JP5022657B2 - Enzyme electrode and manufacturing method thereof - Google Patents

Description

  The present invention relates to an enzyme electrode, and more particularly to an enzyme electrode using an enzyme / metal particle complex produced by an enzyme reaction, a method for producing the same, and an application thereof.

  Enzymes, which are proteinaceous biocatalysts produced in living cells, act strongly under milder conditions than ordinary catalysts. Moreover, the specificity of a substrate is high, and generally each enzyme catalyzes only a certain reaction of a certain substrate. Among enzymes, an enzyme called an oxidoreductase catalyzes a redox reaction of a substrate. If the electric charge generated by the reaction of this oxidoreductase can be taken out to the conductive member, it is possible to create a low overvoltage and high selectivity electrode utilizing the characteristics of the enzyme. However, the redox center of many oxidoreductases is a form confined in the deep part of the three-dimensional structure of protein. Therefore, in order to effectively exchange electrons with the conductive member, the distance between the redox center and the conductive member is large, and as a result, between the active site of the oxidoreductase and the conductive member. It has been generally difficult to perform direct electron transfer.

  Therefore, a method of electrically connecting an enzyme and a conductive member with a substance called a mediator has been widely used (for example, Non-Patent Document 1). When the mediator molecule enters the protein of the enzyme and reaches a distance sufficiently close to the redox center of the enzyme, the mediator molecule exchanges electrons with the redox center of the enzyme for the first time. After that, the mediator that exchanged electrons with the active site of the enzyme, that is, the redox center, transports the charge to the conductive member through diffusion, electron hopping, etc., so that the charge of the enzyme reaction is taken out to the conductive member. Will be.

  By the way, with recent advances in nanotechnology, many methods for preparing metal particles have been reported. Among them, Non-Patent Document 2 describes a method of generating gold particles by a catalytic reaction of glucose oxidase which is an enzyme. In this document, it is suggested by the infrared spectrum and zeta potential measurement that the enzyme is immobilized on the generated gold particles. Furthermore, circular dichroism spectrum measurements suggest that the enzyme is denatured. Apart from this, Non-Patent Document 3 also describes an optical glucose sensor that utilizes the growth of gold particles by hydrogen peroxide generated by the enzymatic reaction of glucose oxidase and the increase in plasmon absorption by this growth.

On the other hand, regarding the complex of enzyme and metal, Non-Patent Document 4 generally describes that the activity of a functional protein containing an enzyme is greatly reduced by adsorbing to particles such as gold and silica. Is disclosed.
Adam Heller J. Phys. Chem. 1992, 96, 3579-3587 Kei Yasui, Nobuo Kimizuka Chem. Lett. 2005, 34, 416-417 Maya Zayats, Ronan Barton, Inna Popov, Itamar Willner Nano Lett. 2004, 5, 21-25 Alexey A. Vertegel, Richard W. Siegel, Jonathan S. Dordick Langmuir 2004, 20, 6800-6807

  Non-Patent Document 1 discloses an enzyme electrode using a mediator. Since the mediator molecule is usually small in size, it can easily enter the vicinity of the redox center in the protein of the enzyme. The charge generated at the redox center of the enzyme due to the enzyme reaction moves to a mediator molecule having an appropriate redox potential, and further, in the vicinity of the conductive member due to diffusion of the mediator molecule or electron hopping between the mediator molecules. Be transported to. Finally, it is taken out to the conductive member by electron transfer between the conductive member and the mediator.

  Here, the diffusion of the mediator to the vicinity of the redox center inside the enzyme is slowed because the protein protein gap is small. That is, the diffusion rate of the mediator is a rate-limiting step in the extraction of charges via the mediator of charges generated at the redox center of the enzyme. Therefore, it is considered that there is a limit to the enzyme electrode using only this mediator in order to further improve the charge extraction speed and increase the current value. That is, in order to obtain higher performance, there is a limit to the sensitivity of a sensor using an enzyme electrode using a mediator and the output of a biofuel cell using this enzyme electrode.

  An object of the present invention is to provide an enzyme electrode and a method for producing the same, in which the charge extraction from the redox center of the enzyme is accelerated. Another object of the present invention is to provide a sensor and a biofuel cell using such an enzyme electrode. Another object of the present invention is to provide a method for detecting and quantifying a substance to be detected using such an enzyme electrode. Another object of the present invention is to provide a method for producing an enzyme / metal particle composite useful as a constituent material of such an enzyme electrode.

Enzyme electrode having a conductive member and an enzyme according to the first invention, the enzyme electrode having an electrically conductive member and an enzyme and a metal particle, the enzyme comprises the metal particles within the enzyme, the metal The particles are formed by reacting the enzyme with the substrate in the presence of a metal precursor capable of forming a metal with the reaction between the enzyme and a substance serving as a substrate for the enzyme. Features. In the fuel cell according to the second aspect of the present invention, a region capable of holding an electrolyte is provided between the anode electrode and the cathode electrode, and at least one of the anode electrode and the cathode electrode is formed of the first electrode. It is comprised by the enzyme electrode in invention.

The method for producing metal particles according to the third aspect of the present invention includes:
Introducing an enzyme into the pores of the porous material,
In the presence of a substrate of the enzyme and a precursor of metal particles,
A method for producing metal particles in pores of a porous material, wherein metal particles are precipitated from a precursor of the metal particles by a reaction between the enzyme and the substrate.

A method for producing an enzyme electrode according to a fourth aspect of the present invention is a method for producing an enzyme electrode having an enzyme and metal particles,
Preparing an enzyme;
The presence of the metal precursor capable of forming a metal by reaction with a substrate for the enzyme and the enzyme, by reacting the said enzyme substrate, and a metal particle in said enzyme, enzyme / metal particles Obtaining a complex;
It is a manufacturing method of the enzyme electrode characterized by having.

