Bioelectrode, bioelectrode production method, electrochemical device, blood sugar level measurement device, and concentration measurement method

Abstract

A bioelectrode comprising: a complex compound composed of a metal and a ligand; a mediator having an anionic functional group; and an electroconductive material.

Description

Bioelectrode, bioelectrode manufacturing method, electrochemical device, blood glucose level measuring device, and concentration measuring method

[0001] The present disclosure relates to a bioelectrode, a method for manufacturing a bioelectrode, an electrochemical device, a blood glucose level measuring device, and a concentration measuring method.

[0002] Electrochemical devices, such as biofuel cells, that use enzymes or microorganisms as electrode catalysts (hereinafter also referred to as biocatalysts) to generate electricity using sugars, alcohols, and other biomass resources as fuel, have been researched and developed. Electrochemical devices that use enzymes or microorganisms generally include a negative electrode (anode) containing a biocatalyst that promotes the oxidation of fuel, and a positive electrode (cathode) containing a biocatalyst that promotes the reduction of oxygen. Electrons extracted by the oxidation of fuel at the negative electrode (for example, the conversion of glucose to gluconolactone) are transferred to the positive electrode, and oxygen is reduced using these electrons to produce water (H ２ O) is produced. The movement of electrons that occurs during this process can be used to generate electricity.

[0003] As an example of a device other than one intended for power generation, for example, a blood glucose level measuring device using glucose dehydrogenase, an enzyme that catalyzes the oxidation reaction of glucose to gluconolactone, has been put into practical use. In recent years, in addition to devices that measure the blood glucose level of collected blood, devices that measure the blood glucose level of interstitial fluid continuously over a long period of time by inserting needle-shaped electrodes into the skin have also become known.

[0004] Electrochemical devices that utilize biocatalysts may use a substance called a mediator that mediates the transfer of electrons between the biocatalyst and the electrode. In electrochemical devices that use a mediator, it is important to suppress the outflow of the mediator and increase the sustainability of the electron transfer function of the mediator. Various techniques for immobilizing the mediator on an electrode have been investigated as a method for increasing the sustainability of the electron transfer function of the mediator. For example, Non-Patent Document 1 describes a method for immobilizing ferrocene as a mediator on the surface of an electrode by electron beam graft polymerization.

[0005] Non-patent document 1: J. Power Sources, 479, 228807 (2020)

[0006] The method described in Non-Patent Document 1 requires steps such as electron beam irradiation and graft polymerization, and therefore leaves room for improvement in terms of production efficiency. In view of the above circumstances, an object of the present disclosure is to provide a bioelectrode that has excellent durability of the electron transfer function due to the mediator, a method for manufacturing a bioelectrode, an electrochemical device, a blood glucose level measuring device, and a concentration measuring method.

[0007] Specific means for solving the above problems include the following embodiments. <1> A bioelectrode comprising: a complex compound composed of a metal and a ligand; a mediator having an anionic functional group; and a conductive material. <2> The bioelectrode according to <1>, wherein the metal comprises cobalt or zinc. <3> The bioelectrode according to <1> or <2>, wherein the ligand comprises an imidazole compound. <4> The bioelectrode according to any one of <1> to <3>, wherein the complex compound comprises a metal organic framework. <5> The bioelectrode according to any one of <1> to <4>, further comprising a biocatalyst. <6> The bioelectrode according to any one of <1> to <5>, further comprising chitosan. <7> An electrochemical device comprising the bioelectrode according to any one of <1> to <6>. <8> A blood glucose level measuring device comprising the bioelectrode according to any one of <1> to <6>. <9> A concentration measurement method comprising: bringing the bioelectrode according to any one of <1> to <6> into contact with a liquid; and measuring the concentration of a substance contained in the liquid.

[0008] According to the present disclosure, there are provided a bioelectrode having excellent durability of the electron transfer function due to the mediator, a method for manufacturing a bioelectrode, an electrochemical device, a blood glucose level measuring device, and a concentration measuring method.

