Biosensor and use thereof

Abstract

The purpose of the present invention is to provide a means with which it is possible to electrochemically measure a coenzyme in real time. Provided is an enzyme electrode including an electrode base material and a formic acid dehydrogenase β subunit (FoDH1B) fixed to the electrode base material via at least one compound selected from the group consisting of (1) and (2): (1) aromatic compounds having an aromatic hydrocarbon ring skeleton or a nitrogen-containing heterocyclic skeleton; and (2) compounds represented by general formula (2a). Formula 2a: HS-(CH2)m-OH [where m is an integer of 1-10].

Description

Biosensors and their uses

[0001] This paper relates to biosensors and their uses.

[0002] Conventionally, various methods, such as electrochemical methods and optical methods, have been known as means for detecting target substances. For example, electrochemical methods include a fructose sensor using fructose dehydrogenase (Non-Patent Document 1) and an oxygen sensor using Cu efflux oxidase (CueO) (Non-Patent Document 2). Fructose dehydrogenase and CueO are known to be enzymes that can directly donate and receive electrons with an electrode via redox substances present within or on the enzyme surface and catalyze redox reactions. On the other hand, the sensors shown in Non-Patent Documents 1 and 2 can only detect target substances up to concentrations of about 0.1 mM, and have difficulty detecting the coenzyme nicotinamide adenine dinucleotide (NADH).

[0003] As a method other than the electrochemical method, a sensor capable of detecting NADH spectroscopically is known. For example, Amplite TM The NADH assay (AAT Bioquest, Inc. (formerly ABD Bioquest, Inc.)) is commercially available (Non-Patent Document 3). However, because this technique uses a spectroscopic detection principle, turbidity in the sample becomes measurement noise, and the measurement requires an incubation time (e.g., 15 minutes to 2 hours).

[0004] An NADH sensor that does not use an enzyme (non-enzymatic NADH sensor) has also been reported (Non-Patent Document 4). However, since this sensor directly oxidizes NADH non-enzymatically, a high potential (e.g., 0.4 V or higher) is applied, resulting in capturing noise signals due to the electrochemical response of bio-related substances other than NADH (e.g., ascorbic acid), making it difficult to accurately measure NADH.

[0005] Suzuki, Y. et al., Diffusion-limited electrochemical D-fructose sensor based on direct electron transfer-type bioelectrocatalysis by a variant of D-fructose dehydrogenase at a porous gold microelectrode, J. Electroanal. Chem., Vol. 877, 2020, 114651.Miyata, M., Diffusion-limited Biosensing of Dissolved Oxygen by Direct Electron Transfer-type Bioelectrocatalysis of Multi-copper Oxidases Immobilized on Porous Gold Microelectrodes, J. Electroanal. Chem., Vol. 860, 2020, Colorimetric / Fluorescent Detection of 113895NADH AmpliteTM NADH Assay Manufacturer: AAT Bioquest, Inc. (Former ABD Bioquest, Inc.), Cosmo Bio Co., Ltd. website product information, https: / / www.cosmobio.co.jp / product / detail / amplite-nadh-assay-abd.asp?entry_id=35294L. Rotariu et al., Poly(allylamine hydrochloride) modified screen-printed carbon electrode for sensitive and selective detection of NADH, Sensors and Actuators B: Chemical, Vol. 191, 2014, pages 491-497 "Creating self-assembled monolayers (SAMs)", Protocol, Functional organic materials, Dojindo Laboratories, Ltd. (https: / / www.dojindo.co.jp / technical / protocol / p53.pdf)Abraham Ulman, Formation and Structure of Self-Assembled Monolayers, Chem. Rev. Vol. 96, 1996, 1533-1554.EnzyFluoTM NAD+ / NADH Assay Kit (EFND-100), BioAssay Systems NAD+ / NADH EFND005.pdf (https: / / bioassaysys.com / wp-content / uploads / EFND.pdf).

[0006] An object of the present invention is to provide a means for electrochemically measuring a substance to be measured in real time, even when the substance to be measured is a coenzyme.

[0007] As a result of extensive research, the present inventors have found that coenzymes can be electrochemically measured in real time by using an electrode substrate modified with formate dehydrogenase β subunit (FoDH1B) via at least one compound selected from the group consisting of the following (1) and (2). The present invention was completed through further research based on this finding. (1) An aromatic compound having an aromatic hydrocarbon ring skeleton or a nitrogen-containing heterocyclic skeleton. (2) A compound represented by the general formula (2a): HS-(CH ２ ) ｍ —OH (2a) [In formula (2a), m represents an integer of 1 to 10.]