The method for producing an enzyme electrode according to the fifth aspect of the present invention is a method for producing an enzyme electrode having an enzyme and metal particles,
Introducing an enzyme into the pores of the conductive porous material;
In the presence of a metal precursor capable of forming a metal by reaction between the enzyme and the enzyme substrate, the enzyme in the pores of the porous material is reacted with the substrate to provide metal particles in the enzyme. Obtaining an enzyme / metal particle complex in the pores of the porous material;
It is a manufacturing method of the enzyme electrode characterized by having.

  According to the present invention, it is possible to obtain an enzyme / metal particle complex in which at least part of the metal particles has entered the inside of the enzyme. And by using this as an enzyme electrode, it becomes possible to speed up the extraction of electrons from the enzyme, and the catalytic current can be improved. When this enzyme / metal particle complex is immobilized in the pores of the porous material, an enzyme electrode with improved environmental stability can be provided.

  In the present invention, as a means for solving the problem, an enzyme / metal particle complex in which a part of metal particles enters the inside of the enzyme is used as the enzyme of the enzyme electrode.

In general, it is known that the activity of a functional protein containing an enzyme is greatly reduced by adsorbing to a particle such as gold or silica (Non-Patent Document 4). Also in Non-Patent Document 2, denaturation of enzyme adsorbed on gold particles without using a stabilizer is suggested from a circular dichroic spectrum. From these backgrounds, a complex in which metal particles and an enzyme are interacted without using a modifier is not usually used as a substance detection or energy generation device. However, as an exception, there is a case where a biological device using characteristics inherent to metal particles, represented by plasmon absorption, is produced. In response to these conventional findings, the present inventors have adopted a method in which metal particles are generated from their precursors by using an enzyme reaction based on the vicinity of the active center of the enzyme as a starting point, and inevitably the redox center of the enzyme. The knowledge that a metal can be obtained in the distance which an electron can transfer between is obtained. Furthermore, the present inventors have obtained the knowledge that this metal can draw out electric charges from the redox center of the enzyme, that is, can function as a so-called lead. Therefore, an enzyme / metal particle complex was prepared by an enzyme reaction, an enzyme electrode was prepared using this complex, and the characteristics of the enzyme electrode prepared using an enzyme not complexed with metal particles were compared. . As a result, it was confirmed that the enzyme electrode using the enzyme / metal particle composite showed higher characteristics than the enzyme electrode prepared using an enzyme not combined with the metal particle, and the present invention was completed. .
Next, a preferred embodiment of the present invention will be described in detail.

  The enzyme electrode of the present invention is an enzyme electrode having a conductive member, an enzyme, and metal particles, and these metal particles and enzyme form an enzyme / metal particle complex in which a part of the metal particles enters the inside of the enzyme. It is characterized by that. Here, the enzyme / metal particle complex in which the metal particles enter the inside of the enzyme has a structure as shown in FIGS. 1 (A) and 1 (B). That is, when the cross-sectional shape is observed, the metal particles straddle the line connecting the points where the enzyme 1, the metal particles 2 and the three substances of the solution surrounding them intersect (shown by a one-dot chain line in the figure). is there. As shown in FIG. 1 (A), a part 3 of metal particles enters a minute gap of enzyme 1, or a part 3 of particle 2 is made of enzyme 1 as shown in FIG. The thing which entered the outer edge over a long range is included.

  The positional relationship between the enzyme and the gold particles as shown in FIG. 1 can be verified, for example, by observing the enzyme / gold particle complex with a transmission electron microscope.

  The structure in which some of the metal particles enter the inside of the enzyme performs an enzyme reaction in the presence of a metal precursor capable of forming a metal by the interaction between the enzyme and the enzyme substrate (enzyme reaction). Can be obtained. By performing the enzyme reaction in the presence of the metal precursor, the substrate reacts inside the enzyme including the active site (active center) of the enzyme, and a metal is formed from the metal precursor accordingly. As these reactions proceed, the metal grows starting from the inside of the enzyme, and in some cases, the metal formed outside the enzyme and the metal inside the enzyme combine to obtain metal particles. The metal particles obtained in this way have a shape in which some of them enter the interior of the enzyme.

  Here, as the substrate of the enzyme, a substrate capable of generating a metal from a metal precursor by a chemical change due to the action of the enzyme is used. As a preferable combination of an enzyme and a substrate, a combination of an enzyme and a substrate that can give a charge to the redox center of the redox enzyme by the action of the enzyme on the substrate can be mentioned. Such enzyme substrates include natural and artificial compounds that can cause an enzyme reaction as a reaction substrate, in addition to the enzyme's original substrate (eg, glucose for glucose oxidase, ethanol for alcohol dehydrogenase). .

  In addition, for the formation of metal particles from the metal precursor, a solution containing an enzyme, an enzyme substrate and a metal precursor is prepared, and at the same time as the metal particles are generated from the metal precursor as the enzyme reaction proceeds, A technique of complexing metal particles and an enzyme is preferably used. In addition, the metal particles at this time are not only the metal particles directly generated by the action of the enzyme on the substrate, but also the products generated by the action of the enzyme on the substrate act on the metal precursor to form a secondary product. Containing metal particles.

Specific examples include a solution to which glucose oxidase, glucose and chloroaurate are added. In this case, chloroauric acid is reduced by FADH ₂ which is the redox center of glucose oxidase produced by the oxidation reaction of glucose in glucose oxidase. In addition, the reduction of chloroauric acid by hydrogen peroxide, which occurs due to the reaction of glucose and oxygen in glucose oxidase, occurs. It is mentioned that the above reaction includes such a case. This does not exclude that the enzyme electrode of the present invention includes an enzyme that is not complexed with metal particles or a metal particle that is not complexed with an enzyme in addition to the enzyme / metal particle complex.

  As the enzyme used for the enzyme / metal particle complex, an oxidoreductase can be preferably used. This enzyme is an enzyme that catalyzes a redox reaction of a substrate.