[0009] 1 is a graph showing FTIR results indicating changes in chemical bonding due to mixing of ZIF67 and 12NQSO. 1 is an SEM image of ZIF67-CNT (before mixing with 12NQSO) produced in Example 1. 2 is an SEM image of ZIF67-CNT (after mixing with 12NQSO) produced in Example 1. 3 is an SEM image of ZIF8-CNT (before mixing with 12NQSO) produced in Example 2. 4 is an SEM image of ZIF8-CNT (after mixing with 12NQSO) produced in Example 2. 5 is a graph showing the results of electrochemical measurements of a bioelectrode. 6 is a graph showing the results of electrochemical measurements of a bioelectrode. 7 is a graph showing the results of electrochemical measurements of a bioelectrode. 8 is a graph showing the concentration dependence of the sensitivity of a bioelectrode to glucose. 9 is a graph showing the selectivity of the sensitivity of a bioelectrode to glucose. 10 is a graph showing the sustainability of the sensitivity of a bioelectrode to glucose. 11 is a graph showing the concentration dependence of the sensitivity of a bioelectrode to lactic acid. 1 is a graph showing the durability of sensitivity of a bioelectrode to lactate.

[0010] Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments. In the following embodiments, components (including element steps, etc.) are not essential unless otherwise specified. The same applies to numerical values ​​and their ranges, and do not limit the present invention. In this disclosure, numerical ranges indicated using "to" include the numerical values ​​before and after "to" as the minimum and maximum values, respectively.

[0011] <Bioelectrode> The bioelectrode of the present disclosure includes a complex compound composed of a metal and a ligand, a mediator having an anionic functional group, and a conductive material.

[0012] As shown in the Examples below, the bioelectrode of the present disclosure exhibits stable current density. The reason for this is thought to be, for example, that the mediator contained in the bioelectrode is immobilized to a conductive material or complex compound via an anionic group by chemical or physical action, allowing the mediator to remain stably within the bioelectrode. Furthermore, immobilization of the mediator to a conductive material or complex compound via an anionic group is thought to control the orientation of the active site of the mediator, thereby enabling efficient electron transfer between the biocatalyst and the electrode.

[0013] In the present disclosure, the term "bioelectrode" refers to an electrode used in an electrochemical device that utilizes a biocatalyst. When the bioelectrode of the present disclosure is used in an electrochemical device together with a biocatalyst, the electron transfer function between the biocatalyst and the electrode via the mediator is well maintained.

[0014] (Complex Compound) The type of complex compound contained in the bioelectrode of the present disclosure is not particularly limited. From the viewpoint of maintaining the mediator in a state where it remains within the bioelectrode, the complex compound is preferably a metal organic framework (MOF). MOFs are complex compounds having a crystalline structure formed by ligands bridging metal ions. MOFs have a large molecular weight and are difficult to dissolve in water. Therefore, the mediator can be effectively maintained in a state where it remains within the bioelectrode together with the conductive material.

[0015] From the viewpoints of thermal stability, chemical stability, and stability in water, the complex compound is more preferably a zeolitic imidazolate framework (ZIF). ZIF is a subfamily of MOFs, and is a complex compound formed by imidazole compounds, which serve as ligands, bridging metal ions. ZIF has a cage-like crystal structure called a sodalite type.

[0016] When the complex compound contained in the bioelectrode has a crystalline structure, at least a part of the crystalline structure may be lost, for example, by at least a part of the metal contained in the complex compound being coordinately bonded to the anionic group of the mediator rather than to the ligand, thereby causing at least a part of the crystalline structure of the complex compound to be lost.

[0017] The type of metal forming the complex compound is not particularly limited. Specific examples of the metal include divalent metals such as cobalt, zinc, nickel, copper, and iron. From the viewpoint of the stability of the complex compound, cobalt and zinc are preferred as the metal. From the viewpoint of maintaining the mediator in a state where it remains within the bioelectrode, it is preferred that the metal contained in the complex compound form a coordinate bond with the anionic group of the mediator while maintaining an appropriate coordinate bond with the ligand. From this viewpoint, cobalt is preferred as the metal forming the complex compound. The metal forming the complex compound may be one type or two or more types.

