JP4632437B2 - Enzyme electrode, device having enzyme electrode, sensor, fuel cell, electrochemical reaction device - Google Patents

Description

  The present invention relates to an enzyme electrode, and more particularly to an enzyme electrode in which a carrier and an enzyme are immobilized on a conductive member having a gap, a method for producing the enzyme electrode, a device using the enzyme electrode, and its use.

  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, and each enzyme generally 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. By introducing a method for connecting the oxidoreductase reaction and the electrode via this mediator, it is possible to control the enzyme reaction by the electrode potential and to function as an energy conversion element. Above all, a device called a biofuel cell is completely different from a fuel cell that uses a metal catalyst such as platinum, and the characteristics of the biocatalyst are reflected in the cell. In the negative electrode, sugar, alcohol, amine, hydrogen, etc. The positive electrode has a feature that, in principle, all substrates used by living organisms, such as oxygen, nitrate ions, and sulfate ions, can be used. In the initial stage of research, these enzymes and mediators were dissolved in the electrolyte solution to ensure the convenience of the experimental system and the freedom of movement of each. However, their effective use and leakage into the system In order to prevent and meet the demand for continuous and long-term use of electrodes, a method of fixing to electrodes has been proposed. As a method for immobilizing the enzyme and mediator on the conductive member, a method using a carrier can be mentioned. The method of chemically and electrostatically immobilizing enzymes and mediators using a carrier generally has a higher ability to hold enzymes and mediators compared to the method of physically adsorbing enzymes, and the outflow from the system. Prevent repeated use of the enzyme electrode.

  One of the important values expressing the performance of the enzyme electrode is the current density, which is the current value per projected area of the conductive member. When this value is high, in the case of a sensor based on the detection of the current value, the detection sensitivity can be improved, the measurement site can be simplified, the detection site can be miniaturized, and it can be used as an electrode for a fuel cell. In some cases, the output can be improved, and when used as an electrochemical reaction device, the reaction time can be shortened. The current density of the enzyme electrode is such that the number of substrate molecules converted by the enzyme molecule per unit time is increased, the electron transfer rate and efficiency between the enzyme / mediator are improved, the electron transfer rate between the mediator / electrode and The efficiency can be increased by improving efficiency, improving the electron transport efficiency of the mediator, increasing the enzyme support density, which is the amount of enzyme supported per projected area of the conductive member, and the like.

  A typical immobilization method using a carrier is a generic immobilization method (FIG. 1) in which an enzyme is encapsulated in a carrier such as a polymer and immobilized on the surface of a conductive member. FIG. 1 conceptually shows a state in which an enzyme is immobilized by entrapping immobilization. The enzyme 2 is entrapped and immobilized in a layer formed from a carrier 3 on a substrate 1 made of a conductive member. For example, a charge flow indicated by 4 can be generated. In this entrapping immobilization method, the charge generated by the enzyme / substrate reaction is taken out to the mediator in the carrier, transported to the vicinity of the conductive member by electron hopping between the mediators, and finally between the mediator and the conductive member. It is detected in an external circuit by charge transfer. Here, for the purpose of increasing the amount of the enzyme supported per projected area of the conductive member, even if the amount of the enzyme supported relative to the amount of the carrier is simply increased, the electron transfer rate between the enzyme / carrier generally decreases, There is a limit to improving the current density. On the other hand, in the comprehensive immobilization method using a mediator, even if the enzyme is immobilized to an amount larger than the enzyme's exclusive area divided by the effective surface area of the conductive member, charge transport between the electrode and the enzyme can be carried through the carrier. Therefore, by increasing the amount of the carrier and increasing the thickness of the carrier, the enzyme loading density per the projected area of the enzyme (the amount of the enzyme as the whole fixed layer) can be increased without increasing the amount of the enzyme supported relative to the amount of the carrier. It is possible to improve the loading amount. However, the diffusion of electrons in the layer containing the carrier is generally slow, and the diffusion rate of electrons through the carrier is limited. Therefore, if the fixed layer of the enzyme carrier exceeds a certain thickness, the charge transport efficiency Decreases. For this reason, it is preferable that the thickness of the fixed layer using the carrier is not more than a certain value. In consideration of this point, as a result, the current density is improved by increasing the amount of enzyme supported per projected area of the conductive member. Have limitations. Regarding the use of an enzyme electrode using the entrapping immobilization method, US Pat. No. 6,531,239 (Heller et al.) Used an enzyme electrode in which an enzyme is entrapped and immobilized with a polymer containing a mediator in the molecule. A fuel cell is disclosed.

In order to improve the enzyme carrying density, it is also effective to increase the effective surface area of the electrode. As a typical technique, there is a technique (FIG. 2) in which an enzyme is physically adsorbed to a conductive member made of particles made of a carbon material and a binder polymer. The enzyme electrode of FIG. 2 has a structure in which a layer in which the enzyme 2 is fixed to carbon particles 5 as a conductive member using a binder polymer 6 is formed on the surface of the conductive substrate 1. In this enzyme electrode, for example, a flow of electric charges can be obtained via the grain boundary 7 of the carbon particle 5 indicated by the arrow 4. In this case, the resistance between the particles 7 of the carbon material is high, and the overall resistance value also increases with the thickness of the conductive member, increasing the internal resistance of the enzyme electrode and decreasing the properties of the enzyme electrode. For this reason, the conductive member is preferably used at a certain thickness or less. As a result, the current density due to an increase in the amount of enzyme supported per projected area of the conductive member (an increase due to an increase in the effective surface area of the electrode). There is a limit to the improvement. Furthermore, in this case, since the carrier as in the entrapping immobilization method is not used, the enzyme retention ability is low, and the repeated use of the enzyme electrode is limited. As this technique, US Pat. No. 4,970,145 (Bennetto et al.) Discloses an enzyme electrode using a conductive member in which carbon particles are immobilized with a resin together with platinum-based metal particles.
US Pat. No. 6,531,939 US Pat. No. 4,970,145

  In the entrapping immobilization method described above, the amount of enzyme supported is maintained while maintaining an electronic connection between the enzyme and the conductive member by increasing the thickness of the enzyme immobilization layer in which the enzyme is immobilized in the layer containing the carrier. However, since the electron diffusion coefficient of the carrier is generally low, the charge transport efficiency decreases when the enzyme fixed layer exceeds a certain thickness, so the thickness of the enzyme fixed layer is In general, there is a limit to increasing the enzyme carrying density per projected area of the enzyme electrode. On the other hand, in the method in which the enzyme is physically adsorbed to the conductive member made of the carbon material and the binder polymer described above, the interparticle resistance of the carbon material is high, and the resistance value includes the conductive member and the enzyme. In order to increase with the thickness of the enzyme immobilization layer and increase the internal resistance of the enzyme electrode, the thickness of the conductive member is preferably used at a certain value or less. Furthermore, in this method using particles made of carbon material, a binder polymer is used for immobilizing the enzyme, no carrier is used in the entrapping immobilization method, and the enzyme retention capacity is low. It is preferable to use it for an application. Therefore, this immobilization method is generally limited in terms of improving the charge transport efficiency and expanding the application of the enzyme electrode.

  Accordingly, an object of the present invention is to provide an enzyme electrode or the like that can improve the current density with a high enzyme carrying density.

Enzyme electrodes according to the present invention is an enzyme electrode having an electrically conductive member and an enzyme, wherein the conductive member comprises a porous structure, and wherein the enzyme is in the hole constituting the porous structure, The conductive member is fixed via a carrier, and the conductive member has at least two working surfaces facing each other, both of the two working surfaces are open, and the conductive member is immersed in a liquid. In this case, the enzyme electrode allows the liquid to pass from one surface to the other surface through the plurality of voids .

In one aspect of the fuel cell according to the present invention, the anode electrode and the cathode electrode have a porous structure, and at least one of the anode electrode and the cathode electrode holds an enzyme in the pores constituting the porous structure. Ri enzyme electrode der that the pore size at the surface side of the enzyme electrode may be greater than the diameter in the interior of the conductive member.

  The enzyme electrode device according to the present invention includes the enzyme electrode having the above-described configuration and wiring connected to a conductive member included in the enzyme electrode.

  The sensor according to the present invention is characterized in that the enzyme electrode device having the above-described configuration is used as a detection site for detecting a substance.

  Another aspect of the fuel cell according to the present invention is a fuel cell in which a region capable of holding an electrolyte solution is provided between an anode electrode and a cathode electrode, and at least one of these anode electrode and cathode electrode has the above-described configuration. It consists of an enzyme electrode device.

That written present invention electrical chemical reactor, Yusuke a reaction region, and the electrode for obtaining the desired product by causing electrochemical reactions in the starting materials introduced into the reaction region, the in that electrical chemical reactor, said electrodes is characterized in that an enzyme electrode device of the above configuration.

According to the present invention, the enzyme is fixed by a carrier in a number of voids communicating with the outside of the conductive member, and the current density is increased by ensuring a high enzyme carrying density in the conductive member having a large specific surface area. An enzyme electrode that can be improved can be provided.
In particular, when the enzyme electrode is formed in a plate shape or a layer shape, even if the thickness is increased, the distance between the enzyme and the conductive member does not increase, and the decrease in the efficiency of electron transfer between the enzyme and the conductive member is suppressed. The

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

  An enzyme electrode according to a preferred embodiment of the present invention includes a conductive member having a gap, an enzyme for exchanging electrons with the conductive member, and a carrier for immobilizing the enzyme in the gap. It is characterized by that. In this electrode, by using a carrier for immobilizing the enzyme, it is possible to stably immobilize the enzyme on the conductive member, and to increase the enzyme carrying density per effective surface area of the conductive member. As a result, stability and current density can be improved. The enzyme electrode has at least two working surfaces facing each other, and has a structure in which liquid can permeate from one surface to the other surface through a number of voids of the conductive member. it can. For example, using a plate-like (or film-like or layer-like) conductive member, holding the opening of the gap on the two opposing faces (front and back faces in the thickness direction), the working surface (interacting with the electrode) A contact surface with the liquid containing the component to be generated), and the liquid can be transmitted from one of these working surfaces to the other. Alternatively, the side surface of the conductive member having such a shape may hold the opening of the gap to allow the liquid to penetrate from the side surface and permeate to the other surface.

  Furthermore, by using a conductive member having a large effective area, high conductivity, and voids compared to the projected area of the conductive member, without increasing the distance between the enzyme and the conductive member, It is possible to improve the thickness of the enzyme electrode with almost no increase in the resistance of the entire electrode, and as a result, it is possible to improve the current density. FIG. 3 is a conceptual diagram (cross section) of a conductive member having a void of the present invention, an enzyme electrode having a conductive member, an enzyme for transferring electrons, and a carrier for immobilizing the enzyme in the void. Figure). In the enzyme electrode of FIG. 3, the enzyme 2 is fixed by the carrier 3 in a large number of voids of the conductive member 8, and for example, charge transfer indicated by an arrow 4 can be obtained. In addition, each space | gap of FIG. 3 is connected with the exterior via the space | gap not shown.

  Furthermore, the enzyme electrode device which can be utilized for various uses can be obtained by connecting the wiring which transmits / receives an electron to an enzyme electrode. In this device, the enzyme electrode can be used as a reaction electrode for the enzyme electrode reaction, and the plate-like (or film-like or layer-like) enzyme electrode can be composed of a single layer or a plurality thereof. . In the case of using a plurality of them, they can be laminated so that the front surface and the back surface face each other. In a plurality of cases, the characteristics of the enzyme electrodes may be uniform, or a combination of enzyme electrodes having different characteristics may be included. For example, as in a fuel cell described later, the anode and the cathode can be arranged alternately. This device can meet the required current, voltage, and output by changing the number of electrodes from a single layer to a multilayer. 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. Furthermore, since the enzyme electrode used in this device has a gap in the conductive member of the electrode, it is possible to permeate the electrolyte through the gap in the conductive member without providing a separate electrolyte flow path. As a result, the device can be simplified. Further, by providing an external mechanism for accelerating the permeation of the electrolytic solution, the supply of the substrate is increased, and as a result, the current density can be increased.

  A sensor according to a preferred embodiment of the present invention is characterized in that a device using an enzyme electrode in a single layer or a multilayer structure is used as a detection site for detecting a substance. As a typical configuration, an enzyme electrode is used as a working electrode in pairs with a counter electrode, and if necessary, a reference electrode can be used to detect a current that can be detected by the enzyme electrode (by the function of the enzyme immobilized on the electrode). Can be used to detect the substance in the liquid in contact with these electrodes. The configuration of the sensor is not particularly limited as long as it can be detected by the enzyme electrode. An example is shown in FIG. The sensor shown in FIG. 4 includes an anode 12, a platinum wire electrode 13, and a silver / silver chloride reference electrode 14, and lead wires 15, 16, and 20 are wired to each electrode, and a potentiostat 18. And connected. This sensor is arranged in a storage region of the electrolyte 11 in the water jacket cell 9 that can be sealed with the lid 10. 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 required, an inert gas such as nitrogen is introduced from the gas blowing port 19 at the outer end of the gas tube 20. Moreover, temperature can be performed by supply of the liquid for temperature control using the temperature control water inflow port 21 and the temperature control water discharge port 22. FIG. In addition to the high selectivity of the substrate specific to the enzyme used as a catalyst for the electrode reaction, this sensor simplifies the detection device due to the high current density due to the enzyme electrode using a conductive member having voids, and / or The detection part can be downsized. 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 a device in which an enzyme electrode is used as a single layer or a multilayer structure is used as at least one of an anode and a cathode. Furthermore, in a configuration in which multiple layers are stacked, the anode and the cathode may be arranged in a predetermined arrangement in the stacking 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 electrolytic solution or a type that does not replenish or circulate the electrolytic solution. As long as the fuel cell can use an enzyme electrode, the type, structure, function, etc. of the fuel are not limited. This fuel cell can obtain a high driving voltage by using a high catalytic action peculiar to an enzyme used as a catalyst for an electrode reaction, and can reduce a substance with a low overvoltage, and can use a conductive member having a gap. A high current density can be obtained by using a conventional enzyme electrode. As a result, the output can be increased and / or the fuel cell can be reduced in size. An example of the fuel cell is shown in FIG. The fuel cell shown in FIG. 8 has a configuration in which an anode 12 connected to an anode lead 15 and a cathode 24 connected to a cathode lead 16 are laminated in an acrylic case 27 via a porous polypropylene film 23. The electrode unit is arranged, the electrolytic solution is introduced from the electrolytic solution introduction port 25, and the electrolytic solution can be discharged from the electrolytic solution discharge port 26, thereby functioning as a fuel cell.

  An electrochemical reaction apparatus according to one preferred embodiment of the present invention is characterized in that a device in which enzyme electrodes are laminated in a single layer or multiple layers is used as a reaction electrode. 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 a 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. As an example, the structure shown in FIGS. 4 and 7 can be cited. When the apparatus of FIGS. 4 and 7 is an electrochemical reaction apparatus, an electrochemical reaction occurs by applying a current or voltage to the lead wire, contrary to the case of using the sensor or fuel cell described above. To obtain the desired product.

