JP2008059805A - Enzyme electrode and biological fuel cell and biosensor provided with the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an enzyme electrode which can attain a high voltage and a high power current and provide a high performance fuel cell and an enzyme sensor provided with the enzyme electrode. <P>SOLUTION: The enzyme electrode is provided with an electrode, an oxidoreductase and an electron mediator to mediate an electron transfer between the electrode and the oxidoreductase. The electron mediator has a plurality of kinds of electron mediators including at least a first electron mediator and a second mediator, and depending on a kind of a substance to be used as each of the electron mediator, a substrate oxidation type enzyme electrode or a substrate reduction type enzyme electrode can be obtained. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an enzyme electrode, and a biofuel cell and biosensor including the 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, 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, since an enzyme is difficult to be directly oxidized and reduced on the electrode surface, the efficiency of the electrode reaction using an electron mediator that mediates electron transfer between the enzyme and the electrode has been increased. The electron mediator transports electrons received from the enzyme that oxidizes the substrate to the electrode, or transports electrons received from the electrode to the 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 from which a sufficient current can be extracted is obtained. The electron mediator is mixed and dispersed in the electrolytic solution together with the enzyme, or fixed to the electrode.

Various materials are used as the electron mediator. For example, Patent Document 1 discloses a transition metal complex containing a bidentate ligand having bipyridine or pyridylimidazole, and Patent Document 2 includes a polymer skeleton and the polymer. A polymer transition metal complex having a spacer covalently bonded to the skeleton and a plurality of transition metal complexes having a specific structure, wherein at least one of the ligands of the transition metal complex is covalently bonded to any of the spacers. Are listed.
Patent Document 3 describes an electron mediator comprising a derivative of a quinone molecule. Specifically, a derivative of sodium anthraquinone-2-sulfonate (AQS) or 2-methyl-1,4-naphtoquinone ( Derivatives of VK ₃ ) are described. Furthermore, in Non-Patent Document 1, metal complexes [Fe (CN) ₆ ] ^{3− / 4−} , [W (CN) ₈ ] ^{3− / 4−} , [Os (CN) _{6 are used} as electron mediators of bilirubin oxidase. ] ^{3− / 4−} and [Mo (CN) ₈ ] ^{3− / 4−} are used.

Special table 2003-514823 gazette Special table 2003-514924 gazette JP 2005-79001 A The chemical Record, Vol.4, 192-203 (2004)

  The potential of the fuel cell is determined by the oxidation-reduction potential of the reaction that occurs at each of the anode and the cathode, and is determined thermodynamically. In order to obtain a high battery voltage, it is necessary to increase the potential difference between the reaction occurring at the anode and the reaction occurring at the cathode.

  In an enzyme electrode in which electron transfer between an enzyme and an electrode is performed via an electron mediator, the obtained electrode potential depends on the redox potential of the electron mediator. Therefore, in the anode electrode (substrate oxidation enzyme electrode), the lower the oxidation start potential of the electron mediator within the range higher than the oxidation-reduction potential of the enzyme that directly passes electrons to the electron mediator (base potential), Energy loss is small, and the voltage obtained when a battery is assembled is high. On the other hand, in the cathode electrode (substrate reduction type enzyme electrode), the higher the nodal reduction start potential of the electron mediator is within a range lower than the redox potential of the enzyme passed from the electron mediator (the noble potential), The energy loss is small, and the voltage obtained when the battery is assembled is high.

  In addition, in an enzyme electrode in which electron transfer is performed between the enzyme and the electrode via the electron mediator, the current obtained depends on the redox reaction rate of the electron mediator. That is, the larger the redox reaction rate of the electron mediator, the larger the current of the enzyme electrode.

Therefore, in the case of a fuel cell having an enzyme electrode (substrate oxidation enzyme electrode) as an anode electrode, the lower the oxidation start potential of the electron mediator, the higher the voltage that can be taken out, and the higher the redox reaction rate of the electron mediator. The larger the value, the higher the current density and the better power generation performance. That is, in a fuel cell including an enzyme electrode as an anode electrode, a high-performance electron mediator is one that has a low oxidation start potential and a high redox reaction rate.
On the other hand, in the case of a fuel cell having an enzyme electrode (substrate reduction enzyme electrode) as a cathode electrode, the higher the reduction start potential of the electron mediator, the higher the voltage that can be taken out, and the higher the redox reaction rate of the electron mediator. The larger the value, the higher the current density and the better power generation performance. That is, in a fuel cell having an enzyme electrode as a cathode electrode, a high-performance electron mediator has a high reduction start potential and a high redox reaction rate.
In addition, an enzyme sensor including an enzyme electrode having a large current or voltage is highly sensitive and can detect a trace component.

  However, each of the electron mediators described in Patent Documents 1 to 3 and Non-Patent Document 1 has a high oxidation-reduction reaction rate, but has a high oxidation start potential or a low oxidation start potential, but has a low redox reaction rate. The redox reaction rate required for the electron mediator of the anode electrode (substrate oxidation enzyme electrode) is not compatible with the oxidation initiation potential. Alternatively, although the oxidation-reduction reaction rate is high, the reduction initiation potential is low, or the reduction initiation potential is high, but the oxidation-reduction reaction rate is low, or the oxidation required for the electron mediator of the cathode electrode (substrate reduction enzyme electrode) The reduction reaction rate and reduction initiation potential are not compatible.

