JP2006058289A - Enzyme electrode, sensor, fuel cell, electrochemical reactor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an enzyme electrode capable of carrying enzymes at high density, and capable of enhancing the current density. <P>SOLUTION: The enzyme, at least one kind of the first mediator for transferring electrons to/from the enzyme, and at least one kind of the second mediator for transferring electrons to/from the first mediator are immobilized onto a conductive member for transferring electrons to/from at least one of the first mediator and the second mediator, via a carrier, so as to obtain the enzyme electrode that is useful as an electrode for a sensor, a fuel cell, an electrochemical reactor or the like. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an enzyme electrode, and more particularly to an enzyme electrode in which a carrier, an enzyme, and a mediator are immobilized on a conductive member, a sensor using the enzyme electrode, a fuel cell, and a method for producing the enzyme electrode.

  Enzymes, which are proteinaceous biocatalysts produced in living cells, act strongly under milder conditions than ordinary catalysts. In addition, the specificity of a substrate, which is a substance that undergoes a chemical reaction under the action of an enzyme, is high. Generally, each enzyme catalyzes only a certain reaction of a certain substrate. If these characteristics of the enzyme can be ideally used for the oxidation-reduction reaction in the electrode, an electrode with low overvoltage and high selectivity can be produced. However, the active center of many oxidoreductases is generally used in a form confined in the deep part of the three-dimensional structure of glycoprotein. It was difficult to perform electron transfer. For this reason, a method for electronically connecting an enzyme and an electrode with a substance called a mediator has been proposed. In the initial stage of research, enzymes and mediators were dissolved in the electrolyte solution to ensure the convenience of the experimental system and the freedom of movement of each, but their effective use, prevention of leakage outside the electrode, In order to meet the demand for continuous and long-term use of electrodes, a method of fixing to electrodes has been proposed. Examples of the method for immobilizing the enzyme and mediator on the conductive member include an adsorption method, a comprehensive method, a crosslinking method, and a covalent bonding method. Among these, the three methods of inclusion, cross-linking, and covalent bonding, in which an enzyme and mediator are immobilized on a conductive member using an appropriate compound, generally have an enzyme and mediator retention capacity compared to the adsorption method. High, prevents outflow from the system and enables repeated use of the enzyme electrode. Furthermore, among these, the entrapment method is preferably used because of its high retention capacity of enzymes and mediators.

  One important value for expressing the performance of the enzyme electrode is a current density that is a current value per projected area of the conductive member. When this value is high, for example, in the case of a sensor based on the detection of the current value, the detection sensitivity can be improved, the measurement part can be simplified, and the detection part can be miniaturized and used as an electrode of a fuel cell. In some cases, the output can be improved, and when used as an electrochemical reaction device, the reaction rate can be improved. Methods for improving the current density of the enzyme electrode include (1) an increase in the turnover number of the enzyme, which is the number of substrate molecules converted by one enzyme molecule per unit time, and (2) the electron transfer rate between the enzyme and the mediator. (3) Improvement of electron transport efficiency of mediator, (4) Improvement of electron transfer speed and efficiency between mediator / conductive member, and (5) Enzyme loading per projected area of conductive member It can be increased by increasing the enzyme carrying density.

  In order to realize the item (5), an immobilization method having a high enzyme retention capacity is advantageous, and the comprehensive method is an effective option. The inclusion method is a method in which an enzyme and a mediator are included in a carrier and immobilized on the surface of a conductive member. FIG. 1 schematically shows the immobilized state of the enzyme by this comprehensive method. In this case, the enzyme 1 and the mediator 2 are included in the carrier 3 on the conductive member 4 (conductive substrate).

  In the comprehensive method, the charge generated on the enzyme by the enzyme / substrate reaction moves from the redox center of the enzyme to the mediator in the carrier, and is transported to the vicinity of the conductive member by electron hopping between the mediators. It is detected in an external circuit by transferring charges between the mediator and the conductive member (the flow of charges is indicated by arrows in FIG. 1). For this reason, even if the enzyme is fixed to an amount equal to or more than the effective area of the conductive member divided by the effective surface area of the conductive member, charge transport between the electrode and the enzyme can be performed through the carrier. Enzyme loading density can be improved. As an example of this comprehensive method, US Pat. No. 6,531,239 (Heller et al.) Discloses a fuel cell comprising an enzyme electrode in which an enzyme is comprehensively immobilized by a polymer containing a mediator in the molecule. .

  Here, it is generally known that the electron transfer rate of the enzyme and the mediator decreases exponentially with the increase of the redox center of the enzyme and the electron transfer distance of the mediator. In the case of the inclusion method, the enzyme and the mediator are not arranged with a distance and orientation, so the electron transfer between the enzyme and the mediator is the distance that the enzyme and the mediator can move as a result of random molecular motion. It is thought that it occurs only when it comes close to. For this reason, the electron transfer rate between the enzyme and the mediator depends on the respective molecular motions, and is not necessarily sufficient, and may be a limiting factor for the current density of the enzyme electrode.

  On the other hand, as an example of the covalent bond method, a mediator is bound on a conductive substrate, the redox center of the enzyme is bound to the mediator, and the enzyme other than the redox center is bound to the redox center bound to the mediator. There is a way to combine them. FIG. 2 schematically shows the enzyme immobilized state by the covalent bond method. In the example of FIG. 2, the mediator 6 and the enzyme 1 are fixed to the surface of the conductive member 4 (conductive substrate) via a compound as a carrier by a covalent bond, and the flow of charges is indicated by arrows.

KATZ et al. Am. Chem. Soc. In 1996, Vol. 118, 10321-10322, an enzyme electrode in which apoglucose oxidase in which a redox center is removed from flavin adenine dinucleotide bound to pyrrolo-kiloquinone as a mediator is reported. In this method, the mediator can be placed close to and oriented at a distance allowing sufficiently fast electron transfer with respect to the redox center of the enzyme, so that high-speed electron transfer between the enzyme / mediator is possible. In this method, a mediator and an enzyme pair are bound like a single molecule on a conductive member, so that it is difficult to immobilize the enzyme to a single layer or more, so there is a limit to the density of the enzyme, There was a limit to improving the current density of the electrode.
US Pat. No. 6,531,939 J. et al. Am. Chem. Soc. 1996, 118, 10321-10322

  The present invention provides an enzyme electrode capable of improving current density. The present invention also provides a sensor, an electrochemical reaction device, a fuel cell, etc. using such an enzyme electrode.

The enzyme electrode according to the present invention is an enzyme electrode having a conductive member, an enzyme, and first and second mediators,
The first mediator and the second mediator are fixed to the conductive member by a carrier, and the first mediator and the second mediator have different redox potentials. Features.

  The sensor of the present invention is a sensor characterized in that the enzyme electrode having the above-described configuration is used as a detection part for detecting a substance. The fuel cell according to the present invention is a fuel cell characterized in that the enzyme electrode having the above-described configuration is used as at least one of an anode and a cathode. The electrochemical reaction apparatus of the present invention is an electrochemical reaction apparatus characterized by using the enzyme electrode having the above-described configuration as a reaction electrode.

  According to the present invention, in an arrangement in which an enzyme is immobilized on a conductive member, the enzyme carrying density per effective surface area of the conductive member can be increased. In the present invention, in addition to the first mediator capable of performing high-speed electron transfer with the enzyme, a second mediator that performs charge transport between the first mediator and the conductive member is further used. In the form, it is possible to perform high-speed electron transfer with the enzyme, and as a result, the enzyme electrode current density can be improved.

  Next, a preferred embodiment of the present invention will be described in detail.

  An enzyme electrode according to the present invention is an enzyme electrode having a conductive member, an enzyme, and first and third mediators, wherein the first mediator and the second mediator are supported by a carrier, and the conductive member. The first mediator and the second mediator have different redox potentials from each other.

  Here, as one mode of the combination of the two mediators, the redox potential of the first mediator is on the negative potential side of the second mediator (in the case of FIG. 4), and the second mediator It is preferable to select a material whose electron transfer reaction rate between the mediator and the conductive member is faster than the electron transfer reaction rate between the first mediator and the conductive member.

  As another aspect of the combination of two mediators, the redox potential of the first mediator is on the positive potential side of the redox potential of the second mediator (in the case of FIG. 5), and the second mediator It is preferable to select a material whose electron transfer reaction rate between the mediator and the conductive member is faster than the electron transfer reaction rate between the first mediator and the conductive member.

  The present invention is particularly useful in the following cases. That is, a certain mediator can efficiently transfer electrons with an enzyme, but cannot efficiently transfer electrons between the mediator and the conductive member.

