通过戊二醛作用，壳聚糖的游离氨基与戊二醛上的一个游离醛共价连接合成席夫碱结构，戊二醛的另一个游离醛与酶相连，实现酶的固定化。
本发明的第五个方面，提供一种辅酶因子的再生方法，所述再生方法包括：
1)向基底电极表面滴涂基底材料；
2)采用电化学沉积法将介质沉积至步骤1)制备得到的负载有基底材料的基底电极上。
步骤1)中，
在一些情况下，所述基底电极包括，但不限于：玻碳(GCE)电极、金电极、石墨电极、碳糊电极，优选为GCE。GCE具有良好的机械稳定性、光稳定性和高导电性。
所述基底材料为碳材料，所述碳材料包括，但不限于：活性炭、石墨烯、纳米碳纤维、纳米碳球、玻璃碳、碳气凝胶、碳纳米管(CNTs)，优选碳纳米管，所述碳纳米管具有大的比表面积和较小内阻的多孔，能够显著提高化学修饰电极的分析性能，同时具有良好的导电性和化学稳定性。
所述碳纳米管既包括多壁、单壁碳纳米管，同时也包括氨基化、羧基化等修饰后的功能化碳纳米管。
步骤2)中，
“介质”是指增加通过与分析物反应获得的还原辅酶的反应性并将电子转移至电极系统或合适的光学指示剂/光学指示剂系统的化合物。
介质可以是任何化学物质(通常是电化学活性的)，其可以参与涉及分析物、辅酶依赖性酶、辅酶及其反应产物的反应方案，以产生可检测的电化学活性反应产物。一般，介质在反应中的参与涉及在与分析物、辅酶依赖性酶、辅酶或作为这些之一的反应产物的物类(例如，辅酶反应为不同的氧化态)中的任何一种相互作用时其氧化态的变化(例如，还原)。多种介质表现出合适的电化学行为。介质也可以在其氧化形式中稳定，可任选地表现出可逆的氧化还原电化学，可表现出在水溶液中良好的溶解性，并且可快速反应以产生电化学活性反应产物。
所述介质的实例包括，但不限于，ABTS、偶氮化合物或偶氮前体、苯醌、普鲁士蓝、亚硝基苯胺或基于亚硝基苯胺的前体、噻嗪或噻嗪衍生物、过渡金属络合物如铁氰化钾、钌络合物如己胺氯化钌、锇衍生物、醌或醌衍生物、吩嗪或基于吩嗪的前体、和吩嗪衍生物和氯化六氨合钌的组合，以及它们的衍生物。
在一些情况下，当辅酶因子是NAD时，介质可以是2,2'-联氮双(3-乙基苯并噻唑啉-6-磺酸)二铵盐(ABTS)。在本发明中，将碳纳米管与ABTS +修饰在GCE电极表面，碳纳米管可以实现对NADH的催化氧化，ABTS +可以增加电子传递。同时ABTS +与NADH发生氧化还原，NADH中吡啶环的4位与ABTS +自由基阳离子发生反应，ABTS +得到氢离子转变为ABTS，NADH被氧化为NAD +，实现NAD +原位再生。经过上述过程后，ABTS失去电子，在电极正极产生ABTS +，进入下一个循环。
本发明的第六个方面，提供上述辅酶因子的再生方法在酶电极和/或酶传感器中的应用。
本发明的第七个方面，提供上述辅酶因子复合物和/或酶电极在制备酶传感器中的应用。
本发明的第八个方面，提供一种酶传感器，所述酶传感器包括至少两个个电极，所述电极至少包括上述辅酶因子复合物和/或上述酶电极。本发明的酶传感器具有较高的检测灵敏度、较高的检测重复性和长期贮存稳定性。
在一些情况下，上述酶传感器中，所述酶传感器包括两个或三个电极组成，相应的，所述酶传感器为双电极或三电极酶传感器。
在一些情况下，由两个电极组成的传感器(即双电极酶传感器)中，所述电极为工作电极和对电极；其中，所述工作电极为上述辅酶因子复合物和/或上述酶电极。
在一些情况下，由三个电极组成的传感器(即三电极酶传感器)中，所述电极为工作电极、对电极和参比电极；其中，所述工作电极为上述辅酶因子复合物和/或上述酶电极。
在一些情况下，在三电极酶传感器中，所述对电极为铂电极；所述参比电极为Ag/AgCl电极。
本发明的第九个方面，提供一种电化学测量待测目标分析物浓度或存在的方法，所述方法包括：将上述酶电极和/或上述酶传感器与具有或怀疑具有待测目标分析物的液体样品接触，测量待测目标分析物的响应电流强度，分析目标分析物的浓度或有无。
在一些情况下，所述目标分析物包括，但不限于氨基酸、葡萄糖、乙醇、甘油、乳酸、苹果酸、丙酮酸、山梨糖醇、甘油三酯和尿酸。
本发明的第十个方面，提供一种苹果酸脱氢酶电极，所述苹果酸脱氢酶电极包括：GCE电极，所述GCE电极上负载碳纳米管，所述碳纳米管表面沉积有ABTS，所述ABTS表面负载有壳聚糖-NAD +复合物和苹果酸脱氢酶。
在一些情况下，所述苹果酸脱氢酶电极的制备方法包括：
1)向GCE电极表面滴碳纳米管；
2)采用电化学沉积法将ABTS沉积至步骤1)制备得到的负载有基底材料的基底电极上；
3)向步骤2)制备得到的材料上滴涂壳聚糖-NAD +复合物；
4)向步骤3)制备得到的材料上负载苹果酸脱氢酶。
在一些情况下，所述苹果酸脱氢酶电极的制备方法包括：
向GCE电极工作面滴涂的碳纳米管分散液(0.1％)，干燥，该电极为CNTs/GCE。
将CNTs/GCE浸入ABTS沉积储备溶液中，以50mV/s的扫描速率在-200～600mV电位范围内进行不同次数的循环伏安(CV)扫描，该电极为ABTS/CNTs/GCE。沉积在电极表面的ABTS阳离子自由基可以参与NADH氧化过程，实现NAD +再生。
其中，所述ABTS沉积储备溶液包含：2.5mmol/L FeCl 3、2.5mmol/L K 3Fe(CN) 6 200mmol/L HCl和1mmol/L ABTS。
在ABTS/CNTs/GCE表面滴加制备的NAD +-壳聚糖复合物，干燥，电极为CTS-NAD +/ABTS/CNTs/GCE。
其中，NAD +-壳聚糖复合物的制备过程包括：
①取5mL NAD +水溶液(1mg/mL)，加入1mL碘乙酸(1mg/mL)，70℃水浴反应1h，得到n1-羧甲基-NAD +。
②在上述NAD +水溶液中加入1mL硫代硫酸钠溶液(1.3mmol/L)，调pH＝11，70℃水浴反应1h，得到n1-羧甲基-NADH，同时强碱条件下发生Dimroth重排得到n6-羧甲基NADH。
③加入1mL甲醛70℃水浴反应1h，得到c6-羧甲基NAD +。
④调整反应体系的pH为中性后加入10mg NHS和10mg EDC，加入10mL壳聚糖溶液(0.1％)，70℃水浴反应1h，得到壳聚糖-NAD +复合物。