  The present invention has been accomplished in view of the above circumstances, and an object of the present invention is to provide an enzyme electrode capable of obtaining a high voltage and a high generated current. In addition, a high-performance fuel cell and enzyme sensor provided with the enzyme electrode are provided.

  A first enzyme electrode provided by the present invention comprises an electrode, an oxidoreductase that oxidizes a substrate, and an electron mediator that receives an electron from the oxidoreductase and transfers the electron to the electrode. The enzyme electrode comprises two or more types including at least a first electron mediator and a second electron mediator as the electron mediator, wherein the first electron mediator has an oxidation start potential of the first electron mediator. And the redox potential of the redox enzyme is larger than the difference between the oxidation start potential of the second electron mediator and the redox potential of the redox enzyme, and the maximum value of the oxidation current (absolute value). ) Is larger than the maximum value (absolute value) of the oxidation current of the second electron mediator.

  The substrate oxidized enzyme electrode has a high current density because a high current density is obtained in a high potential region by the first electron mediator and a certain current density is obtained even in a low potential region by the second electron mediator. And a good current density is secured in a wide potential range from a low potential to a high potential, and a high voltage can be taken out by the second electron mediator.

  In the substrate oxidized enzyme electrode, the specific combination of the first electron mediator and the second electron mediator is not particularly limited. For example, the first electron mediator is represented by the following formula (1). The biosidylamine or bipyridyl represented by the following formula (2) is a first osmium complex in which at least one bidentate ligand composed of bipyridine or a bipyridine derivative is coordinated to osmium. Examples thereof include a combination in which at least one bidentate ligand composed of an amine derivative is a second osmium complex coordinated to osmium.

(In the above formulas (1) and (2), R ^{1 to} R ¹⁶ are each independently H, F, Cl, Br, I, NO ₂ , CN, COOH, SO ₃ H, NHNH ₂ , SH, OH. , NH ₂ or substituted or unsubstituted alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, alkylamino, dialkylamino, alkanoylamino, arylcarboxyamide, hydrazino, alkylhydrazino, hydroxylamino, alkoxylamino, alkylthio , Alkenyl, aryl or alkyl.)

  A second enzyme electrode provided by the present invention is a substrate reduction type comprising an electrode, an oxidoreductase that reduces a substrate, and an electron mediator that receives an electron from the electrode and transfers the electron to the oxidoreductase. An enzyme electrode comprising at least two types including at least a first electron mediator and a second electron mediator as the electron mediator, wherein the first electron mediator has a reduction initiation potential of the first electron mediator and The difference between the redox potential of the redox enzyme is larger than the difference between the reduction start potential of the second electron mediator and the redox potential of the redox enzyme, and the maximum value (absolute value) of the reduction current Is larger than the maximum value (absolute value) of the reduction current of the second electron mediator.

  According to the substrate-reducing enzyme electrode, a high current density is obtained in the low potential region by the first electron mediator, and a certain current density is obtained in the high potential region by the second electron mediator. A good current density is ensured in a wide potential range from a potential to a high potential, and a high voltage can be taken out by the second electron mediator.

In the substrate-reducing enzyme electrode, the specific combination of the first electron mediator and the second electron mediator is not particularly limited. For example, the first electron mediator is [W (CN) ₈ ] ^{3− The} metal complex containing ^{/ 4-} , and the second mediator is a metal complex containing [Mo (CN) ₈ ] ^{3- / 4-} .

  The substrate oxidation enzyme electrode and the substrate reduction enzyme electrode can be used in, for example, a biofuel cell or a biosensor.

  According to the enzyme electrode of the present invention, it is possible to obtain a high voltage and a high current density. Therefore, the fuel cell provided with the enzyme electrode of the present invention has excellent power generation performance. Moreover, the enzyme sensor provided with the enzyme electrode of the present invention has high sensitivity and can detect trace components.

  A first enzyme electrode provided by the present invention is an enzyme electrode comprising an electrode, an oxidoreductase, and an electron mediator that mediates electron transfer between the electrode and the enzyme, and as the electron mediator, 2 or more including at least a first electron mediator and a second electron mediator, and the difference between the oxidation initiation potential of the first electron mediator and the oxidation-reduction potential of the redox enzyme is The oxidation current of the second electron mediator is greater than the difference between the oxidation start potential of the electron mediator and the redox potential of the redox enzyme, and the maximum value (absolute value) of the oxidation current of the first electron mediator is It is a substrate oxidation type enzyme electrode characterized by being larger than the maximum value (absolute value).

  The second enzyme electrode provided by the present invention is an enzyme electrode comprising an electrode, an oxidoreductase, and an electron mediator that mediates electron transfer between the electrode and the enzyme, wherein the electron mediator And at least two kinds including a first electron mediator and a second electron mediator, and the difference between the reduction initiation potential of the first electron mediator and the redox potential of the oxidoreductase is A difference between the reduction initiation potential of the second electron mediator and the redox potential of the redox enzyme, and the maximum value (absolute value) of the reduction current of the first electron mediator is greater than that of the second electron mediator. A substrate-reducing enzyme electrode characterized by being larger than the maximum value (absolute value) of the reduction current.

