New Mediators

Abstract

To provide an electron mediator which can be used simply and conveniently without need for a complicated process for fixation to an electrode.SOLUTION: The present invention relates to a novel mediator, an electrode modification agent including the mediator, an electron transfer accelerator, and an electrode, a battery, a composition and an enzyme sensor, each including the electron transfer accelerator, and methods for using them. The electrode modification agent comprises a compound having a property of sticking to an electrode neither being coupled with a polymer nor polymerizing, and having a structure given by a formula I below.[In the formula, R1 is a hydrogen atom, a C1-6 alkyl group, an aryl which may be substituted with a sulfo group, or -C(=O)-C1-6 alkyl].SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the use of aminoanthraquinone compounds as mediators, electrode modifiers containing the compounds, electrodes containing the compounds, enzyme sensors containing the electrodes, and batteries. The present invention also relates to electrochemical measurements using aminoanthraquinone compounds and redox enzymes, compositions containing aminoanthraquinone compounds and redox enzymes, and electrodes containing aminoanthraquinone compounds.

Background Art

[0002] In enzyme electrodes, mediators are used as mediator substances for transferring electrons generated by redox reactions of enzymes to electrodes. For efficient electron transfer from enzymes to electrodes, it is desirable that the mediator be localized in the vicinity of the electrode. Therefore, various methods for immobilizing mediators on electrodes, such as methods for chemically bonding electrodes and mediators and methods for polymerizing mediators themselves, are used. However, the chemical bonding method has limitations on the side chain functional groups of the mediator, and the method of polymerizing the mediator itself may change the redox potential of the mediator. Conventional methods have lacked versatility. Although it has been reported that the surface functional groups of glassy carbon electrodes are activated by treatment with strong acids and mediators such as quinones are adsorbed and fixed, it has hardly been put into practical use in the industry.

[0003] Furthermore, all of these methods have the problem that complicated processes are required for immobilizing mediators on electrodes, resulting in high costs. Therefore, there has been a demand for an easily usable electron mediator that does not require complicated processes for immobilization on electrodes.

[0004] Patent Document 1 (International Publication No. 2011 / 111531 Pamphlet) discloses a device for detecting an analyte. Specifically, a pH sensor using an anthraquinone-based compound, a phenanthrenequinone-based compound, a benzoquinone-based compound, an azobenzene-based compound, etc. as an analyte-sensitive material is described.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a novel mediator that can at least partially solve the above problems.

Means for Solving the Problems

[0007] As a result of intensive efforts to solve the above problems, the present inventors have found that aminoanthraquinone-based compounds can surprisingly adsorb on the electrode surface and that the compounds can function as mediators, thus completing the present invention. As far as the present inventors know, it has not been reported so far that 1-acetamido-4-hydroxyanthraquinone, 1-amino-4-hydroxyanthraquinone, and 2-amino-3-hydroxyanthraquinone are mediators that adsorb on the electrode, which is a surprising finding.

[0008] This disclosure includes the following embodiments: [1] Having the property of adsorbing on an electrode without being bound to a polymer or without polymerizing, and having the structure of Formula I,

Chemical Formula

[10] The electrode or composition according to Embodiment 9, wherein the enzyme is an oxidoreductase.

[11] The electrode according to Embodiment 10, wherein the oxidoreductase is immobilized.

[12] A sensor comprising the electrode modifier according to Embodiment 1 or the electron transfer promoter according to Embodiment 2, or an enzyme sensor having the electrode according to Embodiment 10 or 11.

[13] The compound has the following structure [Chemical Formula] 1-Acetamido-4-hydroxyanthraquinone having the following structure

Chemical formula

Chemical formula

[14] A method for manufacturing a battery, comprising the step of using the electrode modifier according to Embodiment 1 or the electron transfer promoter according to Embodiment 2.

[15] The method according to Embodiment 14, comprising the step of bringing the electrode modifier or the electron transfer promoter into contact with the electrode of the battery.

[16] A power generation method using the battery according to any one of Embodiments 3 to 6 and 13.

[17] The composition according to any one of Embodiments 7, 9, 10 and 13, the electrode according to any one of Embodiments 8 to 11 and 13, or the sensor according to Embodiments 12 or 13 for use in an electrochemical measurement method.

[18] The electrode modifier according to Embodiment 1 or 13, the electron transfer promoter according to Embodiment 2 or 13, or the composition according to any one of Embodiments 7, 9, 10 and 13, A method for modifying or decorating an electrode, comprising the step of bringing the composition into contact with the electrode.

[0009] This specification incorporates the disclosure of Japanese Patent Application No. 2019-183555, which is the basis of the priority of this application.

Effects of the Invention

[0010] Unlike conventional mediators, the aminoanthraquinone-based compound of the present invention can be adsorbed onto the electrode surface as it is, without requiring special treatments such as acid treatment of the electrode or polymerization of the mediator itself. Therefore, it can be easily immobilized on the electrode. Further, such an electrode can be used for electrochemical measurements. Further, such an electrode can be applied to a battery.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

[0012] (Aminoanthraquinone-based compound) (Electrode adsorbed with aminoanthraquinone-based compound) In one embodiment, the present invention provides an aminoanthraquinone-based compound. In another embodiment, the present invention provides an electrode modifier comprising an aminoanthraquinone-based compound. This electrode modifier can be adsorbed on the electrode surface and can modify the electron-accepting property of the electrode. In another embodiment, the present invention provides an electrode adsorbed with an aminoanthraquinone-based compound or the electrode modifier of the present invention. In another embodiment, the present invention provides an electrochemical measurement composition comprising an aminoanthraquinone-based compound or the electrode modifier of the present invention. The composition may further contain an enzyme. The enzyme can be a redox enzyme. In another embodiment, the present invention provides an electrochemical measurement kit comprising an aminoanthraquinone-based compound or the electrode modifier of the present invention. In this specification, the electrode modifier may be referred to as an electrode adsorbent.

[0013] The aminoanthraquinone-based compound of the present invention can be adsorbed on the electrode surface by contacting with the electrode. At this time, no special operation such as acid treatment and activation of the electrode surface is required to adsorb it on the electrode. In one embodiment, the property of the compound of the present invention to adsorb on the electrode refers to the property that the compound is physically adsorbed on the electrode, and the electrode can be made of materials such as carbon, gold, platinum, etc. Further, an embodiment including adsorbing the compound on a primary material such as carbon powder or a carbon material and then immobilizing the primary material such as the carbon powder or the carbon material on the electrode is included. However, this is an explanation of the property of the compound of the present invention and does not limit the usage method of the compound. That is, in one embodiment, the present invention provides a method of adsorbing the compound of the present invention on a primary material such as carbon powder or a carbon material and then immobilizing the primary material such as the carbon powder or the carbon material on the electrode.

[0014] In one embodiment, the present invention provides a primary material such as carbon powder or a carbon material adsorbed with the compound of the present invention. This primary material such as carbon powder or carbon material can be further applied or used on an electrode. Examples of the primary material include, but are not limited to, carbon, platinum, gold, etc. Examples of the carbon material include carbon black, carbon fiber, single-layer or multi-layer carbon nanotubes, graphene, ketjen black, etc.

[0015] In one embodiment, the property of the compound of the present invention to adsorb to an electrode, carbon powder or carbon material means the property that the compound physically adsorbs directly to the electrode, carbon powder or carbon material, and does not refer to the property of binding the compound to an electrode, carbon powder or carbon material by covalently bonding it to a polymer or a linker. In this specification, such a property may be referred to as the property of adsorbing to an electrode without being bound to a polymer, or the property of adsorbing to carbon powder or a carbon material without being bound to a polymer or without the compound polymerizing. However, this is an explanation of the property of the compound of the present invention and does not limit the method of using the compound. That is, in one embodiment, the present invention provides a method of binding the compound of the present invention to an electrode, carbon powder or carbon material by covalently bonding it to a polymer or a linker. Also, the fact that the compound of the present invention physically adsorbs directly to an electrode, carbon powder or carbon material does not prevent the compound itself (as a monomer) from covalently bonding to the carbon powder or carbon material. In this specification, a polymer refers to a substance in which the same unit is polymerized in a large amount, for example, 10 or more.

[0016] In one embodiment, the carbon powder or carbon material, carbon electrode, gold electrode, platinum electrode for adsorbing the compound of the present invention is not acid-treated. That is, in one embodiment, acid-treated carbon powder or carbon material and carbon electrodes are excluded from the carbon powder or carbon material for adsorbing the compound of the present invention.