Here, the magnitude of the electron transfer reaction rate can be determined by comparing standard rate constants in the electrode reactions of the respective mediators. This standard rate constant is a measure of the kinetic ease of the electrode reaction. The standard speed constant can be calculated from, for example, charge transfer resistance that can be measured by performing impedance measurement. (Tetsugo Osaka, Noboru Oyama, Takeo Osaka, Electrochemical Basic Measurement Manual, Kodansha Scientific)
The enzyme electrode according to a preferred embodiment of the present invention includes an enzyme, at least one kind of first mediator for exchanging electrons with the enzyme, and a second for exchanging electrons with the first mediator. At least one of the mediators, the enzyme, the carrier for immobilizing the first mediator and the second mediator, the carrier in contact with the carrier, and the electrons of the first mediator or the second mediator It has an electroconductive member for performing delivery. The transfer of electrons between the enzyme and the conductive member is performed through at least the first mediator and the second mediator, and further, the transfer of electrons between the enzyme and the conductive member without passing through the mediator is performed. One or both of electrons passing between the mediator and the conductive member without passing through the second mediator may be generated.

  In the enzyme electrode according to the present invention, the enzyme can be stably immobilized on the conductive member by using a carrier for immobilization of the enzyme, and the enzyme carrying density per effective surface area of the conductive member. Can be increased. Furthermore, by using the first mediator capable of performing high-speed electron transfer with the enzyme and the second mediator for transporting charges between the first mediator and the conductive member, the enzyme and the high-speed transfer can be performed. Electron transfer can be performed, and as a result, the stability and current density of the enzyme electrode can be improved. The first mediator in the present invention has a higher electron transfer reaction rate with the enzyme as compared with the second mediator, and as a result, as compared with the enzyme electrode using only the second mediator. High current density can be obtained. On the other hand, the second mediator in the present invention has a higher charge transport capability than that of the first mediator and / or a higher electron transfer reaction rate with the electrode. A higher current density can be obtained as compared with an enzyme electrode using only the mediator. In the present invention, the optimum ratio of the enzyme, the first mediator, the second mediator, and the carrier is high so that the ratio of the first mediator is sufficiently high for electron transfer to and from the enzyme. The ratio of the two mediators is high enough to allow sufficient charge transport between the enzyme / conductive member, the ratio of the carrier is high enough to hold the enzyme and mediator, and the enzyme loading density is kept high enough. For this purpose, it is determined by these correlations. In this example, the weight of the enzyme is 1, the weight of the first mediator is 0.0001 to 1, more preferably 0.0002 to 0.1, the weight of the second mediator is 0.01 to 2, Preferably, 0.3 to 1, and the weight of the carrier is 0.05 to 3, more preferably 0.3 to 1.

  In FIG. 3, an example of the fixed state of the enzyme and the 1st and 2nd mediator in the enzyme electrode of this invention is shown. In this example, the enzyme 1, the first mediator 8, and the second mediator 9 are included in a layer made of a carrier on the surface of the conductive member (conductive substrate) 4. An example of the flow of electrons is indicated by arrows.

  In addition, the first mediator capable of high-speed electron transfer with the enzyme ensures efficient electron transfer between the second mediator that performs charge transport between the first mediator and the conductive member. Therefore, when this enzyme electrode is used as an anode or a negative electrode, the redox potential of the first mediator is on the negative potential side with respect to the second mediator (FIG. 4), and is used as a cathode or a positive electrode. In this case, it is preferable that the redox potential of the first mediator is on the positive potential side of the redox potential of the second mediator (FIG. 5). 4 and 5, the direction of the arrow indicated by E (upper) is the negative potential side, and the opposite direction (lower) is the positive potential side.

  The sensor which is one preferable form of this invention uses an enzyme electrode as a detection site | part for detecting a substance, It is characterized by the above-mentioned. As a typical configuration, an enzyme electrode is used as a working electrode and a set together with a reference electrode and a counter electrode, and the current detectable by the enzyme electrode (by the function of the enzyme fixed to the electrode) is detected, and these electrodes are detected. The structure utilized for the detection of the substance in the liquid which touches can be mentioned. A specific example is a sensor having the configuration shown in FIG. The sensor of FIG. 6 includes an anode (anode) 15, a platinum wire electrode 16, and, if necessary, a silver / silver chloride reference electrode 17, and lead wires 18 to 20 are wired to each electrode. Connected with potentiostat. This sensor is arranged in a storage region of the electrolyte 14 in the water jacket cell 12 that can be sealed with the lid 13. The substrate can be detected in the electrolyte by applying a potential to the working electrode and measuring the steady current. When measurement in an inert gas atmosphere is necessary, an inert gas such as nitrogen is introduced from the gas blowing port 22 at the outer end of the gas tube 23. Moreover, temperature can be performed by supply of the liquid for temperature control using the temperature control water inflow port 24 and the temperature control water discharge port 25. FIG.

  The configuration of the sensor according to the present invention is not particularly limited as long as it can be detected by an enzyme electrode. In addition to the high selectivity of the substrate specific to the enzyme used as the catalyst for the electrode reaction, this sensor has a high stability and current density due to the enzyme electrode using a carrier and multiple mediators. It is possible to simplify the apparatus and downsize the measurement site. In this case, the enzyme electrode can be used as a single layer as a plate or layer, or as a laminated structure of two or more layers. This sensor can detect a substance corresponding to the substrate of the enzyme used for the enzyme electrode. Examples of its use include a glucose sensor, a fructose sensor, a galactose sensor, an amino acid sensor, an amine sensor, a cholesterol sensor, Alcohol sensors, lactic acid sensors, oxygen sensors, hydrogen peroxide sensors, and the like. More specific application examples include blood glucose concentration, sensors that measure lactic acid concentration, sensors that measure the sugar content of fruits, and breathing. Examples include a sensor that measures the alcohol concentration in the air.

  A fuel cell according to a preferred embodiment of the present invention is characterized in that an enzyme electrode is used as at least one of an anode and a cathode. Also in this case, the enzyme electrode can be used as a single layer as a plate or layer, or as a laminated structure of two or more layers. Further, in the case of a laminated structure, the anode and the cathode may be arranged in a predetermined arrangement in the lamination direction. A typical configuration includes a reaction tank capable of storing an electrolytic solution containing a substance serving as a fuel, and an anode and a cathode arranged at a predetermined interval in the reaction tank, and at least one of the anode and the cathode The structure using the enzyme electrode concerning this invention can be mentioned. The fuel cell can be of a type that replenishes or circulates the electrolyte solution or a type that does not replenish or circulate the electrolyte solution. As long as the fuel cell can use an enzyme electrode, the type, structure, function, etc. of the fuel are not limited. At this time, the enzyme used as a catalyst for the enzyme electrode has a high substrate selectivity as compared with a noble metal catalyst (for example, platinum) generally used in the field of electrochemistry, No mechanism for isolating reactants at the other electrode is required, and as a result, the device can be simplified. This fuel cell can obtain a high driving voltage by being able to oxidize and reduce a substance with a low overvoltage by a high catalytic action peculiar to an enzyme used as an electrode reaction catalyst. An enzyme using a carrier and a plurality of mediators Long life and high output are possible due to the high stability and current density of the electrodes.

  An example of the fuel cell is shown in FIG. The configuration of the fuel cell is almost the same as that of the substrate measuring apparatus in the sensor shown in FIG. 6, and the same members are denoted by the same reference numerals. Instead of the sensor shown in FIG. 6, an electrode unit having a structure in which an anode 15 and a cathode 27 are laminated via a porous polypropylene film 26 is used, oxygen gas is introduced into the cell via the tube 23, and a fuel cell To act as.

  The electrochemical reaction apparatus, which is a preferred form of the present invention, obtains the quantitative property of the reaction, which is a characteristic of the electrochemical reaction in addition to the high selectivity and catalytic ability of the substrate specific to the enzyme used as the catalyst for the electrode reaction. As a result, it can be controlled quantitatively with high selectivity, high efficiency, and long life and high output can be achieved with high stability and current density by enzyme electrode using carrier and multiple mediators. It becomes. Also in this case, the enzyme electrode can be used as a single layer as a plate or layer, or as a laminated structure of two or more layers. As a typical configuration, a pair of electrodes and a reference electrode provided as necessary are arranged in a reaction layer capable of storing a reaction liquid, and an electric current is passed between the pair of electrodes in the reaction liquid. An example is a configuration in which an electrochemical reaction is caused in a substance to obtain a desired reaction product, decomposition product, and the like. The enzyme electrode according to the present invention is used for at least one of a pair of electrodes. The apparatus configuration related to the type of reaction solution and reaction conditions is not particularly limited as long as the enzyme electrode can be used. For example, it can be used for obtaining a reaction product by an oxidation-reduction reaction or obtaining a target decomposition product. This electrochemical reaction apparatus can selectively carry out the reaction of a substance corresponding to the enzyme substrate used for the enzyme electrode. Examples of its use include glucose, fructose, galactose, amino acid, amine, cholesterol. Examples include oxidation of alcohol, lactic acid, and reduction reaction of oxygen and hydrogen peroxide. More specific application examples include selective oxidation of cholesterol in the presence of ethanol, reduction reaction of oxygen at low overvoltage, etc. Can be mentioned.