Conventionally, an electron mediator used in an enzyme electrode has a large oxidation-reduction reaction rate and a high current density when the potential difference between the oxidation-reduction potential of the oxidoreductase and the oxidation initiation potential or reduction initiation potential of the electron mediator is large. If the potential difference between the redox potential of the redox enzyme and the oxidation start potential or reduction start potential of the electron mediator is small and a high voltage is obtained, the redox reaction rate tends to be low .
That is, conventional electron mediators have a large oxidation current value but a high oxidation start potential or a low oxidation start potential but a low oxidation current value for a substrate oxidation enzyme electrode. There was nothing that made it possible to achieve both high voltage and high voltage. For substrate-reducing enzyme electrodes, the reduction current value is large but the reduction initiation potential is low, or the reduction initiation potential is high but the reduction current value tends to be small. There was nothing that made it possible.

  Therefore, the inventors of the present invention have intensively studied to realize an enzyme electrode having both high voltage and high current density. As a result, (1) a first electron mediator having a high oxidation start potential and a large oxidation current value is obtained. Or a second electron mediator having a low oxidation start potential and a low oxidation current value, or (2) a first electron mediator having a low reduction start potential and a large reduction current value, and a high reduction start potential. The inventors have found that an enzyme electrode having both high current density and high voltage can be obtained by combining with a second electron mediator having a small reduction current value. In the enzyme electrode of the present invention, a good generated current can be secured in a wide potential range from a low potential to a high potential.

  That is, in the case of (1), a high current density is obtained in the high potential region by the first electron mediator, and a certain current density is obtained in the low potential region by the second electron mediator. It is possible to provide a substrate-oxidized enzyme electrode in which a good current density is secured in a wide potential range over a high potential and a high voltage can be taken out by the second electron mediator. On the other hand, in the case of (2), a high current density is obtained in the low potential region by the first electron mediator, and a certain current density is obtained in the high potential region by the second electron mediator. It is possible to provide a substrate-reducing enzyme electrode in which a good current density is ensured in a wide potential range over a high potential and a high voltage can be taken out by the second electron mediator.

Hereinafter, the enzyme electrode of the present invention will be described separately for (1) a substrate oxidation type and (2) a substrate reduction type.
(1) Substrate oxidized enzyme electrode A substrate oxidized enzyme electrode according to the present invention is an enzyme electrode comprising an electrode, an oxidoreductase, and an electron mediator that mediates electron transfer between the electrode and the enzyme. The electron mediator comprises at least two kinds including at least a first electron mediator and a second electron mediator, and the difference between the oxidation start potential of the first electron mediator and the redox potential of the redox enzyme Is greater than the difference between the oxidation start potential of the second electron mediator and the redox potential of the redox enzyme, and the maximum value (absolute value) of the oxidation current of the first electron mediator is the second It is characterized by being larger than the maximum value (absolute value) of the oxidation current of the electron mediator.

  As described above, the substrate oxidation enzyme electrode provided by the present invention has a higher oxidation initiation potential than the second electron mediator (noble) and a maximum oxidation current (absolute value). A first electron mediator that is larger than the second electron mediator and a combination of the first electron mediator having the above-described relationship with the first electron mediator at the oxidation start potential and the maximum oxidation current value. Therefore, a high current can be obtained by the first electron mediator in the high potential region, and a generated current can be maintained by the second electron mediator even in the low potential region. Further, by using the second electron mediator in which the difference between the oxidation-reduction potential of the oxidoreductase and the oxidation initiation potential is small, it is possible to take out a high voltage with little energy loss. The oxidation current is generally indicated by a negative value, and a large maximum value (absolute value) of the oxidation current means a large negative value.

  The first electron mediator and the second electron mediator are: (difference between the oxidation start potential of the first electron mediator and the redox potential of the redox enzyme)> (the oxidation start potential of the second electron mediator and the redox enzyme) Of the first electron mediator)> (oxidation start potential of the first electron mediator)> (oxidation start potential of the second electron mediator) and (maximum oxidation current of the first electron mediator) > (Maximum oxidation current of the second electron mediator), the combination is not particularly limited, and may be selected and combined from commonly used electron mediators.

  The first electron mediator and the second electron mediator used in the substrate oxidation enzyme electrode can be appropriately selected from the electron mediators used in the conventional substrate oxidation enzyme electrode and used in combination. As a specific combination of the first electron mediator and the second electron mediator, for example, as the first electron mediator, at least a bidentate ligand composed of a bipyridine or a bipyridine derivative represented by the following formula (1) is used. At least one bidentate ligand composed of a bipyridylamine or a bipyridylamine derivative represented by the following formula (2) is coordinated to osmium as the first osmium complex coordinated with one osmium and the second electron mediator. The second osmium combination is listed.

(In the above formula (1) and formula (2), R ^{1 to} R ¹⁶ are each independently H, F, Cl, Br, I, NO ₂ , CN, COOH, SO ₃ H, NHNH ₂ , SH, OH, NH ₂ , or substituted or unsubstituted alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, alkylamino, dialkylamino, alkanoylamino, arylcarboxyamide, hydrazino, alkylhydrazino, hydroxylamino, alkoxylamino, Any of alkylthio, alkenyl, aryl or alkyl.)

  In each of the first osmium complex and the second osmium complex, the coordination number of the bidentate ligand of the above formula (1) or (2) with respect to osmium is not particularly limited, and is 1 to 3 coordinations. Can be made. When a plurality of bidentate ligands of the above formula (1) or (2) are coordinated, these bidentate ligands may be different from each other or the same.