[0017] In certain embodiments, the compounds of the present invention can be used in combination with an enzyme. In certain embodiments, the compounds of the present invention can be used in combination with an oxidoreductase. In certain embodiments, the compounds of the present invention can be used as an electron transfer promoter. In certain embodiments, the aminoanthraquinone-based compounds of the present invention function as a mediator in a redox reaction catalyzed by an oxidoreductase. Examples of oxidoreductases include various oxidoreductases classified in EC group 1, such as glucose oxidase, glucose dehydrogenase, amadoriase (also referred to as fructosylpeptide oxidase or fructosylamino acid oxidase), peroxidase, galactose oxidase, biliverdin oxidase, pyruvate oxidase, D- or L-amino acid oxidase, amine oxidase, cholesterol oxidase, choline oxidase, xanthine oxidase, sarcosine oxidase, D- or L-lactate oxidase (LOD), ascorbic acid oxidase, cytochrome oxidase, alcohol dehydrogenase, cholesterol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, fructose dehydrogenase (FDH), sorbitol dehydrogenase, D- or L-lactate dehydrogenase, malate dehydrogenase, glycerol dehydrogenase, 17B hydroxysteroid dehydrogenase, estradiol 17B dehydrogenase, D- or L-amino acid dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, 3-hydroxysteroid dehydrogenase, diaphorase, catalase, glutathione reductase, cytochrome b5 reductase, adrenodoxin reductase, cytochrome b5 reductase, adrenodoxin reductase, nitrate reductase, phosphate dehydrogenase, biliverdin oxidase, laccase, polyamine oxidase, formate dehydrogenase, pyranose oxidase, pyranose dehydrogenase, tauropine dehydrogenase, etc., but are not limited thereto. Also, a combination of multiple enzymes may be used.Examples of the coenzymes of the above enzyme include nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide (FAD), pyrroloquinoline quinone, and the like. That is, examples of glucose dehydrogenase (GDH) include FAD-dependent GDH, NAD-dependent GDH, PQQ-dependent GDH, and the like. The redox enzymes listed above can be measured for activity using various substrates by, for example, the methods described in Methods in Enzymology (volumes 1 to 602).

[0018] In this specification, functioning as a mediator means that the aminoanthraquinone-based compound contributes to electron transfer. For example, in a system using an electrode, the aminoanthraquinone-based compound of the present invention receives electrons from a redox enzyme to become a reduced form, and then returns to an oxidized form by passing the electrons to the electrode. From this perspective, the aminoanthraquinone-based compound of the present invention that functions as a mediator can also be referred to as an electron transfer mediator, an electron transfer accelerator, or an electron mediator (simply referred to as a mediator). In this specification, these terms are synonymous.

[0019] In certain embodiments, electrodes having acid-treated carbon powder or carbon particles are removed from the electrodes of the present invention.

[0020] In certain embodiments, the redox enzyme used with the electrode of the present invention may be immobilized on the electrode. That is, in this embodiment, the present invention provides an electrode on which a redox enzyme is immobilized and the electrode modifier of the present invention is adsorbed. In certain embodiments, the present invention provides an enzyme sensor comprising an electrode on which a redox enzyme is immobilized and the electrode modifier of the present invention is adsorbed. In certain embodiments, a sensor comprising the electrode modifier or electron transfer accelerator of the present invention is provided.

[0021] The aminoanthraquinone-based compound of the present invention, or an electrode modifier, electrode adsorbent, or electron transfer promoter containing the aminoanthraquinone-based compound, can be adsorbed on the electrode surface without requiring special treatment. Therefore, in certain embodiments, the aminoanthraquinone-based compound, electrode modifier, electrode adsorbent, or electron transfer promoter of the present invention may be adsorbed onto the electrode in advance to produce a modified electrode. In another embodiment, when performing electrochemical measurements, a measurement solution (composition) containing the aminoanthraquinone-based compound, electrode modifier, electrode adsorbent, or electron transfer promoter of the present invention, and an oxidoreductase can be used. In this embodiment, when a composition containing the aminoanthraquinone-based compound, electrode modifier, electrode adsorbent, or electron transfer promoter of the present invention, and an oxidoreductase is physically contacted with the electrode, an electrode on which the aminoanthraquinone-based compound, electrode modifier, electrode adsorbent, or electron transfer promoter of the present invention is adsorbed and the electrochemical properties are modified is produced in situ during measurement.

[0022] In one embodiment, when adsorbing the aminoanthraquinone-based compound, electrode modifier, electrode adsorbent, or electron transfer promoter of the present invention onto an electrode, the electrode is not pretreated with an acid. In another embodiment, when adsorbing the aminoanthraquinone-based compound, electrode modifier, electrode adsorbent, or electron transfer promoter of the present invention onto an electrode, the electrode may be pretreated with an acid. Here, the pretreatment with an acid is defined to include bringing an electrolyte solution containing an acid into contact with the electrode. Also, in this specification, an acid refers to an acid with a pH of 4 or less, for example, less than pH 4, 3 or less, less than pH 3, 2 or less, less than pH 2, or pH 1. Further, in one embodiment, the aminoanthraquinone-based compound, electrode modifier, electrode adsorbent, or electron transfer promoter of the present invention may be adsorbed onto the electrode after being converted to an oxidized form or a reduced form by applying a specific potential. Note that an oxidizing agent or a reducing agent may be used as a method for converting to the oxidized form or the reduced form, and the method is not particularly limited. Additionally, in one embodiment, the aminoanthraquinone-based compound, electrode modifier, electrode adsorbent, or electron transfer promoter of the present invention may be encapsulated in a polymer and adsorbed onto the electrode. Such adsorption may be referred to as embedded or entrapped in this specification. This is distinguished from adsorption fixation, i.e., fixation by physical adsorption. In embedded or entrapped fixation, the compound of the present invention can diffuse within the polymer, whereas in adsorption fixation, the compound of the present invention does not diffuse or hardly diffuses. In another embodiment, the compound, electrode modifier, electrode adsorbent, or electron transfer promoter of the present invention may be adsorbed onto the electrode by electrostatic interaction using an ionic polymer, such as polyethyleneimine or polylysine which are cationic polymers, or polyaniline or polyacrylic acid which are anionic polymers. In another embodiment, the compound, electrode modifier, electrode adsorbent, or electron transfer promoter of the present invention may be adsorbed or fixed by a crosslinking agent to an enzyme and immobilized onto the electrode together with the enzyme. In another embodiment, the adsorbed compound of the present invention may be polymerized by an oxidation-reduction reaction or a crosslinking agent. In one embodiment, the aminoanthraquinone-based compound of the present invention may be included in an electrolyte bath or an electrolyte solution in contact with the electrode.

[0023] In one embodiment, the aminoanthraquinone compound of the present invention can be a compound of the following general formula I or a salt, anhydride or solvate thereof: [Chemical formula] [In the formula, R 1 is hydrogen, C 1-6 alkyl, aryl optionally substituted by sulfo or -C(=O)-C 1-6 alkyl]. When R 1 is hydrogen, it is aminoanthraquinone. When R 1 is C 1-6 alkyl, aryl optionally substituted by sulfo, -C(=O)-C 1-6 alkyl, and an anthraquinone with an alkylamino group, an amide group, or an arylamino group optionally substituted by sulfo added thereto is also included in the aminoanthraquinone compound of the present invention. The same applies to structural isomers having an amino group, an alkylamino group, an amide group, an arylamino group optionally substituted by sulfo, and a hydroxy group at other positions on the anthraquinone. In this specification, "optionally substituted by a certain group" means that it is substituted by the group or not substituted. Examples of the aryl group include, but are not limited to, a phenyl group, a tolyl group, a xylyl group, and a naphthyl group. The tolyl group refers to a CH3C6H4- group derived from toluene. The sulfo group refers to a SO3H- group. In this specification, when referring to sulfo, its salts, such as the Na salt, are also included.

[0024] In one embodiment, the aminoanthraquinone compound of the present invention can be 1-acetamido-4-hydroxyanthraquinone. 1-Acetamido-4-hydroxyanthraquinone has the following structure [Chemical formula] and has a CAS number of 7323-62-8.

[0025] In another embodiment, the aminoanthraquinone compound of the present invention can be 1-amino-4-hydroxyanthraquinone. 1-Amino-4-hydroxyanthraquinone has the following structure [Chemical formula] and has a CAS number of 116-85-8.

[0026] In another embodiment, the aminoanthraquinone compound of the present invention can be 2-amino-3-hydroxyanthraquinone. 2-Amino-3-hydroxyanthraquinone has the following structure [Chemical formula] and has a CAS number of 117-77-1. In another embodiment, the aminoanthraquinone compound of the present invention can be 1-hydroxy-4-toluidinoanthraquinone (CAS number 81-48-1) or Acid Violet 43 (CAS number 4430-18-6). Note that 2-carboxyanthraquinone, acetylaminoanthraquinone, and 2-β-naphthol-methylanthraquinone are not included in the aminoanthraquinone compounds of the present invention. Since 2-carboxyanthraquinone, acetylaminoanthraquinone, and 2-β-naphthol-methylanthraquinone do not have a hydroxyl group, they are considered to have different chemical properties from the aminoanthraquinone compounds of the present invention.

[0027] As used herein, alkyl refers to a straight-chain or branched-chain hydrocarbon having, for example, 6 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl, isobutyl, n-butyl, tert-butyl, isopentyl, n-pentyl, heptyl. Also, -C(=O)-C 1-6 Examples of alkyl include, but are not limited to, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl.

[0028] As used herein, the number of atoms (such as carbon atoms) is represented, for example, as "Cx-Cy alkyl", which refers to an alkyl group having x to y carbon atoms. The same notation is used for other substituents and ranges.