将CTS-NAD +/ABTS/CNTs/GCE工作面浸没于含25％的戊二醛水溶液中，1h后取出，以去离子水充分清洗。随后将电极工作面浸入以苹果酸脱氢酶溶液(240U/mL)内，1h后取出，充分清洗以取出未牢固结合的酶分子，电极为MDH/CTS-NAD +/ABTS/CNTs/GCE。
在一些情况下，苹果酸脱氢酶传感器包括：工作电极、对电极和参比电极，其中，上述苹果酸脱氢酶电极作为工作电极；对电极为铂电极；参比电极为Ag/AgCl电极。
本发明的第十一个方面，提供一种葡萄糖脱氢酶电极，所述葡萄糖脱氢酶电极包括：GCE电极，所述GCE电极上负载碳纳米管，所述碳纳米管表面沉积有ABTS，所述ABTS表面负载有壳聚糖-NAD +复合物和葡萄糖脱氢酶。
在一些情况下，所述葡萄糖脱氢酶电极的制备方法包括：
1)向GCE电极表面滴碳纳米管；
2)采用电化学沉积法将ABTS沉积至步骤1)制备得到的负载有基底材料的基底电极上；
3)向步骤2)制备得到的材料上滴涂壳聚糖-NAD +复合物；
4)向步骤3)制备得到的材料上负载葡萄糖脱氢酶。
在一些情况下，所述葡萄糖脱氢酶电极的制备方法包括：
向GCE电极工作面滴涂的碳纳米管分散液(0.1％)，干燥，该电极为CNTs/GCE。
将CNTs/GCE浸入ABTS沉积储备溶液中，以50mV/s的扫描速率在-200～600mV电位范围内进行不同次数的循环伏安(CV)扫描，该电极为ABTS/CNTs/GCE。沉积在电极表面的ABTS阳离子自由基可以参与NADH氧化过程，实现NAD +再生。
其中，所述ABTS沉积储备溶液包含：2.5mmol/L FeCl 3、2.5mmol/L K 3Fe(CN) 6 200mmol/L HCl和1mmol/L ABTS。
在ABTS/CNTs/GCE表面滴加制备的NAD +-壳聚糖复合物，干燥，电极为CTS-NAD +/ABTS/CNTs/GCE。
其中，NAD +-壳聚糖复合物的制备过程包括：
①取5mL NAD +水溶液(1mg/mL)，加入1mL碘乙酸(1mg/mL)，70℃水浴反应1h，得到n1-羧甲基-NAD +。
②在上述NAD +水溶液中加入1mL硫代硫酸钠溶液(1.3mmol/L)，调pH＝11，70℃水浴反应1h，得到n1-羧甲基-NADH，同时强碱条件下发生Dimroth重排得到n6-羧甲基NADH。
③加入1mL甲醛70℃水浴反应1h，得到c6-羧甲基NAD +。
④调整反应体系的pH为中性后加入10mg NHS和10mg EDC，加入10mL壳聚糖溶液(0.1％)，70℃水浴反应1h，得到壳聚糖-NAD +复合物。
将CTS-NAD +/ABTS/CNTs/GCE工作面浸没于含25％的戊二醛水溶液中，1h后取出，以去离子水充分清洗。随后将电极工作面浸入以葡萄糖脱氢酶溶液(240U/mL)内，1h后取出，充分清洗以取出未牢固结合的酶分子，电极为MDH/CTS-NAD +/ABTS/CNTs/GCE。
在一些情况下，葡萄糖脱氢酶传感器包括：工作电极、对电极和参比电极，其中，上述葡萄糖脱氢酶电极作为工作电极；对电极为铂电极；参比电极为Ag/AgCl电极。
本发明的第十二个方面，提供一种乳酸脱氢酶电极，所述乳酸脱氢酶电极包括：GCE电极，所述GCE电极上负载碳纳米管，所述碳纳米管表面沉积有ABTS，所述ABTS表面包括壳聚糖-NAD +复合物和乳酸脱氢酶。
在一些情况下，所述乳酸脱氢酶电极的制备方法包括：
1)向GCE电极表面滴碳纳米管；
2)采用电化学沉积法将ABTS沉积至步骤1)制备得到的负载有基底材料的基底电极上；
3)向步骤2)制备得到的材料上滴涂壳聚糖-NAD +复合物；
4)向步骤3)制备得到的材料上负载乳酸脱氢酶。
在一些情况下，所述乳酸脱氢酶电极的制备方法包括：
向GCE电极工作面滴涂的碳纳米管分散液(0.1％)，干燥，该电极为CNTs/GCE。
将CNTs/GCE浸入ABTS沉积储备溶液中，以50mV/s的扫描速率在-200～600mV电位范围内进行不同次数的循环伏安(CV)扫描，该电极为ABTS/CNTs/GCE。沉积在电极表面的ABTS阳离子自由基可以参与NADH氧化过程，实现NAD +再生。
其中，所述ABTS沉积储备溶液包含：2.5mmol/L FeCl 3、2.5mmol/L K 3Fe(CN) 6 200mmol/L HCl和1mmol/L ABTS。
在ABTS/CNTs/GCE表面滴加制备的NAD +-壳聚糖复合物，干燥，电极为CTS-NAD +/ABTS/CNTs/GCE。
其中，NAD +-壳聚糖复合物的制备过程包括：
①取5mL NAD +水溶液(1mg/mL)，加入1mL碘乙酸(1mg/mL)，70℃水浴反应1h，得到n1-羧甲基-NAD +。
②在上述NAD +水溶液中加入1mL硫代硫酸钠溶液(1.3mmol/L)，调pH＝11，70℃水浴反应1h，得到n1-羧甲基-NADH，同时强碱条件下发生Dimroth重排得到n6-羧甲基NADH。
③加入1mL甲醛70℃水浴反应1h，得到c6-羧甲基NAD +。
④调整反应体系的pH为中性后加入10mg NHS和10mg EDC，加入10mL壳聚糖溶液(0.1％)，70℃水浴反应1h，得到壳聚糖-NAD +复合物。
将CTS-NAD +/ABTS/CNTs/GCE工作面浸没于含25％的戊二醛水溶液中，1h后取出，以去离子水充分清洗。随后将电极工作面浸入以乳酸脱氢酶溶液(240U/mL)内，1h后取出，充分清洗以取出未牢固结合的酶分子， 电极为MDH/CTS-NAD +/ABTS/CNTs/GCE。
在一些情况下，乳酸脱氢酶传感器包括：工作电极、对电极和参比电极，其中，上述乳酸脱氢酶电极作为工作电极；对电极为铂电极；参比电极为Ag/AgCl电极。
本发明的有益技术效果：
(1)本发明使用简单的NAD +修饰技术，制备了壳聚糖-NAD +复合物(辅酶因子复合物)，并用于脱氢酶电极的制备。该复合物对NAD +腺嘌呤上的氨基进行修饰，不影响NAD +活性功能的发挥，有效提高了固定化NAD +的活性。将该辅酶因子复合物滴涂于电极表面一方面可以实现NAD +在电极表面的固定化，另一方面为脱氢酶提供了电极表面的壳聚糖结合位点，因此该辅酶因子复合物在辅酶依赖型酶电极/生物传感器领域具有广泛的应用价值。
(2)本发明利用ABTS作为电子媒介体，通过电极的电子传递实现NAD +再生。通过电化学手段原位再生NAD +不仅方便、快捷，而且可以避免副产物的影响。