  In the present invention, the carrier immobilizes at least an enzyme on the conductive member. Examples include (1) a polymer compound, (2) an inorganic compound, and (3) an organic compound, which has a covalent bond in the molecule and binds at least an enzyme and a conductive member ( A function of binding enzymes to each other may be added), and at least one of these three types can be used. These carriers preferably have a charge opposite to the surface charge of the enzyme under the conditions of use of the electrode when the enzyme is immobilized on the electrode. This carrier may hold the enzyme by covalent bonding, electrostatic interaction, spatial confinement, etc., and as a result, by physical adsorption on the electrode or a binder polymer used for bonding the electrode. It is possible to hold the enzyme more stably and at a high density as compared with the enzyme holding method. As examples of the polymer compound for the carrier, examples of the conductive polymer compound include polyacetylenes, polyarylenes, polyarylene vinylenes, polyacenes, polyarylacetylenes, polydiacetylenes, polynaphthalenes, polypyrroles. Polyanilines, polythiophenes, polythienylene vinylenes, polyazulenes, polyisothianaphthenes, and other polymers include polystyrene sulfonic acid, polyvinyl sulfate, dextran sulfate, chondroitin sulfate, polyacrylic acid, polymethacrylic acid. Acid, polymaleic acid, polyfumaric acid, polyethyleneimine, polyallylamine hydrochloride, polydiallyldimethylammonium chloride, polyvinylpyridine, polyvinylimidazole, polylysine, deoxyribonucleic acid, ribonucleic acid, pectin , Silicone resins, cellulose, agarose, dextran, chitin, polystyrene, polyvinyl alcohol, nylon. Examples of inorganic compounds for carriers include metal chalcogenide compounds containing at least one element selected from In, Sn, Zn, Ti, Al, Si, Zr, Nb, Mg, Ba, Mo, W, V, and Sr Is mentioned. Examples of organic compounds that are organic compounds and have a covalent bond in the molecule, and the enzyme and the conductive member, and / or the carrier that binds the enzyme and the enzyme include hydroxyl group, carboxyl group, amino group Group, aldehyde group, hydrazino group, thiocyanate group, epoxy group, vinyl group, halogen group, acid ester group, phosphate group, thiol group, disulfide group, dithiocarbamate group, dithiophosphate group, dithiophosphonate group, thioether group, thio Examples thereof include compounds containing at least one functional group selected from a sulfate group and a thiourea group. Representative examples include glutaraldehyde, polyethylene glycol diglycidyl ether, cyanuric chloride, N-hydroxysuccinimide ester, dimethyl-3,3′-dithiopropionimidate hydrochloride, 3,3′-dithio-bis (sulphossucciniimi Zilppropionate), cystamine, alkyldithiol, biphenylenedithiol, benzenedithiol.

  In the present invention, the conductive member holds an enzyme, a mediator, and a carrier for fixing them, and is electrically connected to an external circuit when the enzyme electrode is used. The conductive member may be a material having conductivity, sufficient rigidity during storage and measurement, and sufficient electrochemical stability under the conditions in which the electrode is used. Examples of materials used for the conductive member include metals, conductive polymers, metal oxides, and carbon materials. Examples of this metal include Au, Pt, Ag, Co, Pd, Rh, Ir, Ru, Os, Re, Ni, Cr, Fe, Mo, Ti, Al, Cu, V, Nb, Zr. , Sn, In, Ga, Mg, Pb, Si, and W include those containing at least one element, and these may be alloys or plated. Examples of the conductive polymer include polyacetylenes, polyarylenes, polyarylene vinylenes, polyacenes, polyarylacetylenes, polydiacetylenes, polynaphthalenes, polypyrroles, polyanilines, polythiophenes, polythienylene. Examples include those containing at least one compound among vinylenes, polyazulenes, and polyisothianaphthenes. This metal oxide may be improved or imparted with conductivity by another conductive material. Examples of the metal oxide include those containing at least one element among In, Sn, Zn, Ti, Al, Si, Zr, Nb, Mg, Ba, Mo, W, V, and Sr. Examples of the conductive material at this time include metals, conductive polymers, and carbon materials. This carbon material may be improved or imparted with conductivity by another conductive material. Examples of the carbon material include graphite, carbon black, carbon nanotube, carbon nanohorn, fullerene compound and derivatives thereof. In the present invention, a conductive material having a conductivity of 0.1 S / cm or more and 700000 S / cm or less is preferable. For example, a material of 1 S / cm to 100000 S / cm or a material of 100 S / cm to 100000 S / cm can be used. S (Siemens) is the inverse of Ohm (1 / Ω). Even when a porous structure is used for the enzyme electrode, it is desirable to use a conductive member in the above range.

  The conductive member has a large number of gaps communicating with each other, and has a structure in which these gaps communicate with the outside of the conductive member directly or indirectly through other gaps. Those capable of immobilizing the enzyme and the first and second mediators are preferred. Such a conductive member has a large number of voids communicating with the outside, and has a structure in which walls that partition the void are integrally formed from the constituent materials, and materials that constitute the walls that partition the void. Are preferably bonded firmly. As a constituent material of such a conductive member, the conductive metals, polymers, metal oxides, and carbon materials mentioned above can be used. The plurality of voids of the conductive member are connected one-dimensionally, two-dimensionally or three-dimensionally, and two or more of these connected forms may be mixed. Examples of voids connected in one dimension are columnar voids, examples of voids connected in two dimensions are mesh-like voids, examples of voids connected in three dimensions are spongy, formed after joining fine particles Examples include voids and voids in structural materials created using them as templates. These voids need to be large enough to allow the enzyme to be introduced and / or to sufficiently flow and diffuse the substrate, and small enough to provide a ratio of the effective surface area to the projected area. Examples of the average diameter of the void include a range of 5 nm to 500 μm, more preferably 10 nm to 10 μm. Further, the thickness of the conductive member having voids is small enough to allow the enzyme to be introduced uniformly to the deep part of the conductive member and / or to sufficiently flow and diffuse the substrate, and the projected area of the conductive member. It is necessary that the ratio of the effective surface area to the surface is sufficiently large, and examples of the thickness of the conductive member having the void include 100 nm to 1 cm, more preferably 1 μm to 5 mm. The ratio of the effective surface area to the projected area of the conductive member having the gap needs to be so large that the ratio of the effective surface area to the projected area is sufficiently obtained. As an example, the ratio is 10 times or more, more preferably 100 More than double. Further, the porosity of the conductive member having voids is such that the ratio of the effective surface area to the projected area of the conductive member is sufficiently obtained and / or that a sufficient amount of enzyme and carrier can be introduced. / Or large enough to allow sufficient flow and diffusion of the substrate, and small enough to obtain sufficient mechanical strength, for example, 20% to 99%, more preferably 30% More than 98% is mentioned. In addition, the porosity after enzyme immobilization must be large enough to allow sufficient flow of the electrolyte and diffusion of the substrate, and small enough to obtain a high filling rate of the carrier and enzyme. As an example, 15% It is 98% or less and more preferably 25% or more and 95% or less.

  The metal conductive member forming material having a large number of voids corresponds to foam metal, electrodeposited metal, electrolytic metal, sintered metal, fibrous metal, or one or more of these types. Materials. In addition, as an example of a method for producing a conductive polymer having voids, a material as a mold that forms a void portion is placed in the conductive polymer and molded into a predetermined shape, and then the mold is molded. Method of removing the substance; Method of removing a substance as a template after containing a substance as a template of a void portion in a precursor of a conductive polymer and polymerizing the precursor to form a polymer; A method of forming a layer composed of particles serving as a mold constituting a portion, filling a void between the particles with a polymer to form a layer, and removing the particles from this layer; a mold constituting a portion serving as a void; Forming a layer comprising particles, filling a void between the particles with a polymer precursor to form a layer, polymerizing the precursor to form a polymer layer, and then removing the particles; A method can be mentioned that is used in the production of a resin.

  On the other hand, a conductive member having a large number of voids made of a metal oxide can be obtained by methods such as electrodeposition, sputtering, sintering, chemical vapor deposition (CVD), electrolysis, and combinations thereof. In addition, a conductive member having a large number of voids is obtained by molding fibers, particles, and the like made of graphite, carbon black, carbon nanotubes, carbon nanohorns, fullerene compounds, and derivatives thereof into a predetermined shape and then sintering. be able to.

  Regarding the pore diameter distribution of the porous member, a member having a constant pore diameter (or porosity) in the porous structure in the thickness direction of the conductive member can be used.

  Further, as shown below, the pore size or the porosity of the porous member may have a gradient distribution. FIGS. 8A to 8D show an example in which a porous structure is used as the structure of the conductive member. In the figure, 801 is an electrolyte layer, 802 is a hole, and 803 is a conductive member. Reference numeral 804 denotes a support substrate that can be used as necessary. As shown in the figure, it is preferable that the pore diameter of the pores provided in the conductive member is large on the electrolyte layer side and small on the inside thereof. That is, in the conductive porous member applicable to the present invention, it is desirable that the pore diameter on the surface side of the conductive porous member is larger than the pore diameter inside the conductive porous member. The difference between the two pore diameters is 2 times or more, more preferably 4 times or more, and further preferably 10 times or more. As an upper limit, it is 1000 times or less, for example. The porosity may be the same between two regions having different pore diameters. More preferably, the pore diameter and porosity of the pores provided in the conductive member are both large on the electrolyte layer side (that is, the outer surface side of the conductive member) and small on the inside thereof. .