[0029] As used herein, "optionally substituted" and "substituted or unsubstituted" mean any substitution with one or more substituents, including multiple substitutions.

[0030] The functional group for covalently bonding to an enzyme in the aminoanthraquinone-based compound of the present invention may be modified. An alkyl group having C1 to C20, an amino acid, a peptide, etc. may be inserted as a linker between the aminoanthraquinone-based compound and the functional group. Optionally, a hydroxyl group, an amino group, an alkene, etc. may be included between the linkers. Examples of the functional group include a hydroxyl group, a carboxyl group, an amino group, an aldehyde group, a hydrazino group, a thiocyanate group, an epoxy group, a vinyl group, a halogen group, an acid ester group, a phosphate group, a thiol group, a disulfide group, a dithiocarbamate group, a dithiophosphate group, a dithiophosphonate group, a thioether group, a thiosulfate group, a succinimide group, a maleimide group, and a thiourea group, etc.

[0031] Aminoanthraquinone compounds can exist in an oxidation-reduction state and an ionization state. In the above chemical formula, the aminoanthraquinone compound of the present invention is described in a neutral and reduced form. However, not only this form, but the aminoanthraquinone compound of the present invention can be in an oxidized form, a semi-oxidized form, or a reduced form. Further, the aminoanthraquinone compound of the present invention can be in a neutral form or a cationic form. For the sake of convenience, when referring to the aminoanthraquinone compound of the present invention, for example, the aminoanthraquinone compound of the present invention represented by the above chemical formula, this includes those in a neutral or cationic form, in an oxidized form, a semi-oxidized form, or a reduced form. For example, after adding a neutral and oxidized compound as the aminoanthraquinone compound of the present invention to a measurement system, it may change to an oxidized and cationic compound due to the pH of the solution and electron transfer, but such a compound is also included in the aminoanthraquinone compound of the present invention. Also, when referring to the aminoanthraquinone compound of the present invention, this includes its salts, acid addition salts, anhydrides, and solvates. Examples of salts include, but are not limited to, salts of Group 1 elements and salts of Group 17 elements, such as Na salts, K salts, Cl salts, Br salts, etc. Examples of acid addition salts include, but are not limited to, hydrochloride salts, sulfate salts, sulfite salts, and nitrate salts.

[0032] The aminoanthraquinone-based compound of the present invention may be synthetically produced, obtained from natural products, or may be commercially available. When synthesizing, organic synthesis is performed using conventional organic synthesis techniques, and the product can be confirmed by NMR, IR, mass spectrometry, etc. The implementation of the present invention uses the prior art of chemistry, organic synthesis, biochemistry, molecular biology, and electrochemistry, unless otherwise specified, but these are within the capabilities of those skilled in the art. Such techniques are described in the literature. For example, refer to Organic Chemistry (edited by Jonathan Clayden, written by Nick Greeves, Stuart Warren, and Peter Wothers), Oxford Univ Pr, 2000 and March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure (written by Michael B. Smith and Jerry March), Wiley-Interscience, 6th edition, 2007. These general texts are incorporated herein by reference respectively.

[0033] (Method for immobilizing enzyme or redox enzyme) The enzyme or redox enzyme can be immobilized on a solid phase by any known method. The enzyme, for example, a redox enzyme, may be immobilized on beads, membranes, carbon particles, gold particles, platinum particles, polymers, or the electrode surface. Examples of the immobilization method include a method using a cross-linking reagent, a method of encapsulating in a polymer matrix, a method of coating with a dialysis membrane, a photocrosslinkable polymer, a conductive polymer, a redox polymer, etc. It may be immobilized in a polymer or adsorbed and immobilized on an electrode, or a combination of these may be used. Typically, after immobilizing a redox enzyme on a carbon electrode using glutaraldehyde, it is treated with a reagent having an amine group to block the glutaraldehyde. The amount of the redox enzyme to be immobilized can be an amount that can generate a current required for electrochemical measurement or fuel cell power generation, and can be determined as appropriate.

[0034] (Adsorption Method of Aminoanthraquinone Compounds of the Present Invention) In certain embodiments, the aminoanthraquinone compounds of the present invention may be present in a free state in a solution, and may also be adsorbed, for example, physically adsorbed, on beads, membranes, carbon particles, gold particles, platinum particles, polymers, or electrode surfaces. The fact that the aminoanthraquinone compounds of the present invention are adsorbed on the electrode surface may also be referred to as being immobilized on the electrode surface, but these are synonymous in this specification. Examples of the adsorption method include a method that includes a step of dissolving the aminoanthraquinone compounds of the present invention in a suitable medium and physically contacting the solution with an electrode. In another embodiment, the aminoanthraquinone compounds of the present invention may be sprayed onto the electrode. The amount of the aminoanthraquinone compounds to be adsorbed can be an amount that can generate a current necessary for electrochemical measurement or battery power generation, and can be determined as appropriate.

[0035] In certain embodiments, the aminoanthraquinone compounds of the present invention may be adsorbed on an electrode, and an enzyme, for example, a redox enzyme, may be further immobilized. In this case, the aminoanthraquinone compounds of the present invention may be adsorbed first and then an enzyme, for example, a redox enzyme, may be immobilized, or an enzyme, for example, a redox enzyme, may be immobilized first and then the aminoanthraquinone compounds of the present invention may be adsorbed, or the aminoanthraquinone compounds may be adsorbed simultaneously during the operation of immobilizing an enzyme, for example, a redox enzyme.

[0036] The final concentration of the aminoanthraquinone compound of the present invention added to the sample solution is not particularly limited. For example, it can be 1 pM or more, 2 pM or more, 3 pM or more, 4 pM or more, 5 pM or more, 6 pM or more, 7 pM or more, 8 pM or more, 9 pM or more, 10 pM or more, 1 M or less, 100 mM or less, 20 mM or less, 10 mM or less, 5 mM or less, 1 mM or less, 800 μM or less, 600 μM or less, 500 μM or less, 400 μM or less, 300 μM or less, 200 μM or less, 100 μM or less, 50 μM or less. For example, it can be in the range of 1 pM to 1 M, 1 pM to 100 mM, 1 pM to 20 mM, 1 pM to 10 mM, 1 pM to 5 mM, 2 pM to 1 mM, 3 pM to 800 μM, 4 pM to 600 μM, 5 pM to 500 μM, 6 pM to 400 μM, 7 pM to 300 μM, 8 pM to 200 μM, 9 pM to 100 μM, 10 pM to 50 μM. The final concentration of the aminoanthraquinone compound of the present invention added to the sample solution is not particularly limited. For example, it can be 0.000001 to 0.5% (w / v), 0.000003 to 0.3% (w / v), 0.000005 to 0.1% (w / v), 0.00001 to 0.05% (w / v), 0.00002 to 0.03% (w / v), 0.00003 to 0.01% (w / v). The addition order of the aminoanthraquinone compound and other reagents is not restricted and can be either simultaneous or sequential addition.

[0037] In certain embodiments, the time for performing the redox reaction or the time for performing the electrochemical measurement can be 60 minutes or less, 30 minutes or less, 10 minutes or less, 5 minutes or less, or 1 minute or less. Alternatively, in enzyme sensors, batteries, etc. for long-term measurements, the time for performing the redox reaction can be 60 minutes or more, 120 minutes or more, 1 day or more, 2 days or more, 3 days or more, 1 week or more, 2 weeks or more, 3 weeks or more. For example, when an aminoanthraquinone compound is used together with a redox enzyme, the concentration of each component of the reagent for electrochemical measurement can be adjusted according to the concentration range of the mediator contained in the sample or presumed to be generated in the sample.

[0038] Unless otherwise specified, the enzyme contained in the composition of the present invention or the enzyme immobilized on the electrode of the present invention is a purified enzyme. Cell extracts, cell lysates, and crude enzyme extracts containing enzymes contain various contaminants in addition to the enzymes. For example, in the case of microorganisms, it has been reported that the amount of riboflavin in crude enzyme extracts is about 53 to 133 μM (J Indust Micro Biotech 1999, 22, pp. 8-18). When such crude enzyme extracts or the like are directly used for electrochemical measurements, the contaminants receive electrons or the like, interfering with the transfer of electrons to and from the electrode. Therefore, in the crude enzyme extract, accurate electrochemical measurements are difficult at this riboflavin concentration. Therefore, in the electrochemical measurement method of the present invention, an enzyme from which contaminants have been removed can be used. In this specification, when it is stated that an enzyme is purified or is a purified enzyme, it does not necessarily mean that the protein is a pure product, but rather that contaminants have been removed from the enzyme preparation to such an extent that electrochemical measurements are possible.