  The electrochemical reaction device can obtain the quantitative characteristics 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 electrode reaction catalyst. It is possible to manufacture a reaction apparatus that can be selectively controlled with high efficiency and quantitatively. This electrochemical reaction apparatus can selectively carry out a reaction of a substance corresponding to an enzyme substrate used for an enzyme electrode. Examples of its use include glucose, fructose, galactose, amino acid, amine, Examples include oxidation of cholesterol, alcohol, lactic acid, and reduction reaction of oxygen and hydrogen peroxide. More specific applications include selective oxidation of cholesterol in the presence of ethanol, reduction reaction of oxygen at low overvoltage, etc. Is mentioned.

  Many 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 of structural materials created using them as templates. These voids need to be large enough to allow the enzyme to be introduced and / or sufficiently flow and diffusion of the substrate, and small enough to give a ratio of effective surface area to projected area. Examples of the average diameter of the void include a range of 5 nm to 5 mm, more preferably 10 nm to 500 μm. In addition, the thickness of the conductive member is so small that the enzyme can be uniformly introduced to the deep part of the conductive member and / or the substrate is sufficiently flowed and diffused, and the effective surface area relative to the projected area of the conductive member. The ratio of the thickness of the conductive member having the gap is 100 nm to 1 cm, and 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.

  In addition, a large number of voids in the conductive member are in contact with the electrolyte on the outer surface where the size of the void is from the portion where the outer edge of the conductive member is in contact with the electrolyte, that is, there is an opening communicating with the void in the conductive member. A structure may be provided that decreases as the distance from the portion to the inside of the conductive member increases (a conductive member having a large number of voids having this structure is referred to as a void size (for example, (Referred to as a conductive member having a large number of voids with inclination in the pore diameter)). In order to support an enzyme effective for electrode reaction at a high density, it is effective to use a conductive member having a large number of voids having a pore size of a certain extent or less, but the enzyme is supported at a high density. In this case, the diffusion of the substrate into the enzyme may be a factor that limits the current of the entire electrode, and from the site where the electrolyte contacts the outer edge of the conductive member having a large number of voids, In some cases, sufficient diffusion of the substrate to the enzyme supported at the site where the distance increases is not obtained. In this case, by using a conductive member having a large number of voids having a gradient in the size of the voids, it is possible to secure a sufficient enzyme loading density effective for the electrode reaction, and the substrate to the inside of the conductive member. It is possible to achieve both sufficient diffusion. As the conductive member having a large number of voids having an inclination in the size of the void, a conductive member manufactured so that the size of the void is inclined from the beginning is used, and a plurality of conductive materials having different void sizes are used. A laminated member may be used. A plurality of members having different compositions may be laminated. In addition, as an example of the average diameter of the gap of the conductive member having a large number of gaps having inclinations in the size of the gap, those having a large gap are 100 nm to 5 mm, more preferably 1 μm to 1 mm, and the gap is small. Examples include 5 nm to 500 μm. For example, when the conductive member is plate-shaped, the position where the region where the size of the hole changes is taken as an example, the size of the hole from one surface (front surface) to the other (back surface). Depending on the intended application, such as a configuration in which the pores are continuously or gradually reduced (the back surface has smaller holes than the front surface), and the pores are continuously or gradually decreased from the front and back surfaces toward the center. Can be selected.

As a conductive member used in the concept electrode according to the present invention, a member having a constant pore diameter (or porosity) in a porous structure can be used in the thickness direction.
Further, as shown below, the pore size or the porosity of the porous member may have a gradient distribution.

  FIGS. 9A to 9D 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, the hole diameter of the hole provided in the conductive member is large on the electrolyte layer side (that is, the outer surface side of the conductive member) and small on the inner side (inside the conductive member). It is preferable. 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, it is preferable that the pore diameter and porosity of the pores provided in the conductive member are both large on the electrolyte layer side and small on the inner side.

  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.

  The conductive porous layer constituting the enzyme electrode preferably has a region in which the size of the pores decreases from the electrolyte side surface to the other surface of the porous layer. The pore size of the porous layer according to the present invention is, for example, from one surface side (electrolyte side) of the porous layer to a large porosity region or a small porosity region. It may be a moderate region, a small porosity region, or a large porosity region, a small porosity region, or a large porosity region.

  The carrier is for immobilizing at least the enzyme on the conductive member. Examples include (1) a polymer compound, (2) an inorganic compound, and (3) an organic compound having a covalent bond in the molecule, an enzyme and a conductive member, and / or The enzyme and the enzyme can be combined, and those containing at least one of these three types can be mentioned. These carriers preferably have a charge opposite to the surface charge of the enzyme under the conditions of use of the electrode in order to fix the enzyme to 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.

  Examples of polymer compounds for carriers include conductive polymer compounds such as polyacetylenes, polyarylenes, polyarylene vinylenes, polyacenes, polyarylacetylenes, polydiacetylenes, polynaphthalenes, polypyrroles, polyaniline Examples of polymers, polythiophenes, polythienylene vinylenes, polyazulenes, polyisothianaphthenes, and other polymers include polystyrene sulfonic acid, polyvinyl sulfate, dextran sulfate, chondroitin sulfate, polyacrylic acid, polymethacrylic acid, Polymaleic acid, polyfumaric acid, polyethyleneimine, polyallylamine hydrochloride, polydiallyldimethylammonium chloride, polyvinylpyridine, polyvinylimidazole, polylysine, deoxyribonucleic acid, ribonucleic acid, pectin, Recone resin, 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 are used as a carrier that binds the enzyme and the conductive member and / or the enzyme to 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. Typical examples include glutaraldehyde, polyethylene glycol diglycidyl ether, cyanuric chloride, N-hydroxysuccinimide ester, dimethyl-3,3′-dithiopropionimidate hydrochloride, 3,3′-dithio-bis (sulfosuccinimidyl). Propionate), cystamine, alkyldithiol, biphenylenedithiol, benzenedithiol.

  The mediator promotes the delivery of electrons between the enzyme and the conductive member, and can be used as necessary. This mediator may be chemically bonded to at least one of the carrier and the enzyme. Examples include metal complexes, quinones, heterocyclic compounds, nicotinamide derivatives, flavin derivatives, conductive polymers, conductive fine particle materials, and carbon materials. 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 their derivatives. Examples of quinones include quinone, Examples include benzoquinone, anthraquinone, naphthoquinone, pyrroloquinoline quinone, 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). Here, examples of the conductive polymer include polyacetylenes, polyarylenes, polyarylene vinylenes, polyacenes, polyarylacetylenes, polydiacetylenes, polynaphthalenes, polypyrroles, polyanilines, polythiophenes, polythiophenes Examples include thienylene vinylenes, polyazulenes, and polyisothianaphthenes.

  Here, the conductive fine particle material may include a metal fine particle material. For example, Au, Pt, Ag, Co, Pd, Rh, Ir, Ru, Os, Re, Ni, Cr, Fe, Mo, Ti, Al, Cu, V, Nb, Zr, Sn, In, Ga, Mg, and metal containing at least one element among Pb, conductive polymer fine particles, and these may be alloys, It may be plated. Here, examples of the carbon material include graphite fine particles, carbon black fine particles, fullerene compounds, carbon nanotubes, carbon nanohorns, and derivatives thereof.

  The conductive member has a large number of voids communicating with the outside, and a structure in which the walls that partition the voids are integrally formed from the constituent materials, or the materials that constitute the walls that partition the voids Those that are firmly bonded are preferred. Examples of the constituent material of such a conductive member include conductive metals, polymers, metal oxides, and carbon materials.

  As the metal that can constitute the conductive member, any material that has conductivity, sufficient rigidity during storage and measurement, and sufficient electrochemical stability under the conditions in which the electrode is used can be used. . Examples include Au, Pt, Ag, Co, Pd, Rh, Ir, Ru, Os, Re, Ni, Cr, Fe, Mo, Ti, Al, Cu, V, Nb, Zr, Sn, In, Ga, Examples include Mg, Pb, Si, and W containing at least one element. These may be alloys or plated. Examples of the metal having voids include foam metal, electrodeposited metal, electrolytic metal, sintered metal, fibrous metal, or a material corresponding to one or a plurality of types of these metals. 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 polymer that can be used as the conductive member should be a material that has conductivity, has sufficient rigidity during storage and measurement, and has sufficient electrochemical stability under the conditions in which the electrode is used. Can do. Examples thereof include polyacetylenes, polyarylenes, polyarylene vinylenes, polyacenes, polyarylacetylenes, polydiacetylenes, polynaphthalenes, polypyrroles, polyanilines, polythiophenes, polythienylene vinylenes, polyazulenes. And those containing at least one compound among polyisothianaphthenes. As an example of a method for producing a conductive polymer having voids, a material as a template constituting a portion to be a void is placed in a conductive polymer and molded into a predetermined shape, and then a material as a template is formed. Method of removing; Method of removing a material as a template after polymerizing the precursor into a polymer after containing a material as a template of a void portion in the precursor of the conductive polymer; A method of forming a layer composed of particles serving as a template to be formed, filling a gap between the particles with a polymer to form a layer, and removing the particles from this layer; a particle serving as a template constituting a void portion A method of forming a layer comprising the steps of: forming a layer by filling a void between the particles with a polymer precursor, polymerizing the precursor to form a polymer layer, and then removing the particles; The method currently used for manufacture can be mentioned.

  The metal oxide that can be used as the conductive member can be any material that has sufficient rigidity during storage and measurement and has sufficient electrochemical stability under the conditions in which the electrode is used. 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. Examples of methods for producing metal oxides include electrodeposition, sputtering, sintering, chemical vapor deposition (CVD), electrolysis, and combinations thereof.

  The carbon material used as the conductive member in the present invention may be any material that has sufficient rigidity during storage and measurement and has sufficient electrochemical stability under the conditions in which the electrode is used. 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. Sintering is mentioned as an example of the manufacturing method of the electroconductive member using a carbon material.

  As the enzyme immobilized on the conductive member, an oxidoreductase can be suitably used. This enzyme is an enzyme that catalyzes a redox reaction, and a combination of a plurality of different enzymes can be used in the same enzyme electrode in order to obtain a desired characteristic. 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, cytochrome C, catalase, peroxidase, glutathione reductase, NADH-cytochrome b5 reductase, NADPH-adrenoxin reductase, cytochrome b5 Examples include reductase, adrenodoxin 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 preferable to uniformly fix the carrier in the gap of the conductive member. In order to uniformly fix the carrier in the gap of the conductive member, it is preferable to perform a step of hydrophilizing the surface of the conductive member before introducing the carrier into the conductive member. Examples of the step of hydrophilizing the surface of the conductive member include UV ozone treatment, a step of permeating a water-soluble organic solvent such as alcohol into the voids of the conductive member, and then substituting with water. The process of irradiating an ultrasonic wave in the process of this is mentioned. 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. Examples include a step of immobilizing by performing a step and then performing hydrolysis, polymerization, and crosslinking reaction of the precursor.

  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 conductive member having voids used in the present invention will be described. The particle size of the particles can be measured by observation with a scanning electron microscope, and the film thickness can be measured using a surface roughness meter.

(Preparation Example 1)
The solvent of a commercially available polystyrene latex colloidal dispersion (Nippon Zeon, average particle size 100 nm) is replaced with ethanol, a washed gold substrate is placed in the dispersion, and ethanol is evaporated at 30 ° C. A porous membrane is obtained. By performing this process a plurality of times, a porous film (film thickness 100 μm) made of polystyrene spheres having a target film thickness is created. Heat at 70 ° C. for 30 minutes and then wash with ethanol. Electrodeposition is performed by using this porous membrane as a working electrode and applying a current density of 0.1 mA / cm ² with a galvanostat in a 0.1 M nickel sulfate aqueous solution and a platinum electrode as a counter electrode. The electrodeposition time is controlled by referring to the electrolysis current profile and adjusting the electrolysis time so as to be approximately the same as the thickness of the polystyrene film. After electrodeposition, it is immersed in toluene for 2 days, the latex is removed, and a conductive member having a large number of voids made of nickel is prepared.
(Preparation Example 2)
Printed platinum paste (Tanaka noble metal, platinum particle diameter of 1 μm) on a washed metal plate by screen printing, sintered at 500 ° C. for 1 hour, and a conductive member having a large number of voids made of platinum (film thickness of 100 μm) ) Is prepared.
(Preparation Example 3)
Printed gold paste (Tanaka noble metal, gold particle diameter 1 μm) on a washed gold plate by screen printing method, sintered at 500 ° C. for 1 hour, and conductive member having a large number of voids made of gold (film thickness 100 μm ) Is prepared.
(Preparation Example 4)
Palladium particles (Tanaka noble metal, particle size 1 μm) are dispersed in approximately twice the weight of tarpinol, and ethyl cellulose is added to adjust the viscosity to prepare a palladium paste. A palladium paste is printed on a cleaned gold substrate by a screen printing method and sintered at 500 ° C. for 1 hour to prepare a conductive member (film thickness 100 μm) having a large number of voids made of palladium.
(Preparation Example 5)
A commercially available silica colloid dispersion (Nissan Chemical Co., Ltd., average particle size: 100 nm) was substituted with ethanol, a washed gold substrate was placed in the dispersion, and ethanol was evaporated at 30 ° C., thereby forming a porous film made of silica spheres. Prepare. 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, 0.1M pyrrole, 0.1M lithium perchlorate in acetonitrile solution, using this porous membrane as working electrode, platinum counter electrode, silver / silver chloride reference electrode In the triode cell used, electrolytic polymerization is performed using a potentiostat at a potential of 1.1 V (vsAg / AgCl). The polymerization time is controlled by referring to the electrolysis current profile and adjusting the electrolysis time so as to be approximately the same as the thickness of the silica sphere porous membrane. After the electropolymerization, it is immersed in a 20% hydrofluoric acid solution for 2 days, the silica spheres are removed, and a conductive member (thickness 100 μm) having a large number of voids made of polypyrrole, which is a conductive polymer, is prepared.

(Preparation Example 6)
Instead of pyrrole in Preparation Example 5, 3,4-ethylenedioxythiophene is used to prepare a conductive member (film thickness 100 μm) having a large number of voids of poly (3,4-ethylenedioxythiophene).
(Preparation Example 7)
The solvent of a commercially available poly (3,4-ethylenedioxythiophene) poly (styrenesulfonate) aqueous dispersion (Bayer) was replaced with ethanol (polymer concentration 10 g / L), and prepared in the same manner as in Preparation Example 5. The dropping and drying steps are repeated on the porous film made of silica spheres to fill the voids in the porous film of silica spheres with the polymer. After annealing at 70 ° C. for 30 minutes, the silica spheres were removed by immersion in a 20% hydrofluoric acid solution for 2 days, and poly (3,4-ethylenedioxythiophene) poly (styrene sulfone). A conductive member having a large number of voids.
(Preparation Example 8)
In a 0.5 M aniline, 1 M lithium perchlorate aqueous solution, a porous film made of silica spheres prepared in the same manner as in Preparation Example 5 was used as a working electrode, a platinum wire was used as a counter electrode, and a galvanostat with 0.1 mA. Electrodeposition was performed at a current density of / cm @ 2. At this time, the electrolysis time was adjusted with reference to the profile of the electrolysis current so as to be approximately the same as the thickness of the porous film of silica spheres. After the electropolymerization, it is immersed in a 20% hydrofluoric acid solution for 2 days, the silica sphere is removed, and a conductive member having a large number of voids made of polyaniline is prepared.
(Preparation Example 9)
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. An ITO paste is printed on a cleaned gold substrate by a screen printing method and sintered at 250 ° C. for 1 hour to prepare an ITO porous sintered electrode (film thickness: 100 μm). 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 10)
Commercially available conductive titanium oxide fine particles (Titanium Industry, EC-300, particle size 300 nm) are dispersed in tarpinol, the viscosity is adjusted with ethyl cellulose, and a titanium oxide paste is prepared. A titanium oxide paste is printed on a cleaned gold substrate by a screen printing method and sintered at 450 ° C. for 1 hour to prepare a porous sintered film (film thickness 100 μm) of titanium oxide. Using this membrane as the working electrode and platinum wire as the counter electrode and gold plating solution (Uemura Kogyo 535LC), during electrospray and ultrasonic irradiation, the electrolytic gold plating was set to a current density of 0.1 mA / cm ² with a galvanostat. Then, plating is performed for 1 hour to prepare a conductive member having a large number of voids made of gold-plated porous titanium oxide.

