JP2008096352A - Manufacturing method for enzyme electrode - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an enzyme electrode with excellent electrode performance by immobilizing an electronic mediator and/or oxidoreductase in highly dispersed condition on a conductive base material. <P>SOLUTION: In this manufacturing method for the enzyme electrode comprising the conductive base material connected to an external circuit, the oxidoreductase capable of performing electron transfer to/from the conductive base material, and the electronic mediator capable of mediating electron transfer between the conductive base material and the oxidoreductase, the electron mediator forms a complex connected in the pendant form to a polymer chain of an organic polymer material, and when a polymer-mediator solution, in which the complex is dispersed in a solvent with a dielectric constant of 24 or less, an oxidoreductase solution of dispersed oxidoreductase, and a crosslinking reagent binding the oxidoreductase to the polymer chain of the complex are applied to the surface of the conductive base material, the electron mediator and the oxidoreductase are immobilized on the surface of the conductive base material. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for producing an enzyme electrode.

Enzymes are used in analysis for measuring the abundance of various substances due to their high substrate specificity, such as enzyme sensors. As an enzyme sensor using an enzyme, for example, there is a sensor that measures a current generated by an oxidation-reduction reaction between a target substance (substrate) to be analyzed and an enzyme (oxidoreductase) and quantifies the target substance. Specifically, there is a glucose sensor that utilizes the fact that an electric current generated with an oxidation-reduction reaction between an enzyme that oxidizes glucose and glucose is proportional to the glucose concentration.
Furthermore, recently, research and development of enzymes have been promoted as new catalysts for fuel cells that can replace metal catalysts such as platinum. Enzyme electrodes that use the current generated by the oxidation-reduction reaction between an enzyme and a substrate are expected to be used in a wide range of fields in addition to enzyme sensors and fuel cells.

In general, enzymes are less likely to be directly oxidized and reduced on the electrode surface composed of a conductive base material, so the efficiency of the electrode reaction is improved by using an electron mediator that mediates electron transfer between the enzyme and the electrode. . The electron mediator transports electrons received from an enzyme that oxidizes a substrate to an electrode, or transports electrons received from an electrode to an enzyme that reduces the substrate. By smooth electron transport between the enzyme-electron mediator-electrode, the current value of the enzyme electrode increases, and a biofuel cell capable of taking out a sufficient current is obtained.
In enzyme electrodes, electron mediators can be mixed, dispersed in an electrolyte solution with an enzyme, or fixed to the electrode surface depending on the purpose of use, research purpose, etc. In view of the electrode performance and the like, the enzyme or only the electron mediator is often immobilized on the electrode surface.

  As a method for immobilizing the electron mediator or enzyme on the surface of the electrode (conductive substrate), for example, the enzyme or the electron mediator is formed with an organic polymer material on the conductive substrate, and the organic polymer material is solidified. By immobilizing enzymes and electron mediators in the pores, and ionic organic polymer materials having a charge opposite to that of enzymes and electron mediators at the pH used. A method of immobilizing on a conductive substrate, a method of physically adsorbing and immobilizing an enzyme or electron mediator to a conductive substrate, a functional group of an organic polymer material, etc. and a functional group of an enzyme or electron mediator being covalently bonded By using a cross-linking reagent that forms a covalent bond between the organic polymer material and the enzyme or electron mediator. Method for immobilizing by covalently coupling the chromatography data, and the like.

  In addition, an enzyme electrode in which an organic polymer chain such as polyimidazole and an electron mediator such as an osmium complex bonded in a pendant form is immobilized on the surface of a conductive substrate has been proposed, and exhibits high performance. Are known. Furthermore, an enzyme is also bound to a polymer organic chain (hereinafter sometimes referred to as a polymer-mediator complex) to which such an electron mediator is bound, and the electron mediator and the enzyme are immobilized on the surface of the conductive substrate. Enzyme electrodes have also been proposed.

  Examples of specific enzyme electrodes to which an enzyme or an electron mediator is fixed include those described in Patent Document 1 and Patent Document 2.