[0039] (Measurement of the activity of oxidoreductases) In explaining the measurement of the activity of oxidoreductases, glucose dehydrogenase (GDH) and peroxidase are taken as examples of specific enzymes. GDH (EC 1.1.5.9) catalyzes the reaction of oxidizing the hydroxyl group of glucose to produce glucono-δ-lactone. At this time, the electron acceptor receives electrons and becomes a reduced electron acceptor. The activity of GDH can be measured using the following measurement system that utilizes this principle of action. For example, phenazine methosulfate (PMS) and 2,6-dichlorophenolindophenol (DCIP) are used as electron acceptors. (Reaction 1) D-Glucose + PMS (oxidized form) → D-Glucono-δ-lactone + PMS (reduced form) (Reaction 2) PMS (reduced form) + DCIP (oxidized form) → PMS (oxidized form) + DCIP (reduced form)

[0040] Specifically, first, in (Reaction 1), as D-glucose is oxidized, PMS (reduced form) is generated. Subsequently, in the proceeding (Reaction 2), as PMS (reduced form) is oxidized, DCIP is reduced. The degree of disappearance of this "DCIP (oxidized form)" is detected as the change in absorbance at a wavelength of 600 nm, and the enzyme activity can be determined based on this change amount.

[0041] The activity of GDH can be measured according to the following procedure. Mix 2.05 mL of 100 mM phosphate buffer (pH 7.0), 0.6 mL of 1 M D-glucose solution, and 0.15 mL of 2 mM DCIP solution, and incubate at 37 °C for 5 minutes. Then, add 0.1 mL of 15 mM PMS solution and 0.1 mL of the enzyme sample solution to start the reaction. Measure the absorbance at the start of the reaction and over time, determine the decrease amount per minute (ΔA600) of the absorbance at 600 nm accompanying the progress of the enzyme reaction, and calculate the GDH activity according to the following formula. At this time, the GDH activity is defined as the amount of enzyme that reduces 1 μmol of DCIP per minute in the presence of D-glucose at a concentration of 200 mM at 37 °C as 1 U.

[0042]

Equation

[0043] Note that 3.0 in the formula is the liquid volume (mL) of the reaction reagent + enzyme reagent, 16.3 is the millimolar molar absorption coefficient (cm 2 / μmol) under the conditions of this activity measurement, 0.1 is the liquid volume (mL) of the enzyme solution, 1.0 is the optical path length (cm) of the cell, ΔA600 blank is the decrease amount per minute of the absorbance at 600 nm when the reaction is started by adding 100 mM phosphate buffer (pH 7.0) instead of the enzyme sample solution, and df represents the dilution factor.

[0044] Peroxidase (EC 1.11.1.7) catalyzes the reaction of cleaving the peroxide structure of a compound. At this time, electrons are withdrawn from the electron donor and converted into an oxidized electron donor. The activity of peroxidase can be measured using the following measurement system that utilizes this principle. For example, hydrogen peroxide is used as the compound having a peroxide structure, and pyrogallol is used as the electron donor. 2 Pyrogallol + 3H2O2 → Purpurogallin + 5H2O + CO2 The generated purpurogallin is extracted with ether, and the enzyme activity can be determined by measuring the absorbance at 420 nm.

[0045] The enzyme activity of peroxidase can be measured according to the following procedure. Mix 14.0 ml of distilled water, 2.0 ml of a 5% (w / v) pyrogallol aqueous solution prepared at the time of use, 1.0 ml of a 0.147 M hydrogen peroxide aqueous solution prepared at the time of use, and 2.0 ml of a 0.1 M phosphate buffer (pH 6.0), and pre-warm at 20 °C for about 5 minutes. This is designated as reaction mixture 1. Subsequently, add 1.0 ml of an enzyme solution dissolved in a 0.1 M phosphate buffer (pH 6.0) precooled with ice to initiate the reaction. After reacting precisely for 20 seconds at 20 °C, add 1.0 ml of 2.0 N sulfuric acid to stop the reaction. Extract the generated purpurogallin from the mixture after the reaction has stopped with 15 ml of ether. Repeat this operation 5 times, combine the extracts, and add more ether to make the total volume 100 ml. Measure the absorbance of this solution at 420 nm (this value is designated as OD test).

[0046] For the blank test, reaction mixture 1 is left standing at 20 °C for 20 seconds, then 1.0 ML of 2.0 N sulfuric acid is added and mixed, and then 1.0 ML of the enzyme solution is added for preparation. Perform ether extraction on this solution in the same manner as above and measure the absorbance (this value is designated as OD BLANK).

[0047] Based on the OD test, OD blank, and dilution factor (df) of the enzyme solution, calculate the peroxidase activity according to the following formula. Under the above conditions, the amount of enzyme that produces 1.0 mg of pulprogallin in 20 seconds is defined as 1 U. In the following formula, 0.117 means the absorbance at 420 nm of a 1 mg% pulprogallin ether solution.

Equation

[0048] The kit for electrochemical measurement of the present invention contains an aminoanthraquinone compound of the present invention or an electrode modifier containing the aminoanthraquinone compound in an amount sufficient for at least one assay. Typically, the kit for electrochemical measurement of the present invention contains, in addition to the aminoanthraquinone compound of the present invention, a redox enzyme, a buffer solution necessary for the assay, a substrate standard solution for preparing a calibration curve, and instructions. For example, the redox enzyme may be GDH, and in this case, the substrate standard solution may be a glucose standard solution.

[0049] In one embodiment, the electrochemical measurement kit of the present invention contains the aminoanthraquinone-based compound and the redox enzyme of the present invention as the same reagent. In another embodiment, the electrochemical measurement kit of the present invention contains the aminoanthraquinone-based compound and the redox enzyme as separate reagents. In another embodiment, the redox enzyme may be immobilized on the electrode, and the electrochemical measurement kit of the present invention used for such an electrode contains the aminoanthraquinone-based compound as a single reagent. However, the single reagent here does not mean that the reagent does not contain substances other than the aminoanthraquinone-based compound. The single reagent may contain a suitable medium so that the aminoanthraquinone-based compound of the present invention dissolves in the single reagent. The medium may be any medium as long as the aminoanthraquinone-based compound of the present invention can dissolve therein, and examples include, but are not limited to, water, methanol, ethanol, propanol, acetone, acetonitrile, and the like. The aminoanthraquinone-based compound of the present invention can be provided in various forms, for example, as a powdered solid reagent, as a reagent immobilized on beads or the electrode surface, or as a solution in a suitable storage solution, for example, a light-shielded solution.

[0050] As an example of electrochemical measurement, measurement of glucose concentration can be mentioned. In the case of colorimetric electrochemical measurement, the measurement of glucose concentration can be carried out, for example, as follows. The reaction layer for electrochemical measurement holds a liquid or solid composition containing glucose dehydrogenase (GDH) and at least one substance selected from the group consisting of N-(2-acetamido)iminodiacetic acid (ADA), bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris), sodium carbonate, and imidazole as a reaction accelerator. Here, a pH buffer and a colorimetric reagent (discoloration reagent) are added as necessary. A sample containing glucose is added here and reacted for a certain period of time. During this time, the absorbance corresponding to the maximum absorption wavelength of the dye polymerized or reduced by directly receiving electrons from GDH is monitored. In the case of the rate method, the glucose concentration in the sample can be calculated based on the calibration curve prepared in advance using a glucose solution of a standard concentration from the rate of change of absorbance per unit time, and in the case of the endpoint method, from the change in absorbance until all the glucose in the sample is oxidized.

[0051] As a colorimetric reagent (discoloration reagent) that can be used in this method, for example, 2,6-dichlorophenolindophenol (DCIP) can be added as an electron acceptor, and the glucose can be quantified by monitoring the decrease in absorbance at 600 nm. Also, by adding nitro blue tetrazolium (NTB) as a colorimetric reagent and measuring the absorbance at 570 nm, the amount of formazan produced can be determined and the glucose concentration can be calculated. Needless to say, the colorimetric reagent (discoloration reagent) to be used is not limited to these.

[0052] In certain embodiments, a sensor capable of detecting the compounds of the present invention can be fabricated by utilizing the property of the compounds of the present invention to adsorb onto an electrode. An electrode, such as a carbon electrode, is inserted into a solution containing a compound of the present invention, for example, 1 - acetamido - 4 - hydroxyanthraquinone, 1 - amino - 4 - hydroxyanthraquinone, or 2 - amino - 3 - hydroxyanthraquinone. The electrode is either inserted into another measurement solution after a certain period of time in the original solution or directly inserted into the other measurement solution. The amount of the compound of the present invention can be quantitatively or qualitatively detected by electrochemical measurements, such as cyclic voltammetry (CV) or chronoamperometry.

[0053] (Enzyme sensor) In certain embodiments, the present invention provides an enzyme sensor comprising an electrode on which a redox enzyme is immobilized and to which the electrode modifier of the present invention is adsorbed. Examples of the electrode of the enzyme sensor include carbon electrodes, gold electrodes, platinum electrodes, etc., and a redox enzyme can be coated or immobilized on this electrode. Further, the conductive material may include metal fine particles containing at least one element selected from Co, Pd, Rh, Ir, Ru, Os, Re, Ni, Cr, Fe, Mo, Ti, Al, Cu, V, Nb, Zr, Sn, In, Ga, Mg, Pb, Au, Pt, Ag, and these may be alloys or electroplated materials. Carbon also includes carbon nanotubes, carbon black, graphite, fullerenes, and their derivatives. For the method of immobilizing the redox enzyme, refer to the paragraph of "Method for immobilizing an enzyme or a redox enzyme" above.