  In order to obtain desired properties, one type of enzyme or a plurality of different enzymes may be fixed to the enzyme electrode. Furthermore, as for the enzyme to be complexed with one metal particle, one kind of enzyme may be complexed or a plurality of different enzymes may be complexed.

Examples of enzymes that can be used in the present invention include glucose oxidase, bilirubin oxidase, laccase, pyruvate oxidase, cholesterol oxidase, lactate oxidase, and ascorbate oxidase.
Other examples include cytochrome oxidase, alcohol dehydrogenase, cholesterol dehydrogenase, and aldehyde dehydrogenase. Further examples include formate dehydrogenase, glucose dehydrogenase, lactate dehydrogenase, diaphorase, catalase, peroxidase, and thioredoxin reductase. It is also possible to use an enzyme that is not an oxidoreductase and an oxidoreductase simultaneously. In this case, for example, a product of an enzyme that is not an oxidoreductase can be detected by an oxidoreductase.

  As the metal used in the enzyme / metal particle composite, a material having sufficient electrochemical stability in an aqueous solution and the conditions under which the electrode is used can be suitably used. A combination of a plurality of different metals can also be used. Examples include Au, Pt, Ag, Co, Pd, Rh, Ni, Cr, Fe, Mo, Ti, Cu and W, and alloys containing two or more of these.

  As the metal precursor used for the preparation of the enzyme / metal particle composite, a compound that does not react rapidly even in an aqueous solution and can generate gradual metal particles can be preferably used. For example, metal chloride salts, citrate salts, phosphate salts, borate salts, formate salts, acetate salts, and sulfites. Moreover, you may produce | generate a metal particle from these salts via an intermediate body.

  As the metal particles of the enzyme / metal particle complex, a dispersion of primary particles is most preferably used, but secondary particles in which they are aggregated may be used. The primary particle size of the metal particles is preferably in the range of 2 nm to 50 nm, and more preferably in the range of 2 nm to 20 nm. When the metal particles form secondary particles, the particle size preferably does not exceed 200 nm, and more preferably does not exceed 50 nm. As for the lower limit of the secondary particle diameter, the secondary particle diameter is preferably 2 nm or more. The metal particles can also be referred to as metal fine particles.

  The conductive member plays a role of taking out the electric charge generated by the enzyme reaction to an external circuit. As a constituent material of the conductive member, a material having high conductivity and sufficient electrochemical stability under the conditions in which the electrode is used can be suitably used. Examples of the constituent material of such a conductive member include metals, conductive polymers, metal oxides, and carbon materials. Examples of metals include those containing elements of Au, Pt, Ag, Ni, Cr, Fe, Mo, Ti, Al, Cu, V, In, Ga, and W, which may be alloys. It may be plated. Examples of the conductive polymer include polyacetylenes, polyarylenes, polyarylene vinylenes, polyacenes, polyarylacetylenes, polydiacetylenes, polynaphthalenes, polypyrroles, polyanilines, and polythiophenes. Other examples of the conductive polymer include those containing at least one compound among polythienylene vinylenes, polyazulenes, and polyisothianaphthenes. 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 carbon material include graphite, carbon black, carbon nanotube, carbon nanohorn, fullerene compound and derivatives thereof.

  The mediator promotes the transfer of electrons between the enzyme / metal particle complex and the conductive member, and can be used as necessary. Examples of the mediator include metal complexes, quinones, heterocyclic compounds, nicotinamide derivatives, flavin derivatives, and conductive polymers. Examples of the metal complex include those using a transition metal as a central metal. Examples include Os, Fe, Ru, Co, Cu, Ni, V, Mo, Cr, Mn, Pt, Rh, Pd, and Ir. 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. 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, etc. Further examples include ethylenediamine, porphyrin, phthalocyanine, acetylacetone, quinolinol, ammonia, cyanide, triphenylphosphine oxide and derivatives thereof. Examples of quinones are quinones. Benzoquinone, anthraquinone, naphthoquinone, pyrroloquinoline quinone, tetracyanoquinodimethane, and derivatives thereof Examples of heterocyclic compounds include phenazine, phenothiazine, viologen, and derivatives thereof. Examples of conductors include nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate, and examples of flavin derivatives include flavin adenine dinucleotide (FAD), where the conductivity is high. Examples of molecules include polyacetylenes, polyarylenes, polyarylene vinylenes, polyacenes, polyarylacetylenes, and polydiacetylenes, polynaphthalenes, polypyrroles, polyanilines, polythiophenes, polythienylenes. Vinylenes, polyazulenes, and polyisothianaphthenes can be applied.

  The carrier immobilizes at least one of the enzyme / metal particle complex and the mediator on the conductive member, and can be used as necessary. Examples include (1) a polymer compound, (2) an inorganic compound, and (3) an organic compound having a plurality of bonding groups in the molecule.

  The polymer compound may be used as a carrier for electrostatically immobilizing an enzyme. In this case, the polymer compound has a charge opposite to the surface charge of the enzyme / metal particle complex under the use conditions of the electrode. It is preferable. Examples of this polymer include polyacetylenes, polyarylenes, polyarylene vinylenes, polyacenes, and polyarylacetylenes as examples of conductive polymers. Furthermore, there are polydiacetylenes, polynaphthalenes, polypyrroles, polyanilines, polythiophenes, polythienylene vinylenes, polyazulenes, and polyisothianaphthenes. Examples of other polymers include polystyrene sulfonic acid, polyvinyl sulfate, dextran sulfate, chondroitin sulfate, polyacrylic acid, polymethacrylic acid, polymaleic acid, polyfumaric acid, and polyethyleneimine. Polyallylamine hydrochloride, polydiallyldimethylammonium chloride, polyvinyl pyridine, polyvinyl imidazole, polylysine, deoxyribonucleic acid, ribonucleic acid, and pectin can also be applied. Furthermore, silicone resin, cellulose, agarose, dextran, chitin, polystyrene, polyvinyl alcohol, and nylon are also applicable.