JP 2005-79001 A JP-A-6-174679

In the method of immobilizing an enzyme or electron mediator on the electrode surface as described above, a solution in which the enzyme or electron mediator is dispersed in a solvent is often used. At this time, if the dispersibility of each component in the solution is poor, the enzyme and the electron mediator are immobilized in an aggregated state on the electrode surface. If the dispersibility of the enzyme or the electron mediator on the electrode surface is insufficient, the electron transfer of the enzyme-electron mediator-electrode does not proceed smoothly, so that the resulting enzyme electrode is inferior in electrode performance.
In particular, a polymer-mediator complex in which an electron mediator is bonded to an organic polymer chain has a low dispersibility of the organic polymer chain portion, and the dispersibility of the electron mediator together with the organic polymer chain tends to deteriorate. Furthermore, when an oxidoreductase is also bound to the polymer chain of the polymer-mediator complex, both the dispersibility of the enzyme and the electron mediator deteriorates, and the electron transfer between the enzyme-electron mediator-electrode does not proceed smoothly. This results in an inefficient enzyme electrode with few enzymes and electron mediators that function effectively.

  However, conventionally, even when a polymer-mediator complex is used, a solvent having excellent dispersibility of the polymer-mediator complex is not specified, and a solvent having insufficient dispersibility is used, or Each time, it took time and effort to select a solvent through trial and error.

In the above-mentioned Patent Document 1, in the examples, AEACPVK ₃ (2- {3- [N- (2-Aminoethyl) aminocarbonyl] propyl} -3-methyl-1,4-naphtoquinone), diaphorase (Dp) and glucose dehydrogenase ( GDH) is immobilized on the electrode together with polyvinyl imidazole (PVI) using polyethylene glycol diglycidyl ether (PEGDGE). A buffer solution containing AEACPVK ₃ , Dp, GDH, PEGDGE and PVI is applied onto the glassy carbon electrode. It is applied, reacted and cured.
Moreover, in patent document 2, in the Example, polyvinyl butyral resin which is a water-insoluble polymer and 1,1′-dimethylferrocene which is an electron mediator are dissolved in ethyl carbitol, or polyvinyl butyral resin and dimethylferrocene-TCNQ. An enzyme electrode is prepared using a composition in which (7,7 ′, 8,8′-tetracyanoquinodimethane) complex and TCQN are mixed with ethyl carbitol.

  The present invention has been accomplished in view of the above situation, and an electron mediator and / or an electron mediator bonded to an organic polymer material serving as a carrier for immobilizing an oxidoreductase on the electrode surface are in a highly dispersed state. It aims at providing the enzyme electrode which was fix | immobilized on the electroconductive base material and was excellent in electrode performance.

  The method for producing an enzyme electrode of the present invention comprises a conductive base material connected to an external circuit, an oxidoreductase capable of transferring electrons between the conductive base material, the conductive base material, and the redox enzyme. An enzyme electrode manufacturing method comprising: an electron mediator capable of mediating electron transfer with an enzyme, wherein the electron mediator is bound in a pendant manner to a polymer chain of an organic polymer material. A polymer-mediator solution in which a complex is formed and the polymer-mediator complex is dispersed in a solvent having a dielectric constant of 24 or less is applied to the surface of the conductive substrate. It is fixed on the surface of the conductive substrate.

  According to the present invention, an organic polymer material that is a carrier for immobilizing an electron mediator on an electrode surface can be immobilized on the electrode surface in a highly dispersed state, and accordingly, the electron mediator is also highly dispersed. Immobilized on the electrode surface.

  When the oxidoreductase is also immobilized on the electrode surface together with the electron mediator, the polymer-mediator solution, the oxidoreductase solution in which the oxidoreductase is dispersed, and the polymer-mediator complex in the electron mediator A cross-linking reagent that binds the oxidoreductase to the polymer chain to which the polymer is bound is applied to the surface of the conductive substrate, whereby the polymer-mediator complex is increased on the surface of the conductive substrate. The oxidoreductase can be bound to a molecular chain, and the oxidoreductase can be immobilized on the surface of the conductive substrate together with the electron mediator.