[0018] The type of ligand forming the complex compound is not particularly limited. Specific examples of the ligand include imidazole compounds such as 2-methylimidazole, 5-methylimidazole, benzimidazole, 2-imidazolecarboxylic acid, 4-imidazolecarboxylic acid, 2-nitroimidazole, 4-nitroimidazole, 4,5-dichloroimidazole, 2-ethylimidazole, 5-methylbenzimidazole, 5-chlorobenzimidazole, 5-bromobenzimidazole, and 2-aminobenzimidazole, with 2-methylimidazole being preferred. In the present disclosure, an imidazole compound refers to a compound having an imidazole structure in the molecule. The complex compound may be formed using one or more types of ligands.

[0019] In one embodiment, the complex compound included in the bioelectrode is formed from cobalt and 2-methylimidazole, hereinafter also referred to as ZIF67.

[0020] In one embodiment, the complex compound contained in the bioelectrode is formed from zinc and 2-methylimidazole, hereinafter also referred to as ZIF8.

[0021] (Mediator Having Anionic Group) The mediator contained in the bioelectrode can be selected without particular limitation from compounds that have the property of mediating electron transfer between the biocatalyst and the electrode.

[0022] Specific examples of the anionic group contained in the mediator include a sulfo group, a carboxy group, and a phospho group, with a sulfo group being preferred. The anionic group may form a salt. In the present disclosure, the term "mediator having an anionic group" also includes a state in which the mediator is bonded to another substance via the anionic group.

[0023] Specific examples of mediators having an anionic group include naphthoquinone compounds such as 1,2-naphthoquinone-4-sulfonic acid, anthraquinone compounds such as 2-anthraquinonesulfonic acid, and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). The bioelectrode may contain one or more types of mediators.

[0024] (Conductive Material) The material of the conductive material contained in the bioelectrode is not particularly limited and can be selected from conductive materials such as carbon materials and metals. Examples of carbon materials include graphite, carbon black (Ketjen black, acetylene black, etc.), MgO-templated carbon particles (porous carbon particles produced by a template method), and carbon nanomaterials (carbon nanotubes, graphene, carbon nanohorns, carbon nanowires, etc.). Only one type of carbon material may be used, or two or more types may be used in combination.

[0025] From the viewpoint of maintaining the mediator in a state where it remains within the bioelectrode, the conductive material is preferably in the form of particles or fibers.

[0026] (Biocatalyst) The bioelectrode of the present disclosure may contain a biocatalyst. Specific examples of biocatalysts include enzymes and microorganisms. When the bioelectrode is used as an anode, the bioelectrode contains a biocatalyst that promotes the oxidation of fuel. The biocatalyst that promotes the oxidation of fuel may be a combination of a biocatalyst that makes the fuel oxidizable by hydrolysis or the like, and a biocatalyst that promotes the oxidation. The bioelectrode may contain only one type of biocatalyst, or two or more types of biocatalysts.

[0027] When the fuel is glucose, enzymes include glucose oxidase, glucose dehydrogenase, pyranose oxidase, and cellobiose dehydrogenase. When the fuel is fructose, enzymes include fructose oxidase and fructose dehydrogenase. When the fuel is sucrose, enzymes include a combination of invertase and glucose dehydrogenase. When the fuel is starch, enzymes include a combination of amylase and glucose dehydrogenase. When the fuel is lactic acid, enzymes include lactate oxidase and lactate dehydrogenase. When the fuel is alcohol, enzymes include alcohol oxidase and alcohol dehydrogenase. When the fuel is aldehyde, enzymes include aldehyde oxidase and aldehyde dehydrogenase. When the fuel is pyruvic acid, enzymes include pyruvate oxidase and pyruvate dehydrogenase.

[0028] When a bioelectrode is used as a cathode, the bioelectrode contains a biocatalyst that promotes oxygen reduction. Specific examples of enzymes that promote oxygen reduction include bilirubin oxidase, laccase, polyphenol oxidase, and ascorbic acid oxidase.