  The pore diameter and porosity of the porous body can be measured by nitrogen gas adsorption measurement (BET (Brunauer-Emmett-Teller) method). For example, it can be measured by AUTOSORB-1 (manufactured by Quantachrome Instruments). In addition, when calculating the hole diameter on the surface of the member, some (for example, about 50 to 300) hole diameters can be measured and calculated from an SEM (scanning electron microscope) photograph.

  In the present invention, the mediator promotes the transfer of electrons between the enzyme / mediator / conductive member. The mediator may be chemically bonded to the carrier and / or the enzyme. Examples include metal complexes, quinones, heterocyclic compounds, nicotinamide derivatives, and flavin derivatives. As this metal complex, Os, Fe, Ru, Co, Cu, Ni, V, Mo, Cr, Mn, Pt, Rh, Pd, Mg, Ca, Sr, Ba, Ti, Ir, Zn, Cd are used as the central metal. , Hg, and W containing at least one element can be used. In addition, the ligand of this metal complex contains nitrogen, oxygen, phosphorus, sulfur, carbon atoms and forms a complex with the central metal via at least these atoms, or a cyclopentadienyl ring as a skeleton. The thing which has as is mentioned. Examples include pyrrole, pyrazole, imidazole, 1,2,3- or 1,2,4-triazole, tetrazole, 2,2′-biimidazole, pyridine, 2,2′-bithiophene, 2,2′-bipyridine. 2,2 ′: 6 ′, 2 ″ -terpyridine, ethylenediamine, porphyrin, phthalocyanine, acetylacetone, quinolinol, ammonia, cyanide, triphenylphosphine oxide and derivatives thereof. Examples of quinones include quinone, benzoquinone, anthraquinone, naphthoquinone, pyrroloquinoline quinone (PQQ), tetracyanoquinodimethane, and derivatives thereof. Examples of heterocyclic compounds include phenazine, phenothiazine, viologen, and derivatives thereof. Examples of the nicotinamide derivative include nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate. An example of a flavin derivative is flavin adenine dinucleotide (FAD).

  In the present invention, the enzyme is an enzyme that catalyzes a redox reaction, and may be a combination of a plurality of enzymes. Examples include glucose oxidase, galactose oxidase, bilirubin oxidase, pyruvate oxidase, D- or L-amino acid oxidase, amine oxidase, cholesterol oxidase, choline oxidase, xanthine oxidase, sarcosine oxidase, L-lactate oxidase, ascorbate oxidase, cytochrome Oxidase, alcohol dehydrogenase, glutamate dehydrogenase, cholesterol dehydrogenase, aldehyde dehydrogenase, glucose dehydrogenase, fructose dehydrogenase, sorbitol dehydrogenase, lactate dehydrogenase, malate dehydrogenase, glycerol dehydrogenase, 17B hydroxysteroid dehydrogenase, et Toradiol 17B dehydrogenase, amino acid dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, 3-hydroxysteroid dehydrogenase, diaphorase, catalase, peroxidase, glutathione reductase, NADH-cytochrome b5 reductase, NADPH-adrenoxin reductase, cytochrome b5 reductase, address Examples include nodoxin reductase and nitrate reductase.

  Moreover, as a substrate of this enzyme, the compound corresponding to each enzyme is mentioned, Organic substance, oxygen, hydrogen peroxide, water, nitrate ion is mentioned. Examples of this organic substance include saccharides, alcohols, carboxylic acids, quinones, nicotinamide derivatives, and flavin derivatives. The saccharides may also include cellulose and starch sugar polysaccharides.

In the step of fixing the carrier in the present invention, it is important to uniformly fix the carrier to the conductive member. The step of immobilizing the carrier may include the step of immobilizing the enzyme and / or the step of immobilizing the mediator at the same time. As an example of the process of fixing this carrier,
(1) a step of immersing a conductive member having voids in a carrier solution or dispersion;
(2) A step of applying, injecting, and spraying a carrier solution or dispersion onto a conductive member having a gap,
(3) Dipping a conductive member having voids in a carrier precursor solution or dispersion, or applying, injecting, and spraying a carrier precursor solution or dispersion to a conductive member having voids. Performing the process, and then immobilizing the precursor by hydrolysis, polymerization, and crosslinking reaction,
Etc.

  EXAMPLES The present invention will be described in more detail with reference to examples below, but the method of the present invention is not limited to these examples.

First, a method for preparing a member used in the present invention will be described.
(Preparation Example 1)
A commercially available silica colloid dispersion (Nissan Chemical, average particle size 100 nm) was substituted with ethanol, and a washed metal plate (1 cm square, thickness 0.3 mm, Nilaco) was placed in the dispersion, and ethanol was evaporated at 30 ° C. Thus, a porous film made of silica spheres is prepared. By performing this step a plurality of times, the thickness of the porous membrane made of silica spheres is increased (film thickness 100 μm). After heating at 200 ° C. for 3 hours, washing with ethanol and using this porous membrane as a working electrode in a 0.1 M 3,4-ethylenedioxythiophene, 0.1 M lithium perchlorate acetonitrile solution, a platinum counter electrode Electropolymerization is performed at a potential of 1.1 V (vsAg / AgCl) using a potentiostat in a triode cell using a silver / silver chloride reference electrode. With reference to the electrolysis current profile, the electrolysis time was adjusted so that the polymerization time was approximately the same as the thickness of the silica sphere porous membrane. After electropolymerization, a conductive member (film thickness 100 μm) having a large number of voids made of conductive poly (3,4-ethylenedioxythiophene) by removing silica spheres in a 20% hydrofluoric acid solution for 2 days and removing silica spheres. ) Is prepared.
(Preparation Example 2)
Acicular indium tin oxide (ITO, Sumitomo Metal Mining, 30-100 nm length, aspect ratio of 10 or more) is dispersed in terpinol, the viscosity is adjusted with ethyl cellulose, and an ITO paste is prepared. ITO paste is printed on a washed metal plate (1 cm square, thickness 0.3 mm, Nilaco) by screen printing, and sintered at 450 ° C. for 1 hour, ITO porous sintered electrode (film thickness 100 μm) To prepare. Further, plasma chemical vapor deposition (CVD) is performed thereon to deposit about 10 nm of ITO to prepare a conductive member having a large number of voids made of ITO. (Preparation Example 3)
Natural graphite particles (particle size 11 μm) are 10 wt. % Polyvinylidene fluoride and N-methyl-2-pyrrolidone are mixed to dissolve the polyvinylidene fluoride. Further, the kneaded and prepared graphite paste was formed into a film having a diameter of 11.3 mm and a thickness of 0.5 mm, dried at 60 ° C., then heated at 240 ° C. and further vacuum dried at 200 ° C. A conductive member having a large number of voids between the graphite particles is prepared.
(Preparation Example 4)
A method for preparing N ^6- (2-aminoethyl) FAD represented by the chemical formula (1) will be described. An equimolar amount of ethyleneimine is added to a 10% aqueous solution of FAD to adjust the pH to 6-6.5, and the mixture is reacted at 50 ° C. for 6 hours. The reaction solution is cooled and added to ethanol in an ice bath to collect the precipitate. The collected precipitate is subjected to anion exchange chromatography and reverse-layer high performance chromatography to obtain a purified N ^6- (2-aminoethyl) FAD represented by the chemical formula (1).

(Preparation Example 5)
A method for preparing the ferrocene derivative represented by the chemical formula (2) will be described. In 200 mL of DMF, 4.7 g of 6-aminocaproic acid is dissolved, 2.1 g of ferrocenecarbaldehyde dissolved in 100 mL of DMF is added, and the mixture is stirred at 100 ° C. for 1 hour. 1 g of a saturated aqueous solution of sodium borohydride was added, and the mixture was further stirred at room temperature for 1 hour. The solvent was distilled off under reduced pressure, and a silica column was applied with dichloromethane / methanol = 10/1 solvent to obtain a ferrocene derivative compound represented by chemical formula (2). obtain. The compound is identified by ¹ H-NMR.