  In this case, the organic polymer that is a carrier for immobilizing both the oxidoreductase and the electron mediator on the electrode surface is immobilized on the electrode surface in a highly dispersed state, and accordingly both the electron mediator and the oxidoreductase are Immobilized on the electrode surface in a highly dispersed state.

  Examples of the electron mediator include osmium complexes, and examples of the organic polymer chain to which the electron mediator is bonded include polyvinyl imidazole. Furthermore, examples of the solvent having a dielectric constant of 24 or less include ethanol.

  ADVANTAGE OF THE INVENTION According to this invention, it is possible to provide the enzyme electrode which is excellent in the electron transfer property between oxidoreductase-electron mediator-electrode, and expresses high electrode performance.

  The method for producing an enzyme electrode of the present invention comprises a conductive base material connected to an external circuit, an oxidoreductase capable of transferring electrons between the conductive base material, the conductive base material, and the redox enzyme. An enzyme electrode manufacturing method comprising: an electron mediator capable of mediating electron transfer with an enzyme, wherein the electron mediator is bound in a pendant manner to a polymer chain of an organic polymer material. A polymer-mediator solution in which a complex is formed and the polymer-mediator complex is dispersed in a solvent having a dielectric constant of 24 or less is applied to the surface of the conductive substrate. It is fixed on the surface of the conductive substrate.

Here, an embodiment of a biofuel cell provided with an enzyme electrode (substrate oxidation type) obtained by the present invention will be described with reference to FIG.
First, an oxidase oxidizes a substrate such as glucose as a fuel and receives electrons. Next, the oxidase that has received the electrons usually delivers the electrons to an electron mediator that mediates electron transfer between the oxidase and the electrodes, and the electrons are transferred to the conductive substrate (anode electrode) by the electron mediator. Delivered. And an electric current generate | occur | produces when an electron reaches | attains a cathode electrode through an external circuit from the electroconductive base material which is an anode electrode.
Protons (H ⁺ ) generated in the above process move to the cathode electrode in the electrolytic solution. In the cathode electrode, protons that have moved through the electrolyte solution, electrons that have moved from the anode side through the external circuit, and an oxidizing agent (cathode side substrate) such as oxygen or hydrogen peroxide react with each other. Water is produced.

  In order to obtain a sufficient current value in an enzyme electrode, smooth electron transfer between the oxidoreductase-electron mediator-electrode is important, and the enzyme electrode depends on the mutual positional relationship between the oxidoreductase-electron mediator-electrode. The electrode performance is greatly affected. That is, in order to obtain an enzyme electrode that exhibits excellent electrode performance, it is important to increase the dispersion state when the oxidoreductase or electron mediator is immobilized on the electrode surface.

The present inventors have focused on the fact that the dispersibility of the electron mediator on the electrode surface can be improved by increasing the dispersibility of the organic polymer constituting the polymer-mediator complex on the electrode surface. In the method of immobilizing the electron mediator on the electrode surface by applying a dispersion solution of the polymer-mediator complex to the surface of the conductive substrate, the organic polymer of the polymer-mediator complex in the polymer-mediator solution By improving the dispersibility of the material, the organic polymer material was immobilized on the electrode surface in a highly dispersed state.
Specifically, the use of a solvent having a dielectric constant of 24 or less as the solvent for dispersing the polymer-mediator complex improves the dispersibility of the organic polymer material constituting the polymer-mediator complex. I found it.

In the present invention, since the electron mediator is previously bonded to the organic polymer material that is a carrier for immobilizing the electron mediator on the electrode surface, dispersibility of the organic polymer material on the electrode surface is improved. Thus, the dispersibility of the electron mediator on the electrode surface is also improved.
As described above, the enzyme electrode obtained by the production method of the present invention has a high electron transfer property between the oxidoreductase-electron mediator-electrode because the electron mediator is immobilized on the electrode surface in a highly dispersed state, and the electrode Excellent characteristics. Therefore, for example, by using the enzyme electrode of the present invention as an electrode of a biofuel cell, it is possible to provide a biofuel cell excellent in power generation performance capable of obtaining a large current.