  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 the organic compound having a plurality of bonding groups in the molecule include compounds containing at least the functional groups shown below. The functional group here includes hydroxyl group, carboxyl group, amino group, aldehyde group, hydrazino group, thiocyanate group, epoxy group, vinyl group, halogen group, acid ester group, phosphate group, thiol group, disulfide group. It is. Furthermore, compounds containing a functional group selected from a dithiocarbamate group, a dithiophosphate group, a dithiophosphonate group, a thioether group, a thiosulfate group, and a thiourea group can be given. Typical examples are glutaraldehyde, polyethylene glycol diglycidyl ether, cyanuric chloride, N-hydroxysuccinimide ester, dimethyl-3,3′-dithiopropionimidate hydrochloride. Furthermore, 3,3′-dithio-bis (sulfosuccinimidyl propionate), cystamine, alkyldithiol, biphenylenedithiol, benzenedithiol and the like can be mentioned.

  The enzyme electrode of the present invention may have a structure in which the enzyme / metal particle complex is immobilized in pores of a porous material. The pores of the porous body 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. As the material for the porous body, any material can be used as long as it is conductive, has sufficient rigidity during storage and measurement, and has sufficient electrochemical stability under the conditions in which the electrode is used. This porous body may have improved conductivity by other conductive materials, or may have conductivity. Examples of porous materials include metal oxides containing at least one element of In, Sn, Zn, Ti, Al, Si, Zr, Nb, Mg, Ba, Mo, W, V, and Sr. It is done. Examples of the conductive material include metals, conductive polymers, and carbon materials. Typical examples of these include tin oxide mesoporous materials and anodized alumina subjected to conductive treatment. A material having a pore size of about 2 to 50 nm, which is large enough to introduce an enzyme, is preferably used.

  In addition, according to the method for forming metal particles from the metal precursor used in the present invention, the metal particles can be prepared in a fine space at a high density and in a small number of steps under mild conditions. In general, as a technique for introducing metal particles into a fine space, a solution of a metal precursor such as a metal chloride salt is introduced into the fine space, and then this precursor is treated with a reducing agent, Alternatively, a method in which metal is deposited in a fine space by performing heat reduction has been performed. In this case, if the introduction process of metal precursor into the fine space and the reduction process are performed at the same time, metal particles will be precipitated in the solution regardless of the inside or outside of the pores of the porous material. It is necessary to perform the process and the reduction process as separate processes. For this reason, there is a limit to the amount of metal that can be introduced into the fine space in one cycle of introducing the metal precursor solution, and it is necessary to repeat the cycle in order to immobilize the metal particles at a high density. In the present invention, a substrate that does not easily react with the metal precursor is selected, an enzyme corresponding to this substrate is introduced or immobilized in the pores of the porous body, and then the substrate and the metal precursor are finely divided. Add into the pores. Thereby, metal particles can be formed by selectively depositing metal only at the position where the enzyme exists. That is, the introduction of the metal precursor and the metal deposition step for forming the metal particles can be performed simultaneously. In addition, by changing the reaction time at this time, the size of the metal particles can be controlled, and particles having a large particle diameter can be prepared in one cycle.

  A sensor which is one preferred form for use of the enzyme electrode of the present invention is characterized in that the enzyme electrode is used as a detection site for detecting a substance. As a typical configuration, an enzyme electrode is used as a working electrode, combined with a counter electrode, and a reference electrode is used as necessary. The substrate recognition ability of the enzyme fixed to the electrode, the current generated by the enzyme catalysis can be detected, and the structure used for the qualitative detection and concentration measurement of the substance in the liquid in contact with these electrodes can be mentioned . The configuration of the sensor is not particularly limited as long as it can be detected by the enzyme electrode. In addition to the high substrate selectivity caused by the enzyme, this sensor expands the concentration range that can be detected, simplifies the detection device, and the detection site due to the high current density resulting from the application of the enzyme / metal particle complex. Miniaturization is possible.

  A biofuel cell which is one preferred embodiment of the present invention is characterized in that an enzyme electrode is used as at least one of an anode and a cathode. A typical configuration includes a reaction tank that can store an electrolyte containing a substance that serves as a fuel, and an anode and a cathode that are electrically isolated from each other in the reaction tank. On the other hand, the structure using the enzyme electrode concerning this invention can be mentioned. The biofuel cell can be of a type that replenishes electrolyte, a type that circulates, or a type that does not replenish or circulate electrolyte. As long as the biofuel cell can use an enzyme electrode, the type, structure, function, etc. of the fuel are not limited. This biofuel cell can obtain a high driving voltage by being able to oxidize and reduce a substance with a low overvoltage by a high catalytic action peculiar to an enzyme used as a catalyst for an electrode reaction. Also, an enzyme / metal particle complex can be obtained. It is possible to obtain a high current density resulting from the application of. As a result, a high power density can be obtained.

  The present invention will be described in more detail with reference to the following examples. However, the method of the present invention is not limited to these examples. The experimental temperatures in the following examples are room temperature (25 ° C.) unless otherwise specified. Prior to the examples, preparation examples of the complex polymers used in the examples are described.

Preparation Example 1
A method for synthesizing the complex polymer represented by the following formula (1) will be described.

The following components were added to a 100 mL eggplant flask equipped with a reflux tube.
20 mL ethylene glycol, 0.08 g (NH ₄ ) ₂ [OsCl ₆ ] 0.38 g 4,4'-dimethyl-2,2'-bipyridine.