(Preparation Example 6)
A method for synthesizing an osmium complex-containing polymer represented by chemical formula (3) will be described. To 6 mL of 1-vinylimidazole, 0.50 g of azobisisobutyronitrile is added, and the reaction is performed at 70 ° C. for 2 hours under an argon atmosphere. The reaction solution is air-cooled, and the resulting precipitate is dissolved in methanol and added dropwise to acetone with strong stirring, and the precipitate is collected by filtration to obtain poly-1-vinylimidazole. On the other hand, 1.2 g of 4,4′-dimethyl-2,2′-bipyridine and 1.4 g of ammonium hexachloride osmate are added with 5 mL of ethylene glycol and reacted at 140 ° C. for 24 hours in a nitrogen atmosphere. After the reaction solution is air-cooled, impurities are collected by filtration, and the filtrate is distilled off under reduced pressure to obtain Os (4,4′dimethyl-2,2′-bipyridine) ₂ dichloride chloride salt. 0.13 g of Os (4,4′dimethyl-2,2′-bipyridine) ₂ dichloride chloride salt and 0.20 g of polyvinylimidazole are added to 200 mL of ethanol and refluxed for 3 days under a nitrogen atmosphere. After the filtrate is filtered, it is dropped into 1 L diethyl ether under strong stirring, and the generated precipitate is recovered and dried to obtain an osmium complex polymer represented by chemical formula (3). Identification of the compound is performed by elemental analysis.

(Preparation Example 7)
A method for preparing a ferrocene-containing polymer represented by chemical formula (4) will be described. 1.6 g of 1-vinylimidazole, 0.71 g of 1-vinylferrocene and 6.2 mg of azobisisobutyronitrile are added to 3.0 mL of toluene, and the reaction is carried out at 70 ° C. for 2 hours under an argon atmosphere. The reaction solution is air-cooled, the reaction solution is dropped into 1 L of diethyl ether, and the precipitate is collected by filtration to obtain a ferrocene-containing polymer represented by the chemical formula (4).

(Preparation Example 8)
A method for synthesizing the complex polymer represented by the chemical formula (5) will be described. To 100 g of 40% aqueous glyoxal solution, 370 mL of concentrated aqueous ammonia was added dropwise in an ice bath, stirred at 45 ° C. for 24 hours, air-cooled, the precipitate was filtered, dried in vacuo at 50 ° C. for 24 hours, and 2,2′-biimidazole Get. Identification is performed by thin layer chromatography on silica gel (10% methanol / 90% chloroform). To a solution of 2,2′-biimidazole (4.6 g) in N, N′-dimethylformamide (DMF) in 100 mL was added 2.7 g of sodium hydride in an ice bath under a nitrogen atmosphere, and the mixture was stirred at room temperature for 1 hour. 12.8 g of a DMF solution of methyl paratoluenesulfonate is added dropwise over 20 minutes and stirred at room temperature for 4 hours. The solvent was distilled off at 50 ° C. under vacuum, and the remaining solid was washed with 50 mL hexane and vacuum dried at 160 ° C. to obtain colorless and transparent crystals of N, N′-dimethyl-2,2′-biimidazole. Identification is performed by ¹ H-NMR. To 100 mL of DMF solution of 2,2′-biimidazole in 10 g, 3.3 g of sodium hydride was added in an ice bath under a nitrogen atmosphere, stirred for 1 hour in an ice bath, and methyl iodide (4.6 mL) was added dropwise. The mixture is stirred for 30 minutes in an ice bath and further for 12 hours at room temperature. The reaction solution is poured into 300 mL of ethyl acetate and filtered, and then the solvent is distilled off under reduced pressure under reduced pressure. The residue is dissolved in boiling ethyl acetate and filtered. The filtrate is again boiled in ethyl acetate and saturated with 300 mL of hexane. Let stand in a freezer for 12 hours, grow crystals, collect by suction filtration, recrystallize from ethyl acetate / hexane solvent to obtain N-methyl-2,2′-biimidazole. Identification is performed by ¹ H-NMR. 1 g of N-methyl-2,2′-biimidazole was dissolved in 80 mL of DMF, 0.32 g of sodium hydride was added under a nitrogen atmosphere, stirred for 1 hour in an ice bath, and then 2.5 g of N- (6 -Bromohexyl) phthalimide and 1.0 g of sodium iodide are gradually added and stirred at 80 ° C. for 24 hours under a nitrogen atmosphere. Cool to room temperature, pour into 150 mL of water, extract twice with 150 mL of ethyl acetate, wash with aqueous sodium chloride, dry over sodium sulfate, and evaporate under reduced pressure. Purification with a neutral alumina column (ethyl acetate / hexane 10-40%) gives N-methyl-N ′-(6-phthalimidohexyl) -2,2′-biimidazole. Identification is performed by ¹ H-NMR.

Dissolve 2.5 g of N-methyl-N ′-(6-phthalimidohexyl) -2,2′-biimidazole in 25 mL of ethanol, add 0.39 g of hydrazine hydride, reflux for 2 hours, and cool to room temperature After filtration, the product was collected on a silica gel column with ethanol, and then with 10% ammonia in acetonitrile, and evaporated under reduced pressure to give N- (6-aminohexyl) -N′-methyl-2,2′-biphenyl. Imidazole is obtained. Identification is performed by ¹ H-NMR. 1.1 g of N-methyl-2,2′-biimidazole and 1.4 g of ammonium hexachloride osmate are dissolved in 40 mL of ethylene glycol and stirred at 140 ° C. for 24 hours under a nitrogen atmosphere. 0.8 g of N- (6 -Aminohexyl) -N′-methyl-2,2′-biimidazole dissolved in 5 mL of ethylene glycol is added, and the mixture is further stirred for 24 hours. The filtrate was diluted with 200 mL of water, 40 mL of an anion exchange resin (DOWEX® 1X4) was added, and the mixture was stirred in air for 24 hours. The solution is gradually poured into 150 mL of an aqueous solution of 10.2 g of ammonium hexafluorophosphate, the precipitate is suction filtered, dissolved in acetonitrile, reprecipitated with an aqueous solution of ammonium hexafluorophosphate, washed with water, and washed at 45 ° C. for 24 hours. Vacuum-dried for hours and osmium (III) (N, N′-dimethyl-2,2′-biimidazole) ₂ (N- (6-aminohexyl) -N′-methyl-2,2′-biimidazole) Get hexafluorophosphate. Identification is performed by elemental analysis.

  Add 20 g polyvinylpyridine (average molecular weight 150,000), 5.6 g 6-bromohexanoic acid to 150 mL DMF, stir with a stirrer at 90 ° C. for 24 hours, cool to room temperature, and add to 1.2 L of ethyl acetate with strong stirring. Pour slowly. Decant the solvent, dissolve the solid in methanol, filter, reduce the solvent to about 200 mL by distillation under reduced pressure, reprecipitate in 1 L of diethyl ether, vacuum dry at 50 ° C. for 24 hours, Grind and dry for 48 hours to obtain poly (4- (N- (5-carboxypentyl) pyridinium) -co-4-vinylpyridine). 0.52 g poly (4- (N- (5-carboxypentyl) pyridinium) -co-4-vinylpyridine) was dispersed in 10 mL DMF and 0.18 g O- (N-succinimidyl) -N, N , N ′, N′-tetramethyluronium tetrafluoroborate (TSTU) was added and stirred for 15 minutes, 0.1 mL of N, N-diisopropylethylamine was added, stirred for 8 hours, and 0.89 g of poly (4 -(N- (5-carboxypentyl) pyridinium) -co-4-vinylpyridine) was added and stirred for 5 minutes. Further, 0.1 mL of N, N-diisopropylethylamine was added, and the mixture was stirred at room temperature for 24 hours. Of ethyl acetate. The precipitate is filtered and collected, added to 30 mL acetonitrile, 40 mL DOWEX® 1X4, 100 mL water is added and stirred for 36 hours to dissolve the polymer. After performing suction filtration, the solution is concentrated to 50 mL and extruded with a molecular weight 10,000 cut-off filter (Millipore) at a nitrogen pressure of 275 kPa. Further, the polymer is passed through a DOWEX (registered trademark) 1X4 column with an aqueous solvent and dialyzed in water to obtain a polymer (chloride salt) represented by the chemical formula (5).

(Preparation Example 9)
A method for synthesizing the osmium complex polymer represented by the chemical formula (6) will be described. 0.5 g of azobisisobutyronitrile is added to 6 mL of 1-vinylimidazole, and the reaction is performed at 70 ° C. for 2 hours under an argon atmosphere. The reaction solution is air-cooled, and the resulting precipitate is dissolved in methanol and added dropwise to acetone under strong stirring, and the precipitate is collected by filtration to obtain poly-1-vinylimidazole. 0.76 g of 2,2 ′: 6 ′, 2 ″ -terpyridine and 1.42 g of ammonium hexachloride osmate are added to 5 mL of ethylene glycol and refluxed for 1 hour under an argon atmosphere. Further, 0.60 g of 4,4′dimethyl-2,2′-bipyridine was added to the reaction solution, and the mixture was further refluxed for 24 hours. The reaction solution was air-cooled, and impurities were collected by filtration. An osmium (2,2 ′: 6 ′, 2 ″ -terpyridine) (4,4′-dimethyl-2,2′-bipyridine) chloride chloride salt shown in (5) is obtained. Add 200 mL of ethanol to 0.38 g of (2,2 ′: 6 ′, 2 ″ -terpyridine) (4,4′dimethyl-2,2′-bipyridine) chloride chloride salt and 0.2 g of polyvinylimidazole. After refluxing in a nitrogen atmosphere for 3 days and filtering the filtrate, it was dropped into 1 L diethyl ether under strong stirring, and the produced precipitate was recovered and dried to obtain an osmium complex represented by Chemical Formula 6. Identification of the compound is performed by elemental analysis.