(3)本发明利用NAD +固定化与原位再生技术制备了可以重复使用的脱氢酶电极。并且，该电极与传统脱氢酶电极相比具有较高的检测灵敏度、较高的检测重复性和较好的贮存稳定性，因此具有良好的实际应用价值。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。
图1为本发明实施例1中制备的壳聚糖-NAD +复合物结构变化及对应紫外吸收光谱图；其中，图1(I)为NAD +在260nm处表现出最大的紫外吸收峰；图1(II)为N1-羧甲基-NAD +在250nm处表现出最大的紫外吸收峰；图1(III)为C6-羧甲基NAD +在250nm和340nm处表现出最大的紫外吸收峰；图1(IV)为壳聚糖-NAD +复合物在260nm处表现出最大的紫外吸收峰。
图2为本发明实施例2中苹果酸脱氢酶电极原理图。
图3为本发明实施例2中苹果酸脱氢酶制备过程示意图及扫描电镜图；其中，图3(I)为碳纳米管修饰电极扫描电镜图；图3(II)为沉积ABTS后的电极扫描电镜图；图3(III)为修饰壳聚糖-NAD复合物后的电极扫描电镜图；图3(IV)为苹果酸脱氢酶固定在电极上的扫描电镜图。
图4为本发明实施例2中制备的苹果酸脱氢酶电极重复性结果图。
图5为本发明实施例2中制备的苹果酸脱氢酶电极线性范围结果图。
图6为本发明实施例2中制备的苹果酸脱氢酶电极稳定性结果图。
图7为本发明实施例3中制备的葡萄糖脱氢酶电极线性范围结果图。
图8为本发明实施例3中制备的葡萄糖脱氢酶电极重复性结果图。
图9为本发明实施例3中制备的葡萄糖脱氢酶电极稳定性结果图。
图10为本发明实施例4中制备的乳酸脱氢酶电极线性范围结果图。
图11为本发明实施例4中制备的乳酸脱氢酶电极重复性结果图。
图12为本发明实施例4中制备的乳酸脱氢酶电极稳定性结果图。
图13为本发明实施例5中未修饰ABTS的脱氢酶电极检测苹果酸结果图。
图14为本发明实施例6中使用CNTs-GCE与裸电极检测NADH结果图。
图15为本发明实施例7中固定化NAD +电极对苹果酸的响应与游离NAD +比较图。
图16为本发明实施例8中使用普鲁士蓝作为电子酶介体制得的苹果酸脱氢酶电极测量苹果酸的响应电流强度图。
图17为本发明实施例9中使用琼脂糖作为负载材料制得的苹果酸脱氢酶电极测量苹果酸的响应电流强度图。
图18为本发明实施例10中使用其他种类壳聚糖(高粘度壳聚糖)与标准壳聚糖(中粘度壳聚糖)作为负载材料制得的苹果酸脱氢酶电极测量标准苹果酸的响应电流强度对比图。①壳聚糖(高粘度)溶修饰电极；②标准壳聚糖(中粘度)修饰电极。
具体实施方式
应该指出，以下详细说明都是例示性的，旨在对本发明提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本发明的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。
本发明以碳纳米管为基底材料，以ABTS作为电子媒介体，利用壳聚糖/化学修饰NAD +在碳二亚胺作用下形成的壳聚糖-NAD +复合物薄膜，以及戊二醛对脱氢酶的交联作用，制得一种可以重复使用的脱氢酶电极。壳聚糖是一种具有游离氨基的高分子水溶性多糖，成膜性好，可作为NAD +载体。经过EDC/NHS处理后，壳聚糖上的游离氨基和化学修饰NAD +上的游离羧基形成脲衍生物，完成固定化NAD +。通过戊二醛作用，壳聚糖的游离氨基与戊二醛上的一个游离醛共价连接合成席夫碱结构，戊二醛的另一个游离醛与脱氢酶相连，实现脱氢酶的固定化。
以下通过实施例对本发明做进一步解释说明，但不构成对本发明的限制。应理解这些实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1壳聚糖-NAD +复合物的制备
取NAD +水溶液5mL(1mg/mL)，加入1mL碘乙酸(1mg/mL)，70℃水浴反应1h。在上述NAD +水溶液中加入硫代硫酸钠溶液1mL(1.3mM)，用1M NaOH调pH＝11，70℃水浴反应1h。之后加入1mL甲醛70℃水浴反应1h。使用1M HCl调整反应体系的pH为中性后加入NHS(10mg)，EDC(10mg)，加入0.1％壳聚糖溶液10mL[壳聚糖为中粘度壳聚糖(200-400mPa.s)]，70℃水浴反应1h。对关键步骤进行紫外吸收全波长扫描测试。
壳聚糖-NAD +复合物制备过程中的紫外吸收波长结果如图1所示，我们主要利用NAD +与水溶性好成膜性好的壳聚糖的共价连接作用，实现NAD +固定化。本实验以NAD +为起始，NAD +在260nm处表现出最大的紫外吸收峰(图1-I)。首先在酸性环境下以碘乙酸为烷基化试剂对NAD +腺嘌呤1位氮原子烷基化，得到N1-羧甲基-NAD +。与NAD +相比，N1-羧甲基-NAD +有一个蓝移峰，在250nm处表现出最大的紫外吸收峰(图1-II)。以硫代硫酸钠为还原剂对N1-羧甲基-NAD +进行还原，得到碱性条件下更稳定的N1-羧甲基-NADH。然后在强碱条件下进行Dimroth重排得到6位碳上的氨基被修饰的C6-羧甲基NADH。甲醛在碱性条件下发生Cannizzaro反应，同时重排产物C6-羧甲基NADH被氧化为C6-羧甲基NAD +。如图1-III所示，该化合物在250nm和340nm处表现出最大的紫外吸收峰，NADH在340nm处表现出最大的紫外吸收峰，说明这步反应过程中有NAD +和NADH衍生物共同存在。然后使用EDC/NHS处理C6-羧甲基NAD +和壳聚糖。经过EDC处理后，壳聚糖上的游离氨基和C6-羧甲基NAD +上的游离羧基形成不稳定的脲衍生物，然后通过NHS处理可以增强碳二亚胺交联产物的稳定性，实现NAD +与壳聚糖载体的共价键连接，完成固定NAD +，如图1-IV所示，NAD +大分子在260nm处表现出最大的紫外吸收峰，结果表明，NAD +活性基团不受固定过程的影响。
实施例2苹果酸脱氢酶电极的制备方法
(1)制备壳聚糖-NAD +复合物