  A ligand other than the bidentate ligand of the above formula (1) or (2) may be coordinated with the first and second osmium complexes. For example, a polymer having a coordination site may be coordinated to an osmium atom at the coordination site. Here, the coordination site may form a part of the main chain skeleton of the polymer, or may be bonded in a pendant form directly from the main chain skeleton through a chemical structure serving as a spacer or to the main chain skeleton. The structure may be sufficient. For example, in poly (N-vinylimidazole) and poly (4-vinylpyridine), an imidazole group and a pyridine group can function as a monodentate ligand and can coordinate to osmium, which is a central metal. By fixing the Os complex on the polymer, it becomes easy to fix the Os complex, which is an electron mediator, to the electrode.

  As another form in which the osmium complex is fixed on the polymer, a form in which the polymer is covalently bonded to the ligand of the Os complex can be mentioned. For example, a reactive group possessed by the ligand of the Os complex and a reactive group possessed by the polymer are reacted and covalently bonded. At this time, the polymer and the ligand may be bonded via a chemical structure serving as a spacer.

  Examples of the polymer in which the Os complex is fixed by coordination bond or covalent bond include styrene / maleic anhydride copolymer, methyl vinyl ether / maleic anhydride copolymer, poly (4-vinylbenzyl chloride) copolymer, poly (allylamine) copolymer, poly Any of (4-vinylpyridine) copolymer, poly (4-vinylpyridine), poly (N-vinylimidazole) and poly (4-styrenesulfonate) are preferred. Among these, poly (N-vinylimidazole) and poly (4-vinylpyridine) are preferable from the viewpoint that they can be directly coordinated to the Os complex.

Examples of the other ligand in the osmium complex include at least one selected from Cl, F, Br, I, CN, CO, CH ₃ COO, NH ₃ , NO, pyridine, and imidazole. However, the present invention is not limited to these, and other complexable materials may be used. The ligand determines whether the Os complex is immobilized on the polymer (whether the electron mediator is immobilized on the enzyme electrode surface or dispersed in the electrolyte solution), or the oxidation-reduction potential of the obtained Os complex. It may be selected as appropriate in consideration.

  Specific examples of the first osmium complex and the second osmium complex used in the present invention include those represented by the following formulas (3) and (4).

Here, a method for synthesizing a complex of poly (N-imidazole) and Os complex represented by the above formula (4) [Poly (N-vinylimidozole) complexed with Os (2,2′-bipyridine) ₂ Cl] will be described. To do.

First, (NH ₄ ) ₂ [OsCl ₆ ] (1 mmol) and 2.0 equivalents of 2,2′-bipyridylamine (2 mmol) are refluxed in 1,2-ethanodiol (18 mL) for 1 hour under an argon atmosphere. . After cooling, the reaction mixture is treated with 30 mL of 1M Na ₂ S ₂ O ₄ . The resulting reaction mixture is cooled in an ice bath for 30 minutes, the precipitated Os complex is thoroughly washed with cold water and diethyl ether and vacuum dried to yield cis- [OsCl ₂ (2,2′-bipyridylamine) ₂ ]. can get.
Next, poly-1-vinylimidazole (PVI) and cis- [OsCl ₂ (2,2′-bipyridylamine) ₂ ] obtained above were combined in 200 mL of ethanol for 2 days with PVI (200 mg, 2 .1 mmol), filtered, and the resulting solution is poured into 1.5 L of diethyl ether with stirring to give poly-1-vinylimidazole (PVI) and cis- [OsCl ₂ (2,2 '-Bipyridilamine) ₂ ] can be obtained.

  In the substrate oxidation enzyme electrode of the present invention, the ratio of the first electron mediator and the second electron mediator provided is not particularly limited.

  The oxidoreductase that oxidizes the substrate is not particularly limited, and for example, dehydrogenase, oxidase, or the like can be used. Specific examples include glucose dehydrogenase (GDH), alcohol dehydrogenase (ADH), aldehyde dehydrogenase, glucose oxidase (GOD), alcohol oxidase (AOD), and aldehyde oxidase. When the enzyme electrode of the present invention is used as an electrode for a fuel cell, GDH, ADH, GOD, and AOD are preferably used from the viewpoints of easy fuel acquisition and management and safety. Only one kind of enzyme may be used alone, or two or more kinds of enzymes may be used in combination.

In the substrate oxidase enzyme electrode, biological nutrient sources can be widely used as the substrate for the oxidoreductase, and examples thereof include carbohydrates and fermentation products thereof. In particular, alcohols, sugars and aldehydes are preferably used. It is done. Specific examples include alcohols such as methanol, ethanol, propanol, glycerin and polyvinyl alcohol; sugars such as glucose, fructose and sorbose; aldehydes such as formaldehyde and acetaldehyde. In addition, metabolic intermediate products such as fats, proteins, and sugars, and mixtures thereof can be used.
When the enzyme electrode of the present invention is used as an electrode for a fuel cell, glucose and alcohol are particularly preferred from the viewpoints of easy handling, availability, and low environmental load. Used.