[0029] Examples of microorganisms that can be used as biocatalysts include microorganisms of the genus Shewanella and Geobacter.

[0030] (Other Components) The bioelectrode may contain components other than the conductive material, mediator, and biocatalyst. For example, the bioelectrode may contain a material for immobilizing the biocatalyst to the conductive material. Specific examples of materials for immobilizing the biocatalyst to the conductive material include crosslinkers and binders. These materials may immobilize the biocatalyst directly or indirectly to the conductive material.

[0031] Examples of crosslinking agents include compounds having two or more crosslinkable groups in the molecule. Crosslinking agents bond, for example, between biocatalyst molecules, between binder molecules, or between biocatalyst molecules and binder molecules. Examples of crosslinkable groups of crosslinking agents include hydroxyl groups, epoxy groups, amino groups, aldehyde groups, carboxy groups, and NHS ester groups. Specific examples of crosslinking agents include genipin, polyethylene glycol diglycidyl ether (PEGDGE), 1,2-bis(2-aminoethoxy)ethane (AEE), and glutaraldehyde.

[0032] Binders include chitosan (which may be crosslinked with genipin), polyvinylidene fluoride (PVDF), styrene butadiene rubber (SBR), polytetrafluoroethylene (PTFE), polyvinyl alcohol (PVA), and Nafion. ＴＭ Examples of suitable binders include polymer materials such as polyethyleneimine, polyallylamine, polystyrene sulfonic acid, carrageenan, and polylysine. The binder may form an enzyme immobilization membrane (a membrane made of a polymer that prevents the elution of the enzyme but does not inhibit the permeation of the reactants). Examples of polymers that prevent the elution of the enzyme but do not inhibit the permeation of the reactants include chitosan and Nafion. ＴＭ , polyethyleneimine, polyallylamine, polystyrene sulfonic acid, carrageenan, polylysine, etc.

[0033] <Method for manufacturing a bioelectrode> The method for manufacturing a bioelectrode of the present disclosure includes the steps of: preparing a mixture of a complex compound or a precursor thereof and a conductive material; and mixing the mixture with a mediator having an anionic functional group.

[0034] According to the above method, a bioelectrode having a mediator with excellent durability of electron transfer function can be produced by a simpler method than other methods such as electron beam graft polymerization and electrolytic polymerization. In the above method, the "precursor of the complex compound" means the metal and ligand that constitute the complex compound. The metal may be in a salt state.

[0035] The step of preparing a mixture of a complex compound or a precursor thereof and a conductive material may be carried out simultaneously with the step of mixing the mixture with a mediator having an anionic functional group. That is, the step of preparing a mixture of a complex compound or a precursor thereof and a conductive material may be a step of mixing a complex compound or a precursor thereof, a conductive material, and a mediator having an anionic functional group.

[0036] The method for producing a bioelectrode of the present disclosure may include other steps as needed. For example, it may include a step of supporting a biocatalyst on a conductive material. The method for supporting a biocatalyst on a conductive material is not particularly limited and can be carried out by a known method. For example, a method of contacting a liquid in which a biocatalyst is dissolved or dispersed with a conductive material may be used. The biocatalyst may be fixed to the conductive material by a material such as a crosslinker or binder.

[0037] The details and preferred aspects of each material used in the manufacturing method of the bioelectrode of the present disclosure are the same as the details and preferred aspects of each material used in the bioelectrode described above.

[0038] <Electrochemical Device> The electrochemical device of the present disclosure includes the above-described bioelectrode. The electrochemical device may include the above-described bioelectrode as an anode, a cathode, or both an anode and a cathode. Specific examples of the electrochemical device include a biofuel cell, a bioreactor, a biosensor, etc.

[0039] The structure of a biofuel cell will be described below as an example of an electrochemical device. A biofuel cell has at least a cathode and an anode. When fuel is supplied to the biofuel cell, electrons generated by oxidation of the fuel at the anode are transferred to the cathode and used to reduce oxygen at the cathode. The transfer of electrons generated during this process is used to generate electricity.