取NAD +水溶液5mL(1mg/mL)，加入1mL碘乙酸(1mg/mL)，70℃水浴反应1h。在上述NAD +水溶液中加入硫代硫酸钠溶液1mL(1.3mM)，用1M NaOH调pH＝11，70℃水浴反应1h。之后加入1mL甲醛70℃水浴反应1h。使用1M HCl调整反应体系的pH为中性后加入NHS(10mg)，EDC(10mg)，加入0.1％壳聚糖溶液10mL[壳聚糖为中粘度壳聚糖(200-400mPa.s)]，70℃水浴反应1h。
(2)制备苹果酸脱氢酶电极
所有的电化学测量都是在典型的三电极系统(CHI 760D，CH仪器)中进行的。三电极体系组成包括：铂电极作为对电极，Ag/AgCl电极作为参比电极，玻璃碳电极作为工作电极。实验时，将直径为3mm玻碳电极依次用一定粒径的Al 2O 3浆在抛光布上抛光至镜面，每次抛光后先洗去表面污物，再移入超声水浴中清洗，每次1min，重复三次，最后依次用1:1乙醇、1:1NHO 3超声清洗。彻底洗涤后，在0.20mol/L KNO 3中记录1×10 -3mol/L K 3Fe(CN) 6溶液的循环伏安曲线，以测试电极性能，扫描速度50mV/s，扫描范围0.6～－0.2V。实验室条件下所得循环伏安图中的峰电位差在80mV以下，并尽可能接近64mV，电极方可使用。
向预处理后的电极工作面滴涂5μL 0.1％的碳纳米管分散液，晾干。将电极浸入ABTS沉积储备溶液中(ABTS沉积储备溶液含2.5mM/L FeCl 3、2.5mM/L K 3Fe(CN) 6、200mM/L HCl和1mM/L ABTS，仅限当天使用)，以50mV/s的扫描速率在-200～600mV电位范围内进行不同次数的循环伏安(CV)扫描。此时可以看到界面内的氧化还原峰值随着扫描的圈数一次增大并最后接近重合，说明已有ABTS沉积在电极上。以去离子水充分清 洗工作电极和对电极工作面，向工作面滴涂10μL的壳聚糖-NAD +复合物，再次于室温下晾干后。将新制备的电极工作面浸没于含25％的戊二醛水溶液中，1h后取出，以去离子水充分清洗，使电极表面的壳聚糖修饰层中引入一定数量的活性醛基。随后将电极工作面浸入以苹果酸脱氢酶溶液(240U/mL)内，1h后取出，充分清洗以取出未牢固结合的酶分子。
苹果酸脱氢酶电极工作原理：
如图2所示，苹果酸脱氢酶可以催化苹果酸转化为草酰乙酸，生成NADH。因此，可以通过测定NADH含量来定量分析苹果酸的含量。将碳纳米管与ABTS +修饰在电极表面，碳纳米管可以实现对NADH的催化氧化，ABTS +可以增加电子传递。同时ABTS +与NADH发生氧化还原，NADH中吡啶环的4位与ABTS +自由基阳离子发生反应，ABTS +得到氢离子转变为ABTS，NADH被氧化为NAD +，实现NAD +原位再生。经过上述过程后，ABTS失去电子，在电极正极产生ABTS +，进入下一个循环。
(3)苹果酸脱氢酶电极性能检测
分别配置浓度为1、2、4、6、8、10mmol/L的标准苹果酸溶液，用制得的苹果酸脱氢酶电极测量标准苹果酸的响应电流强度，分析了电极的线性范围。分别在1、2、4、6、8、10mmol/L苹果酸溶液中重复测量电流10次，分析电极的重复性。将制得的苹果酸脱氢酶电极储存在4℃，每天在4mmol/L的苹果酸溶液测定电极响应以分析电极的稳定性。
苹果酸脱氢酶电极制备过程的扫描电镜图3，图3-I为碳纳米管修饰电极，电极表面管束分布明显。沉积ABTS后如图3-II所示，在碳纳米管上电化学沉积ABTS后，电极表面有明显的薄膜结构。如图3-III所示，修饰壳聚糖-NAD复合物后，电极表面的膜结构明显加厚。如图3-IV所示，电极表面有明显的突起，说明苹果酸脱氢酶固定在电极表面。
为了考察脱氢酶电极的重复利用性，分别在1、2、4、6、10mmol/L苹果酸溶液中重复测量电流10次，分析电极的重复性。每次测定后充分清洗电极表面，用于下一次反应。电极重复性试验结果如图4所示。当前响应值的相对标准偏差(RSD)均低于5％，平均为4.40％。这些值表明，实验制备的苹果酸脱氢酶电极具有良好的重现性。
为了考察脱氢酶电极的线性范围，分别配置浓度为1、2、4、6、8、10mmol/L的标准苹果酸溶液，用制得的苹果酸脱氢酶电极测量标准苹果酸的响应电流强度，分析了电极的线性范围。如图5所示，在1-10mmol/L苹果酸含量下，氧化还原峰电流与其浓度的相关系数为0.9836，线性拟合良好。根据3σ原则，当信噪比是3(S/N＝3)，苹果检测极限是0.0278mmol/L。
将制得的苹果酸脱氢酶电极储存在4℃，每天在4mmol/L的苹果酸溶液测定电极响应以分析电极的稳定性。如图6所示，在25天的试验过程中，电流强度在第20天之前没有出现明显的波动，表明电极在20天内保持稳定。
实施例3葡萄糖脱氢酶电极的制备方法
(1)制备葡萄糖脱氢酶电极
所有的电化学测量都是在典型的三电极系统(CHI 760D，CH仪器)中进行的。三电极体系组成包括：铂电极作为对电极，Ag/AgCl电极作为参比电极，玻璃碳电极作为工作电极。实验时，将直径为3mm玻碳电极依次用一定粒径的Al 2O 3浆在抛光布上抛光至镜面，每次抛光后先洗去表面污物，再移入超声水浴中清洗，每次1min，重复三次，最后依次用1:1乙醇、1:1NHO 3超声清洗。彻底洗涤后，在0.20mol/L KNO 3中记录1×10 -3mol/L K 3Fe(CN) 6溶液的循环伏安曲线，以测试电极性能，扫描速度50mV/s，扫描范围0.6～－0.2V。实验室条件下所得循环伏安图中的峰电位差在80mV以下，并尽可能接近64mV，电极方可使用。
向预处理后的电极工作面滴涂5μL 0.1％的碳纳米管分散液，晾干。将电极浸入ABTS沉积储备溶液中，以50mV/s的扫描速率在-200～600mV电位范围内进行不同次数的循环伏安(CV)扫描。此时可以看到界面内的氧化还原峰值随着扫描的圈数一次增大并最后接近重合，说明已有ABTS沉积在电极上。以去离子水充分清洗工作电极和对电极工作面，向工作面滴涂10μL的壳聚糖-NAD +复合物，再次于室温下晾干后。将新制备的电极工作面浸没于含25％的戊二醛水溶液中，1h后取出，以去离子水充分清洗，使电极表面的壳聚糖修饰层中引入一定数量的活性醛基。随后将电极工作面浸入以葡萄糖脱氢酶溶液内，1h后取出，充分清洗以取出未牢固结合的酶分子。
(2)葡萄糖脱氢酶电极性能检测