As the electrode, it is possible to use a conductive carbonaceous material such as graphite, carbon black or activated carbon, a gold electrode, or the like.
The oxidoreductase and electron mediator have an optimum pH by using a buffer solution such as Tris buffer solution, phosphate buffer solution, morpholinopropane sulfonic acid (MOPS) so that the oxidation reaction, which is an electrode reaction, proceeds efficiently and constantly. For example, it is preferable that the pH be maintained in the vicinity of pH 7.
Further, the oxidoreductase and the electron mediator may be used in a state of being dissolved and dispersed in the electrolytic solution, but it is preferable that the oxidoreductase and the electron mediator be immobilized on the electrode surface by an immobilizing material. This is because it is possible to efficiently transfer electrons to the electrode by fixing the oxidoreductase and the electron mediator to the electrode and allowing the enzyme reaction to proceed in the vicinity of the electrode.

  The method for fixing the oxidoreductase and the electron mediator to the electrode is not particularly limited. For example, when the PVI-Os represented by the above formulas (3) and (4) in which an Os complex is bonded to a polymer by a covalent bond or a coordinate bond as described above is used as an electron mediator, an electrode A solution containing PVI-Os, which is an electron mediator, is dropped on the surface of a conductor (for example, a carbon sheet, etc.) serving as a substrate, and then a solution containing an oxidoreductase (GDH, etc.) is dropped. An example is a method in which a crosslinking reagent such as polyethylene glycol diglycidyl ether (hereinafter referred to as PEGDE) is added dropwise to bond an oxidoreductase to the polyvinyl imidazole chain of PVI-Os, which is an electron mediator, via PEGDE, followed by drying. As the crosslinking reagent, a general reagent can be used, and glutaraldehyde or the like can be used in addition to PEGDE.

  Even when an electron mediator in which an Os complex is not bonded to a polymer is used, a polymer or a crosslinking reagent generally used for immobilizing an enzyme, an electron mediator, or the like on an electrode can be used. Specifically, polyvinyl imidazole, polyallylamine, polyamino acid, polypyrrole, polyacrylic acid, polyvinyl alcohol, or the like can be used as the polymer. Moreover, as a crosslinking reagent, polyethylene glycol diglycidyl ether, glutaraldehyde, etc. can be used.

In the enzyme electrode of the present invention, both of the above two types of electron mediators may not be fixed to the electrode surface or dispersed in the electrolyte solution, only one of them may be fixed on the electrode surface and the other may be dispersed in the electrolyte solution. May be.
In order for the electrode reaction to proceed efficiently and constantly, the temperature of the oxidoreductase and the electron mediator is preferably maintained at, for example, about 20 to 30 ° C.

(2) Substrate-reducing enzyme electrode A substrate-reducing enzyme electrode according to the present invention is an enzyme electrode comprising an electrode, an oxidoreductase, and an electron mediator that mediates electron transfer between the electrode and the enzyme. The electron mediator comprises at least two kinds including at least a first electron mediator and a second electron mediator, and the difference between the reduction start potential of the first electron mediator and the redox potential of the redox enzyme Is greater than the difference between the reduction initiation potential of the second electron mediator and the redox potential of the redox enzyme, and the maximum value (absolute value) of the reduction current of the first electron mediator is the second It is characterized by being larger than the maximum value (absolute value) of the reduction current of the electron mediator.

  As described above, the substrate reduction enzyme electrode provided by the present invention has a reduction initiation potential lower (base) than that of the second electron mediator, and the maximum value of the redox current is the second electron. Since the first electron mediator larger than the mediator is used in combination with the second electron mediator having the above-described relationship with the first electron mediator at the reduction initiation potential and the maximum reduction current value, A high current is obtained by the first electron mediator in the low potential region, and a current can be maintained by the second electron mediator even in the high potential region. Further, by using the second electron mediator in which the difference between the redox potential of the oxidoreductase and the reduction start potential is small, it is possible to take out a high voltage with little energy loss.

  The first electron mediator and the second electron mediator are (difference between the reduction start potential of the first electron mediator and the redox potential of the redox enzyme)> (reduction start potential of the second electron mediator and the redox enzyme) Of the first electron mediator) <(reduction start potential of the first electron mediator) <(reduction start potential of the second electron mediator) and (maximum oxidation current of the first electron mediator) > (Maximum oxidation current of the second electron mediator), the combination is not particularly limited, and may be selected and combined from commonly used electron mediators.

The first electron mediator and the second electron mediator used in the substrate-reducing enzyme electrode can be appropriately selected from the electron mediators used in the conventional substrate-reducing enzyme electrode and used in combination. As a specific combination of the first electron mediator and the second electron mediator, for example, a metal complex containing [W (CN) ₈ ] ^{3− / 4−} as the first electron mediator and the second mediator And a combination of metal complexes containing [Mo (CN) ₈ ] ^{3- / 4-} .
The metal complex containing [W (CN) ₈ ] ^{3- / 4-} and the metal complex containing [Mo (CN) ₈ ] ^{3- / 4-} were neutralized by counter ions such as potassium ions. The state may be sufficient, and a hydrate may be sufficient.

  In the substrate-reducing enzyme electrode of the present invention, the ratio of the first electron mediator and the second electron mediator provided is not particularly limited.

  The oxidoreductase that reduces the substrate is not particularly limited, and for example, bilirubin oxidase, laccase, ascorbate oxidase, and the like can be used. From the viewpoint of enzyme reduction performance, bilirubin oxidase is preferably used. Only one kind of enzyme may be used alone, or two or more kinds of enzymes may be used in combination.