  An oxidoreductase solution in which an oxidoreductase is dispersed together with the polymer-mediator solution, and a crosslinking reagent that binds the oxidoreductase to the polymer chain to which the electron mediator is bound in the polymer-mediator complex. When applied to the surface of the conductive substrate, since the oxidoreductase can be bound to the polymer chain of the polymer-mediator complex on the surface of the conductive substrate, the organic of the polymer-mediator complex The oxidoreductase can be immobilized on the surface of the conductive substrate together with the electron mediator using the polymer as a carrier.

  In this way, when the organic polymer material constituting the polymer-mediator complex and the oxidoreductase are reacted and bonded on the electrode surface, the dispersibility of the organic polymer material as a carrier on the electrode surface is improved. This improves the dispersibility of the oxidoreductase on the electrode surface. Therefore, the electron transferability between the oxidoreductase-electron mediator-electrode can be further enhanced.

Hereinafter, the method for producing the enzyme electrode of the present invention will be described in detail.
An electroconductive base material is a base material which comprises an electrode, and is not specifically limited, A general thing can be used. For example, those made of conductive carbon such as graphite, carbon black and activated carbon, and those made of metal such as gold and titanium can be used.

  In the present invention, a polymer-mediator complex in which an electron mediator is previously bonded to a polymer chain of an organic polymer material is used. Then, the oxidoreductase is immobilized on the electrode surface together with the electron mediator by binding the oxidoreductase to the polymer chain of the organic polymer material of the polymer-mediator complex on the surface of the conductive substrate as the electrode. To do.

  The electron mediator is not particularly limited as long as it can bind to the organic polymer chain, and may be appropriately selected from substances generally used according to the oxidoreductase used. Examples thereof include metal complexes having a metal element such as Os, Fe, Ru, Co, Cu, Ni, V, Mo, Cr, Mn, Pt, and W as a central metal; heterocyclic compounds such as viologen, and the like. Among these, an osmium complex having osmium as a central metal is preferable because a redox potential can be controlled by selecting a ligand, and an optimum redox potential can be obtained for the redox enzyme used. Specific examples of preferred Os complexes include those in which bidentate ligands represented by the following formulas (1) and (2) are coordinated to osmium.

(In the above formulas (1) and (2), R ^{1 to} R ⁸ are each independently H, F, Cl, Br, I, NO ₂ , CN, COOH, SO ₃ H, NHNH ₂ , SH, OH. , NH _2, or a substituted or unsubstituted alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, alkylamino, dialkylamino, alkanoylamino, aryl carboxamide, hydrazino, alkyl hydrazino, hydroxylamino, alkoxylamino amino, alkylthio , Alkenyl, aryl or alkyl.)

  In the osmium complex, the ligand coordinated to the central metal is not limited to the bidentate ligand of the above formula, but other bidentate ligands and monodentate coordination such as Cl, I, Br, F, etc. Child etc. are mentioned.

  The organic polymer material to which the electron mediator is bonded to the organic polymer chain is not particularly limited, and examples thereof include styrene / maleic anhydride copolymer, methyl vinyl ether / maleic anhydride copolymer, poly (4-vinylbenzyl chloride) copolymer, Examples include poly (allylamine) copolymers, poly (4-vinylpyridine) copolymers, poly (4-vinylpyridine), polyvinylimidazole, and poly (4-styrenesulfonate). Among them, when a metal complex is used as an electron mediator, a metal From the viewpoint that it can be directly coordinated to the central metal of the complex, polyvinyl imidazole and poly (4-vinylpyridine) are preferable.

  In polyvinylimidazole and poly (4-vinylpyridine), an imidazole group and a pyridine group can function as a monodentate ligand, respectively, and can coordinate to the central metal of the metal complex. Here, the coordination site such as an imidazole group or a pyridine group may form a part of the main chain skeleton of the organic polymer material, or through a chemical structure that becomes a linking group from the main chain skeleton. Or a structure in which the main chain skeleton is directly bonded to the main chain.