The mixture was stirred with a stirrer, and irradiated with 300 W for 20 minutes with a microwave synthesizer under a nitrogen stream. After the obtained solution was cooled to room temperature, 25 mL of water in which 0.4 g of Na ₂ S ₂ O ₄ was dissolved was added. The black purple precipitate produced after stirring at room temperature for 1 hour was filtered, washed with water to remove excess salt, washed with diethyl ether, removed unreacted ligand, and heated to 60 ° C. under reduced pressure. Thus, drying and Os (4,4′-dimethyl-2,2′-bipyridine) ₂ Cl ₂ were obtained.

To a 100 mL three-necked flask equipped with a thermometer and a reflux tube, 15 mL of water, 2.63 g of acrylamide, 0.403 mL of 1-vinylimidazole, 0.069 mL of N, N, N ′, N′-tetramethylethylenediamine were added. . Further, 0.06 g of ammonium persulfate was added under a nitrogen stream. The mixture was heated at 40 ° C. for 30 minutes in a water bath, and then the reaction vessel was air-cooled. The resulting viscous liquid was dropped into 500 mL of methanol with strong stirring and allowed to settle, and the precipitate was collected by centrifugation. A minimum amount of water capable of dissolving the precipitate was added to dissolve, and the aqueous solution was added dropwise to 500 mL of methanol under strong stirring to cause precipitation. The precipitate was collected again by centrifugation, dried by heating to 60 ° C. under reduced pressure, and a polyacrylamide-polyvinylimidazole 7.49 / 1 copolymer was obtained. The formation of molecules and the unit ratio were determined by ¹ HNMR measurement (D ₂ O).

The following components were added to a 100 mL eggplant flask equipped with a reflux tube.
• 25 mL of ethylene glycol, 17.5 mL of ethanol • 0.19 g of Os (4,4′-dimethyl-2,2′-bipyridine) ₂ Cl ₂ prepared previously, 0.22 g of polyacrylamide-polyvinylimidazole Copolymer.

  The mixture was stirred with a stirrer and irradiated with 400 W for 2 hours with a microwave synthesizer under a nitrogen stream. After the obtained solution was cooled to room temperature, a solution containing 20 mL of ethanol was added dropwise to a 500 mL diethyl ether solution with strong stirring. An additional 20 mL of ethanol was added to the resulting viscous precipitate. This was again added dropwise to 500 mL of diethyl ether solution with strong stirring. The viscous precipitate obtained was dried by heating to 60 ° C. under reduced pressure to obtain the target complex polymer described in the formula (1).

Preparation Example 2
A method for synthesizing the complex polymer represented by the following formula (2) will be described.

  3 g of 2,2′-bipyridyl-N, N′-dioxide was placed in a 100 mL eggplant flask equipped with a reflux tube, and 6 mL of concentrated sulfuric acid and 3 mL of fuming sulfuric acid were added thereto on an ice bath. The mixture was stirred with a stirrer to dissolve 2,2′-bipyridyl-N, N′-dioxide. Further, 6 mL of fuming nitric acid (1.52) was added, and then the temperature was raised to 100 ° C. and held for 4 hours. The reaction solution was air-cooled, poured onto crushed ice, and the resulting yellow powder was collected by filtration. The powder was washed with water and dried at 60 ° C. under reduced pressure to obtain 4,4′-dinitro-2,2′-bipyridyl-N, N′-dioxide.

  In a 100 mL eggplant flask equipped with a reflux tube, 4.3 mL of glacial acetic acid, 2.9 mL of acetyl chloride, and 0.27 g of 4,4′-dinitro-2,2′-bipyridyl-N, N′-dioxide were placed. The mixture was heated at 70 ° C. for 4 hours in a hot water bath. The reaction solution was air-cooled, poured onto crushed ice, neutralized with 30% NaOH, and the resulting white precipitate was collected by filtration. The powder was washed with water and dried by heating to 60 ° C. under reduced pressure. Thus, 4,4′-dichloro-2,2′-bipyridyl-N, N′-dioxide was obtained.

  Into a 100 mL eggplant flask equipped with a reflux tube, add 2 mL of chloroform, 0.326 mL of phosphorus trichloride, 0.082 g of 4,4′-dichloro-2,2′-bipyrylyl-N, N′-dioxide, Refluxed on the bath for 75 minutes. The reaction solution is ice-cooled, poured onto crushed ice, and neutralized to pH 7 or higher with 25% NaOH. Chloroform was removed from the solution by distillation under reduced pressure, and the precipitated white powder was collected by filtration and further washed with water. This was dissolved in a small amount of petroleum ether and recrystallized overnight in a freezer to obtain white needle crystals. And it dried by heating to 60 degreeC under pressure reduction. Thus, 4,4′-dichloro-2,2′-bipyridine was obtained.

To a 100 mL eggplant flask equipped with a reflux tube, 20 mL of ethylene glycol, 0.08 g of (NH ₄ ) ₂ [OsCl ₆ ], 0.082 g of 4,4′-dichloro-2,2′-bipyridine was added. 300W was irradiated for 20 minutes with a microwave synthesizer under stirring with a stirrer and nitrogen flow. After the solution was cooled to room temperature, 10 mL of water in which 0.1 g of Na ₂ S ₂ O ₄ was dissolved was added. The black purple precipitate formed after stirring for 1 hour at room temperature was filtered, washed with water to remove excess salts, then washed with diethyl ether to remove unreacted ligands, and heated to 60 ° C. under reduced pressure. Dried. Thus, drying and Os (4,4′-dichloro-2,2′-bipyridine) ₂ Cl ₂ were obtained.

The following components were added to a 100 mL eggplant flask equipped with a reflux tube.
-13 mL ethylene glycol, 9 mL ethanol.
- destination prepared 0.02g of Os (4,4'-dichloro-2,2'- bipyridine) 2 Cl 2.
-0.019 g of polyacrylamide-polyvinylimidazole copolymer prepared in Preparation Example 1.