[0054] In certain embodiments, a glucose sensor is an example of the enzyme sensor of the present invention. This glucose sensor can have the aminoanthraquinone - based compound of the present invention adsorbed on the electrode and glucose dehydrogenase (GDH) or glucose oxidase (GOD) immobilized on the electrode. The glucose sensor can be used for continuous blood glucose measurement and continuous glucose monitoring.

[0055] The electrode, enzyme sensor, and composition for electrochemical measurement of the present invention can be used in various electrochemical measurement methods by using a potentiostat, a galvanostat, or the like. Examples of the electrochemical measurement method include amperometry, such as chronoamperometry, potential step chronoamperometry, voltammetry, such as cyclic voltammetry, differential pulse voltammetry, potentiometry, coulometry, and various other methods. For example, if the measurement target substrate is glucose, the glucose concentration in the sample can be calculated by measuring the current when glucose is reduced by the amperometry method. The applied voltage can be, for example, -1000 mV to +1000 mV (vs. Ag / AgCl), although it depends on the conditions and the settings of the apparatus.

[0056] Whether or not the test compound adsorbs to the electrode can be confirmed by cyclic voltammetry. The sweep rate is changed, for example, in the range from 4 mV / sec to 200 mV / sec, and how the maximum value (I Omax ) of the oxidation current changes is examined. Generally, when the mediator adsorbs to the electrode, it is known that the sweep rate of cyclic voltammetry and the value of I Omax are in a proportional relationship. When the mediator diffuses, I Omax is proportional to the 0.5 power of the sweep rate. From the relationship between I Omax and the sweep rate, it is determined whether the compound is adsorbed or diffused.

[0057] An example of the electrochemical measurement is the electrochemical measurement of glucose. In one embodiment, the present invention provides a method for the electrochemical measurement of glucose, which includes a step of contacting a sample that may contain glucose, an aminoanthraquinone-based compound, and purified glucose oxidase or purified glucose dehydrogenase, and a step of measuring a current. The aminoanthraquinone-based compound may be present in the solution or adsorbed to the electrode, and the enzyme may be immobilized on the electrode.

[0058] For example, the electrochemical measurement of glucose concentration can be carried out as follows. A buffer solution is placed in a thermostatic cell and maintained at a constant temperature. An electrode with immobilized GDH or GOD is used as the working electrode, and a counter electrode (e.g., a platinum electrode) and a reference electrode (e.g., an Ag / AgCl electrode or an Ag / Ag+ electrode) are used. The aminoanthraquinone-based compound of the present invention is added to the reaction solution. A certain voltage is applied to the carbon electrode, and after the current becomes steady, a sample containing glucose is added and the increase in current is measured. The glucose concentration in the sample can be calculated according to the calibration curve prepared with a glucose solution of a standard concentration. The applied potential can be, for example, +800 mV or less, +700 mV or less, +600 mV or less, +500 mV or less, +400 mV or less, +300 mV or less, +200 mV or less, +100 mV or less, +50 mV or less, and can be -200 mV or more, -100 mV or more, -50 mV or more, for example, 0 mV or more, and can be, for example, +800 mV to -200 mV, +800 mV to -100 mV, +800 mV to -50 mV, +600 mV to 0 mV, +500 mV to 0 mV, +400 mV to 0 mV, +300 mV to 0 mV, +200 mV to 0 mV (all with respect to the silver-silver chloride reference electrode). The pH of the measurement solution containing glucose can be in the range of pH 3 to 10. For example, it can be pH 5, pH 6, pH 7, pH 8, pH 9, pH 10, and the solution may contain glycine, acetic acid, citric acid, phosphoric acid, carbonic acid, Good buffer, etc. as a buffer. Even when using enzymes other than GDH and GOD and substrates other than glucose, the measurement can be appropriately carried out by changing the pH.

[0059] As a specific example, an aminoanthraquinone compound such as 1 - acetamido - 4 - hydroxyanthraquinone, 1 - amino - 4 - hydroxyanthraquinone, or 2 - amino - 3 - hydroxyanthraquinone is immobilized in advance on a glassy carbon (GC) electrode, and then 0.2 U to 150 U, for example, 0.5 U to 100 U of GDH or GOD is immobilized, and the response current value to the glucose concentration is measured. 10.0 ml of 100 mM potassium phosphate buffer (pH 6.0) is added into the electrolytic cell. The GC electrode is connected to a potentiostat BAS100B / W (manufactured by BAS), the solution is stirred at 37°C, and +600 mV is applied with respect to the silver / silver chloride reference electrode. A 1 M D - glucose solution is added to these systems so that the final concentrations become 5, 10, 20, 30, 40, 50 mM, and the current value in the steady state is measured for each addition. This current value is plotted against the known glucose concentrations (5, 10, 20, 30, 40, 50 mM) to create a calibration curve. From this, quantification of glucose with the GDH or GOD enzyme - immobilized electrode becomes possible.

[0060] As another example of the electrochemical measurement, the electrochemical measurement of hydrogen peroxide can be mentioned. In one embodiment, the present invention provides a method for the electrochemical measurement of hydrogen peroxide, which includes a step of contacting a sample that may contain hydrogen peroxide with an aminoanthraquinone compound and a purified peroxidase, and a step of measuring a current. The aminoanthraquinone compound may be present in the solution or adsorbed on the electrode, and the enzyme may be immobilized on the electrode.

[0061] For example, the electrochemical measurement of hydrogen peroxide concentration can be carried out as follows. Put a buffer solution in a thermostatic cell and maintain it at a constant temperature. Use an electrode with peroxidase immobilized as the working electrode, and use a counter electrode (for example, a platinum electrode) and a reference electrode (for example, an Ag / AgCl electrode or an Ag / Ag+ electrode). Add the aminoanthraquinone-based compound of the present invention to the reaction solution. Apply a certain voltage to the carbon electrode. After the current becomes steady, add a sample containing hydrogen peroxide and measure the decrease in current. The hydrogen peroxide concentration in the sample can be calculated according to the calibration curve prepared with a hydrogen peroxide solution of a standard concentration. The applied potential can be, for example, +600 mV or less, +500 mV or less, +400 mV or less, +300 mV or less, +200 mV or less, +100 mV or less, 0 mV or less, -100 mV or less, -200 mV or less, -300 mV or less, -400 mV or less, -500 mV or less, and can be -1000 mV or more, -900 mV or more, for example -800 mV or more, and can be, for example, +600 mV to -800 mV, +600 mV to -700 mV, +500 mV to -600 mV, +400 mV to -500 mV, +300 mV to -400 mV, +200 mV to -300 mV, +100 mV to -200 mV, +100 mV to -100 mV (all with respect to the silver-silver chloride reference electrode). The pH of the measurement solution containing hydrogen peroxide can be in the range of pH 3 to 10. For example, it is pH 5, pH 6, pH 7, pH 8, pH 9, pH 10, and the solution may contain glycine, acetic acid, citric acid, phosphoric acid, carbonic acid, Good buffer, etc. as a buffer. Even when using an enzyme other than peroxidase and a substrate other than hydrogen peroxide, the pH can be appropriately changed for measurement.

[0062] As a specific example, an aminoanthraquinone compound, such as 1 - acetamido - 4 - hydroxyanthraquinone, 1 - amino - 4 - hydroxyanthraquinone, or 2 - amino - 3 - hydroxyanthraquinone, is immobilized in advance on a glassy carbon (GC) electrode, and subsequently 0.2 U to 150 U, for example, 0.5 U to 100 U of peroxidase is immobilized, and the response current value with respect to the glucose concentration is measured. 10.0 ml of 100 mM potassium phosphate buffer (pH 7.0) is added into the electrolytic cell. The GC electrode is connected to a potentiostat BAS100B / W (manufactured by BAS), and +600 mV is applied with respect to the silver / silver chloride reference electrode. A hydrogen peroxide solution is added to these systems so that the final concentration becomes 0.05, 0.1, 0.2, 0.5, 1 mM, and the current value in the steady state is measured each time it is added. This current value is plotted against the known hydrogen peroxide concentration (0.05, 0.1, 0.2, 0.5, 1 mM) to create a calibration curve. From this, it becomes possible to quantify hydrogen peroxide with the peroxidase enzyme - immobilized electrode.