  In the substrate-reducing enzyme electrode, an oxidizing agent such as oxygen or hydrogen peroxide can be used as a substrate for the oxidoreductase. From the standpoint that oxygen in the air can be used, oxygen is particularly preferably used.

As the electrode, it is possible to use a conductive carbonaceous material such as graphite, carbon black or activated carbon, a gold electrode, or the like.
The oxidoreductase and electron mediator are optimized for pH by using buffer solutions such as Tris buffer, phosphate buffer, morpholinopropane sulfonic acid (MOPS) so that the reduction reaction, which is an electrode reaction, proceeds efficiently and constantly. For example, it is preferable that the pH be maintained in the vicinity of pH 7.
Further, like the substrate oxidation enzyme electrode, the oxidoreductase and the electron mediator may be used in a state of being dissolved and dispersed in the electrolytic solution, but it is preferably immobilized on the surface of the electrode by an immobilizing material. This is because an oxidoreductase and an electron mediator are fixed to the electrode, and an enzyme reaction is allowed to proceed in the vicinity of the electrode, thereby enabling efficient electron transfer between the electrode and the enzyme.

  The method for immobilizing the oxidoreductase and electron mediator to the electrode is not particularly limited, and a polymer generally used for immobilizing the enzyme, electron mediator or the like as described above to the electrode, such as poly-L- It can be carried out using lysine or the like or a crosslinking reagent such as polyethylene glycol diglycidyl ether (PEGDE).

  In order for the electrode reaction to proceed efficiently and constantly, the temperature of the oxidoreductase and the electron mediator is preferably maintained at, for example, about 20 to 30 ° C.

Hereinafter, a fuel cell provided with the enzyme electrode of the present invention will be described by taking a fuel cell provided with a substrate oxidation enzyme electrode as an example (see FIG. 1).
First, an oxidoreductase oxidizes a substrate such as glucose as a fuel and receives electrons. Next, the oxidoreductase receiving the electrons delivers the electrons to the first electron mediator and the second electron mediator, and the electrons are delivered to the electrode (anode) by the first electron mediator and the second electron mediator. It is. Then, when electrons reach the cathode from the anode through the external circuit, a current is generated. Protons (H ⁺ ) generated in the above process move to the cathode through the proton conducting membrane or the electrolyte without the proton conducting membrane.

  At the cathode, protons that have moved through the electrolyte, electrons that have moved from the anode side through an external circuit, and an oxidant (cathode side substrate) such as oxygen or hydrogen peroxide react to form water. Is generated. As the cathode electrode, the substrate-reducing enzyme electrode of the present invention may be used, or an electrode catalyst generally used in a fuel cell such as platinum or a platinum alloy which is an effective catalyst for the oxidant reduction reaction may be used. A cathode made of a carbonaceous material such as graphite, carbon black or activated carbon, an electrode made of gold or the like, or a conductor made of an electrode catalyst such as platinum or platinum alloy may be used as the cathode electrode. Alternatively, a known oxidoreductase such as laccase or bilirubin oxidase may be used as an electrode catalyst, and an enzyme electrode equipped with one kind of electron mediator may be used as a cathode electrode.

  In order to avoid the influence of impurities (for example, ascorbic acid, uric acid, etc.) that interfere with the electrode reaction at the cathode electrode, an oxygen selective membrane such as dimethylpolysiloxane may be arranged around the cathode electrode.

  On the other hand, in the fuel cell comprising the substrate-reducing enzyme electrode of the present invention, the anode electrode oxidizes a substrate such as glucose as a fuel by the oxidoreductase, and passes the received electrons to the electron mediator. It may be an enzyme electrode that delivers electrons to), or an oxidation reaction of a fuel such as platinum or a platinum alloy (eg, hydrogen, methanol, etc.) that is generally used in fuel cells. Alternatively, an electrode configuration using an electrode catalyst having catalytic activity may be used.

In the cathode, the electrons that have moved from the anode side via the external circuit are transferred from the cathode electrode to the first electron mediator and the second electron mediator, and further, the first electron mediator and the second electron Electrons are transferred from the mediator to the oxidoreductase. A substrate such as oxygen is reduced by oxidoreductase to produce water.
As described above, since the enzyme electrode of the present invention can extract a high current and can generate a high voltage, the power generation performance is large and the loss of voltage is small. It is possible to provide an excellent fuel cell.

The enzyme electrode of the present invention can be used for biosensors such as enzyme sensors, enzyme transistors, etc., in addition to fuel cell electrodes.
When the enzyme electrode of the present invention is used in a biosensor, the presence or concentration of the substrate can be measured by detecting the current or voltage generated by the progress of the redox reaction between the enzyme and the substrate. According to the enzyme electrode of the present invention, since a high current density and a high voltage can be obtained, a highly sensitive biosensor can be provided. Since the enzyme electrode of the present invention has a high redox reaction rate of the electron mediator, the biosensor equipped with the enzyme electrode also has the advantage that the redox reaction of the electron mediator is not easily disturbed by interfering species and has high measurement accuracy. .