  300W was irradiated for 2 hours with a microwave synthesizer under stirring with a stirrer and nitrogen flow. After the solution was cooled to room temperature, a solution obtained by adding 20 mL of ethanol thereto was added dropwise to a 500 mL diethyl ether solution with vigorous stirring. An additional 20 mL of ethanol was added to the resulting viscous precipitate, which was again added dropwise to 500 mL of diethyl ether solution with strong stirring, and the resulting viscous precipitate was dried by heating to 60 ° C. under reduced pressure. . Thus, the target complex polymer described in the formula (2) was obtained.

  Prior to the examples, the method for preparing the porous material used in the examples is described.

Preparation Example 3
A method for preparing carbon-coated alumina nanoholes is described. An Nb film having a thickness of 100 nm is formed by RF sputtering on a mirror-polished n-type phosphorus-doped single crystal Si substrate having a resistance value of 10 ⁻² Ωcm. Thereafter, Al is deposited to a thickness of 500 nm. Anodization is performed by removing the electrode from the entire back side of Si and applying a voltage of DC25V at 3 ° C. in a 0.3 M sulfuric acid aqueous solution. During this step, oxidation is performed while monitoring the anodic oxidation current in order to detect a current indicating that the anodic oxidation has progressed from the Al surface and reached the Nb film. As the reaction proceeds, Al is oxidized and becomes alumina of the insulating layer. At the same time, holes grow and finally the base Nb is electrically connected to the upper part through a conductive path. After the anodizing treatment, alumina nanoholes are prepared by washing with pure water and isopropyl alcohol. It can be seen that the hole diameter at this time is about 20 nm from a scanning electron microscope image. The prepared alumina nanohole is placed in a tubular furnace and raised to 5 ° C. per minute up to the set temperature. During the heat treatment, 2% H ₂ /98% He (volume ratio) is always supplied at 33 sccm, and 1% C ₂ H ₂ /99% He, 1% C ₂ H ₄ /99% He (volume ratio) is used as the hydrocarbon gas. ) At 66 sccm. During hydrocarbon pyrolysis, a total of 100 sccm of gas flows and the mixing ratio is C ₂ H ₄ : H ₂ : He = 1: 1: 1 × 10 ² . Heat to 1000 ° C. over 3 hours and 20 minutes in a 2% H ₂ /98% He (volume ratio) atmosphere and hold at 1000 ° C. for 10 minutes. Thereafter, 1% C ₂ H ₂ /99% He (volume ratio) is allowed to flow for 10 minutes. Then, after hold | maintaining at 1000 degreeC for 1 hour, it is made to cool over 3 hours and 20 minutes. As a result, carbon-coated alumina nanoholes as shown in FIG. 2 are formed.

Example 1
An enzyme electrode using a glucose oxidase / gold particle complex, a glucose sensor using the same, and a biofuel cell will be described.

First, preparation of the anode will be described. The following ingredients were added to a 10 mL sample bottle.
-2 mL of 0.1 M phosphate buffer (pH 6.5).
50 mM glucose, 10 mgmL ⁻¹ glucose oxidase (hereinafter referred to as GOD) Aspergillus niger (190 Umg ⁻¹ , Sigma),
0 (zero: no NaAuCl ₄ added), 4.8 or 8.0, 64 mg mL ⁻¹ NaAuCl ₄
This was left for 3 days to prepare a GOD / gold particle composite. A glassy carbon electrode (BAS) having a diameter of 3 mm was polished with an alumina paste, washed with ultrasonic water, and 6 μL of 10 mg mL ⁻¹ aqueous solution of the complex polymer of Preparation Example 1 was dropped. After drying, 1.2 μL of the previously prepared GOD / gold particle composite was added dropwise, and 1.6 μL of 2.5 mg mL ⁻¹ polyethylene glycol diglycidel ether (hereinafter referred to as PEGDGE) was dropped and dried overnight. . Thereafter, the prepared electrode was washed with 20 mM phosphate buffer pH 7.0 (hereinafter referred to as PBS) to which 0.15 M NaCl was added. The prepared sample is referred to as follows for each NaAuCl ₄ concentration (none, 4.8 or 8.0 mg mL ⁻¹ ). That is, the anode of Comparative Example (without NaAuCl ₄ : Anode A) and the anode of Example (NaAuCl ₄ concentration 4.8 mg mL ⁻¹ : Anode B, 8.0 mg mL ⁻¹ : Anode C).

Next, the preparation of the cathode is described. The following components were mixed in a microtube.
10 mg mL ⁻¹ aqueous solution of complex polymer as described in Preparation Example 2 10.3 μL.
-20 mM phosphate buffer (pH 7.0): 2 μL.
55 mg mL ^-1 bilirubin oxidase (hereinafter referred to as BOD).
-20 mM phosphate buffer (pH 7.0) solution of Myrothecerium verrucaria (15-65 Umg ⁻¹ , Sigma): 1.7 μL.
7 mg mL ^-1 aqueous PEGDGE solution: 2 μL.
6 μL of the solution was dropped onto a glassy carbon electrode having a diameter of 3 mm, which was polished with an alumina paste and washed with ultrasonic water, and dried overnight. Thereafter, the prepared electrode was washed with PBS.

A transmission electron micrograph of the prepared GOD / gold particle composite is shown in FIG. Depending on the added NaAuCl ₄ concentration, the generated gold particles may be primary particles or may form secondary particles in which the primary particles are associated. Under the working conditions of this example, primary particles (FIG. 3A) were formed when the NaAuCl ₄ concentration was 8.0 mg mL ⁻¹ and secondary particles (FIG. 3B) were formed when the NaAuCl ₄ concentration was 4.8 mg mL ⁻¹ . The average particle size of the gold particles was 18 nm (primary particle size) and 45 nm (secondary particle size), respectively. Further, it was confirmed by observation with a transmission electron microscope that an enzyme / metal particle complex was formed in which some of the metal particles entered the inside of the enzyme. (FIG. 3C) A sample prepared with a NaAuCl ₄ concentration of 64 mg mL ⁻¹ had a metallic luster in the solution, and it was estimated that the particles had a size of approximately 300 nm or more.