[0063] Furthermore, a printed electrode can also be used for electrochemical measurement. By this, the amount of solution required for measurement can be reduced. The electrode can be formed on an insulating substrate. Specifically, the electrode can be formed on the substrate by photolithography technology or printing technologies such as screen printing, gravure printing, and flexographic printing. Examples of the material of the insulating substrate include silicon, glass, ceramic, polyvinyl chloride, polyethylene, polypropylene, and polyester, and those with strong resistance to various solvents and chemicals can be used. The area of the working electrode can be set according to the desired response current. For example, in a certain embodiment, the area of the working electrode is 1 mm 2 or more, 1.5 mm 2 or more, 2 mm 2 or more, 2.5 mm 2 or more, 3 mm 2 or more, 4 mm 2 or more, 5 mm 2 or more, 6 mm 2 or more, 7 mm 2 or more, 8 mm 2 or more, 9 mm 2 or more, 10 mm 2 or more, 12 mm2 Above, 15 mm 2 Above, 20 mm 2 Above, 30 mm 2 Above, 40 mm 2 Above, 50 mm 2 Above, 1 cm 2 Above, 2 cm 2 Above, 3 cm 2 Above, 4 cm 2 Above, 5 cm 2 Above, for example, 10 cm 2 Above can be adopted. In certain embodiments, the area of the working electrode is 10 cm 2 Below, 5 cm 2 Below, for example, 1 cm 2 Below can be adopted. The counter electrode may be the same. Also, carbon nanotubes, graphene, Ketjen black, etc. can be immobilized on the working electrode to increase the apparent surface area. In this case, the apparent area can increase by 10 times or more, 50 times or more, 100 times or more, 1000 times or more.

[0064] In certain embodiments, when the electrode modifier, electrode adsorbent or electron transfer promoter of the present invention is used together with an electrode, per 1 cm of the area of the working electrode 2 It can be used in an amount of 0.1 pmol or more, 0.2 pmol or more, 0.3 pmol or more, 0.4 pmol or more, 0.5 pmol or more, 1 pmol or more, 10 mmol or less, 5 mmol or less, 1 mmol or less, 800 μmol or less, 600 μmol or less, 500 μmol or less, 400 μmol or less, 300 μmol or less, 200 μmol or less, 100 μmol or less, 50 μmol or less, for example, 0.1 pmol to 10 mmol, 0.1 pmol to 5 mmol, 0.2 pmol to 1 mmol, 0.3 pmol to 800 μmol, 0.4 pmol to 600 μmol, 0.5 pmol to 500 μmol, 0.6 pmol to 400 μmol, 0.7 pmol to 300 μmol, 0.8 pmol to 200 μmol, 0.9 pmol to 100 μmol, 1 pmol to 50 μmol. These values are based on an area of 1 cm of the working electrode 2This is the case where, when the area of the working electrode is increased or decreased, or when carbon nanotubes, graphene, etc. with a large specific surface area are used, the apparent surface area increases, and the corresponding amount of the electrode modifier of the present invention can be used. In certain embodiments, when the electrode modifier, electrode adsorbent, or electron transfer promoter of the present invention is used by dissolving it in an electrolyte, it can be used at a concentration of 0.1 nmmol / L or more, 1 nmmol / L or more, 10 nmmol / L or more, 100 nmmol / L or more, 1 μmmol / L or more, 10 μmmol / L or more, 100 μmmol / L or more, 1 mmol / L or more, 10 mmol / L or more, 100 mmol / L or more, 1 mol / L or more, 10 mol / L or more, up to the saturation concentration, 100 mol / L or less, for example, at a concentration of 1 mmol to the saturation concentration, 10 mmol to 100 mol / L.

[0065] (The battery of the present invention) In certain embodiments, the present invention provides a battery having the aminoanthraquinone-based compound, electrode modifier, electrode adsorbent, or electron transfer promoter of the present invention. In certain embodiments, in the battery of the present invention, the compound of the present invention is immobilized on the electrode of the battery. In certain embodiments, the battery of the present invention has an oxidoreductase. In certain embodiments, the oxidoreductase may be immobilized on the electrode of the battery. In certain embodiments, the present invention provides a power generation method using the battery of the present invention.

[0066] (The battery of the present invention) In one embodiment, the present invention provides an anode or a cathode for a fuel cell having the aminoanthraquinone-based compound, electrode modifier, electrode adsorbent, or electron transfer promoter of the present invention, and a fuel cell including the anode or the cathode. In one embodiment, the present invention provides a power generation method using the aminoanthraquinone-based compound of the present invention, an electrode adsorbed with the aminoanthraquinone-based compound, or a power generation method in which a redox enzyme such as GDH or GOD is immobilized on an anode electrode and a substrate corresponding to the redox enzyme, for example, glucose, is used as fuel. Optionally, when the redox enzyme shown above is immobilized, a compound that serves as a substrate for the immobilized redox enzyme can be used as fuel. In one embodiment, the present invention provides a flow battery having the aminoanthraquinone-based compound, electrode modifier, electrode adsorbent, or electron transfer promoter of the present invention. The electrolyte solution contained in the electrolyte tank can be appropriately selected according to the battery and may be an aqueous solution or an organic solvent. The pH of the electrolyte solution can be appropriately set in the range of pH 1 to 14.

[0067] In one embodiment, a fuel cell of the present invention comprises an anode or cathode to which the aminoanthraquinone compound of the present invention is adsorbed, a fuel tank, a cathode, an anode having an oxidoreductase, and an electrolyte. Furthermore, the fuel cell of the present invention can optionally have a load resistor disposed between the anode and the cathode, and wiring for this purpose can be provided. In one embodiment, the load resistor is part of the fuel cell of the present invention. In one embodiment, the load resistor is not part of the fuel cell of the present invention, and the fuel cell of the present invention is configured so that it can be connected to an appropriate load resistor. In the fuel cell of the present invention, the oxidoreductase constitutes part of the anode. For example, the oxidoreductase may be in close proximity to or in contact with the anode, or may be immobilized or adsorbed. The fuel tank contains a compound that serves as a substrate for the oxidoreductase immobilized on the electrode. For example, if glucose dehydrogenase is immobilized on the electrode, the fuel can be glucose. In one embodiment, the fuel cell of the present invention can have an ion exchange membrane separating the anode and the cathode. The ion exchange membrane can have pores of 1 nm to 20 nm. The anode can be a common electrode such as a carbon electrode. For example, electrodes made of conductive carbonaceous materials such as carbon black, graphite, activated carbon, carbon nanotubes, and graphene, or electrodes made of metals such as gold and platinum, can be used. Specific examples include carbon paper, carbon cloth, carbon felt, glassy carbon, and HOPG (highly oriented pyrolytic graphite). The cathode pair can be, for example, an electrode in which an electrode catalyst commonly used in fuel cells, such as platinum or a platinum alloy, is supported on a carbonaceous material such as carbon black, graphite, carbon cloth, carbon felt, activated carbon, carbon nanotubes, and graphene, or on a conductor made of gold or platinum, or a conductor made of the electrode catalyst itself, such as platinum or a platinum alloy, is used as the cathode electrode, and an oxidant (cathode-side substrate, oxygen, etc.) can be supplied to the electrode catalyst. In some embodiments, the electrode of the present invention does not include a mercury electrode.

[0068] In another embodiment, as the cathode paired with the anode composed of the substrate-oxidizing enzyme electrode as described above, a substrate-reducing enzyme electrode can be used. Examples of the redox enzyme that reduces the oxidizing agent include known enzymes such as peroxidase, laccase, and bilirubin oxidase. When using a redox enzyme as the catalyst for reducing the oxidizing agent, a known electron transfer mediator may be used as necessary. Examples of the oxidizing agent include oxygen and hydrogen peroxide.

[0069] In one embodiment, in order to avoid the influence of impurities (such as ascorbic acid and uric acid) that interfere with the electrode reaction at the cathode, an oxygen-selective membrane (for example, a dimethylpolysiloxane membrane) can be disposed around the cathode electrode.

[0070] The power generation method of the present invention includes a step of supplying a compound serving as a substrate for the redox enzyme serving as fuel to the anode having the redox enzyme. When fuel is supplied to the anode having the redox enzyme, the substrate is oxidized, and at the same time, the generated electrons are transferred to an electron transfer mediator that mediates the electron transfer between the redox enzyme and the electrode, for example, an aminoanthraquinone-based compound, and the electrons are transferred to the conductive base material (anode electrode) by the electron transfer mediator. When electrons reach the cathode electrode from the anode electrode through the wiring (external circuit), a current is generated. In one embodiment, the power generation method of the present invention includes a step in which a redox reaction of the aminoanthraquinone-based compound occurs. Since the aminoanthraquinone-based compound undergoes binding and dissociation with protons by the transfer of electrons, charge and discharge can be performed well by the redox reaction.

[0071] Protons (H +)( ) moves through the electrolyte solution to the cathode electrode. At the cathode electrode, protons that have moved from the anode in the electrolyte solution, electrons that have moved from the anode side through the external circuit, and an oxidizing agent (cathode side substrate) such as oxygen or hydrogen peroxide react to generate water. Power generation can be carried out using this. Power generation can also be carried out by arranging an electrode containing an aminoanthraquinone-based compound on the cathode side and utilizing the reaction in which the oxidized form of the aminoanthraquinone-based compound receives electrons and reacts to the reduced form.

[0072] (Organic battery of the present invention) In one embodiment, the present invention provides an organic battery having the aminoanthraquinone-based compound, electrode modifier, electrode adsorbent, or electron transfer promoter of the present invention. The electrode modifier, electrode adsorbent, or electron transfer promoter may be adsorbed on the electrode. Examples of electrode materials used for organic batteries include, but are not limited to, quinone, indigo derivatives, benzoquinone compounds having a methoxy group, indigocarmine, pentacene tetrone, and the like. Examples of electrode materials used for organic radical batteries include, but are not limited to, compounds having a nitroxyl radical, for example, an electrode material in which 2,2,6,6-tetramethylpiperidine-N-oxyl is bonded to a polymer such as polymethacrylate or acrylate, and lithium. See, for example, Polymer, Vol. 54, December 2005, p. 886. In an organic battery, the electrode modifier of the present invention can be used as an active material on the anode side and the cathode side.