[Production of enzyme electrode]
(Example 1: Substrate oxidation enzyme electrode)
First, a composite of poly (1-vinylimidazole) and Os (2,2′-bipyridylamine) ₂ Cl (hereinafter referred to as PVI-Os (2)) on the surface of carbon paper (Φ10 mm) using a micropipette. 4 μl of a 15 mg / ml aqueous solution was added dropwise. Subsequently, 15 mg / ml of a complex of poly (1-vinylimidazole) and Os (4,4′-dimethyl-2,2′-bipyridine) ₂ Cl (hereinafter referred to as PVI-Os (1)). 4 μl of the aqueous solution was dropped by a micropipette. Furthermore, 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 allowed to stand overnight at room temperature and dried, and GDH was bonded to the polyvinyl imidazole chains of PVI-Os (1) and PVI-Os (2) via PEGDE. And PVI-Os (1) and PVI-Os (2) as mediators were fixed.
As shown in FIG. 4, a sample electrode was prepared in which the enzyme electrode on which the enzyme and the electron mediator were fixed as described above was fixed to the tip with a nylon net and an O-ring.

(Comparative Example 1: Substrate oxidation enzyme electrode)
In the enzyme electrode of Example 1, 4 μl each of a 15 mg / ml aqueous solution of PVI-Os (2) and a 15 mg / ml aqueous solution of PVI-Os (1) were dropped to obtain a 15 mg / ml aqueous solution of PVI-Os (1). Was added dropwise, and a sample electrode equipped with an enzyme electrode was prepared in the same manner except that a 15 mg / ml aqueous solution of PVI-Os (2) was not dropped.

(Comparative Example 2: Substrate oxidation enzyme electrode)
A sample electrode provided with an enzyme electrode was prepared in the same manner except that 8 μl of a 15 mg / ml aqueous solution of PVI-Os (2) was dropped in the enzyme electrode of Comparative Example 1 instead of PVI-Os (1).

[Evaluation of enzyme electrode]
Using the sample electrode, a three-electrode electrochemical evaluation was performed under the following conditions (see FIG. 5). In addition, Ag / AgCl ₂ was used as a reference electrode, and a Pt wire was used as a counter electrode.

<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>
The results are shown in FIG. As shown in FIG. 2, in the enzyme electrode of Comparative Example 1 using only one kind of PVI-Os (1) as an electron mediator, a large current (about −1400 μA) was generated in the vicinity of 200 mV. However, only a low current was obtained in a low potential range of 0 V or less.

  On the other hand, in the enzyme electrode of Comparative Example 2 using only one type of PVI-Os (2) as the electron mediator, the oxidation initiation potential is about -250 mV, which is lower than that of Comparative Example 1, and in a potential range of less than 0 V. Current was obtained, and voltage loss could be reduced. However, the maximum oxidation current was as low as less than −400 μA, and only a low current of −200 μA was obtained in a potential region such as 100 mV or higher.

On the other hand, PVI-Os (1) and PVI-Os (2) having a higher oxidation starting potential than PVI-Os (2) and a larger oxidation current value than PVI-Os (2). ) Used in combination with the enzyme electrode of Example 1 according to the present invention has a maximum oxidation current of −600 μA, which is inferior to the maximum oxidation current of Comparative Example 1, but the maximum oxidation current of Comparative Example 2 It was better than the value. In addition, the enzyme electrode of Example 1 has an oxidation start potential of about −250 mV as in Comparative Example 2, and an electric current is obtained even in a potential range lower than the oxidation start potential of Comparative Example 1 and less than 0 V, resulting in a voltage loss. Can be reduced.
From the above results, according to the enzyme electrode of Example 1 including two kinds of mediators having different characteristics in the oxidation start potential and the maximum oxidation current value, it is possible to take out current in a low potential region, and voltage loss. It can be seen that a good current can be taken out even in a high potential region even though the current is small.

[Production of enzyme electrode]
(Example 2: Substrate reducing enzyme electrode)
First, ₄ μl of a 5.8 mg / ml aqueous solution of K ₄ Mo (CN) ₈ .H ₂ O was dropped on the surface of carbon paper (Φ10 mm) using a micropipette. Subsequently, ₄ μl of a 5.8 mg / ml aqueous solution of K ₄ W (CN) ₈ .H ₂ O was dropped by a micropipette. Further, 8 μl of bilirubin oxidase (BOD) was added dropwise. Next, 8 μl of a 35 mg / ml aqueous solution of poly-L-lysine was dropped. Carbon which left to dry overnight at ambient temperature, by solidifying a poly -L- _{lysine, K 4 Mo (CN) 8} · H 2 O, the _{K 4 W (CN) 8 ·} H 2 O and BOD Fixed to the paper surface.
As shown in FIG. 4, a sample electrode was prepared in which the enzyme electrode on which the enzyme and the electron mediator were fixed as described above was fixed to the tip with a nylon net and an O-ring.

(Comparative Example 3: Substrate reducing enzyme electrode)
In the enzyme electrode of Example 2, ₄ μl each of 5.8 mg / ml aqueous solution of K ₄ Mo (CN) ₈ · H ₂ O and 5.8 mg / ml aqueous solution of K ₄ W (CN) ₈ · H ₂ O was dropped. However, ₈ μl of a 5.8 mg / ml aqueous solution of K ₄ W (CN) ₈ · H ₂ O was dropped, and a 5.8 mg / ml aqueous solution of K ₄ Mo (CN) ₈ · H ₂ O was not dropped. Except for the above, a sample electrode provided with an enzyme electrode was produced in the same manner.