Next, details of the substrate sensor using the prepared enzyme electrode will be described. As shown in FIG. 4, a three-electrode cell is constructed using the prepared anode as the working electrode 19, the platinum wire as the counter electrode 18, and the silver / silver chloride electrode as the reference electrode 21, and connected to the potentiostat 12, thereby providing a substrate sensor. It was. PBS was used as the electrolytic solution 20, and oxygen was removed from the electrolytic solution by blowing N ₂ gas for 30 minutes or more. A steady current (catalytic current) is observed by applying a potential of 300 mV vs Ag / AgCl to the working electrode 19. The steady-state current values observed from the respective anodes at the 5 mM glucose concentration of the steady-state currents observed using the anodes A, B and C individually as the working electrode 19 are respectively for the anodes A, B and C. 1.1 μA, 2.6 μA, and 4.0 μA. Further, from the anode prepared using gold particles comprising an enzyme generated from NaAuCl ₄ of 64 mg mL-1, significant enzyme current was not observed. Thus, it was observed that using the GOD / gold particle composite under appropriate conditions, the current was nearly quadrupled.

  The catalytic current of the enzyme electrode prepared using the enzyme combined with the metal particles was higher than that of the enzyme electrode prepared using the enzyme not combined with the metal particles. This is presumably because the metal particles generated by the enzymatic reaction have improved the charge extraction rate from the redox center of the enzyme.

  By using an enzyme electrode with an improved charge extraction rate from the redox center of the enzyme, a sensor having a wide measurable concentration range (the upper limit of the measured concentration range is high and the lower limit is low) can be prepared. The reason for this can be explained as follows.

  In general, in a sensor using an enzyme electrode, when the dependence of the enzyme current value on the substrate concentration is observed, a region where the enzyme reaction becomes rate-limiting appears as the substrate concentration increases, and the current value is proportional to the increase in the substrate concentration. A phenomenon that does not increase is observed. Here, in an enzyme electrode produced using an enzyme combined with metal particles, the metal particles take out charges from the redox center of the enzyme and improve the speed. Therefore, it is possible to speed up the charge transport from the active center of the enzyme to the conductive member as compared with a case where an enzyme electrode not combined with metal particles is used (for example, a comparative example). The high-speed charge transport from the active center to the conductive member can improve the concentration of the substrate at which the enzyme reaction is rate-determined, and can improve the upper limit of the measurable concentration range of the sensor using the enzyme electrode.

  In general, in an enzyme electrode, an enzyme current / background current ratio can be mentioned as a main index for determining a measurement lower limit. This background current is generally proportional to the area of the conductive member. For this reason, in order to reduce the background current, it is effective to reduce the area of the conductive member. Furthermore, the reduction of the area of the conductive member reduces the influence of the substrate diffusion rate control, but also reduces the overall enzyme current. Here, in the electrode produced using the enzyme combined with the metal particles, the metal particles take out the charge from the redox center of the enzyme and improve the speed. Therefore, it is possible to speed up the transport of charges from the active center of the enzyme to the conductive member as compared with a case where an enzyme electrode not combined with metal particles is used (for example, a comparative example). Thus, by improving the enzyme current density (current value per unit area of the conductive member) of the enzyme electrode, the same enzyme current value can be given with a smaller area of the conductive member. As a result, the enzyme current / background current ratio can be improved and the measurement lower limit can be lowered.

  FIG. 5 shows a trend of the steady-state current dependence on the glucose concentration in the solution observed using the anode of the example or the comparative example anode. At each anode, the steady-state current increases linearly up to a certain glucose concentration and then saturates. This linearity range is wider in both the upper and lower limits in the enzyme electrode of the example than in the enzyme electrode of the comparative example, and the measurable range of the glucose concentration of the glucose sensor is expanded.

  Next, the details of the biofuel cell using the prepared anode of the example and the anode and cathode of the comparative example in combination will be described. As shown in FIG. 6, the prepared anode is a working electrode 19 and the cathode is a counter electrode 23 to form a biofuel cell. Biofuel cell characteristics are measured using PBS containing 10 mM glucose as the electrolyte 20 and using the anodes A, B, and C individually as the working electrode 19 while bubbling air. The maximum output of the battery is expressed as the product of voltage, current, and fill factor.

  Here, in the electrode produced using the enzyme combined with the metal particle, the metal particle is separated from the redox center of the enzyme as compared with the case where the enzyme electrode not combined with the metal particle is used (for example, comparative example). A high current and output can be obtained because the charge extraction speed is improved.

  FIG. 8 shows the tendency of the characteristics of the current-potential curve of the biofuel cell when the enzyme electrode of the example is used and when the enzyme electrode of the comparative example is used. As a result, the biofuel cell using the enzyme electrode of the example has a higher current value than the biofuel cell using the enzyme electrode of the comparative example, and thus a high output can be obtained.

Example 2
The carbon-coated alumina nanohole substrate of Preparation Example 3 is cut into 1 cm square and hydrophilized by UV-ozone treatment, and then immersed in a PBS solution of ¹ mg mL ⁻¹ GOD for 24 hours in a sample bottle. The substrate was pulled up and washed 10 times with 0.1 M phosphate buffer (pH 6.5). Further, the substrate is immersed in 0 (no addition of NaAuCl ₄ ) or 8 mg mL ⁻¹ NaAuCl ₄ in 0.1 M phosphate buffer (pH 6.5), and 50 mM glucose is added and left for 3 days. After the substrate is pulled up and washed 10 times with PBS, contact is made with the copper wire to be the lead using carbon paste (Asahi Chemical Research Laboratories). Silicone rubber (Shin-Etsu Silicone) is coated and insulated on conductive parts such as carbon paste and copper wire that come into contact with the solution. In Example 2, the prepared samples are referred to as comparative anodes and example anodes for each NaAuCl ₄ concentration (none, 8.0 mg mL ⁻¹ ).

