Anode electrode

Abstract

To provide an anode electrode and a manufacturing method for the same, and an electrochemical device including the anode electrode, capable of increasing the current density in an electrode by a simple method.SOLUTION: In an aspect of the present disclosure, an anode electrode includes a conductive layer and a reagent layer in contact with the conductive layer. The reagent layer includes a reagent layer main body containing a conductive material, and an enzyme crosslinking body and an electron transfer accelerator that are carried by the reagent layer main body. The enzyme crosslinking body is a reaction product of an anode enzyme including at least one kind of amino group selected from a primary amino group and a secondary amino group, and a polyalkylene glycol derivative including one or more epoxy group in one molecule and having a number-average molecular weight of 600 or more.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to an anode electrode and a method for manufacturing the same, as well as an electrochemical device including the anode electrode. [Background technology]

[0002] In recent years, with the rise in health consciousness, there has been a growing need for sensors that can selectively detect organic substances in biological fluids such as sweat, saliva, urine, tears, and blood in order to monitor the physical condition. In particular, non-invasive measurements that do not injure the subject's body, such as those that do not require blood sampling, have attracted attention.

[0003] Patent Document 1 discloses a sensor for measuring the glucose concentration in a liquid, which includes a reagent layer formed by mixing a mediator (electron transfer promoter) having a hydrophilic functional group with glucose dehydrogenase (enzyme) and applying the mixture. However, there was a problem in that the mediator (electron transfer promoter) and the enzyme were eluted into the measurement solution, making it difficult to obtain repeatable responses. Patent Document 2 discloses that a mixed solution containing a polymer containing a mediator (electron transfer promoter), an enzyme, and a multi-epoxidized compound is used to form an electrode in order to prevent elution of the mediator and enzyme. It discloses that the use of this mixed solution results in a higher current density and allows the high current density to be maintained for a longer period of time than when a mixed solution containing a mediator alone, an enzyme, and a multi-epoxidized compound is used. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2017-194484 [Patent Document 2] Japanese Patent Publication No. 2023-128506 Summary of the Invention [Problem to be solved by the invention]

[0005] However, the use of the polymer obtained by binding the mediator to the base polymer is disadvantageous from the viewpoint of productivity. Therefore, it is desirable to achieve an increase in current density at the electrode by a simpler method. When the enzyme and mediator are water-soluble, the decrease in current density due to their elution is a significant problem.

[0006] The present disclosure provides an anode electrode and a method for producing the same, which enable an increase in current density in the electrode in a simple manner, and an electrochemical device including the anode electrode. [Means for solving the problem]

[0007] In one aspect, the present disclosure provides a liquid crystal display device including a conductive layer and a reagent layer in contact with the conductive layer, the reagent layer includes a reagent layer main body containing a conductive material, and an enzyme crosslinker and an electron transfer promoter supported on the reagent layer main body; The enzyme crosslinker relates to an anode electrode, which is a reaction product between an anode enzyme containing at least one amino group selected from a primary amino group and a secondary amino group and a polyalkylene glycol derivative containing one or more epoxy groups in one molecule and having a number-average molecular weight of 600 or more.

[0008] In one aspect, the present disclosure provides a method for producing an anode electrode of the present disclosure, comprising: applying a solution containing an electron transfer promoter to the reagent layer main body and drying the applied solution to cause the reagent layer main body to support the electron transfer promoter; The method for producing an anode electrode includes applying to the reagent layer main body a solution for preparing an enzyme crosslinker, the solution comprising an anode enzyme containing at least one type of amino group selected from a primary amino group and a secondary amino group and a polyalkylene glycol derivative containing one or more epoxy groups in one molecule and having a number average molecular weight of 600 or more, and drying the applied solution for preparing an enzyme crosslinker.

[0009] In one aspect, the present disclosure relates to an electrochemical device including an anode electrode of the present disclosure. [Effects of the Invention]

[0010] According to the present disclosure, it is possible to provide an anode electrode that enables an increase in current density in a simple manner, a method for manufacturing the same, and an electrochemical device that includes the anode electrode. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a graph showing the results of chronoamperometry measurements repeatedly performed using the anode electrodes of Examples 1 to 3 and Comparative Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0012] In one aspect, the anode electrode of the present disclosure is an anode electrode used in an electrochemical device or the like. The anode electrode includes a conductive layer, a reagent layer, and a substrate. The reagent layer includes a reagent layer main body, and an enzyme (hereinafter also referred to as an "anode enzyme") and an electron transfer promoter (hereinafter also referred to as an "anode mediator") supported on the reagent layer main body. The present disclosure is based on the new finding that an anode electrode with a high current density can be provided by supporting an anode enzyme, together with an anode mediator, in the form of a reaction product (hereinafter also referred to as an "enzyme crosslinker") between the anode enzyme containing at least one type of amino group selected from a primary amino group and a secondary amino group and a polyalkylene glycol derivative containing one or more epoxy groups per molecule and having a number-average molecular weight of 600 or more (hereinafter also referred to as the "immobilizing agent of the present disclosure").