分别配置浓度为0.55、11、22、33、44、55mmol/L的标准葡萄糖溶液，用制得的葡萄糖脱氢酶电极测量标准葡萄糖的响应电流强度，分析了电极的线性范围。分别在1、10、20、30、40、50mmol/L葡萄糖溶液中重复测量电流10次，分析电极的重复性。将制得的葡萄糖脱氢酶电极储存在4℃，每天在30mmol/L的葡萄糖溶液测定电极响应以分析电极的稳定性。
为了考察脱氢酶电极的线性范围，分别配置浓度为0.55、11、22、33、44、55mmol/L的标准葡萄糖溶液，用制得的葡萄糖脱氢酶电极测量标准葡萄糖的响应电流强度，分析了电极的线性范围。如图7所示，在0.55-55mmol/L葡萄糖含量下，氧化还原峰电流与其浓度的相关系数为0.9954，线性拟合良好。根据3σ原则，当信噪比是3(S/N＝3)，苹果检测极限是0.00347mmol/L。
为了考察脱氢酶电极的重复利用性，分别在1、10、20、30、40、50mmol/L葡萄糖溶液中重复测量电流10次，分析电极的重复性。每次测定后充分清洗电极表面，用于下一次反应。电极重复性试验结果如图8所示。当前响应值的相对标准偏差(RSD)均低于5％，平均为3.80％。这些值表明，实验制备的葡萄糖脱氢酶电极具有良好的重现性。
将制得的葡萄糖脱氢酶电极储存在4℃，每天在30mmol/L的葡萄糖溶液测定电极响应以分析电极的稳定性。如图9所示，在20天的试验过程中，电流强度在第15天之前没有出现明显的波动，表明电极在15天内保持稳定。
实施例4乳酸脱氢酶电极的制备方法
(1)制备乳酸脱氢酶电极
所有的电化学测量都是在典型的三电极系统(CHI 760D，CH仪器)中进行的。三电极体系组成包括：铂电极作为对电极，Ag/AgCl电极作为参比电极，玻璃碳电极作为工作电极。实验时，将直径为3mm玻碳电极依次用一定粒径的Al 2O 3浆在抛光布上抛光至镜面，每次抛光后先洗去表面污物，再移入超声水浴中清洗，每次1min，重复三次，最后依次用1:1乙醇、1:1NHO 3超声清洗。彻底洗涤后，在0.20mol/L KNO 3中记录1×10 -3mol/L K 3Fe(CN) 6溶液的循环伏安曲线，以测试电极性能,扫描速度50mV/s，扫描范围0.6～－0.2V。实验室条件下所得循环伏安图中的峰电位差在80mV以下，并尽可能接近64mV，电极方可使用。
向预处理后的电极工作面滴涂5μL 0.1％的碳纳米管分散液，晾干。将电极浸入ABTS沉积储备溶液中，以50mV/s的扫描速率在-200～600mV电位范围内进行不同次数的循环伏安(CV)扫描。此时可以看到界面内的氧化还原峰值随着扫描的圈数一次增大并最后接近重合，说明已有ABTS沉积在电极上。以去离子水充分清洗工作电极和对电极工作面，向工作面滴涂10μL的壳聚糖-NAD +复合物，再次于室温下晾干后。将新制备的电极工作面浸没于含25％的戊二醛水溶液中，1h后取出，以去离子水充分清洗，使电极表面的壳聚糖修饰层中引入一定数量的活性醛基。随后将电极工作面浸入以乳酸脱氢酶溶液内，1h后取出，充分清洗以取出未牢固结合的酶分子。
(2)乳酸脱氢酶电极性能检测
分别配置浓度为2、4、6、8、10mmol/L的标准乳酸溶液，用制得的乳酸脱氢酶电极测量标准乳酸的响应电流强度，分析了电极的线性范围。分别在2、3、4、5、6、8mmol/L乳酸溶液中重复测量电流10次，分析电极的重复性。将制得的乳酸脱氢酶电极储存在4℃，每天在6mmol/L的乳酸溶液测定电极响应以分析电极的稳定性。
为了考察脱氢酶电极的线性范围，分别配置浓度为2、4、6、8、10mmol/L的标准乳酸溶液，用制得的乳酸脱氢酶电极测量标准乳酸的响应电流强度，分析了电极的线性范围。如图10所示，在2-10mmol/L乳酸含量下，氧化还原峰电流与其浓度的相关系数为0.9977，线性拟合良好。根据3σ原则,当信噪比是3(S/N＝3)，乳酸检测极限是0.0609mmol/L。
为了考察脱氢酶电极的重复利用性，分别在2、3、4、5、6、8mmol/L乳酸溶液中重复测量电流10次，分析电极的重复性。每次测定后充分清洗电极表面，用于下一次反应。电极重复性试验结果如图11所示。当前响应值的相对标准偏差(RSD)均低于5％，平均为2.14％。这些值表明，实验制备的乳酸脱氢酶电极具有良好的重现性。
将制得的乳酸脱氢酶电极储存在4℃，每天在6mmol/L的乳酸溶液测定电极响应以分析电极的稳定性。如图12所示，在28天的试验过程中，电流强度在第21天之前没有出现明显的波动，表明电极在21天内保持稳定。
实施例5苹果酸脱氢酶电极的制备过程中ABTS的作用研究(未修饰ABTS，NAD +无法再生)
(1)制备壳聚糖-NAD +复合物
取NAD +水溶液5mL(1mg/mL)，加入1mL碘乙酸(1mg/mL)，70℃水浴反应1h。在上述NAD +水溶液中加入硫代硫酸钠溶液1mL(1.3mM)，用1M NaOH调pH＝11，70℃水浴反应1h。之后加入1mL甲醛70℃水浴反应1h。使用1M HCl调整反应体系的pH为中性后加入NHS(10mg)，EDC(10mg)，加入0.1％壳聚糖溶液10mL，70℃水浴反应1h。
(2)制备苹果酸脱氢酶电极
所有的电化学测量都是在典型的三电极系统(CHI 760D，CH仪器)中进行的。三电极体系组成包括：铂电极作为对电极，Ag/AgCl电极作为参比电极，玻璃碳电极作为工作电极。实验时，将直径为3mm玻碳电极依次用一定粒径的Al 2O 3浆在抛光布上抛光至镜面，每次抛光后先洗去表面污物，再移入超声水浴中清洗，每次1min，重复三次，最后依次用1:1乙醇、1:1NHO 3超声清洗。彻底洗涤后，在0.20mol/L KNO 3中记录1×10 -3mol/L K 3Fe(CN) 6溶液的循环伏安曲线，以测试电极性能，扫描速度50mV/s，扫描范围0.6～－0.2V。实验室条件下所得循环伏安图中的峰电位差在80mV以下，并尽可能接近64mV，电极方可使用。
向预处理后的电极工作面滴涂5μL 0.1％的碳纳米管分散液，晾干。向工作面滴涂10μL的壳聚糖-NAD +复合物，再次于室温下晾干后。将新制备的电极工作面浸没于含25％的戊二醛水溶液中，1h后取出，以去离子水充分清洗，使电极表面的壳聚糖修饰层中引入一定数量的活性醛基。随后将电极工作面浸入以苹果酸脱氢酶溶液(240U/mL)内，1h后取出，充分清洗以取出未牢固结合的酶分子。配置浓度为10mmol/L的标准苹果酸溶液，用制得的苹果酸脱氢酶电极测量标准苹果酸的响应电流强度。