(Preparation Example 11)
The cleaned gold substrate is immersed in a 0.01 M zinc nitrate water / ethanol = 9/1 solution, and a platinum wire is used as a counter electrode and a silver / silver chloride electrode as a reference electrode, 85 ° C., −1.2 V (vsAg / AgCl) is applied for 1.5 hours to grow acicular zinc oxide crystals. After cleaning the substrate, carbon coating is performed. The substrate is placed in a tubular furnace and raised by 5 ° C. per minute to the set temperature. During the heat treatment, 2% hydrogen / 98% helium is always flowed at 33 sccm, and 1% ethylene / 99% helium and 1% ethylene / 99% helium are flowed at 66 sccm as the hydrocarbon gas. At the time of hydrocarbon pyrolysis, conditions of a total gas amount of 100 sccm and ethylene: hydrogen: helium = 1: 1: 100 are used. The mixture was heated to 1000 ° C. over 200 minutes in a 2% hydrogen / 98% helium atmosphere, held for 10 minutes, and then 1% ethylene / 99% helium was allowed to flow for 10 minutes. Then, after hold | maintaining at 1000 degreeC for 1 hour, it is made to cool over 200 minutes and the electroconductive member which has many space | gap consisting of the needle-like crystal of the zinc oxide coated with carbon is prepared.
(Preparation Example 12)
An aluminum plate (thickness: 100 μm) is electropolished by an electropolishing method and then anodized with 0.3 M sulfuric acid at 25 V for 1 hour to produce porous alumina having a period of 60 nm. Using a platinum counter electrode for this electrode, electroplating gold plating (Uemura Kogyo 535LC) was performed with a galvanostat for 1 hour at a current density of 0.1 mA / cm ² during jetting and ultrasonic irradiation, and many gold plated A conductive member made of porous alumina having a plurality of voids is prepared.
(Preparation Example 13)
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 in which a number of voids are formed between the graphite particles is prepared.
(Preparation Example 14)
Instead of the graphite particles of Preparation Example 13, a conductive member having a large number of voids between a large number of carbon black particles bonded to each other using carbon black (Lion, Carbon ECP600JD) is prepared.
(Preparation Example 15)
Using a similar process, 20% by weight of single-walled carbon nanotubes (carbon nanotech research institute) is added to the carbon black of Preparation Example 14, and a number of voids are formed between a number of carbon nanotubes bonded to each other. A conductive member is prepared.

Next, the preparation of mediator molecules is described.
(Preparation Example 16)
A method for synthesizing the complex polymer represented by the following formula (1) 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) of 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 dried in vacuo 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 and 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 distilled 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, cooled to room temperature, and filtered. 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, and 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 of formula (1) (chloride salt).

(Preparation Example 17)
A method for synthesizing the complex polymer represented by the following formula (2) 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, impurities were collected by filtration, and the filtrate was distilled off under reduced pressure. (2,2 ′: 6 ′, 2 ″ -terpyridine) (4,4′-dimethyl-2,2′-bipyridine) chloride chloride salt 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 the formula (2). Identification of the compound is performed by elemental analysis.

(Preparation Example 18)
A method for synthesizing the complex polymer represented by the following formula (3) will be described.

Add 1.9 g of 2,2′-bipyridyl-N, N′-dioxide to 7.5 mL of concentrated sulfuric acid, slowly drop 1.6 g of fuming nitric acid in a salt ice bath, stir for 5 minutes, and then on crushed ice And the precipitated solid is collected by filtration to give 4,4′-dinitro-2,2′-bipyridyl-N, N′-dioxide. 0.5 g of 4,4′-dinitro-2,2′-bipyridyl-N, N′-dioxide was placed in 2.0 g of acetyl chloride and refluxed for 1 hour. After cooling the reaction solution, excess chloride was added. Acetyl is distilled off and the product is recrystallized from chloroform to give 4,4′-dichloro-2,2′-bipyridine. Identification is performed by ¹ H-NMR.

  Dissolve 24 g of acetylamide, 7 mL of 1-vinylimidazole in 150 mL of water, add 0.69 mL of an aqueous solution of N, N, N ′, N′-tetramethylethylenediamine, and dissolve 0.6 g of ammonium persulfate. 150 mL of the aqueous solution is added, and the reaction is carried out at 40 ° C. for 30 minutes under an argon atmosphere. The reaction solution is air-cooled, and the resulting precipitate is precipitated in 2 L of methanol. The precipitate is redissolved in 300 mL water and reprecipitated in 2 L methanol. The product was separated and allowed to stand in methanol at 4 ° C. for 12 hours, and then the solvent was distilled off under reduced pressure to obtain a polyacrylamide-polyvinylimidazole 7/1 copolymer.

  To 1.5 g of 4,4′-dichloro-2,2′-bipyridine and 1.4 g of ammonium hexachloride osmate, 5 mL of ethylene glycol is added and refluxed in an argon atmosphere for 1 hour. After the reaction solution is air-cooled, impurities are collected by filtration, and the filtrate is distilled off under reduced pressure to obtain osmium (4,4′-dichloro-2,2′-bipyridine) 2 dichloride chloride.

  Add 200 mL of ethanol to 1.0 g of osmium (4,4'-dichloro-2,2'-bipyridine) 2 dichloride chloride, 0.90 g of polyacrylamide-polyvinylimidazole 7/1 copolymer, and under nitrogen atmosphere 3 After refluxing for a day and filtering, the filtrate is dropped into 1 L of diethyl ether under strong stirring, and the generated precipitate is recovered and dried to obtain an osmium complex represented by the formula (3). Identification of the compound is performed by elemental analysis.

(Preparation Example 19)
A ferrocene derivative represented by the following formula (4) and a method for preparing glucose oxidase modified with the ferrocene derivative are described.

Dissolve 4.1 g of diethylenetriamine in 200 mL of DMF, add 2.1 g of ferrocenecarbaldehyde dissolved in 100 mL of DMF, and stir 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, a silica column was applied with dichloromethane / methanol = 10/1 solvent, the dimer was removed, and the formula (4) was obtained. A ferrocene derivative compound is obtained. The compound is identified by ¹ H-NMR. In a sample tube, 0.052 g of glucose oxidase (Aspergillus niger) is added to 1.3 mL of a 0.1 M aqueous sodium hydrogen carbonate solution, and 0.7 mL of 7 mg / mL sodium periodate is added. Stir for hours. The solution is added to 2 mL of 0.2 M citrate buffer, and 0.01 g of a ferrocene derivative compound represented by the formula (4) is further added thereto, and the mixture is stirred for 15 hours and centrifuged. The supernatant is filtered through a filter (Millipore) having a pore size of 0.2 μm, and applied to a gel filtration column (Sephadex® G25) to remove unreacted ferrocene derivative and to obtain glucose oxidase to which the ferrocene derivative is bound.

(Preparation Example 20)
The preparation method of the complex polymer (M = Ru) represented by the following formula (5) will be described.

  0.21 g of ruthenium trichloride, 0.31 g of 2,2′-bipyridine was added with 20 mL of ethylene glycol, refluxed for 24 hours under an argon atmosphere, the reaction solution was air-cooled, impurities were collected by filtration, and the filtrate was distilled under reduced pressure. Leaving ruthenium (2,2′-bipyridine) 2 dichloride chloride salt.

  0.1 g of ruthenium (2,2′-bipyridine) 2 dichloride chloride is added to 30 mL of DMF solution of 0.11 g polyvinylpyridine (average molecular weight 150,000), stirred at 90 ° C. for 24 hours, cooled to room temperature, Slowly pour into 1.2 L of stirring ethyl acetate. 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 diethyl ether, vacuum dry at 50 ° C. for 24 hours, and further crush the solid And dried for 48 hours to obtain a ruthenium complex polymer represented by the formula (5). Identification is performed by elemental analysis.

(Preparation Example 21)
A method for preparing the complex polymer (M = Co) represented by the formula (5) is described below.

  In place of 0.21 g of ruthenium trichloride of Preparation 20 0.13 g of cobalt dichloride, 0.10 g of ruthenium (2,2′-bipyridine) 2 dichloride chloride salt instead of 0.088 g of cobalt ( 2,2′-bipyridine) 2 dichloride chloride salt is used to obtain a cobalt complex polymer represented by formula (5).

(Preparation Example 22)
Describes (2-aminoethyl) FAD PREPARATION - N ^6. 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 was cooled, the precipitate is recovered in ethanol in an ice bath. The collected precipitate is subjected to anion exchange chromatography and reverse layer high performance chromatography to obtain purified N ^6- (2-aminoethyl) FAD.

(Preparation Example 23)
A method for preparing phenothiazine-modified glucose oxidase represented by the following formula (6) will be described.

  Add 0.40 g of phenothiazine and 3.0 g of polyethylene glycol (molecular weight 3000) to 50 mL of 0.01 M potassium hydroxide aqueous solution, add 0.040 g of ethylene oxide while stirring in an ice bath, and then stir at room temperature for 6 hours. After that, ultrafiltration is performed to remove the remaining raw material phenothiazine, followed by vacuum distillation to obtain polyethylene glycol-modified phenothiazine. Dissolve 3.2 g of polyethylene-modified phenothiazine in 50 mL of tetrahydrofuran (THF), add 0.11 g of methanesulfonyl chloride and 0.10 g of triethylamine, and stir at room temperature for 2 hours. The solvent is distilled off to obtain methanesulfonylated polyethylene glycol modified phenothiazine. This is dissolved in 100 mL of 5% aqueous ammonia and stirred at room temperature for 2 days to obtain an aminated polyethylene glycol modified phenothiazine. The carboxyl group on the surface of glucose oxidase (Aspergillus niger) was activated with 10 mM N-hydroxysuccinimide, 10 mM 1-ethyl-3- (3- (dimethylamino) propyl) carbodiimide in phosphate buffer, and aminated polyethylene glycol The modified phenothiazine was added and stirred at 25 ° C. for 24 hours, and then excess aminated polyethylene glycol-modified phenothiazine was removed by ultrafiltration to obtain phenothiazine-modified glucose oxidase.

In addition, the preparation of the enzyme is described.
(Preparation Example 24)
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 0.1 M 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 25)
Describes how to prepare cytochrome oxidase. 1 kg of the washed bovine heart muscle meat is stirred with 4 L of 0.02 M phosphate buffer (pH 7.4) for 6 minutes and centrifuged at 2500 G for 20 minutes. The supernatant is collected, and the precipitate is again stirred with 2 L of 0.02 M phosphate buffer (pH 7.4) for 3 minutes and centrifuged at 2500 G for 20 minutes. The supernatant is collected, combined with the previous supernatant, adjusted to pH 5.6 with 1M acetic acid, and centrifuged at 2500 G for 20 minutes. The precipitate is redispersed with 1 L of pure water and centrifuged at 2500 G for 20 minutes. The precipitate is redispersed with 450 mL of 0.02 M phosphate buffer (pH 7.4), 125 mL of 10% NaCl solution and 90 g of ammonium sulfate are added, and the mixture is allowed to stand at room temperature for 2 hours. Add 41 g ammonium sulfate and centrifuge at 7000 G for 20 minutes. Collect the supernatant (500 mL), add 50 g ammonium sulfate and centrifuge at 7000 G for 20 minutes. The precipitate was collected, dissolved in 200 mL of 0.1 M phosphate buffer (pH 7.4) containing 2% NaCl, 66 mL of saturated aqueous ammonium sulfate solution was added, and the mixture was allowed to stand at 0 ° C. for 12 hours, and 20% at 7000 G. Centrifuge for minutes. Collect the supernatant (200 mL), add 31 mL of saturated aqueous ammonium sulfate, and centrifuge at 7000 G for 20 minutes. The precipitate is recovered, dissolved in 100 mL of 0.1 M phosphate buffer (pH 7.4) containing 2% NaCl, and centrifuged at 7000 G for 20 minutes to recover the precipitate. Further, the steps of dissolving the precipitate in 100 mL phosphate buffer / adding 31 mL saturated aqueous ammonium sulfate solution / centrifugating / recovering the precipitate were repeated four times. pH 7.4) Dissolve in 30 mL to obtain a cytochrome oxidase solution.

(Preparation Example 26)
The solvent of a commercially available polystyrene latex colloid dispersion (Neon, average particle size: 100 nm) is replaced with ethanol, a washed gold substrate is placed in the dispersion, and ethanol is evaporated at 30 ° C. A porous membrane is obtained. By carrying out this step a plurality of times, a porous membrane (thickness 150 μm) made of polystyrene spheres with the desired thickness is prepared, heated at 70 ° C. for 30 minutes, and then washed with ethanol. Electrodeposition is performed by using this porous membrane as a working electrode, applying a current density of 0.1 mA / cm ² with a galvanostat in a 0.1 M nickel sulfate aqueous solution and using a platinum electrode as a counter electrode. The electrodeposition time is controlled by referring to the profile of the electrolysis current and adjusting the electrolysis time so as to be approximately the same as the film thickness of the polystyrene film. After electrodeposition, it is immersed in toluene for 2 days, the polystyrene sphere is removed, and a conductive member having a large number of voids made of nickel is prepared.

  Furthermore, preparation examples 27 to 29, 21, 33, 34 and 36 describe a method for preparing a conductive member having an inclination in the size of the gap used in the present invention. The particle diameter can be measured by observation with a scanning electron microscope, the size of the void can be measured by gas adsorption measurement, and the film thickness can be measured by using a surface roughness meter.

(Preparation Example 27)
The solvent of a commercially available polystyrene latex colloidal dispersion (Nippon ZEON, average particle size 100 nm, 200 nm) was replaced with ethanol, and a washed gold substrate was first set up in the dispersion solution with an average particle size of 100 nm, 30 ° C. By evaporating ethanol, a porous film made of polystyrene spheres is obtained. By performing this process a plurality of times, a porous film (film thickness of about 50 μm) made of 100 nm polystyrene spheres is prepared. Subsequently, on the prepared porous film made of polystyrene spheres having an average particle diameter of 100 nm, a porous film made of polystyrene spheres having an average particle diameter of 200 nm (film thickness of about 100 μm, total of about 150 μm) is the same as that at 100 nm. Prepare by procedure. Heat at 70 ° C. for 30 minutes and then wash with ethanol. Using this porous film, a conductive member having a large number of voids having a gradient in the size of the voids made of nickel is prepared using the same method as in Preparation Example 26.