[0040] The electrochemical device of the present disclosure may include a biocatalyst. Examples of the biocatalyst include the enzymes and microorganisms described above. The biocatalyst may be immobilized on an electrode or may be unimmobilized on an electrode (e.g., present in the electrolyte).

[0041] The type of fuel used in biofuel cells is not particularly limited as long as it is a substance whose oxidation is promoted by a biocatalyst. Specific examples of fuel include sugars, alcohols, aldehydes, amino acids, amines, organic acids such as acetic acid, lactic acid, and uric acid, and hydrogen.

[0042] The biofuel cell may use one or more fuels, either directly oxidizable by the biocatalyst or oxidizable by hydrolysis or other processes (e.g., starch hydrolyzed to glucose).

[0043] <Blood Glucose Level Measurement Device> The blood glucose level measurement device of the present disclosure includes the bioelectrode described above. The blood glucose level measurement device of the present disclosure exhibits excellent sensitivity to glucose over a wide concentration range. The blood glucose level measurement device may include an enzyme that uses glucose as a substrate, such as glucose dehydrogenase, as a biocatalyst. The method for measuring blood glucose levels using the blood glucose level measurement device of the present disclosure is not particularly limited and can be selected from known methods. In one embodiment, the blood glucose level measurement device of the present disclosure is used for continuous measurement of blood glucose levels in subcutaneous interstitial fluid. The blood glucose measurement period is not particularly limited and can be selected from a range of 1 hour to 2 weeks, for example.

[0044] <Concentration Measurement Method> The concentration measurement method of the present disclosure includes contacting the above-described bioelectrode with a liquid and measuring the concentration of a substance contained in the liquid. The concentration measurement method of the present disclosure may measure the concentration of a substance whose oxidation is promoted by a biocatalyst contained in the bioelectrode. Specific examples of substances whose concentrations are to be measured include sugars, alcohols, aldehydes, amino acids, amines, and organic acids such as acetic acid, lactic acid, and uric acid. The liquid containing the substance whose concentration is to be measured may be a liquid derived from a living body, such as blood or sweat, or a liquid not derived from a living body. The method for measuring the concentration of a substance contained in a liquid brought into contact with a bioelectrode is not particularly limited, and can be performed by a known method.

[0045] The present disclosure will be described in more detail below based on examples, but the present disclosure is not limited to these examples.

[0046] Example 1 (1) Preparation of ZIF67-CNT 60 mg of carbon nanotubes (CNT) as a conductive material was added to 10 mL of methanol (MtOH) in which 0.24 g of polyvinylpyrrolidone (PVP) was dissolved, and the mixture was stirred for 30 minutes. The collected CNT was washed with MtOH, added to 5 mL of MtOH, and ultrasonically dispersed for 10 minutes to obtain a CNT dispersion. 0.3 g of Co(NO ３ ) ２ was dissolved in 2 mL of MtOH to obtain a Co ion solution. 5.765 g of 2-methylimidazole (2-Mim) was dissolved in 20 mL of MtOH to obtain a 2-Mim solution.

[0047] The CNT dispersion and Co ion solution were added to the 2-Mim solution and stirred overnight. The precipitate was then washed several times with MtOH and dried overnight at 60°C to obtain powdered ZIF67-CNT. Scanning electron microscope (SEM) observation of the ZIF67-CNT revealed the formation of a complex compound with the crystalline structure of ZIF67.

[0048] (2) Mixture of ZIF67-CNT and mediator: 20 mg of ZIF67-CNT was added to a solution (320 μM) of 1,2-naphthoquinone-4-sulfonic acid (12NQSO) dissolved in 20 mL of water, and the mixture was incubated at room temperature for 24 hours. The precipitate was then washed several times with water and dispersed in 1 mL of water to obtain a mixture of ZIF67-CNT and mediator (ZIF67-CNT / 12NQSO).