实验结果：
如图13循环伏安曲线所示，在苹果酸溶液中随扫描圈数的增加，电流逐渐减小。说明随反应进行，固定在电极表面的NAD +逐渐消耗，不能实现再生，因此修饰在电极表面的ABTS对NAD +的再生起关键作用。
实施例6 CNTs电极的检测NADH的研究
(1)制备苹果酸脱氢酶电极
所有的电化学测量都是在典型的三电极系统(CHI 760D，CH仪器)中进行的。三电极体系组成包括：铂电极作为对电极，Ag/AgCl电极作为参比电极，玻璃碳电极作为工作电极。实验时，将直径为3mm玻碳电极依次用一定粒径的Al 2O 3浆在抛光布上抛光至镜面，每次抛光后先洗去表面污物，再移入超声水浴中清洗，每次1min，重复三次，最后依次用1:1乙醇、1:1NHO 3超声清洗。彻底洗涤后，在0.20mol/L KNO 3中记录1×10 -3mol/L K 3Fe(CN) 6溶液的循环伏安曲线，以测试电极性能，扫描速度50mV/s，扫描范围0.6～－0.2V。实验室条件下所得循环伏安图中的峰电位差在80mV以下，并尽可能接近64mV，电极方可使用。
向预处理后的电极工作面滴涂5μL 0.1％的碳纳米管分散液，晾干。记作CNTs-GCE。
分别配置浓度为2、6、10mmol/L的NADH溶液，用制得的CNTs-GCE测量标准NADH的响应电流强度。使用裸电极检测NADH电信号，与上述CNTs电极结果进行比较。
实验结果：
使用CNTs-GCE检测NADH结果如图14所示，结果表明，随NADH浓度增加，响应电流逐渐增加，说明制备的CNTs-GCE对NADH具有很好的线性响应，并且，CNTs-GCE电极响应电流强度明显高于GCE裸电极响应电流强度，电位明显低于裸电极响应电位，说明CNTs是制备脱氢酶电极的理想基底材料。
实施例7固定化NAD +与游离NAD +比较
(1)制备壳聚糖-NAD +复合物
取NAD +水溶液5mL(1mg/mL)，加入1mL碘乙酸(1mg/mL)，70℃水浴反应1h。在上述NAD +水溶液中加入硫代硫酸钠溶液1mL(1.3mM)，用1M NaOH调pH＝11,70℃水浴反应1h。之后加入1mL甲醛70℃水浴反应1h。使用1M HCl调整反应体系的pH为中性后加入NHS(10mg)，EDC(10mg)，加入0.1％壳聚糖溶液10mL，70℃水浴反应1h。
(2)制备苹果酸脱氢酶电极
所有的电化学测量都是在典型的三电极系统(CHI 760D，CH仪器)中进行的。三电极体系组成包括：铂电极作为对电极，Ag/AgCl电极作为参比电极，玻璃碳电极作为工作电极。实验时，将直径为3mm玻碳电极依次用一定粒径的Al 2O 3浆在抛光布上抛光至镜面，每次抛光后先洗去表面污物，再移入超声水浴中清洗，每次 1min，重复三次，最后依次用1:1乙醇、1:1NHO 3超声清洗。彻底洗涤后，在0.20mol/L KNO 3中记录1×10 -3mol/L K 3Fe(CN) 6溶液的循环伏安曲线，以测试电极性能，扫描速度50mV/s，扫描范围0.6～－0.2V。实验室条件下所得循环伏安图中的峰电位差在80mV以下，并尽可能接近64mV，电极方可使用。
向预处理后的电极工作面滴涂5μL 0.1％的碳纳米管分散液，晾干。将电极浸入ABTS沉积储备溶液中(ABTS沉积储备溶液含2.5mM/L FeCl 3、2.5/LmM K 3Fe(CN) 6、200mM/L HCl和1mM/L ABTS，仅限当天使用)，以50mV/s的扫描速率在-200～600mV电位范围内进行不同次数的循环伏安(CV)扫描。此时可以看到界面内的氧化还原峰值随着扫描的圈数一次增大并最后接近重合，说明已有ABTS沉积在电极上。以去离子水充分清洗工作电极和对电极工作面，向工作面滴涂10μL的壳聚糖-NAD +复合物，再次于室温下晾干后。将新制备的电极工作面浸没于含25％的戊二醛水溶液中，1h后取出，以去离子水充分清洗，使电极表面的壳聚糖修饰层中引入一定数量的活性醛基。随后将电极工作面浸入以苹果酸脱氢酶溶液(240U/mL)内，1h后取出，充分清洗以取出未牢固结合的酶分子。配置10mmol/L的标准苹果酸溶液，用制得的苹果酸脱氢酶电极测量标准苹果酸的响应电流强度。
另外制备一支未修饰固定化NAD +的电极，其他步骤与上述过程相同。在10mmol/L的标准苹果酸溶液中添加1mg/mL游离NAD +，并用该游离NAD +电极检测标准苹果酸溶液的响应电流，与上述固定化NAD +电极检测结果进行比较。
实验结果如图15所示，使用未固定化NAD +的电极在游离NAD +环境中检测苹果酸时，该电极可在0.2V左右出现响应信号，但信号强度较低。与游离NAD +电极的电信号相比，本专利制备的固定化NAD +电极工作信号明显增强，固定化NAD +电极明显优于游离NAD +电极，说明该电极制备技术是制备脱氢酶电极及脱氢酶生物传感器的理想方法。
实施例8普鲁士蓝再生NAD +的脱氢酶电极制备(其他种类电子酶介体/介质无法使NAD +再生)
(1)制备壳聚糖-NAD +复合物
取NAD +水溶液5mL(1mg/mL)，加入1mL碘乙酸(1mg/mL)，70℃水浴反应1h。在上述NAD +水溶液中加入硫代硫酸钠溶液1mL(1.3mM)，用1M NaOH调pH＝11，70℃水浴反应1h。之后加入1mL甲醛70℃水浴反应1h。使用1M HCl调整反应体系的pH为中性后加入NHS(10mg)，EDC(10mg)，加入0.1％壳聚糖溶液10mL，70℃水浴反应1h。
(2)制备苹果酸脱氢酶电极
所有的电化学测量都是在典型的三电极系统(CHI 760D，CH仪器)中进行的。三电极体系组成包括：铂电极作为对电极，Ag/AgCl电极作为参比电极，玻璃碳电极作为工作电极。实验时，将直径为3mm玻碳电极依次用一定粒径的Al 2O 3浆在抛光布上抛光至镜面，每次抛光后先洗去表面污物，再移入超声水浴中清洗，每次1min，重复三次，最后依次用1:1乙醇、1:1NHO 3超声清洗。彻底洗涤后，在0.20mol/L KNO 3中记录1×10 -3mol/L K 3Fe(CN) 6溶液的循环伏安曲线，以测试电极性能，扫描速度50mV/s，扫描范围0.6～－0.2V。实验室条件下所得循环伏安图中的峰电位差在80mV以下，并尽可能接近64mV，电极方可使用。