(Preparation Example 10)
A method for preparing apoglucose oxidase from which FAD has been removed is described. Glucose oxidase (Aspergillus niger) is dissolved in 0.25 M sodium phosphate buffer (pH 6, 3 mL) containing 30% glycerol. The solution is cooled to 0 ° C. and adjusted to pH 1.7 with a pH 1.1 0.025 M sodium phosphate buffer-sulfuric acid solution containing 30% glycerol. This solution is passed through a (Sephadex® G-25) column with 0.1M sodium phosphate solution (pH 1.7) containing 30% glycerol as a solvent and collected while detecting at a wavelength of 280 nm. Dextran-coated charcoal is added to the solution and adjusted to pH 7 by adding 1M sodium hydroxide solution and then stirred at 4 ° C. for 1 hour. The purified solution is centrifuged, filtered through a 0.45 μm filter, and dialyzed using 0.1 M sodium phosphate buffer to obtain apoglucose oxidase.
(Preparation Example 11)
A method for preparing apo D-amino acid oxidase from which FAD has been removed will be described. The D-amino acid oxidase (pig kidney) was exchanged four times against 0.1 M pyrophosphate buffer (pH 8.5) containing 1 M potassium bromide and 3.0 mM ethylenediaminetetraacetic acid at 4 ° C. Then, dialysis was performed for 2 days to confirm that the color of holo D-amino acid oxidase disappeared. Subsequently, apo D-amino acid oxidase is obtained by excluding potassium bromide by dialysis for 2 days while exchanging the solution three times against 0.1 M pyrophosphate buffer (pH 8.5).
(Preparation Example 12)
A method for preparing PQQ-modified FAD is described. 1 mM N ^6- (2-aminoethyl) FAD described in Preparation Example 4 in 10 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide in 0.01 M HEPES buffer solution (pH 7.3) PQQ-modified FAD is obtained by adding 10 mM PQQ, stirring for 2 hours, ultrafiltration and removing excess PQQ.
(Preparation Example 13)
A method for preparing ferrocene-modified FAD is described. A ferrocene-modified FAD is obtained by using the ferrocene derivative described in Preparation Example 5 in place of PQQ in Preparation Example 12.
(Preparation Example 14)
A method for preparing PQQ-modified glucose oxidase is described. 0.2 mg / mL PQQ-modified FAD described in Preparation Example 12 was added to 0.1 mg phosphate buffer (pH 7.0) of apoglucose oxidase described in Preparation Example 10 at 4 mg / mL, and the mixture was heated at 25 ° C. for 4 hours. Excess PQQ-modified FAD is removed by stirring at 4 ° C. for 12 hours and ultrafiltration to prepare PQQ-modified glucose oxidase.
(Preparation Example 15)
A method for preparing ferrocene-modified glucose oxidase is described. Ferrocene-modified glucose oxidase is obtained by using the ferrocene derivative described in Preparation Example 13 in place of the PQQ-modified FAD of Preparation Example 14.
(Preparation Example 16)
A method for preparing ferrocene-modified D-amino acid oxidase is described. Ferrocene-modified D-amino acid oxidase is obtained by using the apoamino acid oxidase described in Preparation Example 11 in place of the apoglucose oxidase described in Preparation Example 10 of Preparation Example 15.
(Preparation Example 17)
A method for preparing an enzyme electrode for a cathode used in a fuel cell is described. In a sample tube, the osmium polymer described in Preparation Example 9 was dissolved in 5 mL of water at a concentration of 10 mg / mL, added to 1 mL of 0.2 M citrate buffer (pH 5), and further 30 mg / mL laccase (Coriolus). 1 mL of aqueous solution. After stirring, 2 mL of a 10 mg / mL aqueous solution of polyethylene glycol diglycider ether is added and stirred. Then cut nickel alloy foam metal (Mitsubishi Materials, elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5mm, gold plating thickness 0.5μm, pore diameter 50μm) into 1cm square. Then, after washing and UV ozone treatment, it is immersed in the previously prepared enzyme and osmium polymer solution, pulled up, and dried in a desiccator for 2 days to prepare an enzyme electrode.

Next, a method for producing the enzyme electrode of the present invention will be described.
Example 1
In a sample tube, prepare 0.1 mL of sodium bicarbonate, 1 mL of 40 mg / mL aqueous solution of PQQ-modified glucose oxidase described in Preparation Example 14, add 0.5 mL of 7 mg / mL aqueous sodium periodate, and add 1 mL in the dark. Stir for hours. A metal plate (1 cm square) was prepared by adding 0.42 g of potassium hexacyanoferrate (III) to 0.4 mL of an aqueous solution of 10% polypyrrole in 6 mL and 2.5 mg / mL polyethylene glycol diglycidel ether. , Thickness 0.3 mm, Niraco) was washed, UV ozone treated was immersed, pulled up, dried in a desiccator for 2 days to prepare an enzyme electrode.
(Example 2)
Instead of the 10% polypyrrole solution of Example 1 and using 10% polyaniline solution instead of 0.42 g potassium hexacyanoferrate (III), 0.63 g potassium octacyanotungstate (IV), respectively. Then, an enzyme electrode is prepared.
(Example 3)
Prepare 1 mL of an aqueous solution of PQQ-modified glucose oxidase as described in Preparation Example 14 in 0.1 M sodium bicarbonate, 40 mg / mL in a sample tube, add 0.5 mL of 7 mg / mL sodium periodate aqueous solution and add 1 mL in the dark. Stir for hours. To this was added 6 mL of an aqueous solution of an osmium complex polymer shown in Preparation Example 6 of 10 mg / mL and 0.4 mL of 2.5 mg / mL polyethylene glycol diglycidel ether, and a metal plate (1 cm square, thickness 0.3 mm). , Nilaco), UV ozone treated, soaked, lifted and dried in a desiccator for 2 days to prepare an enzyme electrode.
Example 4
In a sample tube, prepare 0.1 mL of sodium bicarbonate, 1 mL of 40 mg / mL aqueous solution of PQQ-modified glucose oxidase described in Preparation Example 14, add 0.5 mL of 7 mg / mL aqueous sodium periodate, and add 1 mL in the dark. Stir for hours. To this, 6 mL of an aqueous solution of polyvinylimidazole described in Preparation Example 6 of 4 mg / mL, 0.4 mL of potassium hexacyanoferrate (III) was added to 0.4 mL of a 2.5 mg / mL aqueous solution of polyethylene glycol diglycider ether. A metal plate (1 cm square, thickness 0.3 mm, Nilaco) is washed and immersed in a product that has been subjected to UV ozone treatment, pulled up, and dried in a desiccator for 2 days to prepare an enzyme electrode.
(Example 5)
An enzyme electrode is prepared by using 0.63 g potassium octacyanotungstate (IV) instead of 0.42 g potassium hexacyanoferrate (III) in Example 4.

(Example 6)
An enzyme electrode is prepared by using the ferrocene-modified glucose oxidase described in Preparation Example 15 in place of the PQQ-modified glucose oxidase of Example 5.
(Example 7)
An enzyme electrode is prepared by using the ferrocene-modified amino acid oxidase described in Preparation Example 16 in place of the ferrocene-modified glucose oxidase of Example 6.
(Example 8)
An enzyme electrode is prepared by using polyvinyl pyridine (average molecular weight 150,000) instead of polyvinyl imidazole described in Preparation Example 6 of Example 4.
Example 9
An enzyme electrode is prepared by using poly-L-lysine (average molecular weight 150,000) instead of the aqueous solution of polyvinyl imidazole described in Preparation Example 6 of Example 5.
(Example 10)
An enzyme electrode is prepared by using the ferrocene-containing polymer described in Preparation Example 7 in place of the osmium complex polymer shown in Preparation Example 6 of Example 3.

(Example 11)
Instead of the gold plate of Example 1, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Example 12)
Instead of the gold plate of Example 2, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Example 13)
Instead of the gold plate of Example 3, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Example 14)
Instead of the gold plate of Example 4, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Example 15)
Instead of the gold plate of Example 5, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).

(Example 16)
Instead of the gold plate of Example 6, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Example 17)
Instead of the gold plate of Example 7, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Example 18)
Instead of the gold plate of Example 8, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Example 19)
Instead of the gold plate of Example 9, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Example 20)
Instead of the gold plate of Example 10, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).

(Example 21)
Instead of the metal plate of Example 3, an enzyme electrode is prepared by using a conductive member having a void of a poly (3,4-ethylenedioxythiophene) electrode described in Preparation Example 1.
(Example 22)
An enzyme electrode is prepared by using the ITO conductive member described in Preparation Example 2 in place of the metal plate of Example 3.
(Example 23)
Instead of the gold plate of Example 3, an enzyme electrode is prepared by using a transparent conductive glass (Asahi Glass, 8Ω / sq) of fluorine-doped tin oxide.
(Example 24)
An enzyme electrode is prepared by using the conductive member having the graphite voids shown in Preparation Example 3 instead of the metal plate of Example 3.