[0049] (3) Preparation of Bioelectrodes ZIF67-CNT / 12NQSO was placed on the surface of a glassy carbon electrode and dried. A mixture of FAD-coenzyme-dependent glucose dehydrogenase (FAD-GDH), chitosan, and genepin was then placed on the ZIF67-CNT / 12NQSO to obtain a bioelectrode. The FAD-GDH, chitosan, and genepin mixture was prepared by mixing a solution (10 μL) containing 1% by weight of chitosan and 1% by weight of acetic acid, a 25 mg / mL FAD-GDH solution (20 μL), and a solution (10 μL) of 30 mg of genepin dissolved in 1 mL of ethanol.

[0050] (4) FTIR Analysis To investigate the changes in chemical bonding due to the mixing of ZIF67 and 12NQSO, FTIR analysis was performed on the mixture of ZIF67 and 12NQSO (ZIF / 12NQSO), ZIF67, and 2-methylimidazole. The results are shown in Figure 1. As shown in Figure 1, the transmittance T (%) of the peak derived from the Co-N bond resulting from the coordination bond between cobalt and the ligand observed in ZIF67 was significantly reduced in ZIF / 12NQSO. Furthermore, a peak derived from the Co-O bond, indicating the interaction between Co and the oxygen atom contained in the sulfo group of 12NQSO, appeared in ZIF / 12NQSO. These results suggest that when ZIF67 and 12NQSO are mixed, a portion of the Co forming ZIF67 forms a coordination bond with the sulfo group of 12NQSO.

[0051] (5) Electron Microscopy Observation SEM images of ZIF67-CNT before mixing with 12NQSO and ZIF67-CNT / 12NQSO after mixing with 12NQSO were obtained. The results are shown in Figures 2A and 2B. As shown in Figures 2A and 2B, the crystalline structure of ZIF67 was confirmed in ZIF67-CNT before mixing with 12NQSO. On the other hand, the crystalline structure of ZIF67 was not confirmed in ZIF67-CNT / 12NQSO after mixing with 12NQSO. The collapse of the original structure of ZIF67 is thought to be due to the partial collapse of the metal-ligand coordination of ZIF67, resulting in the formation of new bonds between the metal and 12NQSO, as explained in the FTIR results in Figure 1.

[0052] Example 2 (1) Preparation of ZIF8-CNT 60 mg of carbon nanotubes (CNT) as a conductive material was added to 10 mL of methanol (MtOH) in which 0.24 g of polyvinylpyrrolidone (PVP) was dissolved, and the mixture was stirred for 30 minutes. The collected CNT was washed with MtOH and added to 5 mL of MtOH to obtain a CNT dispersion. 0.3 g of Zn(NO ３ ) ２ ・6H ２ Zn ion solution was obtained by dissolving 0 in 2 mL of MtOH, and 5.765 g of 2-methylimidazole (2-Mim) was dissolved in 20 mL of MtOH to obtain a 2-Mim solution.

[0053] The CNT dispersion and Zn ion solution were added to the 2-Mim solution and stirred overnight. The precipitate was then washed several times with MtOH and dried overnight at 60°C to obtain powdered ZIF8-CNT. Scanning electron microscope (SEM) observation of the ZIF8-CNT revealed the formation of a complex compound with the crystalline structure of ZIF8.

[0054] (2) Mixture of ZIF8-CNT and mediator A mixture of ZIF8-CNT and a mediator (ZIF8-CNT / 12NQSO) was obtained in the same manner as in Example 1, except that ZIF8-CNT was used instead of ZIF67-CNT.

[0055] (3) Preparation of Bioelectrode A bioelectrode was prepared in the same manner as in Example 1, except that ZIF8-CNT / 12NQSO was used instead of ZIF67-CNT / 12NQSO.

[0056] (4) Electron Microscope Observation SEM images were taken of ZIF8-CNT before mixing with 12NQSO and ZIF8-CNT / 12NQSO after mixing with 12NQSO. The results are shown in Figures 3A and 3B. As shown in Figures 3A and 3B, the crystalline structure of ZIF8 was confirmed in ZIF8-CNT before mixing with 12NQSO. Unlike Example 1, the crystalline structure of ZIF8 was also confirmed in ZIF8-CNT / 12NQSO after mixing with 12NQSO.