向预处理后的电极工作面滴涂5μL 0.1％的碳纳米管分散液，晾干。将电极浸入普鲁士蓝沉积储备溶液中(以去离子水配置含2.5mM/L FeCl 3，2.5mM/L K 3Fe(CN) 6，100mM/L KCl，200mM/L HCl及100mM/L的EDTA·Na 2作为储备溶液，溶液3日内有效，以现用现配为佳)，以50mV/s的扫描速率在-200～600mV电位范围内进行不同次数的循环伏安(CV)扫描。此时可以看到界面内的氧化还原峰值随着扫描的圈数一次增大并最后接近重合，说明已有普鲁士蓝沉积在电极上。以去离子水充分清洗工作电极和对电极工作面，向工作面滴涂10μL的壳聚糖-NAD +水溶液，再次于室温下晾干后。将新制备的电极工作面浸没于含25％的戊二醛水溶液中，1h后取出，以去离子水充分清洗，使电极表面的壳聚糖修饰层中引入一定数量的活性醛基。随后将电极工作面浸入以苹果酸脱氢酶溶液(240U/mL)内，1h后取出，充分清洗以取出未牢固结合的酶分子。配置10mmol/L的标准苹果酸溶液，用制得的苹果酸脱氢酶电极测量标准苹果酸的响应电流强度。
实验结果如图16所示，电极检测无信号，说明普鲁士蓝作为电子酶介体/介质不能使固定在电极表面的NAD +再生。
实施例9琼脂糖-NAD +复合物制备脱氢酶电极的研究(其他种类NAD +大分子复合物无法在电极修饰时使用)
(1)制备琼脂糖-NAD +复合物
取NAD +水溶液5mL(1mg/mL)，加入1mL碘乙酸(1mg/mL)，70℃水浴反应1h。在上述NAD +水溶液中加入 硫代硫酸钠溶液1mL(1.3mM)，用1M NaOH调pH＝11，70℃水浴反应1h。之后加入1mL甲醛70℃水浴反应1h。使用1M HCl调整反应体系的pH为中性后加入NHS(10mg)，EDC(10mg)，加入1％琼脂糖凝胶1g，70℃水浴反应1h。
(2)制备苹果酸脱氢酶电极
所有的电化学测量都是在典型的三电极系统(CHI 760D，CH仪器)中进行的。三电极体系组成包括：铂电极作为对电极，Ag/AgCl电极作为参比电极，玻璃碳电极作为工作电极。实验时，将直径为3mm玻碳电极依次用一定粒径的Al 2O 3浆在抛光布上抛光至镜面，每次抛光后先洗去表面污物，再移入超声水浴中清洗，每次1min，重复三次，最后依次用1:1乙醇、1:1NHO 3超声清洗。彻底洗涤后，在0.20mol/L KNO 3中记录1×10 -3mol/L K 3Fe(CN) 6溶液的循环伏安曲线，以测试电极性能，扫描速度50mV/s，扫描范围0.6～－0.2V。实验室条件下所得循环伏安图中的峰电位差在80mV以下，并尽可能接近64mV，电极方可使用。
向预处理后的电极工作面滴涂5μL 0.1％的碳纳米管分散液，晾干。将电极浸入ABTS沉积储备溶液中(ABTS沉积储备溶液含2.5mM/L FeCl 3、2.5mM/L K 3Fe(CN) 6、200mM/L HCl和1mM/LABTS，仅限当天使用)，以50mV/s的扫描速率在-200～600mV电位范围内进行不同次数的循环伏安(CV)扫描。此时可以看到界面内的氧化还原峰值随着扫描的圈数一次增大并最后接近重合，说明已有ABTS沉积在电极上。以去离子水充分清洗工作电极和对电极工作面，向工作面滴涂琼脂糖-NAD +复合物，再次于室温下晾干后。将电极表面的琼脂糖-NAD +复合物浸没于含25％的戊二醛水溶液中，1h后取出，以去离子水充分清洗，使电极表面的琼脂糖修饰层中引入一定数量的活性醛基。随后将琼脂糖-NAD +复合物浸入以苹果酸脱氢酶溶液(240U/mL)内，1h后取出，充分清洗以取出未牢固结合的酶分子。配置10mmol/L的标准苹果酸溶液，用制得的苹果酸脱氢酶电极测量标准苹果酸的响应电流强度。
实验结果如图17所示，电极检测无信号，说明琼脂糖-NAD +复合物不能应用于脱氢酶电极的制备。
实施例10壳聚糖种类对苹果酸脱氢酶电极性能的影响
(1)制备高粘度壳聚糖-NAD +复合物
取NAD+水溶液5mL(1mg/mL)，加入1mL碘乙酸(1mg/mL)，70℃水浴反应1h。在上述NAD+水溶液中加入硫代硫酸钠溶液1mL(1.3mM)，用1M NaOH调ph＝11，70℃水浴反应1h。之后加入1mL甲醛70℃水浴反应1h。使用1M HCl调整反应体系的pH为中性后加入NHS(10mg)，EDC(10mg)，加入0.1％其他种类的壳聚糖溶液(其他种类的壳聚糖为高粘度壳聚糖，其粘度为30-3000mpa.s)10mL，70℃水浴反应1h。
(2)制备苹果酸脱氢酶电极
所有的电化学测量都是在典型的三电极系统(CHI 760D，CH仪器)中进行的。三电极体系组成包括：铂电极作为对电极，Ag/AgCl电极作为参比电极，玻璃碳电极作为工作电极。实验时，将直径为3mm玻碳电极依次用一定粒径的Al 2O 3浆在抛光布上抛光至镜面，每次抛光后先洗去表面污物，再移入超声水浴中清洗，每次1min，重复三次，最后依次用1:1乙醇、1:1NHO 3超声清洗。彻底洗涤后，在0.20mol/L KNO 3中记录1×10 -3mol/L K 3Fe(CN) 6溶液的循环伏安曲线，以测试电极性能，扫描速度50mV/s，扫描范围0.6～－0.2V。实验室条件下所得循环伏安图中的峰电位差在80mV以下，并尽可能接近64mV，电极方可使用。
向预处理后的电极工作面滴涂5μL 0.1％的碳纳米管分散液，晾干。将电极浸入ABTS沉积储备溶液中(ABTS沉积储备溶液含2.5mM/L FeCl 3、2.5mM/L K 3Fe(CN) 6 200mM/L HCl和1mM/L ABTS，仅限当天使用)，以50mV/s的扫描速率在-200～600mV电位范围内进行不同次数的循环伏安(CV)扫描。此时可以看到界面内的氧化还原峰值随着扫描的圈数一次增大并最后接近重合，说明已有ABTS沉积在电极上。以去离子水充分清洗工作电极和对电极工作面，向工作面滴涂10μL的壳聚糖-NAD+水溶液，再次于室温下晾干后。将新制备的电极工作面浸没于含25％的戊二醛水溶液中，1h后取出，以去离子水充分清洗，使电极表面的壳聚糖修饰层中引入一定数量的活性醛基。随后将电极工作面浸入以苹果酸脱氢酶溶液(240U/mL)内，1h后取出，充分清洗以取出未牢固结合的酶分子。配置10mmol/L的标准苹果酸溶液，用制得的苹果酸脱氢酶电极测量标准苹果酸的响应电流强度，并且与实施例2中的中粘度壳聚糖制备的电极相比较。