[0013] The details of the mechanism by which the effects of the present disclosure are manifested are speculated as follows.

[0014] It is presumed that the polyalkylene glycol chain of the enzyme crosslinker is strongly physically adsorbed to the conductive material of the reagent layer body, thereby increasing the amount of the anode enzyme immobilized on the reagent layer body and improving the current density. It is also presumed that, for the same reason, migration of the anode enzyme out of the reagent layer body is suppressed, thereby maintaining a high current density for a long period of time. It is also presumed that, because the anode mediator in the reagent layer is encapsulated within the enzyme crosslinker, even if the anode mediator is a monomolecular molecule, elution into an aqueous solution containing the substrate (target of measurement) is suppressed. It is presumed that these factors enable the anode electrode of the present disclosure to achieve a high current density and maintain that high current density for a long period of time. The ability to maintain a high current density over a long period of time can be evaluated by the ratio of the current density obtained by the nth chronoamperometry measurement (CA measurement) to the initial current density obtained by the first CA measurement. A high ratio indicates good repeatability of the current density, and that a high current density can be maintained over a long period of time.

[0015] Next, an example of the anode electrode of the present disclosure will be described.

[0016] An example of an anode electrode according to the present disclosure includes a substrate, a conductive layer formed on one main surface of the substrate, and a reagent layer formed in contact with the conductive layer. The reagent layer includes a reagent layer main body formed from a conductive material, and an anode mediator and an enzyme crosslinker supported on the reagent layer main body.

[0017] The term "supporting" the enzyme crosslinker and the anode mediator means "fixing" and means that the enzyme crosslinker and the anode mediator are in a state in which they do not easily migrate from the reagent layer. There are no particular restrictions on the position of the enzyme crosslinker as long as it is supported on the main body of the reagent layer, but from the viewpoint of effectively obtaining the effect of promoting oxidation or reduction by the enzyme crosslinker, it is preferable that the enzyme crosslinker be fixed on the surface of the reagent layer where contact with the substrate begins or in the vicinity thereof.

[0018] The crosslinked enzyme may be supported directly on the conductive material that constitutes the main body of the reagent layer, or may be supported indirectly via an anode mediator.

[0019] There are no particular restrictions on the material of the substrate, but from the viewpoint of disposability, paper, cloth, etc. made from natural materials are preferred, and Japanese paper is more preferred.

[0020] A conductive layer for electrically connecting the electrodes is formed on one main surface of the substrate using a paste containing a commonly used conductive material, such as carbon paste.

[0021] The conductive material constituting the reagent layer main body can be selected from conductive materials such as carbon materials and metals. However, because the polyalkylene glycol chains of the enzyme crosslinkers can be physically adsorbed inside the pores, thereby increasing the amount of enzyme crosslinkers immobilized in the reagent layer, a porous conductive material is preferred, and porous carbon or porous nickel is more preferred. From the viewpoints of disposability and biocompatibility, porous carbon is even more preferred, and porous carbon having many pores (pores with diameters of 10 to 1,000 nm) and forming a three-dimensional network structure, including interconnected pores, is even more preferred. A preferred commercially available porous carbon product is, for example, Knobel (registered trademark) (manufactured by Toyo Tanso Co., Ltd.). Furthermore, the reagent layer containing porous carbon may contain metal powder to improve conductivity. The reagent layer main body is formed using a paste containing the conductive material.

[0022] The specific surface area of ​​the porous conductive material constituting the reagent layer body is preferably 100 m from the viewpoint of increasing the amount of the enzyme crosslinked body to be immobilized. 2 / g or more, more preferably 400m 2 / g or more, and from the viewpoint of increasing the effective electrode surface area of ​​the anode mediator and the enzyme crosslinker, it is preferably 1000 m 2 / g or less, more preferably 700m 2The specific surface area of ​​the porous conductive material is a BET specific surface area, which can be measured by a nitrogen adsorption / desorption measurement method.