(Comparative Example 1)
A metal plate (1 cm square, 0.3 mm thick, Niraco), which has been washed and UV ozone treated, is immersed in a 0.02 M cystamine aqueous solution for 2 hours, pulled up and washed with water to prepare a cystamine modifying member. PQQ modification is performed by immersing in 0.01 M HEPES buffer solution of 3 mM PQQ, 10 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide for 1 hour and washing with water. 1 mM N ^6- (2-aminoethyl) FAD described in Preparation Example 4 in 10 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide in 0.01 M HEPES buffer solution (pH 7.3) The previous PQQ-modified electrode is immersed for 2 hours and washed with water to perform FAD modification. Furthermore, it was immersed in a 0.1 M phosphate buffer solution (pH 7.0) of apoglucose oxidase described in Preparation Example 10 at 4 mg / mL for 4 hours at 25 ° C. and then pulled up for 12 hours at 4 ° C. 0) Prepare an enzyme electrode by immersing in 1 hour.
(Comparative Example 2)
An enzyme electrode is prepared by removing potassium hexacyanoferrate (III) from the enzyme solution of Example 1.
(Comparative Example 3)
An enzyme electrode is prepared by using glucose oxidase (Aspergillus niger) instead of the PQQ-modified glucose oxidase described in Preparation Example 14 of Example 1.
(Comparative Example 4)
An enzyme electrode is prepared by using a 10% polyaniline solution instead of the 10% polypyrrole solution of Comparative Example 2.
(Comparative Example 5)
Instead of the 10% polypyrrole solution of Comparative Example 3, 10% polyaniline solution is used instead of 0.42 g potassium hexacyanoferrate (III) and 0.63 g potassium octacyanotungsten (IV) is used. Then, an enzyme electrode is prepared.
(Comparative Example 6)
An enzyme electrode is prepared by removing potassium hexacyanoferrate (III) from the enzyme solution of Example 4.
(Comparative Example 7)
An enzyme electrode is prepared by using glucose oxidase (Aspergillus niger) instead of the PQQ-modified glucose oxidase described in Preparation Example 14 of Example 3.
(Comparative Example 8)
An enzyme electrode is prepared by using glucose oxidase (Aspergillus niger) instead of PQQ-modified glucose oxidase described in Preparation Example 14 of Example 4.
(Comparative Example 9)
An enzyme electrode is prepared by using glucose oxidase (Aspergillus niger) instead of PQQ-modified glucose oxidase described in Preparation Example 14 of Example 5.
(Comparative Example 10)
An enzyme electrode is prepared by removing potassium octacyanotungsten (IV) from the enzyme solution of Example 6.

(Comparative Example 11)
An enzyme electrode is prepared by removing potassium octacyanotungstate (IV) from the enzyme solution of Example 7.
(Comparative Example 12)
An enzyme electrode is prepared by using D-amino acid oxidase (pig kidney) instead of the ferrocene-modified amino acid oxidase described in Preparation Example 16 of Example 7.
(Comparative Example 13)
An enzyme electrode is prepared by using the osmium complex polymer of Preparation Example 8 instead of the osmium complex polymer described in Preparation Example 6 of Example 3.
(Comparative Example 14)
An enzyme electrode is prepared by using potassium hexacyanomanganate (III) instead of potassium hexacyanoferrate (III) in Example 4.
(Comparative Example 15)
An enzyme electrode is prepared by using the ferrocene-modified glucose oxidase described in Preparation Example 15 instead of the PQQ-modified glucose oxidase described in Preparation Example 14 of Comparative Example 13.
(Comparative Example 16)
An enzyme electrode is prepared by using potassium hexacyanomanganese (III) instead of potassium octacyanotungsten (IV) in Example 6.
(Comparative Example 17)
An enzyme electrode is prepared by using polyvinyl pyridine (average molecular weight 150,000) instead of polyvinyl imidazole described in Preparation Example 6 of Comparative Example 6.
(Comparative Example 18)
An enzyme electrode is prepared by using polyvinyl pyridine (average molecular weight 150,000) instead of polyvinyl imidazole described in Preparation Example 6 of Comparative Example 8.
(Comparative Example 19)
An enzyme electrode is prepared by removing potassium octacyanotungsten (IV) from the enzyme solution of Example 9.
(Comparative Example 20)
An enzyme electrode is prepared by using glucose oxidase (Aspergillus niger) instead of the PQQ-modified glucose oxidase described in Preparation Example 14 of Example 9.

(Comparative Example 21)
An enzyme electrode is prepared by using glucose oxidase (Aspergillus niger) instead of PQQ-modified glucose oxidase described in Preparation Example 14 of Example 10.
(Comparative Example 22)
Instead of the gold plate of Comparative Example 2, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Comparative Example 23)
Instead of the gold plate of Comparative Example 3, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Comparative Example 24)
Instead of the gold plate of Comparative Example 4, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Comparative Example 25)
Instead of the gold plate of Comparative Example 5, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Comparative Example 26)
Instead of the gold plate of Comparative Example 6, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Comparative Example 27)
Instead of the gold plate of Comparative Example 7, foam metal made of nickel alloy (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Comparative Example 28)
In place of the gold plate of Comparative Example 8, foam metal made of nickel alloy (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Comparative Example 29)
Instead of the gold plate of Comparative Example 9, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Comparative Example 30)
Instead of the gold plate of Comparative Example 10, a metal foam made of nickel alloy (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).

(Comparative Example 31)
Instead of the gold plate of Comparative Example 11, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Comparative Example 32)
Instead of the gold plate of Comparative Example 12, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Comparative Example 33)
Instead of the gold plate of Comparative Example 13, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Comparative Example 34)
Instead of the gold plate of Comparative Example 14, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Comparative Example 35)
Instead of the gold plate of Comparative Example 15, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Comparative Example 36)
Instead of the gold plate of Comparative Example 16, foam metal made of nickel alloy (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Comparative Example 37)
Instead of the gold plate of Comparative Example 17, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Comparative Example 38)
In place of the gold plate of Comparative Example 18, foam metal made of nickel alloy (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Comparative Example 39)
Instead of the gold plate of Comparative Example 19, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Comparative Example 40)
Instead of the gold plate of Comparative Example 20, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).

(Comparative Example 41)
Instead of the gold plate of Comparative Example 21, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, gold plating thickness 0.5 μm, An enzyme electrode is prepared by using a pore diameter of 50 μm).
(Comparative Example 42)
Instead of the gold plate of Example 6, an enzyme electrode is prepared by using a conductive member having a gap of a poly (3,4-ethylenedioxythiophene) electrode described in Preparation Example 1.
(Comparative Example 43)
Instead of the metal plate of Example 7, an enzyme electrode is prepared by using a conductive member having a gap of a poly (3,4-ethylenedioxythiophene) electrode described in Preparation Example 1.
(Comparative Example 44)
An enzyme electrode is prepared by using the ITO conductive member described in Preparation Example 2 instead of the metal plate of Example 6.
(Comparative Example 45)
An enzyme electrode is prepared by using the ITO conductive member described in Preparation Example 2 instead of the metal plate of Example 7.
(Comparative Example 46)
An enzyme electrode is prepared by using a fluorine-doped tin oxide transparent conductive glass (Asahi Glass, 8 Ω / sq) instead of the gold plate of Example 6.
(Comparative Example 47)
An enzyme electrode is prepared by using a fluorine-doped tin oxide transparent conductive glass (Asahi Glass, 8 Ω / sq) instead of the metal plate of Example 7.
(Comparative Example 48)
An enzyme electrode is prepared by using a conductive member having a graphite gap shown in Preparation Example 3 instead of the metal plate of Example 6.
(Comparative Example 49)
An enzyme electrode is prepared by using a conductive member having a graphite gap shown in Preparation Example 3 instead of the metal plate of Example 7.
(Example 25)
Sensors are prepared using the enzyme electrodes described in Examples 1 to 24 and Comparative Examples 1 to 49. An enzyme electrode with a lead attached to an enzyme electrode is used as a working electrode, a silver-silver chloride electrode as a reference electrode, a platinum electrode as a counter electrode (FIG. 6), and an enzyme electrode using glucose oxidase as an electrolyte. 15 mM glucose, 20 mM phosphate buffer (pH 7.2) containing 0.1 M NaCl, while the enzyme electrode using D-amino acid oxidase is 0.1 M containing 15 mM D-alanine. Use pyrophosphate buffer (pH 8.5). In the measurement, each electrode is connected to a potentiostat (Toho Giken, Model 2000), and a steady current when a potential of 0.5 V (vsAg / AgCl) is applied to the working electrode in a nitrogen atmosphere is recorded. A water jacket cell is used for the container, and the measurement temperature is maintained at 37 ° C. by a thermostatic bath. The results are shown in Table 1.