(Preparation Example 28)
The solvent of a commercially available polystyrene latex colloid dispersion (three types of Nippon Zeon, average particle size 100 nm, 200 nm, and 300 nm) was replaced with ethanol, and a washed gold substrate was first placed in a dispersion solution with an average particle size of 100 nm, By evaporating ethanol at 30 ° C., a porous film made of polystyrene spheres is obtained. By performing this step a plurality of times, a porous film (film thickness of about 50 μm) made of 100 nm polystyrene spheres is prepared. Subsequently, on the prepared porous film made of polystyrene spheres having an average particle diameter of 100 nm, a porous film made of polystyrene spheres having an average particle diameter of 200 nm (film thickness of about 50 μm, total of about 100 μm) is the same as that at 100 nm. Prepare by procedure. Further, on the prepared porous film made of polystyrene spheres having an average particle diameter of 100 and 200 nm, a porous film made of polystyrene spheres having an average particle diameter of 300 nm (film thickness of about 50 μm, total of about 150 μm) is the same as in the case of 100 nm. Prepare according to the procedure. Heat at 70 ° C. for 30 minutes and then wash with ethanol. Using this porous film, a conductive member having a large number of voids having a gradient in the size of the voids made of nickel is prepared using the same method as in Preparation Example 26.

(Preparation Example 29)
A commercially available silica colloid dispersion (Nissan Chemical, average particle size 100 nm, three types of 300 nm) is substituted with ethanol, and a washed gold substrate is first placed in a dispersion solution with an average particle size of 100 nm, and ethanol is evaporated at 30 ° C. A porous membrane made of silica spheres is prepared. By performing this process a plurality of times, the thickness of the porous film made of silica spheres is increased (film thickness 50 μm). Subsequently, on the prepared porous film made of silica spheres having an average particle diameter of 100 nm, a porous film made of silica spheres having an average particle diameter of 300 nm (film thickness of about 50 μm, total of about 100 μm) is the same as that at 100 nm. Prepare by procedure. After heating at 200 ° C. for 3 hours, it was washed with ethanol, and this porous membrane was used as a working electrode in a 0.1 M 3,4-ethylenedioxythiophene, 0.1 M lithium perchlorate acetonitrile solution. 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. The polymerization time is controlled by referring to the electrolysis current profile and adjusting the electrolysis time so as to be approximately the same as the thickness of the silica sphere porous membrane. After electropolymerization, immersed in a 20% hydrofluoric acid solution for 2 days, the silica spheres are removed, and the size of the void made of poly (3,4-ethylenedioxythiophene), which is a conductive polymer, has an inclination. A conductive member having a large number of voids is prepared.

(Preparation Example 30)
Commercially available conductive titanium oxide fine particles (titanium industry, particle size of about 250 nm) are dispersed in tarpinol, the viscosity is adjusted with ethyl cellulose, and a titanium oxide paste is prepared. A titanium oxide paste is printed on a cleaned gold substrate by a screen printing method and sintered at 450 ° C. for 1 hour to prepare a porous sintered film (film thickness 100 μm) of titanium oxide. Using this membrane as the working electrode, platinum wire as the counter electrode, and gold plating solution (Uemura Kogyo 535LC), during electrospray and ultrasonic irradiation, electrolytic gold plating was set to a current density of 0.1 mA / cm ² with a galvanostat. Then, plating is performed for 1 hour to prepare a conductive member having a large number of voids made of gold-plated porous titanium oxide.
(Preparation Example 31)
Commercially available conductive titanium oxide fine particles (titanium industry, particle size of about 250 nm, two types of 400 nm) are each dispersed in tarpinol, the viscosity is adjusted with ethyl cellulose, and a titanium oxide paste is prepared. First, a titanium oxide paste having a particle diameter of 250 nm is printed on the cleaned gold substrate by screen printing (after baking, a film thickness of about 50 μm) and calcined at 150 ° C. for 5 minutes. A titanium oxide paste having a particle diameter of about 400 nm is printed thereon and sintered at 450 ° C. for 1 hour to prepare a titanium oxide porous sintered film (total film thickness 100 μm). Using this porous film, a conductive member having a large number of voids having a gradient in the size of the voids made of gold-plated porous titanium oxide is prepared using the same method as in Preparation Example 29.
(Preparation Example 32)
Nickel alloy foam metal (Mitsubishi Materials, MA600, thickness 0.5 mm, hole diameter 50 μm) is joined by spot welding with three layers to prepare a conductive member having a number of voids made of nickel alloy.
(Preparation Example 33)
Nickel alloy foam metal (Mitsubishi Materials, MA600, thickness 0.5mm, hole diameter 50, 150μm) is joined by spot welding with a total of three sheets in the order of one 50μm hole diameter and two 150μm. A conductive member having a large number of voids having an inclination in the size of the voids is prepared.
(Preparation Example 34)
Spot welding of nickel alloy foam metal (Mitsubishi Materials, MA600, thickness 0.5mm, hole diameter 50, 150μm, 300μm), 50μm, 150μm, 300μm one by one in order To form a conductive member having a large number of voids having a gradient in the size of the voids made of a nickel alloy.

(Preparation Example 35)
Two carbon fibers (Toray, Torayca cloth, thickness 0.2mm, fiber density 40 / 25cm) are stacked and cut into 1cm squares, and carbon paste (made by SPI) is applied on all sides to integrate them, and many carbon fibers A conductive member having a void is prepared.
(Preparation Example 36)
Two types of carbon fibers (Toray, Torayca cloth, thickness 0.2 mm, fiber density 40/25 cm, and 22.5 / 25 cm) are stacked one by one and cut into 1 cm squares, and carbon paste (made by SPI) is cut into four sides A conductive member is prepared which is integrated by coating and has a large number of voids having inclinations in the size of the voids made of carbon fibers.

  Next, a method for producing the enzyme electrode of the present invention will be described.

Example 1
Gold-plated stainless steel foam metal (Mitsubishi Materials, SUS316L (containing elements Fe, Cr, Ni, Mo, Si, O, Mn, C, P, S, Au) thickness 0.5 mm, gold plating thickness 0. 5 μm, pore diameter 50 μm) is cut into 1 cm square, washed and dried, and then subjected to UV ozone treatment to make it hydrophilic. 1 mL of 1.0 mg / mL glucose oxidase (Aspergillus niger), 1 wt% Triton X-100 (registered trademark) aqueous solution was prepared and mixed with 9 mL of 0.1 M pyrrole and 0.1 M lithium perchlorate aqueous solution. As an electrolyte, 1.1 V (vs Ag / AgCl) was applied for 1 second and 0.35 V was applied for 30 seconds in a nitrogen atmosphere with a foam metal as a working electrode, a platinum wire as a counter electrode, and a silver / silver chloride electrode as a reference electrode. A glucose oxidase enzyme electrode using polypyrrole as a carrier and mediator is prepared by applying a pulse 100 times at intervals to perform electrochemical polymerization and washing with water.
(Example 2)
In place of the 1.0 mg / mL glucose oxidase (Aspergillus niger) of Example 1, 245 U / mL quinohemoprotein-alcohol dehydrogenase (Gluconobacter sp-33) was used, so that an alcohol dehydrogenase enzyme electrode using polypyrrole as a carrier and mediator To prepare.
(Example 3)
A glucose oxidase enzyme electrode using poly (3,4-ethylenedioxythiophene) as a carrier and mediator is prepared by using 3,4-ethylenedioxythiophene instead of pyrrole in Example 1.
(Example 4)
By using 3,4-ethylenedioxythiophene instead of pyrrole in Example 2, an alcohol dehydrogenase enzyme electrode using poly (3,4-ethylenedioxythiophene) as a carrier and mediator is prepared.
(Example 5)
A glucose oxidase enzyme electrode using polyaniline as a carrier and mediator is prepared by using aniline instead of pyrrole in Example 1.
(Example 6)
By using aniline instead of pyrrole in Example 2, an alcohol dehydrogenase enzyme electrode using polyaniline as a carrier and mediator is prepared.

(Example 7)
Of water 5mL sample tube, to dissolve the osmium polymer described in Preparation Example 17 at a concentration of 10 mg / mL, in addition to citrate buffer 1mL of 0.2 M, further 30 mg / mL of laccase (Coriolus hirsutus) aqueous solution Add 1 mL. After stirring, 2 mL of a 10 mg / mL aqueous solution of polyethylene glycol diglycider ether is added and stirred. Thereafter, stainless steel metal foam was gold-plated (Mitsubishi Materials Corp., SUS316L (containing elements Fe, Cr, Ni, Mo, Si, O, Mn, C, P, S, Au) thickness 0.5 mm, gold plating thickness 0.5 [mu] m, pore size 50 [mu] m) and cut into 1cm square, washing, enzyme prepared in advance those subjected to UV ozone treatment, dipped in osmium polymer solution, pulled up, dried in a desiccator for 2 days, the enzyme electrode Prepare.
(Example 8)
The aqueous solution 5mL osmium polymer with sample tube shown in Preparation Example 18 of 10 mg / mL was prepared, further phosphate buffer 1 mL, bilirubin oxidase solution of 46mg / mL 1mL, 7mg / mL polyethylene glycol diglycidyl ether solution of 1 mL of is added. This was followed by gold-plated stainless steel metal foam (Mitsubishi Materials, SUS316L, contained elements Fe, Cr, Ni, Mo, Si, O, Mn, C, P, S, Au, thickness 0.5 mm, gold plating thickness 0.5 [mu] m, pore size 50 [mu] m) and cut into 1cm square, washing, enzyme prepared in advance those subjected to UV ozone treatment, dipped in osmium polymer solution, pulled up, dried in a desiccator for 2 days, the enzyme electrode Prepare.
Example 9
Prepare 1 mL of 0.1 M sodium bicarbonate aqueous solution of ferrocene-modified glucose oxidase as shown in Preparation Example 19 at 40 mg / mL in a sample tube, add 0.5 mL of 7 mg / mL sodium periodate aqueous solution, and stir in the dark for 1 hour. To do. This polyvinyl imidazole solution of 4 mg / mL 6 mL, plus the polyethylene glycol diglycidyl ether solution 0.4mL of 2.5 mg / mL, stainless steel metal foam was gold-plated (Mitsubishi Materials Corp., SUS316L, containing Element Fe, Cr, Ni, Mo, Si, O, Mn, C, P, S, Au, thickness 0.5 mm, gold plating thickness 0.5 μm, pore diameter 50 μm) are cut into 1 cm square, washed, and UV ozone treated The soaked material is dipped, pulled up and dried in a desiccator for 2 days to prepare an enzyme electrode.

(Example 10)
Prepare 1 mL of 0.1 M sodium bicarbonate and 40 mg / mL aqueous solution of glucose oxidase (Aspergillus niger) in a sample tube, add 0.5 mL of 7 mg / mL sodium periodate aqueous solution, and stir in the dark for 1 hour . To this, 6 mL of an aqueous solution of the ruthenium complex polymer shown in Preparation Example 20 of 10 mg / mL and 0.4 mL of a 2.5 mg / mL aqueous solution of polyethylene glycol diglycider ether were added to a foam metal made of stainless steel subjected to gold plating. (Mitsubishi Materials, SUS316L, containing elements Fe, Cr, Ni, Mo, Si, O, Mn, C, P, S, Au, thickness 0.5 mm, gold plating thickness 0.5 μm, pore diameter 50 μm) to 1 cm square A material that has been cut, washed, and UV ozone treated is immersed, pulled up, and dried in a desiccator for 2 days to prepare an enzyme electrode.
(Example 11)
Using the cobalt complex polymer shown in Preparation Example 21 instead of the ruthenium complex polymer shown in Preparation Example 20 of Example 10, an enzyme electrode is prepared using the same method.
(Example 12)
34 units of glucose dehydrogenase, 27 units of diaphorase, 0.22 mg of vitamin K3, 0.15 mg of nicotinamide adenine dinucleotide (NADH), 0.13 mg of polyvinylpyridine (average molecular weight) 150,000) was added to 5 mL of phosphate buffer, and 0.4 mL of 2.5 mg / mL polyethylene glycol diglycidel ether aqueous solution was added and stirred, followed by gold-plated stainless steel foam metal (Mitsubishi Materials, Cutting and cleaning SUS316L (containing elements Fe, Cr, Ni, Mo, Si, O, Mn, C, P, S, Au) thickness 0.5 mm, gold plating thickness 0.5 μm, pore diameter 50 μm, into 1 cm square, Perform UV ozone treatment Immersed things, pulling, dried in a desiccator for 2 days to prepare an enzyme electrode.
(Example 13)
An enzyme electrode is prepared in the same manner using 0.027 mg of anthraquinone instead of 0.022 mg of vitamin K3 of Example 12.
(Example 14)
An enzyme electrode is prepared in the same manner using the phenothiazine-modified glucose oxidase shown in Preparation Example 23 instead of the ferrocene-modified glucose oxidase shown in Preparation Example 19 of Example 9.
(Example 15)
Prepare 1 mL of an aqueous solution of 0.1 M sodium bicarbonate and 40 mg / mL glucose oxidase (Aspergillus niger) in a sample tube, add 0.5 mL of a 7 mg / mL aqueous sodium periodate solution, and stir in the dark for 1 hour. To this was added 6 mL of an aqueous solution of an osmium complex polymer shown in Preparation Example 16 of 10 mg / mL and 0.4 mL of 2.5 mg / mL polyethylene glycol diglycidel ether, and a foam metal made of stainless steel subjected to gold plating ( Mitsubishi Materials, SUS316L (containing elements Fe, Cr, Ni, Mo, Si, O, Mn, C, P, S, Au) thickness 0.5mm, gold plating thickness 0.5μm, pore diameter 50μm) cut to 1cm square Then, after washing and UV ozone treatment are immersed, they are pulled up and dried in a desiccator for 2 days to prepare an enzyme electrode.
(Example 16)
To 1.8 mL of 0.1 M phosphate buffer, add 0.25 mL of 1 M N- (3- (trimethoxysilyl) propyl) ethylenediamine solution and 0.25 mL of 0.01 M chloroauric acid solution, and ultrasonic For 10 minutes. Hydrochloric acid is added to adjust the pH to 7, and 0.013 mL of a 0.1 M sodium borohydride solution is added. The sol is then stirred for 24 hours to prepare a silica sol containing gold fine particles. Dissolve 10 mg of glucose oxidase in 6 mL of 0.05 M phosphate buffer (pH 7.0), add 1.6 g of polyvinylpyridine, and stir until uniform. Stainless steel foam metal (Mitsubishi Materials, SUS316L (containing elements Fe, Cr, Ni, Mo, Si, O, Mn, C, P S, Au) thickness 0.5mm, gold plating thickness 0.5μm, pore diameter 50μm) cut into 1cm square, washed, soaked with UV ozone treatment, soaked up, dried in desiccator for 2 days, enzyme Prepare the electrode.
(Example 17)
An enzyme electrode is prepared in the same manner using palladium chloride in place of chloroauric acid in Example 16.
(Example 18)
Under a nitrogen atmosphere, 0.25 mL of titanium (IV) isopropoxide is dissolved in a small amount of isopropanol, 1.8 mL of 0.1 M phosphate buffer and 0.25 mL of 0.01 M chloroauric acid solution are added, and ultrasonic waves are added. For 1 hour. 0.1M hydrochloric acid is added to adjust the pH to 7, and after adding 0.01M mL of 0.1M sodium borohydride solution, the sol is stirred for 24 hours to prepare a titania sol containing gold fine particles. Dissolve 10 mg of glucose oxidase in 6 mL of 0.05 M phosphate buffer (pH 7.0), add 1.6 g of polyvinyl pyridine, and stir until homogeneous, then add to the titania sol containing the fine gold particles prepared above. The mixture was stirred until it became homogeneous, and the resulting mixture was subjected to gold-plated stainless steel foam metal (Mitsubishi Materials, SUS316L (containing elements Fe, Cr, Ni, Mo, Si, O, Mn, C, P , S, Au) thickness 0.5 mm, gold plating thickness 0.5 μm, pore diameter 50 μm) cut into 1 cm square, washed, soaked with UV ozone treatment, pulled up, dried in a desiccator for 2 days, An enzyme electrode is prepared.
(Example 19)
An enzyme electrode is prepared in the same manner as in Example 18 except that palladium chloride is used instead of chloroauric acid.
(Example 20)
Dissolve 20 mg polylysine hydrochloride (average molecular weight 70,000) in 8 mL 0.1 M phosphate buffer, add 40 mg bilirubin oxidase and 27 mg potassium octacyanotungstate, stir at 0 ° C. for 1 hour, Stainless steel foam metal (Mitsubishi Materials, SUS316L (containing elements Fe, Cr, Ni, Mo, Si, O, Mn, C, P, S, Au) thickness 0.5 mm, gold plating thickness 0.5 μm, empty A piece having a pore size of 50 μm) cut into 1 cm square, washed and subjected to UV ozone treatment is immersed, pulled up and dried in a desiccator for 2 days to prepare an enzyme electrode.