[0023] The average diameter of the pores of the porous conductive material is preferably 100 nm or less, more preferably 80 nm or less, from the viewpoint of electron transfer between the enzyme and the mediator, and is preferably 10 nm or more, more preferably 30 nm or more, from the viewpoint of increasing the amount of immobilized enzyme crosslinked product. The average diameter of the pores of the porous conductive material can be calculated by nitrogen adsorption / desorption measurement, as in the case of determining the BET specific surface area.

[0024] The thickness of the reagent layer main body is preferably thicker from the viewpoint of reducing the internal resistance of the electricity storage device, specifically, preferably 30 μm or more, more preferably 40 μm or more, whereas the thickness of the reagent layer main body is preferably 120 μm or less, more preferably 100 μm or less from the viewpoint of substrate permeability.

[0025] The reagent layer itself may contain a binder (binder resin) to the extent that its conductivity is not impaired. Examples of binders that can be used include polyvinylidene fluoride (PVDF), vinylidene fluoride-hexafluoropropylene copolymer, styrene-butadiene rubber (SBR), and polyacrylonitrile, either alone or in combination.

[0026] The anode mediator is not particularly limited as long as it is a redox substance that can transfer electrons to the electrode, and any conventionally known compound that can be reversibly oxidized and reduced can be used. The anode mediator is preferably an aromatic compound that can be reversibly oxidized and reduced, more preferably at least one selected from benzoquinone, 1,2-naphthoquinone, 1,4-naphthoquinone, thionine acetate, Azure A, Azure B, Azure C, phenothiazine, methylene blue, and Prussian blue, even more preferably at least one selected from thionine acetate, Azure A, Azure B, and Azure C, and even more preferably thionine acetate.

[0027] For example, the oxidized form of thionine acetate combines with hydrogen produced by the reaction between the anode enzyme and the substrate to become the reduced form, and the reagent layer of the anode electrode accepts electrons produced by the reaction, returning thionine acetate to the oxidized form. Meanwhile, the cathode electrode accepts electrons from the anode electrode, and oxygen in the air is reduced by these electrons. In this way, a current flows from the cathode electrode to the anode electrode in response to the reaction between the anode enzyme and the substrate. Therefore, for example, when lactic acid is the substrate, an enzyme sensor capable of measuring the concentration of lactic acid can be provided as an electrochemical device.

[0028] The anode enzyme is not particularly limited as long as it contains at least one amino group selected from a primary amino group and a secondary amino group as an active hydrogen-containing group that is highly reactive with an epoxy group and has specificity for a substrate. From the viewpoint of electron transfer efficiency, the anode enzyme is preferably at least one selected from oxidases and dehydrogenases, more preferably at least one selected from glucose oxidase, glucose dehydrogenase, fructose dehydrogenase, invertase, glucose dehydrogenase, amylase, glucose dehydrogenase, L-lactate oxidase, L-lactate dehydrogenase, and alcohol dehydrogenase, and from the viewpoint of easy availability, even more preferably L-lactate oxidase.

[0029] The fixative of the present disclosure is not particularly limited as long as it is a polyalkylene glycol derivative having one or more epoxy groups in one molecule and a number-average molecular weight of 600 or more. From the viewpoint of improving the stability of repeated current density, however, it is preferably a polyalkylene glycol derivative having epoxy groups at both ends, and more preferably a polyethylene glycol derivative (polyethylene glycol diglycidyl ether) having epoxy groups at both ends. Since at least one amino group selected from a primary amino group and a secondary amino group readily reacts with an epoxy compound at room temperature, if an anode enzyme and the immobilizing agent of the present disclosure coexist, at least one amino group selected from a primary amino group and a secondary amino group reacts with an epoxy group to form an enzyme crosslinked product.

[0030] From the viewpoint of increasing the amount of immobilization of the enzyme crosslinked product in the reagent layer to improve the initial current density, and effectively preventing the detachment of the anode enzyme and the anode mediator from the reagent layer main body due to the decrease in solubility in the measurement solution containing the substrate and improving the repetitive stability of the current density, the number average molecular weight of the immobilizing agent of the present disclosure is 600 or more, preferably 800 or more, more preferably 1000 or more, still more preferably 1500 or more. Further, from the viewpoint of the reactivity between the amino group and the polyalkylene glycol derivative in the solution for preparing the enzyme crosslinked product, the number average molecular weight of the immobilizing agent of the present disclosure is preferably 10000 or less, more preferably 8000 or less. The number average molecular weight of the immobilizing agent of the present disclosure was determined by the SEC (Size Exclusion Chromatography) method under the following conditions. <SEC conditions><0000​​​​​​​​​​​​​​​​​​The biosensor includes a cathode electrode and an anode electrode of the present disclosure. When a substrate is supplied to the biosensor, electrons generated by oxidation of the substrate at the anode electrode are transferred to the cathode electrode and used to reduce oxygen at the cathode electrode. The transfer of electrons generated during this process is used to measure the substrate, for example, to measure the substrate concentration.