  A sensor using an enzyme electrode described in Examples 1 to 24 using a first mediator for exchanging electrons with an enzyme and a second mediator for exchanging electrons with the first mediator. These show higher current densities than the corresponding conductive members, carriers, and enzyme electrodes using enzymes. This is thought to be because the introduction of the first mediator has improved the electron transfer rate between the enzyme and the mediator, and the density of the enzyme immobilized on the conductive member has been improved by performing the comprehensive immobilization. It is done. On the other hand, the redox potential of the second mediator used in the enzyme electrode of Comparative Example 13-16 was −0.19 V (vsAg / AgCl, Examples 13 and 15) and −1.02 V (from the literature values, respectively). vsAg / AgCl, Examples 14 and 16), which are positioned more negative than the potential of the corresponding first mediator (the most negative -0.14 V vsAg / AgCl), so between mediator 1 / mediator 2 It is considered that no electron transfer is performed and a low current density is exhibited. From this, it is possible to increase the sensitivity of the sensor by introducing an enzyme electrode using a first mediator for exchanging electrons with an enzyme and a second mediator for exchanging electrons with the first mediator. It is confirmed.

(Example 26)
Fuel cells are prepared using the enzyme electrodes described in Examples 1 to 24 and Comparative Examples 1 to 49. A bipolar cell configuration (FIG. 7) in which a lead is attached to an enzyme electrode as a working electrode, the enzyme electrode described in Preparation Example 17 as a counter electrode, and a porous polypropylene film (thickness 20 μm) sandwiched between both electrodes. In a cell using 0.2 M citrate buffer (pH 5.0) as the electrolyte and an enzyme electrode using glucose oxidase as the anode fuel, 15 mM glucose is used as the enzyme electrode using D-amino acid oxidase. In the cell used, measurement is performed using 15 mM D-alanine and saturated oxygen as the cathode fuel. In the measurement, each electrode is connected to a potentiostat (Toho Giken, Model 2000), the voltage is changed from −1.2 V to 0.1 V at a rate of 1 mV / sec, and the voltage-current characteristics are measured. A water jacket cell is used for the container, and the measurement temperature is maintained at 37 ° C. by a thermostatic bath. The results are shown in Table 2.

  A fuel cell using an enzyme electrode using a first mediator for delivering electrons to and from the enzymes described in Examples 1 to 24, and a second mediator for delivering electrons to and from the first mediator, These show higher short-circuit current densities than the corresponding conductive members, carriers, and enzyme electrodes using enzymes. This is thought to be because the introduction of the first mediator has improved the electron transfer rate between the enzyme and the mediator, and the density of the enzyme immobilized on the conductive member has been improved by performing the comprehensive immobilization. It is done. On the other hand, the redox potential of the second mediator used in the enzyme electrode of Comparative Example 13-16 was −0.19 V (vsAg / AgCl, Comparative Examples 13 and 15) and −1.02 V (from the literature values, respectively). vsAg / AgCl, Comparative Examples 14 and 16), which are located more negative than the corresponding mediator potential (the most negative -0.14 V vsAg / AgCl), so that the electron transfer between mediator 1 / mediator 2 is It is considered that the low current density is exhibited. With respect to the maximum output, the use of multiple types of mediators causes voltage loss. Therefore, in the fuel cell using the enzyme electrode described in some examples, the corresponding conductive member, carrier, and enzyme must be replaced. Some have a lower output than the fuel cell using the enzyme electrode described in the comparative example used. However, as a whole, the fuel cell using the enzyme electrode described in the examples has a higher output than the fuel cell using the enzyme electrode described in the comparative example using the corresponding conductive member, carrier and enzyme. Therefore, the introduction of the first mediator for exchanging electrons with the enzyme and the second mediator for exchanging electrons with the first mediator has led to high output of the fuel cell. It is confirmed that

(Example 27)
Electrochemical reaction apparatuses are prepared using the enzyme electrodes described in Examples 1 to 24 and Comparative Examples 1 to 49. Using a triode cell (FIG. 6) with an enzyme electrode as a working electrode, a silver-silver chloride electrode as a reference electrode, and a platinum wire as a counter electrode, 0.1 M NaCl, 20 mM phosphate buffer (pH 7.2), 10 mM An electrolytic solution containing glucose, 10 mM D-alanine is used, and a potential of 0.4 V vs Ag / AgCl is applied for 100 minutes in a water jacket cell under a nitrogen atmosphere, and the product is quantified by high performance liquid chromatography. The results are shown in Table 3.

  From the reaction electrolyte, the electrochemical reaction apparatus using the enzyme electrode of Comparative Examples 13-16 was excluded, and gluconolactone was found from the reaction liquid of the reaction apparatus using the enzyme electrode using an enzyme with glucose as a substrate. No pyruvate is detected, no pyruvate is detected, no gluconolactone is detected from the reaction solution of the reaction apparatus using an enzyme electrode using an enzyme with D-alanine as a substrate, and any enzyme It can be seen that also in the reaction apparatus using electrodes, the reaction proceeds in a selective manner with respect to the substrate. On the other hand, the redox potential of the second mediator used in Comparative Examples 13-16 was −0.19 V (vsAg / AgCl, Comparative Examples 13 and 15) and −1.02 V (vsAg / AgCl, respectively) from literature values. Comparative Examples 14 and 16), which are positioned more negative than the potential of the corresponding mediator (the most negative one is −0.14 V vs Ag / AgCl), the electron transfer between the mediators ½ is not performed and is low It is considered to indicate the amount of charge. Therefore, the introduction of the first mediator for exchanging electrons with the enzyme and the second mediator for exchanging electrons with the first mediator makes it possible to accelerate the reaction in the electrochemical reaction apparatus. It is confirmed that the sensitivity can be increased.

It is a conceptual diagram of the enzyme electrode of a comprehensive method. It is a conceptual diagram of the enzyme electrode of a covalent bond method. It is a conceptual diagram of the enzyme electrode of this invention. It is a conceptual diagram (anode or negative electrode) of the electric potential relationship of the mediator of this invention. It is a conceptual diagram (cathode or positive electrode) of the electric potential relationship of the mediator of this invention. It is explanatory drawing of a triode cell. It is explanatory drawing of a bipolar cell. It is an example of the porous electroconductive member applicable to this invention.

Explanation of symbols

1 Enzyme 2 Mediator 3 Carrier 4 Conductive member 5 Charge flow 6 Mediator 7 Carrier 8 First mediator 9 Second mediator 10 Electron flow 11 Potential (direction of arrow is negative)
12 Water jacket cell 13 Water jacket cell lid 14 Electrolytic solution 15 Anode (anode, anode)
16 Platinum wire electrode 17 Silver silver chloride reference electrode 18 Anode lead 19 Cathode lead 20 Reference electrode lead 21 Potentiostat 22 Gas inlet 23 Gas tube 24 Temperature controlled water inlet 25 Temperature controlled water outlet 26 Porous polypropylene film 27 Cathode 801 Electrolyte Layer 802 Hole 803 Conductive member 804 Support substrate

Claims (11)

An enzyme electrode having a conductive member, an enzyme, and first and second mediators,
The first mediator and the second mediator are fixed to the conductive member by a carrier, and the first mediator and the second mediator have different redox potentials. Characteristic enzyme electrode. The redox potential of the first mediator is on the negative potential side of the redox potential of the second mediator, and the electron transfer reaction rate between the second mediator and the conductive member is The enzyme electrode according to claim 1, wherein the enzyme electrode has a faster electron transfer reaction rate between the first mediator and the conductive member.   The redox potential of the first mediator is on the positive potential side with respect to the redox potential of the second mediator, and the electron transfer reaction rate between the second mediator and the conductive member is The enzyme electrode according to claim 1, wherein the enzyme electrode has a faster electron transfer reaction rate between the first mediator and the conductive member.   The first mediator is a mediator for exchanging electrons with the enzyme, and the second mediator is a mediator for exchanging electrons with the first mediator. Item 4. The enzyme electrode according to any one of Items 1 to 3.   The enzyme electrode according to claim 1, wherein the redox potential of the first mediator is on the negative potential side of the second mediator and is used as an anode.   The enzyme electrode according to claim 1, wherein the redox potential of the second mediator is on the positive potential side of the second mediator and is used as a cathode.   The enzyme electrode according to claim 1, wherein the conductive member has a porous structure.   The enzyme according to any one of claims 1 to 7, wherein the first mediator and the second mediator comprise at least one selected from metal complexes, quinones, heterocyclic compounds, nicotinamide derivatives, and flavin derivatives. electrode.   9. A sensor using the enzyme electrode according to claim 1 as a detection site for detecting a substance.   9. A fuel cell, wherein the enzyme electrode according to claim 1 is used as at least one of an anode and a cathode.   An electrochemical reaction apparatus using the enzyme electrode according to claim 1 as a reaction electrode.

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