Next, details of the substrate sensor using the prepared enzyme electrode will be described. As shown in FIG. 4, the prepared anode is connected to a potentiostat using the working electrode 19, the platinum wire as the counter electrode 18, and the silver / silver chloride electrode as the reference electrode 21, thereby obtaining a substrate sensor. PBS is used as the electrolytic solution 20, and oxygen is removed from the electrolytic solution by blowing N ₂ gas for 30 minutes or more. A stationary current (enzyme catalyst current) is observed by applying a potential of 600 mV vsAg / AgCl to the working electrode 19. FIG. 7 shows the tendency of the steady-state current depending on the glucose concentration in the solution observed using the anodes D and F individually as the working electrode 19. As a result, when the anode of the comparative example is used, the enzyme catalyst current is small or not observed, whereas when the anode of the example is used, the current value is large and within a certain glucose concentration range. Increases linearly with glucose concentration. This result shows that the redox center of the enzyme and the carbon on the wall surface of the substrate are not effectively electrically connected to the anode of the comparative example in which gold particles are not generated simply by supporting the enzyme in alumina nanoholes. It is shown that. For this reason, it shows that it is difficult to effectively extract the enzyme catalyst current resulting from the oxidation of glucose to an external circuit. On the other hand, in the anode of the example, the GOD / gold particle composite is formed in the carbon-coated alumina nanohole, whereby the redox center of the enzyme and the carbon of the substrate wall are electrically connected, and the enzyme catalytic current is Observed. Further, by introducing an enzyme into the pores of the porous material, and in the presence of the enzyme substrate and the metal precursor, the metal particles can be precipitated from the metal precursor by a reaction involving the enzyme substrate in the enzyme.

  INDUSTRIAL APPLICABILITY The present invention can provide a novel enzyme electrode, a sensor using the same, and a biofuel cell. For example, the present invention is extremely useful as a member that can be used in a fuel cell using a biosensor or an enzyme substrate as a fuel.

It is a conceptual diagram about the enzyme / metal particle composite_body | complex in which the metal particle entered the inside of the said enzyme. It is a conceptual diagram which shows the structure of a carbon covering alumina nanohole. 2 is a transmission electron micrograph of gold particles prepared in Example 1. FIG. It is the schematic of a 3 electrode cell. 3 is a graph showing the relationship between the steady-state current / substrate concentration of the substrate sensor of Example 1. It is the schematic of a 2 electrode cell. 6 is a graph showing the relationship between the steady current / substrate concentration of the substrate sensor of Example 2. 4 is a graph showing a tendency of characteristics of a current-potential curve of the battery of Example 1.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Enzyme 2 Metal fine particle 3 Metal fine particle which entered inside of enzyme 4 Anodized alumina 5 Carbon 6 Nb (conductive path)
7 Nb film 8 Si substrate 9 Reference electrode lead 10 Working electrode lead 11 Counter electrode lead 12 Potentiostat 13 Gas supply port 14 Temperature control cell cover 15 Gas supply pipe 16 Temperature control cell 17 Temperature control water supply port 18 Platinum Counter electrode 19 Anode 20 Electrolyte 21 Reference electrode 22 Temperature controlled water outlet 23 Cathode

Claims (11)

In an enzyme electrode having a conductive member, an enzyme, and metal particles,
The enzyme includes the metal particles inside the enzyme ,
The metal particles are formed by reacting the enzyme and the substrate in the presence of a metal precursor capable of forming a metal in association with a reaction between the enzyme and a substance serving as a substrate for the enzyme. An enzyme electrode characterized by the above.   2. The enzyme according to claim 1, wherein the metal particles and the enzyme form a complex of an enzyme and metal particles having a structure in which a part of the metal particles enter the inside of the enzyme. electrode. The enzyme electrode according to claim 1 or 2 , wherein the conductive member has a porous structure, and a complex of the enzyme and the metal particles is formed in pores of the porous structure. The enzyme electrode according to any one of claims 1 to 3 metal is gold constituting the metal particles. The enzyme electrode according to any one of claims 1 to 4 , wherein a secondary particle size of the metal particles is 200 nm or less. The enzyme electrode according to claim 2 , further comprising a mediator that mediates charge transfer between the complex and the conductive member. The enzyme electrode according to claim 6 , further comprising a carrier that immobilizes at least one of the complex and the mediator on the conductive member. In the fuel cell which provided the area | region which can hold | maintain electrolyte solution between an anode electrode and a cathode electrode, at least one of the said anode electrode and the said cathode electrode is an enzyme electrode of any one of Claims 1 thru | or 7. A fuel cell characterized by being. Introducing an enzyme into the pores of the porous material,
In the presence of a substrate of the enzyme and a precursor of metal particles,
A method for producing metal particles in pores of a porous material, wherein metal particles are precipitated from a precursor of the metal particles by a reaction between the enzyme and the substrate. A method for producing an enzyme electrode having an enzyme and metal particles,
Preparing an enzyme;
Enzyme / metal particles comprising metal particles in the enzyme by reacting the enzyme with the substrate in the presence of a metal precursor capable of forming a metal by reaction of the enzyme with a substrate of the enzyme Obtaining a complex;
A method for producing an enzyme electrode, comprising: A method for producing an enzyme electrode having an enzyme and metal particles,
Introducing an enzyme into the pores of the conductive porous material;
In the presence of a metal precursor capable of forming a metal by reaction between the enzyme and the enzyme substrate, the enzyme in the pores of the porous material is reacted with the substrate to provide metal particles in the enzyme. Obtaining an enzyme / metal particle complex in the pores of the porous material;
A method for producing an enzyme electrode, comprising:

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