(Example 21)
34 units glucose dehydrogenase (Thermoplasma acidophilum), 27 units diaphorase (Spinacia oleracea), 0.22 mg vitamin K3, 0.15 mg NADH were added to 5 mL phosphate buffer and 1% bovine serum albumin 0.1%. Add 0.4 mL of 5 mL, 2.5 mg / mL glutaraldehyde solution and stir. Gold-plated stainless steel foam metal (Mitsubishi Materials, SUS316L (containing elements Fe, Cr, Ni, Mo, Si, O, Mn, C, P, S, Au) thickness 0.5 mm, gold plating thickness 0. 5 μm, pore diameter 50 μm) is cut into 1 cm square, washed, and subjected to UV ozone treatment, soaked in a 0.02 M aqueous solution of aminoethanethiol for 2 hours, and then washed with water. Thereafter, it is immersed in an enzyme-carrying solution, pulled up, and dried in a desiccator for 2 days to prepare an enzyme electrode.
(Example 22)
Gold-plated stainless steel foam metal (Mitsubishi Materials, SUS316L (containing elements Fe, Cr, Ni, Mo, Si, O, Mn, C, P, S, Au) thickness 0.5 mm, gold plating thickness 0. 5 μm, pore diameter 50 μm) is cut into 1 cm square, washed, and UV ozone treated, soaked in 0.02M cystamine aqueous solution for 2 hours, pulled up and washed with water to prepare a cystamine modified electrode. 3 mM pyrroloquinoline quinone (PQQ), 10 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide in 0.01 M N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) buffer PQQ modification is performed by immersing in a liquid solution for 1 hour and washing with water. 1 mM N ^6- (2-aminoethyl) FAD described in Preparation Example 22, 10 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide in 0.01 M HEPES buffer solution (pH 7.3) FAD modification is performed by immersing the previous PQQ-modified electrode for 2 hours and washing with water. Further, 4 mg / mL of apoglucose oxidase described in Preparation Example 24 in 0.1 M phosphate buffer (pH 7.0) was dipped and pulled up at 25 ° C. for 4 hours and 4 ° C. for 12 hours, and then phosphate buffer (pH 7. 0) Prepare an enzyme electrode by dipping in 1 hour.
(Example 23)
0.06 mM sulfo-N-hydroxysuccinimide-modified gold microparticles (Nanoprobes), 0.018 HEPES buffer solution in which 0.68 mM N ^6- (2-aminoethyl) FAD described in Preparation Example 22 was dissolved ( pH 7.9) is stirred at room temperature for 1 hour and at 4 ° C. for 12 hours to react gold fine particles with N ^6- (2-aminoethyl) FAD. Unreacted N ^6- (2-aminoethyl) FAD is removed using a spin column (Sigma) to prepare FAD-modified gold fine particles. Further, 0.1 mg phosphoric acid containing 30% glycerol, 0.1% bovine serum albumin, and 0.1% sodium azide containing 3 mg / mL apoglucose oxidase described in Preparation Example 24 and 4.8 μM FAD-modified gold fine particles. The buffer solution is stirred at room temperature for 4 hours and at 4 ° C. for 12 hours, and then the glucose oxidase-modified gold microparticles are separated by centrifugation. Gold-plated stainless steel foam metal (Mitsubishi Materials, SUS316L (containing elements Fe, Cr, Ni, Mo, Si, O, Mn, C, P, S, Au) thickness 0.5 mm, gold plating thickness 0. 5 μm, pore diameter 50 μm) cut to 1 cm square, washed and UV ozone treated, soaked in 0.02M cystamine aqueous solution for 2 hours, pulled up and washed with water to prepare cystamine modified electrode, 1 μM glucose oxidase An enzyme electrode is prepared by immersing the modified gold fine particles in a phosphate buffer solution at 4 ° C. for 12 hours.
(Example 24)
Gold-plated stainless steel foam metal (Mitsubishi Materials, SUS316L (containing elements Fe, Cr, Ni, Mo, Si, O, Mn, C, P, S, Au) thickness 0.5 mm, gold plating thickness 0. 5 μm, pore diameter 50 μm) is cut into 1 cm square, washed and UV ozone treated, soaked in an ethanol solution of 1 mM cystamine for 2 hours, pulled up and washed with ethanol to prepare a cystamine-modified substrate. This substrate was treated with 1 mM 1,2-dehydro-1,2-methanofullerene [60] -61-carboxylic acid (Material Technologies Research Limited), 5 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide. A fullerene-modified base material is prepared by immersing in ethanol: dimethylsulfoxide (DMSO) = 1: 1 solution at room temperature for 4 hours and washing the base material with a mixed solvent of ethanol and DMSO. Add 0.8 mL of 2.5 mg / mL glutaraldehyde to 10 mL of phosphate buffer of 30 mg / mL glucose oxidase (Aspergillus niger), stir, and add the fullerene-modified substrate at room temperature for 1 hour at 4 ° C. for 12 hours. It is immersed for a period of time, pulled up, washed with a phosphate buffer, and then dried in a desiccator for 2 days to prepare an enzyme electrode.
(Example 25)
Gold-plated stainless steel foam metal (Mitsubishi Materials, SUS316L (containing elements Fe, Cr, Ni, Mo, Si, O, Mn, C, P, S, Au) thickness 0.5 mm, gold plating thickness 0. 5 μm, pore diameter 50 μm) is cut into 1 cm square, washed, and subjected to UV ozone treatment, immersed in a 0.02M cystamine aqueous solution for 2 hours, pulled up, and washed with water. Immerse in 0.3M microperoxidase-11 (MP11), 10 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide in 0.01M HEPES buffer solution for 3 hours, pull up, 0.01M HEPES An enzyme electrode is prepared by immersion in a buffer solution (pH 7.3) for 1 hour.

(Example 26)
Gold-plated stainless steel foam metal (Mitsubishi Materials, SUS316L (containing elements Fe, Cr, Ni, Mo, Si, O, Mn, C, P, S, Au) thickness 0.5 mm, gold plating thickness 0. 5 μm, pore diameter 50 μm) is cut into 1 cm square, washed, and UV ozone treated, soaked in a 0.02 M cystamine aqueous solution for 2 hours, pulled up, washed with water, and a cystamine modified electrode is prepared. The cystamine-modified electrode was immersed in a 0.01 M HEPES buffer solution containing 3 mM N-succinimidyl-3-maleimidopropionate and 10 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide for 1 hour. The modification is performed by washing with 0.01 M HEPES buffer solution. This electrode was immersed in 0.1 M phosphate buffer (pH 7.0) containing 4 mg / mL cytochrome C at 25 ° C. for 4 hours and at 4 ° C. for 12 hours, pulled up, and in phosphate buffer (pH 7.0). The maleimide is modified with the thiol group of the enzyme by soaking for 1 hour. Furthermore, this electrode was immersed in a 0.1 M phosphate buffer solution (pH 7.0) containing cytochrome oxidase described in Preparation Example 25 at 4 mg / mL for 4 hours at 25 ° C. and then pulled up for 12 hours at 4 ° C. After coupling with cytochrome C / cytochrome oxidase by immersing in the solution (pH 7.0) for 1 hour, 0.1 mM phosphate buffer solution (pH 7.0) of 10 mM glutaraldehyde at 25 ° C. for 10 minutes. An immobilized enzyme electrode is prepared by immersion for 1 hour at 4 ° C.
(Example 27)
In place of the stainless steel foam metal subjected to gold plating in Example 4, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, A gold plating thickness of 0.5 μm and a pore diameter of 50 μm is used, and the enzyme electrode is prepared in the same manner as the other configurations.
(Example 28)
Instead of the stainless steel foam metal subjected to gold plating in Example 8, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, A gold plating thickness of 0.5 μm and a pore diameter of 50 μm is used, and the enzyme electrode is prepared in the same manner as the other configurations.
(Example 29)
Instead of the foam metal made of stainless steel subjected to gold plating in Example 15, a foam metal made of nickel alloy (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, A gold plating thickness of 0.5 μm and a pore diameter of 50 μm is used, and the enzyme electrode is prepared in the same manner as the other configurations.
(Example 30)
Instead of the stainless steel foam metal subjected to gold plating in Example 18, a nickel alloy foam metal (Mitsubishi Materials, containing elements Ni, Cr, Ti, Nb, Al, Mn, Si, C, thickness 0.5 mm, A gold plating thickness of 0.5 μm and a pore diameter of 50 μm is used, and the enzyme electrode is prepared in the same manner as the other configurations.

(Example 31)
In place of the stainless steel foam metal subjected to gold plating in Example 4, a conductive member having nickel voids described in Preparation Example 1 was used, and the enzyme electrode was prepared in the same manner as in the other configurations.
(Example 32)
In place of the stainless steel foam metal subjected to gold plating in Example 8, a conductive member having nickel voids described in Preparation Example 1 is used, and the enzyme electrode is prepared in the same manner as in the other configurations.
(Example 33)
Instead of the stainless steel foam metal subjected to gold plating in Example 15, a conductive member having nickel voids described in Preparation Example 1 was used, and the enzyme electrode was prepared in the same manner as in the other configurations.
(Example 34)
Instead of the stainless steel foam metal subjected to gold plating in Example 18, a conductive member having nickel voids described in Preparation Example 1 was used, and the enzyme electrode was prepared in the same manner as in the other configurations.
(Example 35)
In place of the stainless steel foam metal subjected to gold plating in Example 4, a stainless steel net (Nilaco, contained elements Fe, Cr, Ni, Mo, Si, O, Mn, C, P, S, 400 mesh) was used. The enzyme electrode is prepared in the same manner as the other configurations.
(Example 36)
In place of the stainless steel foam metal subjected to gold plating in Example 8, a stainless steel net (Nilaco, contained elements Fe, Cr, Ni, Mo, Si, O, Mn, C, P, S, 400 mesh) was used. The enzyme electrode is prepared in the same manner as the other configurations.
(Example 37)
Instead of the stainless steel foam metal subjected to gold plating in Example 15, a stainless steel net (Nilaco, containing elements Fe, Cr, Ni, Mo, Si, O, Mn, C, P, S, 400 mesh) was used. The enzyme electrode is prepared in the same manner as the other configurations.
(Example 38)
Instead of the stainless steel foam metal subjected to gold plating in Example 18, a nickel alloy mesh stainless steel mesh (Niraco, containing elements Fe, Cr, Ni, Mo, Si, O, Mn, C, P, S, 400 The enzyme electrode is prepared in the same manner with the other configurations.
(Example 39)
An electroconductive member having platinum voids described in Preparation Example 2 is used instead of the stainless steel foam metal subjected to gold plating in Example 4, and an enzyme electrode is prepared in the same manner as in the other configurations.
(Example 40)
In place of the stainless steel foam metal subjected to gold plating in Example 8, a conductive member having platinum voids described in Preparation Example 2 was used, and the enzyme electrode was prepared in the same manner as in the other configurations.

(Example 41)
In place of the stainless steel foam metal subjected to gold plating in Example 15, a conductive member having a platinum void described in Preparation Example 2 was used, and an enzyme electrode was prepared in the same manner as in the other configurations.
(Example 42)
Instead of the stainless steel foam metal subjected to gold plating in Example 18, a conductive member having platinum voids described in Preparation Example 2 was used, and the enzyme electrode was prepared in the same manner as in the other configurations.
(Example 43)
Instead of the stainless steel foam metal subjected to gold plating in Example 4, a conductive member having gold voids described in Preparation Example 3 was used, and the enzyme electrode was prepared in the same manner as in the other configurations.
(Example 44)
Instead of the stainless steel foam metal subjected to gold plating in Example 8, a conductive member having gold voids described in Preparation Example 3 was used, and the enzyme electrode was prepared in the same manner as in the other configurations.
(Example 45)
Instead of the stainless steel foam metal subjected to gold plating in Example 15, a conductive member having gold voids described in Preparation Example 3 is used, and the enzyme electrode is prepared in the same manner as in the other configurations.
(Example 46)
Instead of the stainless steel foam metal subjected to gold plating in Example 18, a conductive member having gold voids described in Preparation Example 3 was used, and the enzyme electrode was prepared in the same manner as in the other configurations.
(Example 47)
Instead of the stainless steel foam metal subjected to gold plating in Example 24, the conductive member having gold voids described in Preparation Example 3 is used, and the enzyme electrode is prepared in the same manner as in the other configurations.
(Example 48)
In place of the stainless steel foam metal subjected to gold plating in Example 4, a conductive member having a void of palladium described in Preparation Example 4 is used, and the enzyme electrode is prepared in the same manner as in the other configurations.
(Example 49)
Instead of the stainless steel foam metal subjected to gold plating in Example 8, a conductive member having a void of palladium described in Preparation Example 4 is used, and the enzyme electrode is prepared in the same manner as in the other configurations.
(Example 50)
Instead of the stainless steel foam metal subjected to gold plating in Example 15, a conductive member having a void of palladium described in Preparation Example 4 is used, and the enzyme electrode is prepared in the same manner as in the other configurations.