(Comparative Example 4: Substrate reducing enzyme electrode)
In the enzyme electrode of Comparative Example 3, except that 8 μl of a 5.8 mg / ml aqueous solution of K ₄ Mo (CN) ₈ · H ₂ O was dropped instead of K ₄ W (CN) ₈ · H ₂ O in the same manner. A sample electrode provided with an enzyme electrode was prepared.

[Evaluation of enzyme electrode]
Using the sample electrode, a three-electrode electrochemical evaluation was performed under the following conditions (see FIG. 5). In addition, Ag / AgCl ₂ was used as a reference electrode, and a Pt wire was used as a counter electrode.

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

<Result>
The results are shown in FIG. As shown in FIG. 3, in the enzyme electrode of Comparative Example 3 using only one kind of K ₄ W (CN) ₈ .H ₂ O as an electron mediator, the maximum reduction current (about 100 μA) in a low potential range of 300 mV or less. However, the reduction initiation potential was as low as about 500 mV, and almost no current was obtained in the high potential range of 400 mV or higher.

On the other hand, in the enzyme electrode of Comparative Example 4 using only one kind of K ₄ Mo (CN) ₈ .H ₂ O as an electron mediator, the reduction initiation potential is about 700 mV, which is higher than that of Comparative Example 3, and 400 mV Current could be obtained even in the high potential range, and voltage loss could be reduced. However, the maximum reduction current is as low as about 60 μA, and only a current as low as about 50 μA was obtained in a potential region of 400 mV or less.

In _{contrast, K 4 Mo (CN) 8} · H 2 low reduction onset potential than O, and, the _{K 4 Mo (CN) 8 ·} H is larger maximum value of the reduction current than ₂ O _K 4 W The enzyme electrode of Example 2 according to the present invention using (CN) ₈ · H ₂ O and K ₄ Mo (CN) ₈ · H ₂ O in combination has a maximum reduction current of about 340 μA, A higher value than both the reduction current maximum values of Comparative Example 3 and Comparative Example 4 was exhibited. In addition, the enzyme electrode of Example 2 has a reduction initiation potential of about 700 mV, as in Comparative Example 4, and is higher than the reduction initiation potential of Comparative Example 3, and a current is obtained even in a high potential range of 500 mV or more. In the vicinity of 550 mV, a high current of about 130 μA flows and voltage loss can be reduced. In the enzyme electrode of Example 2, the current of 130 μA obtained in the high potential region of 550 mV was higher than the maximum reduction current value of Comparative Example 3 and Comparative Example 4.
From the above results, according to the enzyme electrode of Example 2 including two kinds of mediators having different characteristics in the oxidation start potential and the maximum oxidation current value, it is possible to take out a high current in a high potential region, Although the loss is small, it can be seen that a very high current can be extracted even in a low potential region.

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 graph which shows the evaluation result in an Example. It is a figure which shows the preparation methods of the sample electrode in an Example. It is a figure which shows the evaluation system of the three-electrode-type electrochemical evaluation method in an Example.

Claims (6)

A substrate oxidizing enzyme electrode comprising: an electrode; an oxidoreductase that oxidizes a substrate; and an electron mediator that receives electrons from the oxidoreductase and transmits the electrons to the electrode,
The electron mediator comprises at least two kinds including at least a first electron mediator and a second electron mediator,
In the first electron mediator, the difference between the oxidation start potential of the first electron mediator and the redox potential of the redox enzyme is such that the oxidation start potential of the second electron mediator and the redox enzyme of the redox enzyme An enzyme electrode characterized by being larger than a difference from a potential and having a maximum oxidation current (absolute value) larger than a maximum oxidation current (absolute value) of the second electron mediator. The first electron mediator is a first osmium complex in which at least one bidentate ligand composed of bipyridine or a bipyridine derivative represented by the following formula (1) is coordinated to osmium,
The second electron mediator is a second osmium complex in which at least one bidentate ligand composed of a bipyridylamine or a bipyridylamine derivative represented by the following formula (2) is coordinated to osmium. The enzyme electrode according to 1.

(In the above formulas (1) and (2), R ^{1 to} R ¹⁶ are each independently H, F, Cl, Br, I, NO ₂ , CN, COOH, SO ₃ H, NHNH ₂ , SH, OH. , NH ₂ or substituted or unsubstituted alkoxycarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkoxy, alkylamino, dialkylamino, alkanoylamino, arylcarboxyamide, hydrazino, alkylhydrazino, hydroxylamino, alkoxylamino, alkylthio , Alkenyl, aryl or alkyl.) A substrate-reducing enzyme electrode comprising: an electrode; an oxidoreductase that reduces a substrate; and an electron mediator that receives electrons from the electrode and transfers the electrons to the oxidoreductase,
The electron mediator comprises at least two kinds including at least a first electron mediator and a second electron mediator,
In the first electron mediator, the difference between the reduction start potential of the first electron mediator and the redox potential of the redox enzyme is such that the reduction start potential of the second electron mediator and the redox potential of the redox enzyme An enzyme electrode characterized by being larger than the difference from the potential and having a maximum reduction current (absolute value) greater than a maximum reduction current (absolute value) of the second electron mediator. The first electron mediator is a metal complex containing [W (CN) ₈ ] ^{3- / 4-} , and the second mediator is a metal complex containing [Mo (CN) ₈ ] ^{3- / 4-.} The enzyme electrode according to claim 3.   A biofuel cell comprising the enzyme electrode according to claim 1.   A biosensor comprising the enzyme electrode according to claim 1.

Images