[0057] Comparative Example 1 A bioelectrode was produced in the same manner as in Example 1, except that CNT that had not been mixed with the Co ion solution and the 2-Mim solution was used instead of ZIF67-CNT.

[0058] Comparative Example 2 A bioelectrode was produced in the same manner as in Example 1, except that ZIF67 obtained by mixing a Co ion solution and a 2-Mim solution was used instead of ZIF67-CNT.

[0059] Comparative Example 3 A bioelectrode was produced in the same manner as in Example 1, except that 12NQSO (5 mM) was used instead of ZIF67-CNT.

[0060] <Electrochemical Measurement> Electrochemical measurements were carried out on the bioelectrodes prepared in Examples 1 and 2 and Comparative Examples 1 to 3. The results are shown in Figures 4 and 5. Specifically, measurements were carried out in a static solution at 25°C using a three-electrode potentiostat with a silver-silver chloride electrode as the reference electrode and a platinum wire as the counter electrode. The solution used was 8 mL of 0.1 M phosphate buffer solution to which glucose had been added to make a 0.1 M solution. Conditions for cyclic voltammetry shown in Figure 4: Potential sweep range: -0.2 V to 0.4 V (vs. silver-silver chloride electrode); Potential sweep rate: 25 mV s －１ Conditions for chronoamperometry shown in Figure 5 Applied potential: 0.3 V (vs. silver-silver chloride electrode)

[0061] As shown in Figures 4 and 5, the bioelectrode of the example containing ZIF and CNT together with 12NQSO was superior in the magnitude of current density and the sustainability of current density compared to the bioelectrode of the comparative example not containing ZIF or CNT.

[0062] A bioelectrode was prepared in the same manner as in Example 1, except that the mediator concentration was changed to 80 μM, and electrochemical measurements (chronoamperometry, applied potential: 0.3 V (vs. silver-silver chloride electrode), solution temperature: 25°C) were performed. 12NQSO, 1,2-naphthoquinone (12NQ), thionine (TH), toluidine blue (TB), and methylene blue (MB) were used as mediators. The results are shown in Figure 6. As shown in Figure 6, the bioelectrode of Example 1 using a mediator with an anionic group (12NQSO) exhibited a higher current density than the bioelectrodes using mediators without anionic groups (12NQ, TH, TB, and MB).

[0063] <Evaluation of Glucose Sensitivity Dependence> The bioelectrode fabricated in Example 1 was used to evaluate its sensitivity to glucose at various concentrations. The results are shown in Figure 7. Specifically, electrochemical measurements of the bioelectrode (chronoamperometry, applied potential: 0.3 V (vs. silver-silver chloride electrode), solution temperature: 25°C) were performed using the following procedure. A glucose solution was added to 4 mL of 0.1 M phosphate buffer to achieve the desired final concentration, and the mixture was stirred with a stirrer until it stopped. After stirring was stopped, electrochemical measurements were performed. The current value 200 seconds after the start of measurement was recorded. This procedure was repeated while varying the glucose concentration. As shown in Figure 7, the bioelectrode fabricated in Example 1 exhibited excellent sensitivity to a wide range of glucose concentrations. Because the blood glucose levels of healthy individuals range from 4.9 mM to 6.9 mM (blood) and 3.9 mM to 6.6 mM (interstitial fluid), the bioelectrode fabricated in Example 1 is useful as an electrode for a blood glucose measurement device.

[0064] <Evaluation of Selectivity of Sensitivity to Glucose> The selectivity to glucose was evaluated using the bioelectrode prepared in Example 1. The results are shown in Figure 8. Specifically, electrochemical measurements of the bioelectrode (chronoamperometry, applied potential: 0.3 V (vs. silver-silver chloride electrode), solution temperature: 25°C) were performed using the following procedure. To 4 mL of 0.1 M phosphate buffer, fructose (5 mM), maltose (5 mM), sucrose (5 mM), lactose (5 mM), dopamine (0.1 mM), serotonin (1 mM), ascorbic acid (0.1 mM), uric acid (0.4 mM), and glucose (5 mM) were added in this order. After the addition of each substance, the solution was stirred with a stirrer. Measurement was initiated after stirring was stopped, and the current value was recorded for 200 seconds from the start. Measurement was paused between the addition of each substance and the start of measurement. As shown in Figure 8, the bioelectrode prepared in Example 1 exhibited high selectivity to glucose.