(Example 51)
Instead of the stainless steel foam metal subjected to gold plating in Example 18, a conductive member having a void of palladium described in Preparation Example 4 is used, and the enzyme electrode is prepared in the same manner as in the other configurations.
(Example 52)
In place of the stainless steel foam metal subjected to gold plating in Example 4, a conductive member having a void of the polypyrrole electrode described in Preparation Example 5 is used, and the enzyme electrode is prepared in the same manner as in the other configurations.
(Example 53)
In place of the stainless steel foam metal subjected to gold plating in Example 8, a conductive member having a void of the polypyrrole electrode described in Preparation Example 5 is used, and the enzyme electrode is prepared in the same manner as in the other configurations.
(Example 54)
Instead of the stainless steel foam metal subjected to gold plating in Example 15, the macroporous polypyrrole electrode described in Preparation Example 5 is used, and the enzyme electrode is prepared in the same manner as in the other configurations.
(Example 55)
In place of the stainless steel foam metal subjected to gold plating in Example 18, the conductive member having the voids of the polypyrrole electrode described in Preparation Example 5 is used, and the enzyme electrode is prepared in the same manner as in the other configurations.
(Example 56)
A conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used instead of the stainless steel foam metal subjected to gold plating in Example 1, and the other configurations were the same. Then, an enzyme electrode is prepared.
(Example 57)
A conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used instead of the stainless steel foam metal subjected to gold plating in Example 2, and the other configurations were the same. Then, an enzyme electrode is prepared.
(Example 58)
A conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used instead of the stainless steel foam metal subjected to gold plating in Example 3, and the other configurations were the same. Then, an enzyme electrode is prepared.
(Example 59)
A conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used instead of the stainless steel foam metal subjected to gold plating in Example 4, and the other configurations were the same. Then, an enzyme electrode is prepared.
(Example 60)
A conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used instead of the stainless steel foam metal subjected to gold plating in Example 5, and the other configurations were the same. Then, an enzyme electrode is prepared.

(Example 61)
A conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used instead of the stainless steel foam metal subjected to gold plating in Example 6, and the other configurations were the same. Then, an enzyme electrode is prepared.
(Example 62)
A conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used instead of the stainless steel foam metal subjected to gold plating in Example 7, and the other configurations were the same. Then, an enzyme electrode is prepared.
(Example 63)
A conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used instead of the stainless steel foam metal subjected to gold plating in Example 8, and the other configurations were the same. Then, an enzyme electrode is prepared.
(Example 64)
A conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used instead of the stainless steel foam metal subjected to gold plating in Example 9, and the other configurations were the same. Then, an enzyme electrode is prepared.
(Example 65)
A conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used instead of the stainless steel foam metal subjected to gold plating in Example 10, and the other configurations were the same. Then, an enzyme electrode is prepared.
Example 66
A conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used instead of the stainless steel foam metal subjected to gold plating in Example 11, and the other configurations were the same. Then, an enzyme electrode is prepared.
(Example 67)
A conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used instead of the stainless steel foam metal subjected to gold plating in Example 12, and the other configurations were the same. Then, an enzyme electrode is prepared.
(Example 68)
A conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used instead of the stainless steel foam metal subjected to gold plating in Example 13, and the other configurations were the same. Then, an enzyme electrode is prepared.
(Example 69)
Instead of the stainless steel foam metal subjected to gold plating in Example 14, a conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used, and the other configurations were made the same. Prepare the enzyme electrode.
(Example 70)
A conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used instead of the stainless steel foam metal subjected to gold plating in Example 15, and the other configurations were the same. Then, an enzyme electrode is prepared.

(Example 71)
Instead of the stainless steel foam metal subjected to gold plating in Example 16, a conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used, and the other configurations were the same. Then, an enzyme electrode is prepared.
(Example 72)
A conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used in place of the stainless steel foam metal subjected to gold plating in Example 17, and the other configurations were the same. Then, an enzyme electrode is prepared.
(Example 73)
A conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used instead of the stainless steel foam metal subjected to gold plating in Example 18, and the other configurations were the same. Then, an enzyme electrode is prepared.
(Example 74)
A conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used in place of the stainless steel foam metal subjected to gold plating in Example 19, and the other configurations were the same. Then, an enzyme electrode is prepared.
(Example 75)
A conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used instead of the stainless steel foam metal subjected to gold plating in Example 20, and the other configurations were the same. Then, an enzyme electrode is prepared.
(Example 76)
Instead of the stainless steel foam metal subjected to gold plating in Example 4, a conductive member having voids of poly (3,4-ethylenedioxythiophene) poly (styrenesulfonate) described in Preparation Example 7 is used. Then, the enzyme electrode is prepared in the same manner as the other components.
(Example 77)
Instead of the stainless steel foam metal subjected to gold plating in Example 8, a conductive member having voids of poly (3,4-ethylenedioxythiophene) poly (styrene sulfonate) described in Preparation Example 7 is used. Then, the enzyme electrode is prepared in the same manner as the other components.
(Example 78)
Instead of the stainless steel foam metal subjected to gold plating in Example 15, a conductive member having voids of poly (3,4-ethylenedioxythiophene) poly (styrenesulfonate) described in Preparation Example 7 was used. Then, the enzyme electrode is prepared in the same manner as the other components.
(Example 79)
Instead of the stainless steel foam metal subjected to gold plating in Example 18, a conductive member having voids of poly (3,4-ethylenedioxythiophene) poly (styrenesulfonate) described in Preparation Example 7 was used. Then, the enzyme electrode is prepared in the same manner as the other components.
(Example 80)
In place of the stainless steel foam metal subjected to gold plating in Example 4, a conductive member having a polyaniline void described in Preparation Example 8 was used, and the enzyme electrode was prepared in the same manner as in the other configurations.

(Example 81)
In place of the stainless steel foam metal subjected to gold plating in Example 8, a conductive member having a polyaniline void described in Preparation Example 8 was used, and an enzyme electrode was prepared in the same manner as the other components.
(Example 82)
In place of the stainless steel foam metal subjected to gold plating in Example 15, a conductive member having a polyaniline void described in Preparation Example 8 was used, and an enzyme electrode was prepared in the same manner as the other components.
(Example 83)
In place of the stainless steel foam metal subjected to gold plating in Example 18, a conductive member having a polyaniline void described in Preparation Example 8 was used, and an enzyme electrode was prepared in the same manner as in the other configurations.
(Example 84)
In place of the stainless steel foam metal subjected to gold plating in Example 4, a conductive member having an ITO gap described in Preparation Example 9 was used, and the enzyme electrode was prepared in the same manner as in the other configurations.
(Example 85)
In place of the stainless steel foam metal subjected to gold plating in Example 8, a conductive member having an ITO gap described in Preparation Example 9 was used, and an enzyme electrode was prepared in the same manner as in the other configurations.
(Example 86)
In place of the stainless steel foam metal subjected to gold plating in Example 15, a conductive member having an ITO gap described in Preparation Example 9 was used, and an enzyme electrode was prepared in the same manner as the other components.
(Example 87)
In place of the stainless steel foam metal subjected to gold plating in Example 18, a conductive member having an ITO gap described in Preparation Example 9 was used, and an enzyme electrode was prepared in the same manner as in the other configurations.
(Example 88)
In place of the gold-plated stainless steel foam metal of Example 4, the conductive member having the voids of the gold-plated porous titanium oxide described in Preparation Example 10 was used, and the enzyme electrode was prepared in the same manner with the other configurations. Prepare.
Example 89
In place of the gold-plated stainless steel foam metal of Example 8, the conductive member having the voids of the gold-plated porous titanium oxide described in Preparation Example 10 was used, and the enzyme electrode was prepared in the same manner with the other configurations. Prepare.
(Example 90)
Instead of the stainless steel foam metal subjected to gold plating in Example 15, the conductive member having the voids of the gold-plated porous titanium oxide described in Preparation Example 10 was used, and the enzyme electrode was prepared in the same manner as in the other configurations. Prepare.

(Example 91)
Instead of the stainless steel foam metal subjected to gold plating in Example 18, the conductive member having voids of the gold-plated porous titanium oxide described in Preparation Example 10 was used, and the enzyme electrode was prepared in the same manner as in the other configurations. Prepare.
(Example 92)
Instead of the stainless steel foam metal subjected to gold plating in Example 24, a conductive member having voids of gold-plated porous titanium oxide described in Preparation Example 10 was used, and the enzyme electrode was formed in the same manner as in the other configurations. Prepare.
(Example 93)
Instead of the stainless steel foam metal subjected to gold plating in Example 1, the conductive member having voids of the carbon-coated zinc oxide needle-like crystals described in Preparation Example 11 was used, and the other configurations were made the same. Prepare the enzyme electrode.
(Example 94)
Instead of the stainless steel foam metal subjected to gold plating in Example 2, the conductive member having voids of carbon-coated zinc oxide needle crystals described in Preparation Example 11 was used, and the other configurations were made the same. Prepare the enzyme electrode.
(Example 95)
Instead of the stainless steel foam metal subjected to gold plating in Example 3, a conductive member having voids of carbon-coated zinc oxide needle-like crystals described in Preparation Example 11 was used, and the other configurations were made the same. Prepare the enzyme electrode.
(Example 96)
Instead of the stainless steel foam metal subjected to gold plating in Example 4, a conductive member having voids of carbon-coated zinc oxide needle crystals described in Preparation Example 11 was used, and the other configurations were made the same. Prepare the enzyme electrode.
(Example 97)
Instead of the stainless steel foam metal subjected to gold plating in Example 5, the conductive member having voids of the carbon-coated zinc oxide needle crystals described in Preparation Example 11 was used, and the other configurations were made the same. Prepare the enzyme electrode.
(Example 98)
Instead of the stainless steel foam metal subjected to gold plating in Example 6, the conductive member having the voids of the carbon-coated zinc oxide needle crystals described in Preparation Example 11 was used, and the other configurations were made the same. Prepare the enzyme electrode.
Example 99
Instead of the stainless steel foam metal subjected to gold plating in Example 7, the conductive member having the voids of the carbon-coated zinc oxide needle crystals described in Preparation Example 11 was used, and the other configurations were made the same. Prepare the enzyme electrode.
(Example 100)
Instead of the stainless steel foam metal subjected to gold plating in Example 8, a conductive member having voids of carbon-coated zinc oxide needle-like crystals described in Preparation Example 11 was used, and the other configurations were made the same. Prepare the enzyme electrode.

(Example 101)
Instead of the stainless steel foam metal subjected to gold plating in Example 9, the conductive member having voids of the carbon-coated zinc oxide needle crystals described in Preparation Example 11 was used, and the other configurations were made the same. Prepare the enzyme electrode.
(Example 102)
Instead of the stainless steel foam metal subjected to gold plating in Example 10, the conductive member having the voids of the carbon-coated zinc oxide needle crystals described in Preparation Example 11 was used, and the other configurations were made the same. Prepare the enzyme electrode.
(Example 103)
Instead of the stainless steel foam metal subjected to gold plating in Example 11, the conductive member having the voids of the carbon-coated zinc oxide needle-like crystal described in Preparation Example 11 was used, and the other configurations were made the same. Prepare the enzyme electrode.
(Example 104)
Instead of the stainless steel foam metal subjected to gold plating in Example 12, the conductive member having voids of the carbon-coated zinc oxide needle-like crystals described in Preparation Example 11 was used, and the other configurations were made the same. Prepare the enzyme electrode.
(Example 105)
Instead of the stainless steel foam metal subjected to gold plating of Example 13, the conductive member having the voids of the carbon-coated zinc oxide needle-like crystal described in Preparation Example 11 was used, and the other configurations were made the same. Prepare the enzyme electrode.
(Example 106)
Instead of the stainless steel foam metal subjected to gold plating in Example 14, the conductive member having voids of the carbon-coated zinc oxide needle-like crystals described in Preparation Example 11 was used, and the other configurations were made the same. Prepare the enzyme electrode.
(Example 107)
Instead of the stainless steel foam metal subjected to gold plating in Example 15, the conductive member having the voids of the carbon-coated zinc oxide needle crystals described in Preparation Example 11 was used, and the other configurations were made the same. Prepare the enzyme electrode.
(Example 108)
Instead of the stainless steel foam metal subjected to gold plating in Example 16, the conductive member having voids of carbon-coated zinc oxide needle crystals described in Preparation Example 11 was used, and the other configurations were made the same. Prepare the enzyme electrode.
(Example 109)
Instead of the stainless steel foam metal subjected to gold plating in Example 17, the conductive member having the voids of the carbon-coated zinc oxide needle crystals described in Preparation Example 11 was used, and the other configurations were made the same. Prepare the enzyme electrode.
(Example 110)
Instead of the stainless steel foam metal subjected to gold plating in Example 18, the conductive member having the voids of the carbon-coated zinc oxide needle crystals described in Preparation Example 11 was used, and the other configurations were made the same. Prepare the enzyme electrode.

(Example 111)
Instead of the stainless steel foam metal subjected to gold plating in Example 19, the conductive member having voids of the carbon-coated zinc oxide needle crystals described in Preparation Example 11 was used, and the other configurations were made the same. Prepare the enzyme electrode.
(Example 112)
Instead of the stainless steel foam metal subjected to gold plating in Example 20, the conductive member having voids of carbon-coated zinc oxide needle crystals described in Preparation Example 11 was used, and the other configurations were made the same. Prepare the enzyme electrode.
(Example 113)
In place of the gold-plated stainless steel foam metal of Example 4, a conductive member having voids of gold-plated alumina nanoholes as described in Preparation Example 12 is used, and the enzyme electrode is prepared in the same manner as the other components. .
(Example 114)
In place of the stainless steel foam metal subjected to gold plating in Example 8, a conductive member having voids of gold-plated alumina nanoholes described in Preparation Example 12 was used, and the enzyme electrode was prepared in the same manner as the other components. .
(Example 115)
In place of the gold-plated stainless steel foam metal of Example 15, a conductive member having voids of gold-plated alumina nanoholes as described in Preparation Example 12 was used, and the enzyme electrode was prepared in the same manner as the other components. .
(Example 116)
In place of the gold-plated stainless steel foam metal of Example 18, a conductive member having gold-plated alumina nanohole voids described in Preparation Example 12 was used, and the other configurations were similarly used to prepare an enzyme electrode. .
(Example 117)
In place of the gold-plated stainless steel foam metal of Example 24, a conductive member having voids of gold-plated alumina nanoholes as described in Preparation Example 12 was used, and the enzyme electrode was prepared in the same manner as the other components. .
(Example 118)
In place of the stainless steel foam metal subjected to gold plating in Example 4, the conductive member having the graphite voids described in Preparation Example 13 was used, and an enzyme electrode was prepared in the same manner as in the other configurations.
(Example 119)
In place of the stainless steel foam metal subjected to gold plating in Example 8, a conductive member having a graphite void described in Preparation Example 13 was used, and an enzyme electrode was prepared in the same manner as the other components.
(Example 120)
Instead of the stainless steel foam metal subjected to gold plating in Example 15, a conductive member having a graphite void described in Preparation Example 13 was used, and an enzyme electrode was prepared in the same manner as in the other configurations.

(Example 121)
In place of the stainless steel foam metal subjected to gold plating in Example 18, a conductive member having a graphite void described in Preparation Example 13 was used, and an enzyme electrode was prepared in the same manner as in the other configurations.
(Example 122)
In place of the stainless steel foam metal subjected to gold plating in Example 4, a conductive member having a carbon black void described in Preparation Example 14 is used, and the enzyme electrode is prepared in the same manner as in the other configurations.
(Example 123)
Instead of the stainless steel foam metal subjected to gold plating in Example 8, a conductive member having a carbon black void described in Preparation Example 14 is used, and the enzyme electrode is prepared in the same manner as in the other configurations.
(Example 124)
In place of the stainless steel foam metal subjected to gold plating in Example 15, a conductive member having carbon black voids described in Preparation Example 14 was used, and an enzyme electrode was prepared in the same manner as in the other configurations.
(Example 125)
Instead of the stainless steel foam metal subjected to gold plating in Example 18, a conductive member having a carbon black void described in Preparation Example 14 was used, and the enzyme electrode was prepared in the same manner as in the other configurations.
(Example 126)
Instead of the stainless steel foam metal subjected to gold plating in Example 1, a conductive member having carbon nanotube voids described in Preparation Example 15 was used, and the enzyme electrode was prepared in the same manner as in the other configurations.
(Example 127)
In place of the stainless steel foam metal subjected to gold plating in Example 2, a conductive member having a carbon nanotube void described in Preparation Example 15 is used, and the enzyme electrode is prepared in the same manner as in the other configurations.
(Example 128)
Instead of the stainless steel foam metal subjected to gold plating in Example 3, a conductive member having a carbon nanotube void described in Preparation Example 15 is used, and the enzyme electrode is prepared in the same manner as in the other configurations.
(Example 129)
In place of the stainless steel foam metal subjected to gold plating in Example 4, a conductive member having voids of carbon nanotubes described in Preparation Example 15 is used, and an enzyme electrode is prepared in the same manner as in the other configurations.
(Example 130)
In place of the stainless steel foam metal subjected to gold plating in Example 5, a conductive member having voids of carbon nanotubes described in Preparation Example 15 is used, and an enzyme electrode is prepared in the same manner as in the other configurations.