[0073] The electrode modifier containing the aminoanthraquinone-based compound of the present invention and the electrode adsorbed with the electrode modifier can be used for various electrochemical measurements. Further, the electrode can be used as an enzyme sensor by immobilizing an oxidoreductase. Furthermore, the electrode modifier containing the aminoanthraquinone-based compound of the present invention and the electrode adsorbed with the electrode modifier can be used for fuel cells and organic batteries. These are examples, and the uses of the electrode modifier containing the aminoanthraquinone-based compound of the present invention or the electrode adsorbed with the electrode modifier are not limited thereto.

[0074] The aminoanthraquinone-based compound of the present invention may be adsorbed onto the electrode as it is, or may be linked to the electrode by chemical modification using a cross-linking reagent or the like. Examples of the cross-linking reagent include, but are not limited to, 1-pyrenebutyric acid N-hydroxysuccinimide ester. In addition, the aminoanthraquinone-based compound can also be directly modified on the electrode by graft polymerization.

[0075] The present invention will be further illustrated by the following examples. However, the technical scope of the present invention is not limited by these examples in any way.

Example

[0076] Materials and Methods Unless otherwise specified, materials and reagents were either commercially available or obtained or prepared according to conventional techniques in the art and procedures described in known literature. Single-walled carbon nanotubes manufactured by Sigma, Meijo NanoCarbon, or Zeon Nanotechnology were used. Multi-walled carbon nanotubes manufactured by Sigma, Meijo NanoCarbon, or Kanto Chemical were used. The carbon nanotubes used were those appropriately dispersed by sonication in an aqueous solution containing a low-molecular surfactant, a water-soluble polymer, a water-soluble polysaccharide, or the like. As the surfactant, Triton X-100, sodium dodecyl sulfate, or the like is used, but not limited thereto. The compounds 1-acetamido-4-hydroxyanthraquinone, 1-amino-4-hydroxyanthraquinone, and 2-amino-3-hydroxyanthraquinone were obtained as commercial products from Tokyo Chemical Industry Co., Ltd.

[0077] Example 1. Confirmation of Adsorptivity between Aminoanthraquinone-Based Compound and Carbon Electrode Cyclic voltammetry (CV) was performed using a printed electrode with three types of aminoanthraquinone-based compounds (1-acetamido-4-hydroxyanthraquinone, manufactured by Tokyo Chemical Industry Co., Ltd., product code A1130), 1-amino-4-hydroxyanthraquinone, manufactured by Tokyo Chemical Industry Co., Ltd., product code A0314), and 2-amino-3-hydroxyanthraquinone, manufactured by Tokyo Chemical Industry Co., Ltd., product code A0315). Specifically, a carbon working electrode (12.6 mm2 ) On SCREEN-PRINTED ELECTRODES (manufactured by DropSens, product number DRP-C110) with a printed silver reference electrode, 15 μl of a 10% ethanol aqueous solution of 1-acetamido-4-hydroxyanthraquinone, 1-amino-4-hydroxyanthraquinone, or 2-amino-3-hydroxyanthraquinone with a final concentration of 10 μg / ml and 45 μl of 20 mM potassium phosphate buffer containing 1.5 M potassium chloride were dropped. Then, the electrode was connected to an ALS electrochemical analyzer 814D (manufactured by BAS) using a dedicated connector (manufactured by DropSens, DRP-CAC). Cyclic voltammetry was performed by sweeping the potential within a predetermined range using the working electrode, reference electrode, and counter electrode printed on the electrode. The range of the potential to be swept was +300 mV to +1000 mV (vs. Ag / AgCl) for 1-acetamido-4-hydroxyanthraquinone, +200 mV to +800 mV (vs. Ag / AgCl) for 1-amino-4-hydroxyanthraquinone, and +200 mV to +600 mV (vs. Ag / AgCl) for 2-amino-3-hydroxyanthraquinone. The sweep rate was changed in the range of 4 mV / sec to 200 mV / sec, and how the maximum value of the oxidation current (I Omax ) changed was investigated. Generally, when the mediator is adsorbed on the electrode, it is known that the sweep rate of cyclic voltammetry and the value of I Omax are in a proportional relationship. When the mediator is diffusing, I Omax is proportional to the 0.5 power of the sweep rate.

[0078] Cyclic voltammetry using 1-acetamido-4-hydroxyanthraquinone was performed, and the results of plotting the sweep rate and I Omax are shown in Fig. 1. The value of R 2 was 0.9952. On the other hand, when the results were plotted with the 0.5 power of the sweep rate on the horizontal axis, the value of R 2 decreased to 0.9765. That is, it was confirmed that the sweep rate and I Omax were in a proportional relationship, indicating that 1-acetamido-4-hydroxyanthraquinone was adsorbed on the carbon electrode.

[0079] The results of conducting a similar test using 1-amino-4-hydroxyanthraquinone instead of 1-acetamido-4-hydroxyanthraquinone are shown in Fig. 2. R 2 The value of R was 0.9954. On the other hand, when the results were plotted with the square root of the sweep rate on the horizontal axis, the value of R 2 decreased to 0.9681. That is, it was confirmed that the sweep rate and IOMax were in a proportional relationship, indicating that 1-amino-4-hydroxyanthraquinone was adsorbed on the carbon electrode.

[0080] Also, the results of conducting a similar test using 2-amino-3-hydroxyanthraquinone are shown in Fig. 3. R 2 The value of R was 0.9987. On the other hand, when the results were plotted with the square root of the sweep rate on the horizontal axis, the value of R 2 decreased to 0.9687. That is, it was confirmed that the sweep rate and IOMax were in a proportional relationship, indicating that 2-amino-3-hydroxyanthraquinone was adsorbed on the carbon electrode.

[0081] Comparative Example (p-phenylenediamine) The results of cyclic voltammetry obtained by dropping 10 μl of a 10% ethanol solution of p-phenylenediamine with a final concentration of 100 μg / ml and 10 μl of 100 mM potassium phosphate buffer (pH 7.0) onto the above-described printed electrode and sweeping the voltage in the range from -200 mV to +400 mV (vs. Ag / Ag+) are shown in Fig. 4. R 2 The value of R was 0.9565. On the other hand, when plotted with the square root of the sweep rate on the horizontal axis, the value of R 2 increased to 0.9899, from which it was confirmed that I Omax was proportional to the square root of the sweep rate. This indicates that p-phenylenediamine was not adsorbed on the electrode and was diffusing.

[0082] Example 2. Electrochemical Evaluation Using Aminoanthraquinone Compounds Cyclic voltammetry was performed using a printed electrode with FAD-dependent GDH and one of the aminoanthraquinone compounds of the present invention (1-amino-4-hydroxyanthraquinone). Specifically, a round carbon electrode (DEP-Chip EP-PP, manufactured by Bio Device Technology Co., Ltd.) on which a carbon working electrode (2.64 mm 2 ) and a silver / silver chloride reference electrode are printed was connected to an ALS Electrochemical Analyzer 814D (manufactured by BAS) using a dedicated connector. Then, 2 μl of a 2000 U / ml FADGDH-AA (manufactured by Kikkoman Biochemifa Co., Ltd., product number 60100) solution, 8 μl of a 50 mM potassium phosphate buffer (pH 7.0) containing 1.5 M potassium chloride, and 10 μl of a 10% ethanol aqueous solution of 1-amino-4-hydroxyanthraquinone prepared to 10 μg / ml were dropped onto the electrode. Subsequently, cyclic voltammetry was performed with the voltage swept in the range from -400 mV to 800 mV (vs. Ag / AgCl). The sweep rate was 50 mV / sec. Subsequently, 2 μl of a 500 mM glucose aqueous solution was added, and cyclic voltammetry was performed in the same manner.

[0083] The oxidation current at +600 mV application was 123 nA without glucose addition, whereas it was 716 nA with glucose addition. Since a response current to glucose was observed, it was shown that 1-amino-4-hydroxyanthraquinone functions as a mediator.

[0084] Subsequently, cyclic voltammetry was performed on the printed electrode using peroxidase (manufactured by Toyobo Co., Ltd., product number PEO-301, derived from Wasabia japonica) and three aminoanthraquinone compounds (1-acetamido-4-hydroxyanthraquinone, 1-amino-4-hydroxyanthraquinone, 2-amino-3-hydroxyanthraquinone). Specifically, 5 μl of a 1100 U / ml peroxidase solution, 25 μl of a 20 mM potassium phosphate buffer (pH 7.0) containing 1.5 M potassium chloride, and 15 μl of a 10% ethanol solution of a 10 μg / ml aminoanthraquinone compound were dropped onto the electrode, and cyclic voltammetry was carried out with a scan rate of 30 mV / sec and a scan range from +200 mV to +800 mV (vs. Ag / AgCl). Furthermore, 5 μl of a 3 mM aqueous hydrogen peroxide solution was added, and cyclic voltammetry was similarly carried out. As a control experiment, the same test was carried out under conditions without the aminoanthraquinone compound, and the current values were compared. Note that wild-type enzyme may be used as the peroxidase.