[0033] The substrate is not particularly limited as long as it is a substance whose oxidation is promoted by the enzyme contained in the anode electrode, and specific examples include lactic acid, glucose, fructose, sucrose, alcohol, etc. When the anode enzyme is lactate oxidase, the substrate is preferably L-lactic acid.

[0034] Like the anode electrode, the cathode electrode includes a substrate, a conductive layer formed on one main surface of the substrate, and a reagent layer formed on the conductive layer. The conductive layer of the cathode electrode may be made of the same material as that of the anode electrode and can be prepared by a conventionally known method. The reagent layer of the cathode electrode preferably includes a reagent layer main body and a cathode enzyme supported on the reagent layer main body. The material of the reagent layer main body can be selected from conductive materials such as carbon materials and metals. From the viewpoint of increasing the amount of the cathode enzyme supported, a porous conductive material is preferred, more preferably porous carbon or porous nickel, and even more preferably porous carbon. The reagent layer main body of the cathode electrode can be prepared by a conventionally known method. The cathode enzyme is not particularly limited as long as it promotes the reduction of oxygen in the air. However, from the viewpoints of disposability and biocompatibility, organic substances are preferred, such as bilirubin oxidase and laccase. The cathode enzyme may be one type or two or more types.

[0035] The cathode electrode may further contain a redox substance (cathode mediator). An example of the cathode mediator is ABTS (2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid).

[0036] Next, an example of a method for manufacturing the anode electrode according to the present disclosure will be described.

[0037] The conductive layer can be produced by a conventionally known method, for example, by applying a carbon paste to one main surface of the substrate by screen printing or the like, baking the coating by heating, and repeating this process until the desired thickness is achieved. The reagent layer main body can also be produced by a conventionally known method, for example, by applying a paste containing a conductive material to one main surface of the substrate on which the conductive layer has been formed by screen printing or the like, baking the coating by heating, and repeating this process until the desired thickness is achieved. The reagent layer main body is formed so that at least a portion of it overlaps with the conductive layer.

[0038] Next, the enzyme crosslinker and the anode mediator are supported on the reagent layer main body. Specifically, the method for producing an anode electrode of the present disclosure includes applying a solution containing the anode mediator to the reagent layer main body and drying the applied solution. The method for producing an anode electrode of the present disclosure also includes applying a solution for preparing an enzyme crosslinker, the solution containing an anode enzyme containing at least one amino group selected from a primary amino group and a secondary amino group, and a polyalkylene glycol derivative (immobilizing agent) containing one or more epoxy groups per molecule and having a number-average molecular weight of 600 or more, and drying the applied solution. There are no particular restrictions on the order in which the enzyme crosslinker and the anode mediator are supported; however, from the viewpoint of effectively preventing the anode mediator from separating from the reagent layer main body, it is preferable to support the enzyme crosslinker after supporting the anode mediator.

[0039] The amount of immobilizing agent used per 1 U of enzyme is preferably 3 nmol or more, more preferably 6 nmol or more, from the viewpoint of improving crosslink density, and is preferably 30 nmol or less, more preferably 15 nmol or less, from the viewpoint of increasing interfacial resistance. [Example]

[0040] Hereinafter, the present disclosure will be described in more detail with reference to examples, but these are merely illustrative examples and the present disclosure is not limited to these examples.

[0041] Japanese paper (Hanshiya Gasenshi Shounsha) was prepared as a substrate, and carbon paste (JELCON CH-8, Jujo Chemical) was applied to one main surface of the Japanese paper and baked for 15 minutes at 120°C. This carbon paste application and baking was repeated six times, resulting in a total of six carbon layers being laminated to create a conductive layer.

[0042] Next, porous carbon Knobel (manufactured by Toyo Tanso Co., Ltd., specific surface area 560 m 2 A paste containing 1000 sachets of carbon nanotubes (1000 sachets / g, catalog value) with an average pore diameter of 40 nm (catalog value) was prepared, which was then applied to the conductive layer and baked at 60°C for 24 hours. This process was repeated three times to produce a 40 μm-thick reagent layer body consisting of a total of three porous carbon layers.