(Example 131)
Instead of the stainless steel foam metal subjected to gold plating in Example 6, a conductive member having carbon nanotube voids described in Preparation Example 15 was used, and the enzyme electrode was prepared in the same manner as in the other configurations.
(Example 132)
In place of the stainless steel foam metal subjected to gold plating in Example 7, a conductive member having carbon nanotube voids described in Preparation Example 15 is used, and the enzyme electrode is prepared in the same manner as in the other configurations.
(Example 133)
In place of the stainless steel foam metal subjected to gold plating in Example 8, a conductive member having carbon nanotube voids as described in Preparation Example 15 was used, and an enzyme electrode was prepared in the same manner as the other components.
(Example 134)
In place of the stainless steel foam metal subjected to gold plating in Example 9, a conductive member having voids of carbon nanotubes described in Preparation Example 15 is used, and an enzyme electrode is prepared in the same manner as in the other configurations.
(Example 135)
In place of the stainless steel foam metal subjected to gold plating in Example 10, a conductive member having voids of carbon nanotubes described in Preparation Example 15 is used, and an enzyme electrode is prepared in the same manner as in the other configurations.
(Example 136)
Instead of the stainless steel foam metal subjected to gold plating in Example 11, a conductive member having a carbon nanotube void described in Preparation Example 15 was used, and an enzyme electrode was prepared in the same manner as the other components.
(Example 137)
Instead of the stainless steel foam metal subjected to gold plating in Example 12, a conductive member having carbon nanotube voids described in Preparation Example 15 was used, and the enzyme electrode was prepared in the same manner as the other components.
(Example 138)
Instead of the stainless steel foam metal subjected to gold plating in Example 13, a conductive member having carbon nanotube voids described in Preparation Example 15 was used, and the enzyme electrode was prepared in the same manner as in the other configurations.
(Example 139)
In place of the stainless steel foam metal subjected to gold plating in Example 14, a conductive member having voids of carbon nanotubes described in Preparation Example 15 is used, and the enzyme electrode is prepared in the same manner as in the other configurations.
(Example 140)
In place of the stainless steel foam metal subjected to gold plating in Example 15, a conductive member having carbon nanotube voids described in Preparation Example 15 was used, and an enzyme electrode was prepared in the same manner as in the other configurations.

(Example 141)
Instead of the stainless steel foam metal subjected to gold plating in Example 16, a conductive member having carbon nanotube voids described in Preparation Example 15 is used, and the enzyme electrode is prepared in the same manner as in the other configurations.
(Example 142)
Instead of the stainless steel foam metal subjected to gold plating in Example 17, the conductive member having the carbon nanotube voids described in Preparation Example 15 was used, and the enzyme electrode was prepared in the same manner as the other components.
(Example 143)
In place of the stainless steel foam metal subjected to gold plating in Example 18, a conductive member having a carbon nanotube gap described in Preparation Example 15 was used, and an enzyme electrode was prepared in the same manner as in the other configurations.
(Example 144)
In place of the stainless steel foam metal subjected to gold plating in Example 19, a conductive member having a carbon nanotube void described in Preparation Example 15 was used, and an enzyme electrode was prepared in the same manner as in the other configurations.
(Example 145)
In place of the gold-plated stainless steel foam metal of Example 20, a conductive member having carbon nanotube voids described in Preparation Example 15 is used, and the other configuration is used to prepare an enzyme electrode.
(Example 146)
In place of the gold-plated stainless steel foam metal of Example 4, a conductive member having a large number of voids made of nickel described in Preparation Example 26 is used, and the enzyme electrode is prepared in the same manner as the other components. .
(Example 147)
Instead of the stainless steel foam metal subjected to gold plating in Example 8, a conductive member having a number of voids made of nickel described in Preparation Example 26 is used, and the enzyme electrode is prepared in the same manner as in the other configurations. .
(Example 148)
Instead of the stainless steel foam metal subjected to gold plating in Example 15, a conductive member having a large number of voids described in Preparation Example 26 is used, and the enzyme electrode is prepared in the same manner as in the other configurations. .
(Example 149)
In place of the stainless steel foam metal subjected to gold plating in Example 18, a conductive member having a large number of voids described in Preparation Example 26 was used, and the enzyme electrode was prepared in the same manner as in the other configurations. .
(Example 150)
Instead of the stainless steel foam metal subjected to gold plating in Example 4, a conductive member having a large number of voids having a gradient in the size of the voids described in Preparation Example 27 was used. Similarly, an enzyme electrode is prepared.

(Example 151)
Instead of the stainless steel foam metal subjected to gold plating in Example 8, a conductive member having a large number of voids inclined in the size of the voids described in Preparation Example 27 was used. Similarly, an enzyme electrode is prepared.
(Example 152)
Instead of the stainless steel foam metal subjected to gold plating in Example 15, a conductive member having a large number of voids having a gradient in the size of the voids described in Preparation Example 27 was used. Similarly, an enzyme electrode is prepared.
(Example 153)
Instead of the stainless steel foam metal subjected to gold plating in Example 18, a conductive member having a large number of voids having a gradient in the void size described in Preparation Example 27 was used, and other configurations were used. Similarly, an enzyme electrode is prepared.
(Example 154)
Instead of the stainless steel foam metal subjected to gold plating in Example 4, a conductive member having a large number of voids having a gradient in the void size described in Preparation Example 28 was used. Similarly, an enzyme electrode is prepared.
(Example 155)
Instead of the stainless steel foam metal subjected to gold plating in Example 8, a conductive member having a large number of voids having a gradient in the void size described in Preparation Example 28 was used. Similarly, an enzyme electrode is prepared.
(Example 156)
Instead of the stainless steel foam metal subjected to gold plating in Example 15, a conductive member having a large number of voids inclined in the size of the voids described in Preparation Example 28 was used. Similarly, an enzyme electrode is prepared.
(Example 157)
Instead of the stainless steel foam metal subjected to gold plating in Example 18, a conductive member having a large number of voids having a gradient in the size of the voids described in Preparation Example 28 was used. Similarly, an enzyme electrode is prepared.
(Example 158)
A conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used instead of the stainless steel foam metal subjected to gold plating in Example 4, and the other configurations were the same. Then, an enzyme electrode is prepared.
(Example 159)
A conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used instead of the stainless steel foam metal subjected to gold plating in Example 8, and the other configurations were the same. Then, an enzyme electrode is prepared.
(Example 160)
A conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used instead of the stainless steel foam metal subjected to gold plating in Example 15, and the other configurations were the same. Then, an enzyme electrode is prepared.

(Example 161)
A conductive member having a void of poly (3,4-ethylenedioxythiophene) described in Preparation Example 6 was used instead of the stainless steel foam metal subjected to gold plating in Example 18, and the other configurations were the same. Then, an enzyme electrode is prepared.
(Example 162)
Instead of the stainless steel foam metal subjected to gold plating in Example 4, a conductive material having a large number of voids having a gradient in the size of the voids made of poly (3,4-ethylenedioxythiophene) described in Preparation Example 29 An enzyme electrode is prepared in the same manner with other components using a sex member.
(Example 163)
Instead of the stainless steel foam metal subjected to gold plating in Example 8, a conductive material having a large number of voids having a gradient in the size of the voids made of poly (3,4-ethylenedioxythiophene) described in Preparation Example 29 An enzyme electrode is prepared in the same manner with other components using a sex member.
(Example 164)
Instead of the stainless steel foam metal subjected to gold plating in Example 15, a conductive material having a large number of voids having inclinations in the size of the voids made of poly (3,4-ethylenedioxythiophene) described in Preparation Example 29 An enzyme electrode is prepared in the same manner with other components using a sex member.
(Example 165)
Instead of the gold-plated stainless steel foam metal of Example 18, a conductive material having a large number of voids inclined in the size of the voids made of poly (3,4-ethylenedioxythiophene) described in Preparation Example 29 An enzyme electrode is prepared in the same manner with other components using a sex member.
(Example 166)
A conductive member having a large number of voids made of gold-plated porous titanium oxide described in Preparation Example 30 was used instead of the stainless steel foam metal subjected to gold plating in Example 4, and the other configurations were made in the same manner. An enzyme electrode is prepared.
(Example 167)
A conductive member having a large number of voids made of gold-plated porous titanium oxide described in Preparation Example 30 was used instead of the stainless steel foam metal subjected to gold plating in Example 8, and the other configurations were made in the same manner. An enzyme electrode is prepared.
(Example 168)
Instead of the stainless steel foam metal subjected to gold plating in Example 15, a conductive member having a large number of voids made of gold-plated porous titanium oxide described in Preparation Example 30 was used, and the other configurations were the same. An enzyme electrode is prepared.
(Example 169)
Instead of the stainless steel foam metal subjected to gold plating in Example 18, a conductive member having a large number of voids made of gold-plated porous titanium oxide described in Preparation Example 30 was used, and the other configurations were made in the same manner. An enzyme electrode is prepared.
(Example 170)
A conductive member having a large number of voids made of gold-plated porous titanium oxide described in Preparation Example 30 was used instead of the stainless steel foam metal subjected to gold plating in Example 24, and the other configurations were made the same. An enzyme electrode is prepared.

(Example 171)
Instead of the stainless steel foam metal subjected to gold plating in Example 4, a conductive member having a large number of voids having a gradient in the size of the voids made of gold-plated porous titanium oxide described in Preparation Example 31 was used. The enzyme electrode is prepared in the same manner as the other configurations.
(Example 172)
Instead of the stainless steel foam metal subjected to gold plating in Example 8, a conductive member having a large number of voids having a gradient in the size of the voids made of gold-plated porous titanium oxide described in Preparation Example 31 was used. The enzyme electrode is prepared in the same manner as the other configurations.
(Example 173)
Instead of the stainless steel foam metal subjected to gold plating in Example 15, a conductive member having a large number of voids having inclinations in the size of the voids made of gold-plated porous titanium oxide described in Preparation Example 31 was used. The enzyme electrode is prepared in the same manner as the other configurations.
(Example 174)
Instead of the stainless steel foam metal subjected to gold plating in Example 18, a conductive member having a large number of voids having a gradient in the size of the voids made of gold-plated porous titanium oxide described in Preparation Example 31 was used. The enzyme electrode is prepared in the same manner as the other configurations.
(Example 175)
Instead of the stainless steel foam metal subjected to gold plating in Example 24, a conductive member having a large number of voids having a gradient in the size of the voids made of gold-plated porous titanium oxide described in Preparation Example 31 was used. The enzyme electrode is prepared in the same manner as the other configurations.
(Example 176)
Instead of the stainless steel foam metal subjected to gold plating in Example 4, a conductive member having a large number of voids made of a nickel alloy described in Preparation Example 32 was used, and the enzyme electrode was prepared in the same manner as the other components. To do.
(Example 177)
In place of the stainless steel foam metal subjected to gold plating in Example 8, a conductive member having a large number of voids made of the nickel alloy described in Preparation Example 32 was used, and the enzyme electrode was prepared in the same manner as the other components. To do.
(Example 178)
Instead of the stainless steel foam metal subjected to gold plating in Example 15, a conductive member having a number of voids made of the nickel alloy described in Preparation Example 32 was used, and the enzyme electrode was prepared in the same manner as in the other configurations. To do.
(Example 179)
Instead of the stainless steel foam metal subjected to gold plating in Example 18, a conductive member having a large number of voids made of the nickel alloy described in Preparation Example 32 was used, and the enzyme electrode was prepared in the same manner as the other components. To do.
(Example 180)
In place of the stainless steel foam metal subjected to gold plating in Example 4, a conductive member having a large number of voids having a gradient in the size of the voids described in Preparation Example 33 is used. The enzyme electrode is prepared in the same manner.

(Example 181)
Instead of the stainless steel foam metal subjected to gold plating in Example 8, a conductive member having a large number of voids having a gradient in the size of the voids described in Preparation Example 33 is used. The enzyme electrode is prepared in the same manner.
(Example 182)
Instead of the stainless steel foam metal subjected to gold plating in Example 15, a conductive member having a large number of voids having a gradient in the size of the voids described in Preparation Example 33 was used. The enzyme electrode is prepared in the same manner.
(Example 183)
Instead of the stainless steel foam metal subjected to gold plating in Example 18, a conductive member having a large number of voids having a gradient in the size of the voids described in Preparation Example 33 was used. The enzyme electrode is prepared in the same manner.
(Example 184)
Instead of the stainless steel foam metal subjected to gold plating in Example 4, a conductive member having a large number of voids having a gradient in the size of the voids described in Preparation Example 34 was used, and the other configuration. The enzyme electrode is prepared in the same manner.
(Example 185)
Instead of the stainless steel foam metal subjected to gold plating in Example 8, a conductive member having a large number of voids having a gradient in the size of the voids described in Preparation Example 34 was used. The enzyme electrode is prepared in the same manner.
(Example 186)
Instead of the stainless steel foam metal subjected to gold plating in Example 15, a conductive member having a large number of voids having a gradient in the size of the voids described in Preparation Example 34 was used. The enzyme electrode is prepared in the same manner.
(Example 187)
Instead of the stainless steel foam metal subjected to gold plating in Example 18, a conductive member having a large number of voids having a gradient in the size of the voids described in Preparation Example 34 was used. The enzyme electrode is prepared in the same manner.
(Example 188)
An enzyme electrode was prepared in the same manner except that a conductive member having a large number of voids made of carbon fibers described in Preparation Example 35 was used instead of the stainless steel foam metal subjected to gold plating in Example 4. To do.
(Example 189)
Instead of the stainless steel foam metal subjected to gold plating in Example 8, a conductive member having a large number of voids composed of carbon fibers described in Preparation Example 35 was used, and the enzyme electrode was prepared in the same manner as the other components. To do.
(Example 190)
An enzyme electrode was prepared in the same manner except that a conductive member having a large number of voids made of carbon fibers described in Preparation Example 35 was used instead of the stainless steel foam metal subjected to gold plating in Example 15. To do.