In the polymer-mediator complex, the electron mediator is bonded in a pendant manner to the polymer chain of the organic polymer material. Here, the term “bonded to the polymer chain in a pendant manner” refers to a state in which an electron mediator is bonded to the main chain or side chain of the organic polymer material. The bond of the electron mediator to the organic polymer chain may be direct, or may be indirect via a spacer or the like. In addition, the type of bond between the electron mediator and the polymer chain is not particularly limited, and examples thereof include coordination bond and covalent bond.
Only one type of electron mediator and organic polymer material may be used, or two or more types may be used in combination.

  The production method of the present invention is greatly characterized in that a solvent having a dielectric constant of 24 or less is used as a solvent for dispersing the polymer-mediator complex in which the electron mediator is bonded to an organic polymer chain. Since the solvent having such a dielectric constant has high dispersibility of the organic polymer material, a polymer-mediator solution in which the polymer-mediator complex is highly dispersed can be obtained. The optimal dielectric constant of the solvent varies depending on the polymer-mediator complex to be dispersed, particularly the organic polymer material of the polymer-mediator complex. Therefore, if the solvent is appropriately selected according to the polymer-mediator complex to be used Good.

  Examples of the solvent having a dielectric constant of 24 or less include hexane, ethanol, propanol, tetrahydrofuran, and the like. For example, when polyvinyl imidazole is used as the polymer organic material, ethanol is particularly suitable when polyvinyl imidazole is used as the polymer material and an Os complex is used as the electron mediator.

The oxidoreductase is not particularly limited and may be appropriately selected depending on the substrate. For example, dehydrogenase, oxidase, or the like can be used as the substrate oxidizing enzyme. Specific examples include glucose dehydrogenase (GDH), alcohol dehydrogenase (ADH), aldehyde dehydrogenase, glucose oxidase (GOD), alcohol oxidase (AOD), and aldehyde oxidase. GDH, ADH, GOD, and AOD are preferably used from the viewpoint of ease of management and safety.
Further, GDH, ADH, GOD, AOD and the like have an advantage that a substrate as a fuel is easily available when the enzyme electrode according to the present invention is used in a biofuel cell. Examples of the substrate-reducing enzyme include laccase and bilirubin oxidase.
Only one type of oxidoreductase may be used alone, or two or more types may be used in combination.

  When oxidoreductase is bound to the polymer chain of the polymer-mediator complex, the oxidoreductase is usually applied to the surface of the conductive substrate as an oxidoreductase solution dispersed in a solvent. In the oxidoreductase solution, the solvent in which the oxidoreductase is dispersed is not particularly limited, but usually, from the viewpoint of maintaining the activity of the oxidoreductase and preventing aggregation of the oxidoreductase, a Tris buffer solution, a phosphate buffer solution A buffer solution such as morpholinopropane sulfonic acid (MOPS) is used, and the pH of the oxidoreductase solution is maintained at an optimum value, for example, about pH 7.

  The cross-linking reagent for binding the oxidoreductase to the polymer chain of the organic polymer material is not particularly limited and can be appropriately selected according to the organic polymer material or oxidoreductase used. Examples thereof include polyethylene glycol diglycidyl (PEGDE) and glutaraldehyde having a reactive group capable of covalently bonding an oxidoreductase and an organic polymer material.

  For example, as a polymer-mediator complex, an imidazole group of polyvinyl imidazole (PVI) is coordinated to an Os complex (hereinafter sometimes referred to as PVI-Os; see the following formula (3)), and GDH as an oxidoreductase When PEGDE is used as a crosslinking reagent, GDH can be bound to PVI of PVI-Os via PEGDE on the electrode surface.

  The crosslinking reagent is also usually applied to the electrode surface in a state dispersed in a solvent. The solvent in which the crosslinking reagent is dispersed is not particularly limited, and examples thereof include an organic solvent such as ethanol and an aqueous solvent.

The cross-linking reagent may be dispersed together with the polymer-mediator complex in the polymer-mediator solution, or may be dispersed together with the oxidoreductase in the oxidoreductase solution.
The oxidoreductase may be immobilized on the electrode surface by a method other than the method of binding to the polymer chain of the polymer-mediator complex as described above, or may not be immobilized on the electrode surface and the electrolyte solution. It may be dispersed within.