[0065] <Evaluation of Sustained Sensitivity to Glucose> The bioelectrode prepared in Example 1 was used to evaluate the sustainability of sensitivity to glucose. The results are shown in FIG. 9 . Specifically, electrochemical measurements of the bioelectrode (chronoamperometry, applied potential: 0.3 V (vs. silver-silver chloride electrode), solution temperature: 25°C) were performed according to the following procedure. The solution used was 4 mL of 0.1 M phosphate buffer to which a glucose solution was added to give a final concentration of 20 mM or 5 mM. As shown in FIG. 9 , the sensitivity of the bioelectrode prepared in Example 1 was maintained at a glucose concentration (20 mM) equivalent to the blood glucose level of a diabetic patient and a glucose concentration (5 mM) equivalent to the blood glucose level of a healthy subject, even 54 hours after the start of the measurement.

[0066] Example 3 A bioelectrode was obtained in the same manner as in Example 1, except that a mixture of lactate oxidase (LOx), chitosan, and genepin was used instead of the mixture of FAD-GDH, chitosan, and genepin used to prepare the bioelectrode in Example 1. The mixture of LOx, chitosan, and genepin was prepared by mixing a solution (10 μL) containing 1% by mass of chitosan and 1% by mass of acetic acid, a 25 mg / mL LOx solution (20 μL), and a solution (5 μL) in which 30 mg of genepin was dissolved in 1 mL of ethanol.

[0067] <Evaluation of concentration dependence of sensitivity to lactic acid> The bioelectrode prepared in Example 3 was used to evaluate sensitivity to lactic acid at various concentrations. The evaluation of concentration dependence of sensitivity to lactic acid was carried out in the same manner as the evaluation of concentration dependence of sensitivity to glucose described above. The results are shown in Figure 10. As shown in Figure 10, the bioelectrode prepared in Example 3 exhibited excellent sensitivity to lactic acid over a wide concentration range.

[0068] <Evaluation of Sustained Sensitivity to Lactic Acid> Using the bioelectrode prepared in Example 3, the sustainability of sensitivity to lactic acid (2 mM or 20 mM) was evaluated. The results are shown in Figure 11. The evaluation of the sustainability of sensitivity to lactic acid was carried out in the same manner as the evaluation of the sustainability of sensitivity to glucose described above. The results are shown in Figure 11. As shown in Figure 11, the sensitivity of the bioelectrode prepared in Example 3 to lactic acid was sustained even 10 hours after the start of measurement.

[0069] The disclosure of Japanese Patent Application No. 2023-140572 is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards mentioned herein are incorporated by reference into this specification to the same extent as if each individual document, patent application, and technical standard was specifically and individually indicated to be incorporated by reference.

Claims

1. A bioelectrode comprising: a complex compound consisting of a metal and a ligand; a mediator having an anionic functional group; and a conductive material.

2. The bioelectrode of claim 1, wherein the metal comprises cobalt or zinc.

3. The bioelectrode of claim 1, wherein the ligand comprises an imidazole compound.

4. The bioelectrode of claim 1, wherein the complex compound comprises a metal-organic framework.

5. The bioelectrode of claim 1, further comprising a biocatalyst.

6. The bioelectrode of claim 1, further comprising chitosan.

7. An electrochemical device comprising a bioelectrode according to any one of claims 1 to 6.

8. A blood glucose measuring device comprising the bioelectrode according to any one of claims 1 to 6.

9. A concentration measurement method comprising: bringing the bioelectrode according to any one of claims 1 to 6 into contact with a liquid; and measuring the concentration of a substance contained in the liquid.

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