[0085] The difference in the reduction current between the presence and absence of hydrogen peroxide at +400 mV was 152 nA under the condition without the aminoanthraquinone compound, while it was 266 nA in the presence of 1-acetamido-4-hydroxyanthraquinone, 284 nA in the presence of 1-amino-4-hydroxyanthraquinone, and 241 nA in the presence of 2-amino-3-hydroxyanthraquinone. Since the response current to hydrogen peroxide increased in the presence of the aminoanthraquinone compound compared to the absence, it was shown that these compounds function as mediators also for peroxidase. Therefore, it was shown that the mediator of the present invention can be used not only for the anode electrode but also for the cathode electrode.

[0086] Example 3. Electrochemical evaluation when an aminoanthraquinone compound is chemically modified on the electrode 80 μl of a multi-walled carbon nanotube solution (1.4 wt%, MWCNT solution) was applied to a 5 mm square carbon cloth (manufactured by Toyo Technica Co., Ltd.) in multiple portions. After thoroughly drying this, it was washed with ultrapure water. Subsequently, 100 μg of 1-pyrenebutyric acid N-hydroxysuccinimide was applied and thoroughly dried. Further, 20 μg of 1-amino-4-hydroxyanthraquinone, which is the aminoanthraquinone-based compound of the present invention dissolved in ethanol, was applied. After drying, 20 μl of 20 mg / ml peroxidase was applied and dried at room temperature. The fabricated electrode was washed with ultrapure water. Using the washed electrode as the working electrode, silver / silver chloride as the reference electrode, and platinum as the counter electrode, the three electrodes were immersed in 10 ml of 100 mM potassium phosphate buffer (pH 7), and cyclic voltammetry was performed. The sweep rate was 20 mV / sec, and the sweep range was from +0 mV to +700 mV (vs. Ag / AgCl). A hydrogen peroxide solution was dropped to a final concentration of 1 mM, and the difference in the reduction current before and after the addition of hydrogen peroxide was observed. As a result, at +0.3 V, a larger reduction current flowed when hydrogen peroxide was added compared to when it was not added, by 0.54 mA / cm 2 A large reduction current flowed.

[0087] On the other hand, when an electrode was fabricated by applying 20 μl each of the multi-walled carbon nanotube dispersion solution to the front and back of a 0.5 mm square carbon cloth, drying at 60 °C for 1 hour or more, applying 20 μl of peroxidase with a final concentration of 5 mg / ml and a 5% glutaraldehyde solution, and drying at room temperature for 2 hours, as a result of comparing the cyclic voltammograms before and after the addition of the hydrogen peroxide solution, no difference in the reduction current was observed in the range from +200 mV to +700 mV (vs. Ag / AgCl).

[0088] Example 4. Construction of a fuel cell 80 μl of the multi-walled carbon nanotube solution was applied to a 5 mm x 5 mm carbon cloth (manufactured by Toyo Corporation) in several portions and dried at 60°C. After washing with pure water, the cloth was further dried to adsorb and fix the 1-amino-4-hydroxyanthraquinone of the present invention, and then washed with ultrapure water. Next, 20 μl of 20 mg / ml peroxidase (manufactured by Toyobo Co., Ltd., product number PEO-301) was applied, dried at 25°C, and the carbon cloth was exposed to 25% glutaraldehyde vapor for 20 minutes to crosslink and fix the peroxidase, forming a cathode electrode. Multi-walled carbon nanotubes were also applied to the carbon cloth and dried using the same procedure. After adsorption and immobilization of N-isopropyl-N'-phenyl-p-phenylenediamine as a mediator, 20 μl of 20 mg / ml FAD-dependent glucose dehydrogenase (GLD1, Funakoshi Co., Ltd.) was applied and dried at 25°C. The carbon cloth was then exposed to 25% glutaraldehyde vapor for 20 minutes to crosslink and immobilize the enzyme. This was used as the anode electrode. The cathode and anode electrodes were immersed in 100 mM potassium phosphate buffer (pH 7) containing 200 mM D-glucose and connected to a variable resistor and potentiostat. The open-circuit voltage was measured. The result was 0.37 V. This indicates that 1-amino-4-hydroxyanthraquinone functions as an active material for the cathode electrode. When the cathode and anode electrodes were immersed in 100 mM potassium phosphate buffer (pH 7) containing 200 mM D-glucose and 1 mM hydrogen peroxide, the open circuit voltage was 0.5 V, and when connected at 10 kΩ, the current was 0.15 mA / cm. 2 Therefore, a fuel cell could be constructed using the electrode on which 1-amino-4-hydroxyanthraquinone of the present invention was adsorbed and fixed.

[0089] Moreover, single-walled carbon nanotubes can be used instead of multi-walled carbon nanotubes. [Industrial Applicability]

[0090] By using an electrode modifier containing the aminoanthraquinone compound of the present invention or an electrode adsorbed with the electrode modifier, various electrochemical measurements such as those of a battery can be performed.

[0091] All publications, patents, and patent applications mentioned or cited in this specification are hereby incorporated herein by reference in their entirety.

Claims

1. An electrode modifier comprising a compound having the property of adsorbing to an electrode without binding to a polymer or without polymerizing, and having the structure of Formula I, 【Chemical 1】 [wherein, R 1 is hydrogen, C 1-6 alkyl, aryl which may optionally be substituted by sulfo or -C(=O)-C 1-6 alkyl] wherein the compound has the structure of Formula I.

2. An electron transfer promoter comprising a compound having the property of adsorbing to an electrode without binding to a polymer or without polymerizing, and having the structure of Formula I, [Chemical 2] [wherein, R 1 is hydrogen, C 1-6 alkyl, aryl which may optionally be substituted by sulfo or -C(=O)-C 1-6 alkyl] wherein the compound has the structure of Formula I.

3. A battery comprising the electrode modifier according to Claim 1 or the electron transfer promoter according to Claim 2.

4. The battery according to Claim 3, wherein the compound is immobilized on the electrode of the battery.

5. The battery according to Claim 3 or 4, comprising a redox enzyme.

6. The battery according to Claim 5, wherein the redox enzyme is immobilized on the electrode.

7. An electrode modification composition comprising the electrode modifier according to Claim 1 or the electron transfer promoter according to Claim 2.

8. An electrode comprising the electrode modifier according to Claim 1 or the electron transfer promoter according to Claim 2.

9. The electrode according to Claim 8, having an enzyme.

10. The electrode modification composition according to Claim 7, comprising an enzyme.

11. The electrode according to Claim 9, wherein the enzyme is a redox enzyme.

12. The electrode modification composition according to Claim 10, wherein the enzyme is a redox enzyme.

13. The electrode according to Claim 11, wherein the redox enzyme is immobilized.

14. A sensor comprising the electrode modifier according to Claim 1 or the electron transfer promoter according to Claim 2.

15. An enzyme sensor having the electrode according to Claim 11 or 13.

16. The compound is 1-acetamido-4-hydroxyanthraquinone having the following structure 【Chemical Formula 3】 wherein the compound has the following structure, 1-amino-4-hydroxyanthraquinone having the following structure 【Chemical Formula 4】 wherein the compound has the following structure, or 2-amino-3-hydroxyanthraquinone having the following structure 【Chemical Formula 5】 wherein the compound has the following structure, or a salt, anhydride or solvate thereof, The electrode modifier according to Claim 1.

17. The compound is 1-acetamido-4-hydroxyanthraquinone having the following structure ​ wherein the compound has the following structure, 1-amino-4-hydroxyanthraquinone having the following structure [Chemical Formula 7] wherein the compound has the following structure, or 2-amino-3-hydroxyanthraquinone having the following structure 【Chemical 8】 wherein the compound has the following structure, or a salt, anhydride or solvate thereof, The electron transfer promoter according to Claim 2.

18. A method for manufacturing a battery, comprising the step of using the electrode modifier according to Claim 1 or the electron transfer promoter according to Claim 2 for the electrode of the battery.

19. A power generation method using the battery according to any one of claims 3 to 6.

20. An electrochemical measurement method, comprising bringing the electrode modification composition according to any one of claims 7, 10, and 12 into contact with an electrode and using the electrode.

21. An electrochemical measurement method using the electrode according to any one of claims 8, 9, 11, and 13.

22. An electrochemical measurement method using the sensor according to claim 14 or 15.

23. A method for modifying or decorating an electrode, comprising a step of bringing the electrode modifier according to claim 1 or 16 into contact with the electrode.

24. A method for modifying or decorating an electrode, comprising a step of bringing the electron transfer promoter according to claim 2 or 17 into contact with the electrode.

25. A method for modifying or decorating an electrode, comprising a step of bringing the electrode modification composition according to any one of claims 7, 10, and 12 into contact with the electrode.

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