  In the present invention, the method for applying the polymer-mediator solution, oxidoreductase solution, and crosslinking reagent solution to the surface of the conductive substrate and the order of application are not particularly limited. As a method for applying these solutions, for example, a dropping method or the like can be used. From the viewpoint of electron transfer between the electrode, electron mediator, and oxidoreductase, the order of application to the surface of the conductive substrate is polymer-mediator along the order of electron transfer between the oxidoreductase, electron mediator, and electrode. It is preferable to apply the solution first, and it is preferable to apply the oxidoreductase solution subsequent to the application of the polymer-mediator solution, and then apply the crosslinking reagent.

  The enzyme electrode obtained by the present invention can be used for biosensors and the like in addition to biofuel cells. For example, when the enzyme electrode obtained according to the present invention is incorporated into a biofuel cell as a substrate oxidation enzyme electrode, general fuels (substrates) can be widely used. For example, alcohols, aldehydes, In addition to hydrocarbon compounds composed of sugars and ketones, hydrogen and the like can be mentioned. Among them, at least one selected from hydrocarbon compounds composed of alcohols, aldehydes, saccharides, and ketones is preferable from the viewpoint of ease of handling and the like. Specific examples include alcohols such as methanol, ethanol, propanol and glycerine; aldehydes such as formaldehyde and acetaldehyde; saccharides such as glucose, fructose and sorbose; ketones and the like. In addition, metabolic intermediate products such as fats, proteins, and sugars, and mixtures thereof can be used.

  The electrolyte solution in which the fuel or oxidant as the substrate is dispersed or dissolved is used in the tris buffer solution, phosphate buffer solution, morpholinopropane sulfonic acid so that the oxidation reaction or reduction reaction that is an electrode reaction proceeds efficiently and constantly. It is preferably maintained at an optimum pH, for example, around pH 7, by a buffer solution such as (MOPS). Moreover, in order to make an electrode reaction advance efficiently and stably, it is preferable to maintain at about 20-30 degreeC, for example.

  As the cathode paired with the anode (substrate oxidation enzyme electrode) as described above, for example, an electrode generally used in a fuel cell such as platinum or a platinum alloy which is an effective catalyst for a reduction reaction of an oxidizing agent. Cathode is used as a cathode electrode with a catalyst supported on a carbonaceous material such as graphite, carbon black or activated carbon, or a conductor made of gold, titanium or the like, or a conductor made of an electrode catalyst such as platinum or platinum alloy. The oxidant may be supplied to the electrode catalyst.

Alternatively, the cathode that is paired with the anode composed of the substrate oxidation enzyme electrode as described above may be used as the substrate reduction enzyme electrode. Examples of the oxidoreductase that reduces the oxidant include known ones such as laccase and bilirubin oxidase. When an oxidoreductase is used as a catalyst for reducing the oxidant, a known electron mediator may be used as necessary.
Examples of the oxidizing agent include oxygen and hydrogen peroxide. In order to avoid the influence of impurities that hinder the electrode reaction at the cathode, an oxygen selective membrane such as dimethylpolysiloxane may be disposed around the cathode electrode.

[Preparation of substrate oxidation enzyme electrode]
(Example 1)
First, a composite of poly (1-vinylimidazole) and Os (2,2′-bipyridylamine) ₂ Cl (hereinafter referred to as PVI-Os) is used on the surface of carbon paper (10 mmΦ) using a micropipette. 8 μl of a PVI-Os 15 mg / ml ethanol solution in which the following formula (3)) was dispersed in ethanol (dielectric constant 24) was dropped. Subsequently, 8 μl of 5 mg / mL phosphate buffer solution of glucose dehydrogenase (GDH) was dropped. Next, 8 μl of a 3 mg / ml aqueous solution of polyethylene glycol diglycidyl ether (PEGDE) was dropped.