(Example 191)
Instead of the stainless steel foam metal subjected to gold plating in Example 18, a conductive member having a large number of voids composed of carbon fibers described in Preparation Example 35 was used, and the enzyme electrode was prepared in the same manner as the other components. To do.
(Example 192)
In place of the stainless steel foam metal subjected to gold plating in Example 4, a conductive member having a large number of voids inclined in the size of the voids described in Preparation Example 36 was used. The enzyme electrode is prepared in the same manner.
(Example 193)
In place of the stainless steel foam metal subjected to gold plating in Example 8, a conductive member having a large number of voids having a gradient in the size of the voids described in Preparation Example 36 was used. The enzyme electrode is prepared in the same manner.
(Example 194)
Instead of the stainless steel foam metal subjected to gold plating in Example 15, a conductive member having a large number of voids with inclination in the size of the voids described in Preparation Example 36 was used. The enzyme electrode is prepared in the same manner.
(Example 195)
In place of the stainless steel foam metal subjected to gold plating in Example 18, a conductive member having a large number of voids inclined in the size of the voids described in Preparation Example 36 was used. The enzyme electrode is prepared in the same manner.

(Comparative Examples 1-26)
For each of Examples 1 to 26, a gold plate (1 cm square, thickness 0.3 mm, Nilaco) was used as the conductive member instead of the gold-plated stainless steel foam metal, and the other configurations were the same. Prepare the enzyme electrode.
(Example 196)
Sensors are prepared using the enzyme electrodes described in Examples 1 to 195 and Comparative Example 1-26. An overview of a triode cell as a measuring instrument is shown in FIG. An enzyme electrode with a lead attached is used as a working electrode, a silver-silver chloride electrode as a reference electrode, a platinum wire as a counter electrode, and air is supplied through a gas tube into a water jacket cell with a lid. The measurement temperature is maintained at 37 ° C. by a constant temperature circulation tank. In the measurement, each electrode is connected to a potentiostat (Toho Giken, Model 2000), and the steady current when the potential shown in Table 1 is applied to the working electrode is recorded. As the electrolyte, measurement is performed using the electrolyte shown in Table 1 corresponding to the enzyme supported on each enzyme electrode. For the sensors described in symbols S12, 13, 21, 25, 67, 68, 104, 105, 137, 138, 157, 158, 166, and 170 in Table 2, as a counter electrode, a platinum wire modified with polydimethylsiloxane Is used. For the sensors described in symbols S1 to 30, 35 to 38, 118 to 145, and 176 to 195 in Table 2, not only a single layer electrode but also an electrode in which five enzyme electrodes are stacked is prepared and measured. As shown in FIGS. 5 (A) and 5 (B) and FIGS. 6 (A) and 6 (B), the sensor using all the enzyme electrodes has a linear current with increasing substrate concentration. It is clear that it shows an increase in density and functions as a sensor. Table 2 shows the results of current density observed from each sensor.

  Examples 1 to 149, 158 to 161, 166 to 170, 176 to 179, sensors using an enzyme electrode having a conductive member having a void described in 188 to 191 are all compatible carriers, mediators, enzymes, It shows a higher current density than a sensor using a smooth gold electrode using a substrate. In particular, a sensor in which electrodes are stacked in five layers exhibits a current density that is much higher than that of a smooth gold electrode, which is nearly 30 times the maximum. This confirms that the sensitivity of the sensor can be increased by introducing a conductive member having a gap. Furthermore, enzyme electrodes using conductive members having a large number of gaps with inclinations in the gap sizes described in Examples 150 to 157, 162 to 165, 171 to 175, 180 to 187, and 192 to 195 are compared. The current density is higher than that of an enzyme electrode using a conductive member having a large number of voids that do not have an inclination in the size of the voids listed as objects. This confirms that the sensitivity of the sensor can be further increased by introducing a conductive member having a large number of gaps that are inclined in the size of the gap.

(Example 197)
From the enzyme electrode of the example, a fuel cell is prepared using the enzyme electrode combinations shown in Table 4, the electrolyte solution combinations shown in Table 3, and the substrate types and concentrations for the enzymes shown in Table 1. An overview of a bipolar cell that is a measuring instrument is shown in FIG. An anode and a cathode are disposed with a porous polypropylene film (thickness 20 μm) interposed therebetween, and this is disposed in an electrolyte solution in a water jacket cell with a lid. Air is supplied from the gas inlet through the gas tube into the electrolyte solution of the enzyme electrode that uses an enzyme with oxygen as a substrate. The measurement temperature is maintained at 37 ° C. by a constant temperature circulation tank. Each lead is connected to a potentiostat (Toho Giken, Model 2000), the voltage is changed from -1.2 V to 0.1 V, and the voltage-current characteristics are measured. In the fuel cell in which the enzyme electrode using the enzyme shown in Table 3 is used for one or both electrodes, the electrolyte composition is the electrolyte composition shown in Table 3, and the enzyme shown in Table 3 is the anode and cathode. When an enzyme electrode that is not used for either one is used, conditions of 0.1 M, NaCl, 20 mM, phosphate buffer, and oxygen saturation are used. When an enzyme electrode containing glucose dehydrogenase / diaphorase is used, an electrochemical measurement cell with a diaphragm (made by Hokuto Denko) is used as a measurement cell when MP-11 is used. Separate the tank and perform the measurement. Further, for the fuel cells indicated by symbols FC1 to 25, FC29 to 31, FC98 to 121, and 145 to 159 in Table 4, not only the measurement with the enzyme electrode arranged in a single layer but also the anode and the cathode respectively. Measurement is also performed when five layers are stacked. The results are shown in Table 4.

  The fuel cells using the enzyme electrodes having conductive members having gaps indicated by symbols FC1 to 124, 131 to 133, 137 to 140, 145 to 147, and 154 to 156 in Table 4 are all compatible carriers and mediators. It exhibits a higher current density than a fuel cell combining a smooth gold electrode using an enzyme and a substrate, and shows a maximum power higher than that of a fuel cell using a corresponding smooth gold electrode in most fuel cells. In particular, a fuel cell in which electrodes are stacked in five layers exhibits a current density that is much higher than that of a smooth gold electrode, nearly 30 times the maximum current density and a maximum power of about 25 times maximum. From this, it is confirmed that the output of the fuel cell can be increased by introducing a conductive member having a gap. Furthermore, an enzyme electrode using a conductive member having a large number of gaps with inclinations in the gap sizes indicated by symbols FC125 to 130, 134 to 136, 141 to 144, 148 to 153, and 157 to 159 in Table 4 above Compared with fuel cells that use enzyme electrodes that use conductive members that have a large number of voids that have no inclination in the size of the voids listed in the comparison object, the fuel cell using the Indicates. In addition, even in a fuel cell in which electrodes are stacked in five layers, a fuel cell using a conductive member having a large number of voids having inclinations in the size of the voids is inclined to the size of the voids listed as comparison targets. Compared with an enzyme electrode using a conductive member having a large number of voids, it has a higher current density and maximum power. This confirms that the fuel cell can be further increased in power by introducing a conductive member having a large number of voids having inclinations in the size of the voids.

(Example 198)
Further, flow cells are created for the fuel cells indicated by symbols FC1 to 9, FC12 to 18, FC21 to 25, FC29 to 31, FC98 to 112, FC115 to 121, and 145 to 159 in Table 4 above. As shown in FIG. 8, the anode and the cathode are packed in an acrylic case so that the anode and the cathode are alternately sandwiched with a porous polypropylene film (thickness 20 μm, porosity 80%). A 1 mm gold wire was brought into contact with the anode and the cathode to ensure electrical contact. Each lead was fixed, and the gold wire was fixed to the acrylic case using silicone resin. Tubes were connected to holes formed at both ends of the acrylic case, and measurement was performed using a precision pump while allowing the electrolyte to permeate at a flow rate of 0.25 mL / sec. And the temperature of electrolyte solution is maintained at 37 degreeC through a thermostat. The composition of the electrolytic solution is the same as that used in Example 197. Table 5 shows the measurement results.

  Compared with the fuel cell shown in Table 4 using a corresponding conductive member, carrier, mediator, enzyme, and substrate, the fuel cell that has been made into a flow cell has a current density that is nearly 2.5 times as high as the output. Indicates. From this, it is confirmed that introduction of the flow cell structure can increase the output of the fuel cell. In addition, even in a fuel cell having a flow cell structure, a fuel cell using a conductive member having a large number of gaps with inclinations in the gap sizes indicated by symbols FCF50 to 55 and 59 to 61 in Table 5 is compared. Compared to an enzyme electrode using a conductive member having a large number of voids that have no inclination in the size of the voids listed as objects, the current density and maximum power are high. From this, it is confirmed that by introducing a conductive member having a large number of air gaps having a gradient in the size of the air gap, even a fuel cell having a flow cell structure can achieve higher output.

(Example 199)
From the enzyme electrode of the example, an electrochemical reaction apparatus is prepared using the enzyme electrode shown in Table 6. As shown in FIG. 4, using a triode cell 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, 10 mM glucose, Using a 10 mM ethanol electrolyte, a potential of 0.3 V vs Ag / AgCl is applied for 100 minutes under a nitrogen atmosphere in a water jacket cell, and the product is quantified by high performance liquid chromatography. In the reactors described in symbols CR10, 11, 18, 53, 54, 83, 84, 110, 111, 127, 128, and 135 in Table 6, a platinum wire modified with polydimethylsiloxane is used as a counter electrode. The results are shown in Table 6.

  From the reaction electrolyte, gluconolactone is detected, acetaldehyde is not detected, and alcohol is used as the substrate from the reaction solution of the reaction apparatus using an enzyme electrode having an enzyme (glucose oxidase, glucose dehydrogenase) whose substrate is glucose. Acetaldehyde is detected from the reaction solution of the reaction apparatus using the enzyme electrode having the enzyme (alcohol dehydrogenase), and gluconolactone is not detected. In any reaction apparatus using the enzyme electrode, the substrate is selectively selected. It can be seen that the reaction is in progress. Further, it can be seen that in any reaction apparatus, there is a high correlation between the amount of reaction charge and the produced substance, and the reaction proceeds quantitatively. Reactors using enzyme electrodes using conductive members having voids indicated by symbols CR1 to 120, 127 to 129, 133 to 136, 141 to 143, and 150 to 152 in Table 6 are all shown as comparison targets. Shows a higher reaction charge amount than a smooth gold electrode using a corresponding carrier, mediator, enzyme, and substrate, which confirms that the reaction time can be shortened by introducing a conductive member having voids. . Furthermore, an enzyme electrode using a conductive member having a large number of gaps having inclinations to the gap sizes indicated by symbols CR121 to 126, 130 to 132, 137 to 140, 144 to 149, and 153 to 155 in Table 6 is provided. The chemical reaction device used has a higher reaction charge and generation compared to the chemical reaction device that uses an enzyme electrode that uses a conductive member with a large number of voids that have no inclination in the size of the voids shown in the comparison target. This indicates that the reaction time can be further shortened by the introduction of a conductive member having a large number of voids having inclinations in the size of the voids.

(Example 200)
Further, a flow cell is created for the electrochemical reactors indicated by symbols CR1 to 9, CR12 to 17, CR19 to 24, CR28 to 30, CR95 to 109, CR112 to 117, 141 to 155 in the above table. Using enzyme electrode as working electrode and platinum wire mesh (Niraco, 150 mesh) as counter electrode, as shown in FIG. 8, the working electrode and counter electrode are alternately sandwiched between porous polypropylene films (thickness 20 μm, porosity 80%) Assemble 5 sets of acrylic case, and contact the gold electrode with a diameter of 0.1mm to the enzyme electrode and the platinum wire with a diameter of 0.1mm from the hole made in the case to ensure electrical contact. Each wire was fixed to the acrylic case using silicone resin. Tubes were connected to holes formed at both ends of the acrylic case, and measurement was performed while circulating the electrolyte at a flow rate of 0.5 mL / second using a precision pump. The composition of the electrolyte was 0.1M NaCl, 20 mM phosphate buffer, 10 mM glucose, 10 mM ethanol, and a voltage of 1.5 V was applied for 100 minutes under a nitrogen atmosphere, and the product was subjected to high performance liquid chromatography. Quantify with. The results are shown in Table 7.

  The electrochemical reaction apparatus formed into a flow cell is shown in Table 6 using corresponding conductive members, carriers, mediators, enzymes, and substrates, which are shown as comparison targets without impairing the substrate specificity of the reaction and the quantitativeness of the reaction. Compared with the electrochemical reaction device, the reaction charge amount and the reaction product generation amount nearly three times as much are shown. This confirms that the reaction time can be shortened by introducing the flow cell structure. In addition, even in a chemical reaction apparatus incorporating a flow cell structure, a chemical reaction apparatus using a conductive member having a large number of voids inclined in the size of the voids indicated by symbols CRF49 to 54 and 58 to 60 in Table 5 is as follows. Compared with a chemical reaction apparatus using a conductive member having a large number of voids that do not have an inclination in the size of the voids listed as comparison objects, a high reaction charge amount and a reaction product generation amount are shown. From this, it is confirmed that the reaction time can be further shortened even in a chemical reaction apparatus in which a flow cell structure is introduced by introducing a conductive member having a large number of voids having inclinations in the size of the voids.

It is a conceptual diagram of the enzyme electrode using the comprehensive immobilization method. It is a conceptual diagram of the enzyme electrode which used the carbon particle for the member. It is a conceptual diagram of the enzyme electrode using the electroconductive member which has a space | gap. It is a figure which shows the structure of a triode cell. It is a figure which shows the relationship between the substrate concentration-current density of a sensor. It is a figure which shows the relationship between the substrate concentration-current density of a sensor. It is a figure which shows the structure of a bipolar cell. It is a figure which shows the structure of a five-layer type flow cell. It is an example of the porous electroconductive member applicable to this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Conductive member 2 Enzyme 3 Carrier 4 Charge flow 5 Carbon particle 6 Binder polymer 7 Graphite grain boundary 8 Conductive member 9 having a gap Water jacket cell 10 Water jacket cell lid 11 Electrolytic solution 12 Anode (anode, anode)
13 Platinum wire electrode 15 Anode lead 16 Cathode lead 17 Reference electrode lead 18 Potentiostat 19 Gas inlet 20 Gas tube 21 Temperature controlled water inlet 22 Temperature controlled water outlet 23 Porous polypropylene film 24 Cathode 25 Electrolyte inlet 26 Electrolyte Discharge port 27 Acrylic case 801 Electrolyte layer 802 Hole 803 Conductive member 804 Support substrate

Claims (6)

An enzyme electrode having a conductive member and an enzyme,
The conductive member has a porous structure, and the enzyme is fixed in a hole constituting the porous structure via a carrier,
The conductive member has at least two working surfaces facing each other, both of the two working surfaces are open, and when the conductive member is immersed in a liquid, from one of these surfaces An enzyme electrode through which the liquid permeates through the plurality of voids on the other surface.   An enzyme electrode device comprising: the enzyme electrode according to claim 1; and a wiring connected to a conductive member of the enzyme electrode.   The enzyme electrode device according to claim 2, wherein the enzyme electrode device has a structure in which a plurality of the enzyme electrodes are stacked with their working surfaces facing each other.   A sensor using the enzyme electrode device according to claim 2 or 3 as a detection site for detecting a substance.   A fuel cell provided with a region capable of holding an electrolyte solution between an anode electrode and a cathode electrode, wherein at least one of the anode electrode and the cathode electrode comprises the enzyme electrode device according to claim 2 or 3. A fuel cell. In an electrochemical reaction apparatus having a reaction region and an electrode for causing an electrochemical reaction to a raw material introduced into the reaction region to obtain a target product,
An electrochemical reaction apparatus, wherein the electrode is the enzyme electrode device according to claim 2 or 3.

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