[0043] [Example 1] 20 μL of a 50 mM thionine acetate (mediator, Tokyo Chemical Industry Co., Ltd.) methanol solution was applied to the reagent layer main body, followed by drying under reduced pressure for 90 minutes. Next, 20 μL (40 U enzyme amount) of an L-lactate oxidase (Toyobo Co., Ltd.) solution containing polyethylene glycol diglycidyl ether 1 (PEGDGE1000, Polysciences, Inc.) was applied to the reagent layer main body as an enzyme crosslinker preparation solution, followed by drying under reduced pressure for 90 minutes. A 0.1 mol / L phosphate buffer (pH 7.4) was used as the solvent to prepare the enzyme crosslinker preparation solution. The amount of polyethylene glycol diglycidyl ether 1 added was such that 0.46 μmol was applied per electrode (11.5 nmol per 1 U enzyme amount). In this way, the anode electrode of Example 1 was obtained.

[0044] [Example 2] An anode electrode of Example 2 was prepared in the same manner as in Example 1, except that polyethylene glycol diglycidyl ether 2 (Sigma-Aldrich, product number 731811) was used instead of polyethylene glycol diglycidyl ether 1.

[0045] [Example 3] An anode electrode of Example 3 was produced in the same manner as in Example 1, except that polyethylene glycol diglycidyl ether 3 (Sigma-Aldrich, product number 731803) was used instead of polyethylene glycol diglycidyl ether 1.

[0046] [Comparative Example 1] An anode electrode of Comparative Example 1 was prepared in the same manner as in Example 1, except that polyethylene glycol diglycidyl ether 4 (Sigma-Aldrich, product number 47696) was used instead of polyethylene glycol diglycidyl ether 1.

[0047] The anode electrodes of Examples 1 to 3 and Comparative Example 1 were immersed in a 1 mol / L phosphate buffer solution (pH 7.0) in a 37°C thermostatic bath, and chronoamperometry measurements were performed. Specifically, the electrodes were held at the natural potential for 30 seconds, then held at a potential of 0.2 V for 300 seconds, and the current density after 300 seconds was measured. This was designated the initial current density. This current density measurement was repeated five times. The ratio of the current density at the fifth measurement to the initial current density was calculated and shown in Table 1 below as an index of the current density repetition stability.

[0048] [Table 1]

[0049] As shown in Table 1, in Examples 1 to 3, in which the enzyme was supported on the reagent layer body in the form of a reaction product with the immobilizing agent of the present disclosure, the initial current density was significantly higher than in Comparative Example 1, and it was confirmed that the repeatability of the current density was also higher than in Comparative Example 1. [Industrial Applicability]

[0050] The anode electrode of the present disclosure is useful as an electrode for a biosensor and can be used in any field requiring a biosensor, such as various industrial fields including medicine, food, the environment, etc. For example, when lactic acid is used as a substrate, it can be applied to an enzyme sensor that measures the concentration of lactic acid.

Claims

1. a conductive layer and a reagent layer in contact with the conductive layer, the reagent layer includes a reagent layer main body containing a conductive material, and an enzyme crosslinker and an electron transfer promoter supported on the reagent layer main body; The anode electrode, wherein the enzyme crosslinker is a reaction product between an anode enzyme containing at least one type of amino group selected from a primary amino group and a secondary amino group and a polyalkylene glycol derivative containing one or more epoxy groups in one molecule and having a number average molecular weight of 600 or more.

2. The anode electrode of claim 1 , wherein the reagent layer body comprises porous carbon.

3. The anode electrode of claim 1 , wherein the anode enzyme is lactate oxidase.

4. 2. The anode electrode according to claim 1, wherein the electron transfer promoter is thionine acetate.

5. 2. The anode electrode according to claim 1, wherein the polyalkylene glycol derivative is a polyethylene glycol derivative having epoxy groups at both ends.

6. A method for manufacturing an anode electrode according to any one of claims 1 to 5, applying a solution containing an electron transfer promoter to the reagent layer main body and drying the applied solution to cause the reagent layer main body to support the electron transfer promoter; a method for manufacturing an anode electrode, the method comprising: applying to the reagent layer main body a solution for preparing an enzyme crosslinker, the solution comprising an anode enzyme containing at least one kind of amino group selected from a primary amino group and a secondary amino group, and a polyalkylene glycol derivative containing one or more epoxy groups in one molecule and having a number average molecular weight of 600 or more; and drying the applied solution for preparing an enzyme crosslinker.

7. An electrochemical device comprising the anode electrode according to any one of claims 1 to 5.

8. 8. The electrochemical device of claim 7, wherein the electrochemical device is an enzyme sensor that measures the concentration of a desired substrate.

9. The electrochemical device of claim 7, wherein the electrochemical device is a biofuel cell.

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