This was left to stand overnight at room temperature and dried, and GDH as an enzyme and PVI-Os as a mediator were fixed on the surface of the carbon sheet by binding GDH to the polyvinylimidazole chain of PVI-Os via PEGDE. .
As shown in FIG. 3, the carbon paper in which the oxidoreductase and the electron mediator are fixed as described above is fixed to the tip of a member having a carbon core connected to an external circuit with a nylon net and an O-ring, A substrate oxidized enzyme electrode (sample electrode) was used.

(Comparative Example 1)
In Example 1, a substrate oxidized enzyme electrode was produced in the same manner as in Example 1 except that an aqueous solution of PVI-Os 15 mg / ml in which PVI-Os was dispersed in water (dielectric constant 80) was used.

[Evaluation of enzyme electrode]
Using the substrate oxidized enzyme electrode as a sample electrode, a power generation test (3-electrode electrochemical evaluation, see FIG. 4) was performed under the following conditions. In addition, Ag / AgCl ₂ was used as a reference electrode, and a Pt wire was used as a counter electrode. Moreover, the electrochemical surface area of each enzyme electrode was calculated | required from the oxidation-reduction peak obtained by the cyclic voltammetry method. The results are shown together in FIG.

<Test conditions>
・ Temperature: Room temperature (about 27 ℃)
Electrolyte solution: aqueous solution (pH 7.0) containing MOPS 30 mM, CaCl ₂ 3 mM, KCl 100 mM, and glucose 80 mM

<Result>
As shown in FIG. 2, the enzyme electrode of Example 1 in which an organic polymer material (PVI-Os) bonded with an electron mediator is dispersed in a solvent having a dielectric constant of 24 or less has a dielectric constant of 24 from PVI-Os. Compared with the enzyme electrode of Comparative Example 1 dispersed in a large solvent, both the current value and the electrochemical surface area were large, and the electrode characteristics were excellent. This is because PVI that functions as a carrier that binds an electron mediator and an oxidoreductase and is immobilized on the electrode surface is dispersed using a solvent having a dielectric constant of 24 or less, whereby the dispersibility of PVI in the dispersion solution is increased. As a result, PVI was adhered to the electrode surface in a highly dispersed state, and as a result, the dispersibility of the Os complex and GDH bonded to PVI on the electrode surface was also improved.

It is a conceptual diagram which shows one example of the biofuel cell provided with the enzyme electrode of this invention. It is a graph which shows the evaluation result in an Example. It is a figure which shows the preparation methods of the enzyme electrode in an Example. It is a figure which shows the evaluation system of the electric power generation test (three-electrode-type electrochemical evaluation) in an Example.

Claims (5)

A conductive substrate connected to an external circuit, an oxidoreductase capable of transferring electrons between the conductive substrate, and an electron transfer between the conductive substrate and the oxidoreductase. A method for producing an enzyme electrode comprising:
The electron mediator is bonded in a pendant manner to a polymer chain of an organic polymer material to form a polymer-mediator complex,
The polymer-mediator solution in which the polymer-mediator complex is dispersed in a solvent having a dielectric constant of 24 or less is applied to the surface of the conductive substrate, thereby fixing the electron mediator to the surface of the conductive substrate. A method for producing an enzyme electrode, comprising:   The polymer-mediator solution, the oxidoreductase solution in which the oxidoreductase is dispersed, and the cross-link that binds the oxidoreductase to the polymer chain to which the electron mediator is bound in the polymer-mediator complex. By applying a reagent to the surface of the conductive substrate, the oxidoreductase is bound to the polymer chain of the polymer-mediator complex on the surface of the conductive substrate, and the oxidation media is combined with the electron mediator. The method for producing an enzyme electrode according to claim 1, wherein a reductase is immobilized on the surface of the conductive substrate.   The method for producing an enzyme electrode according to claim 1 or 2, wherein the electron mediator is an osmium complex.   The method for producing an enzyme electrode according to any one of claims 1 to 3, wherein the organic polymer material to which the electron mediator is bonded is polyvinylimidazole.   The method for producing an enzyme electrode according to any one of claims 1 to 4, wherein the solvent having a dielectric constant of 24 or less is ethanol.

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