实验结果：
循环伏安曲线表明(图18)，其他种类的壳聚糖(高粘度)溶修饰电极(①)的电信号明显小于标准壳聚糖(中粘度)修饰电极(②)的电信号，说明壳聚糖的种类在电极修饰过程中起到重要作用。
应注意的是，以上实例仅用于说明本发明的技术方案而非对其进行限制。尽管参照所给出的实例对本发明进行了详细说明，但是本领域的普通技术人员可根据需要对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围。

Claims (10)

  1. 一种乳酸脱氢酶电极，其特征在于，所述乳酸脱氢酶电极包括：
    基底电极，以及由基底电极负载的辅酶因子复合物；
    优选的，所述基底电极包括玻碳电极(GCE)、金电极、石墨电极、碳糊电极，进一步优选为GCE；
    所述辅酶因子复合物由辅酶因子和负载材料复合得到；
    所述辅酶因子为天然辅酶或人工辅酶；
    所述人工辅酶通过将天然辅酶的非活性部位进行化学修饰得到；
    所述负载材料为高分子载体；优选的，所述负载材料包括壳聚糖、琼脂糖、海藻酸钠和聚乙二醇；优选的，所述负载材料为壳聚糖；更优选的，壳聚糖为中粘度壳聚糖(200-400mPa.s)；
    优选的，所述天然辅酶包括NAD、NADP、PQQ、硫代-NAD或硫代-NADP；
    优选的，所述人工辅酶包括人工NAD(P)/NAD(P)H化合物，所述人工NAD(P)/NAD(P)H化合物是天然NAD/NADH或天然NADP/NADPH的化学衍生物。
  2. 如权利要求1所述乳酸脱氢酶电极，其特征在于，包括：
    基底电极，所述基底电极上负载基底材料，所述基底材料表面沉积有介质，所述介质表面包覆辅酶因子复合物和乳酸脱氢酶；
    优选的，所述基底材料选自碳材料，所述碳材料包括活性炭、石墨烯、纳米碳纤维、纳米碳球、玻璃碳、碳气凝胶、碳纳米管(CNTs)，进一步优选为碳纳米管；所述碳纳米管包括多臂、单臂碳纳米管，并且包括氨基化、羧基化等修饰后的功能化碳纳米管；
    优选的，所述介质包括2,2'-联氮双(3-乙基苯并噻唑啉-6-磺酸)二铵盐(ABTS)、偶氮化合物或偶氮前体、苯醌、普鲁士蓝、亚硝基苯胺或基于亚硝基苯胺的前体、噻嗪或噻嗪衍生物、过渡金属络合物如铁氰化钾、钌络合物如己胺氯化钌、锇衍生物、醌或醌衍生物、吩嗪或基于吩嗪的前体、和吩嗪衍生物和氯化六氨合钌的组合，以及它们的衍生物；
    优选的，所述辅酶因子复合物为NAD +-壳聚糖复合物，所述NAD +-壳聚糖复合物是通过对NAD +腺嘌呤上的氨基进行修饰得到人工NAD辅酶，然后人工NAD辅酶与壳聚糖共价连接制备得到；
    优选的，当辅酶是NAD/NADH时，介质为2,2'-联氮双(3-乙基苯并噻唑啉-6-磺酸)二铵盐(ABTS)。
  3. 权利要求1或2所述乳酸脱氢酶电极的制备方法，其特征在于，所述乳酸脱氢酶电极制备方法包括：
    1)向基底电极表面滴涂基底材料；
    2)采用电化学沉积法将介质沉积至步骤1)制备得到的负载有基底材料的基底电极上；
    3)向步骤2)制备得到的材料上滴涂辅酶因子复合物；
    4)向步骤3)制备得到的材料上负载乳酸脱氢酶。
  4. 如权利要求3所述的制备方法，其特征在于，所述步骤4)中，将酶负载至步骤3)制备得到的材料上采用戊二醛对酶的交联作用得以实现；或，
    所述步骤3)中，所述辅酶因子复合物为NAD +-壳聚糖复合物时，其制备方法包括：
    1)向NAD +水溶液中加入碘乙酸，加热反应得n1-羧甲基-NAD +；
    2)在n1-羧甲基-NAD +水溶液中加入硫代硫酸钠溶液，调节pH至碱性，加热反应得到n1-羧甲基-NADH，同时在强碱条件下发生Dimroth重排得到n6-羧甲基-NADH；
    3)加入甲醛加热反应，得c6-羧甲基NAD +；
    4)调整反应体系的pH为中性后加入NHS和EDC，加入壳聚糖溶液，加热反应得壳聚糖-NAD +复合物。
  5. 如权利要求4所述的制备方法，其特征在于，所述步骤1)中，加热反应为水浴加热，水浴反应条件为在60～80℃条件下反应0.5～3h，优选为70℃水浴反应1h；或，
    所述步骤2)中，pH调节至强碱性，优选为10-11，加热反应优选为水浴加热，水浴反应条件为在60～80℃条件下反应0.5～3h，优选为70℃水浴反应1h；或，
    所述步骤3)中，加热反应优选为水浴加热，水浴反应条件为在60～80℃条件下反应0.5～3h，优选为70℃水浴反应1h；或，
    所述步骤4)中，加热反应优选为水浴加热，水浴反应条件为在60～80℃条件下反应0.5～3h，优选为70℃水浴反应1h。
  6. 权利要求1或2所述乳酸脱氢酶电极在制备乳酸脱氢酶传感器中的应用。
  7. 一种乳酸脱氢酶传感器，其特征在于，所述乳酸脱氢酶传感器包括至少两个电极，其中一个电极至少为权利要求1或2所述乳酸脱氢酶电极。
  8. 如权利要求7所述乳酸脱氢酶传感器，其特征在于，所述乳酸脱氢酶传感器包括两个或三个电极组成；
    优选的，由两个电极组成的乳酸脱氢酶传感器中，所述电极为工作电极和对电极；其中，所述工作电极为权利要求1或2所述乳酸脱氢酶电极；
    优选的，由三个电极组成的酶传感器中，所述电极为工作电极、对电极和参比电极；其中，所述工作电极为权利要求1或2所述乳酸脱氢酶电极；
    进一步优选的，在三电极酶传感器中，所述对电极为铂电极；所述参比电极为Ag/AgCl电极。
  9. 如权利要求8所述乳酸脱氢酶传感器，其特征在于，所述乳酸脱氢酶电极包括：GCE电极，所述GCE电极上负载碳纳米管，所述碳纳米管表面沉积有ABTS，所述ABTS表面负载有壳聚糖-NAD +复合物和乳酸脱氢酶。
  10. 一种电化学测量乳酸浓度或存在的方法，其特征在于，所述方法包括：将权利要求7-9任一项所述乳酸脱氢酶传感器与具有或怀疑具有乳酸的液体样品接触，测量待测目标分析物的响应电流强度，分析目标分析物的浓度或有无。
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