[0008] The present disclosure encompasses, for example, the following representative inventions. Item 1. An enzyme electrode comprising an electrode substrate and a formate dehydrogenase β subunit (FoDH1B) immobilized on the electrode substrate via at least one compound selected from the group consisting of the following (1) and (2): (1) an aromatic compound having an aromatic hydrocarbon ring skeleton or a nitrogen-containing heterocyclic skeleton; (2) a compound represented by general formula (2a): HS-(CH ２ )m-OH (2a) [In formula (2a), m represents an integer of 1 to 10.] Item 2. The compound (1) is an aromatic compound represented by the following general formula (1a): [In formula (1a), ring A represents an aromatic hydrocarbon ring or a nitrogen-containing heterocycle, n represents an integer of 0 or more, and n R１ are the same or different and each represent an amino group, a thiol group, C １～５ alkyl group of C ２～５ an alkenyl group of C ２～５ an alkynyl group of C １～５ an alkyloxy group of C ２～５ or an alkenyloxy group of C ２～５ These groups each independently have an amino group, a thiol group, a C １～５ alkyl group of C ２～５ an alkenyl group of C ２～５ an alkynyl group of C １～５ an alkyloxy group of C ２～５ and an alkenyloxy group of C ２～５ and / or the compound (2) is represented by the general formula (2a), wherein m is an integer of 1 to 3. Item 3. The enzyme electrode according to Item 1 or 2, wherein, in the general formula (1a), n is an integer of 1 to 3, the aromatic hydrocarbon ring is a benzene ring or a condensed polycyclic aromatic hydrocarbon ring having two, three, or four rings, and the nitrogen-containing heterocycle is a monocyclic nitrogen-containing heterocycle. Item 4. The enzyme electrode according to any one of Items 1 to 3, wherein the compound (1) is an aromatic compound represented by the following general formula (1b): [In formula (1b), X １ , X ２ , X ３ , X ４ and X ５ each independently represents a carbon atom or a nitrogen atom, where p is 1 in the case of a carbon atom and 0 in the case of a nitrogen atom; R １－１ , R １－２ , R １－３ , R １－４ and R １－５ are each independently a hydrogen atom, an amino group, or C １～５ alkyl group of C ２～５ an alkenyl group of C ２～５ an alkynyl group of C １～５ an alkyloxy group of C ２～５ or an alkenyloxy group of C ２～５and the alkyl group, the alkenyl group, the alkynyl group, the alkyloxy group, the alkenyloxy group, and the alkynyloxy group each independently have an amino group and a C １～５ Item 5. In the general formula (1b), X may be substituted with at least one group selected from the group consisting of alkyloxy groups. １ , X ２ , X ３ , X ４ and X ５ each independently represents a carbon atom or a nitrogen atom, where p is 1 for a carbon atom and 0 for a nitrogen atom; R １－１ , R １－２ , R １－３ , R １－４ and R １－５ Item 6. The enzyme electrode according to Item 4, wherein each independently represents a hydrogen atom, an amino group, a methoxy group, or an ethoxy group. １ , X ２ , X ３ , X ４ and X ５ each represents a carbon atom, p is 1, and R １－１ , R １－２ , R １－４ and R １－５ are all hydrogen atoms, and R １－３ is an amino group or a methoxy group, or X １ , X ２ , X ４ and X ５ Each represents a carbon atom, and X １ , X ２ , X ４ and X ５ p in is 1, and X ３ is a nitrogen atom, and X ３ p in is 0, and R １－１ , R １－２ , R １－４ and R １－５ are all hydrogen atoms or X ２ , X ３ , X ４ and X ５ Each represents a carbon atom, and X ２ , X ３, X ４ and X ５ p in is 1, and X １ is a nitrogen atom, and X １ p in is 0, and R １－２ , R １－３ , R １－４ and R １－５ are both hydrogen atoms. Item 7. The enzyme electrode according to Item 1 or 2, wherein in general formula (2a), m is an integer of 2 or 3. Item 8. A biosensor comprising the enzyme electrode according to any one of Items 1 to 7 as a working electrode. Item 9. A method for electrochemically measuring a coenzyme, using the enzyme electrode according to any one of Items 1 to 7 or the biosensor according to Item 8. Item 10. The method for electrochemically measuring a coenzyme according to Item 9, wherein the coenzyme is NADH.

[0009] The present invention provides an enzyme electrode capable of electrochemically measuring coenzymes in real time. The present invention also provides a biosensor including the enzyme electrode. The present invention also provides an electrochemical measurement method using the enzyme electrode or biosensor. The use of the enzyme electrode or biosensor enables electrochemical measurement of coenzymes in real time.

[0010] The results of test examples (Examples 1 to 4, Reference Example, Comparative Example 1) are shown. In Examples 1 to 4, coenzyme (NADH) could be detected in real time without calibration or incubation. The results of test examples (Examples 5 to 6, Reference Example, Comparative Examples 2 to 3) are shown. In Examples 5 and 6, coenzyme could be detected in real time without calibration or incubation. The test results show that NADH could be detected in a concentration-dependent manner. The test results show that a linear response was observed in the NADH concentration range up to 600 μM. The differences from known methods are shown.

[0011] Hereinafter, embodiments included in the present disclosure will be described in more detail. In the present disclosure, the terms "contain" and "comprise" also encompass the meanings of "consist essentially of" and "consist of."

[0012] The present disclosure encompasses an enzyme electrode comprising an electrode substrate modified with formate dehydrogenase β subunit (FoDH1B) via at least one compound selected from the group consisting of the following (1) and (2): In other words, the present disclosure encompasses an enzyme electrode comprising: an electrode substrate; and formate dehydrogenase β subunit (FoDH1B) immobilized on the electrode substrate via at least one compound selected from the group consisting of the following (1) and (2): In the present disclosure, the electrode may be referred to as the "enzyme electrode of the present disclosure." (1) An aromatic compound having an aromatic hydrocarbon ring skeleton or a nitrogen-containing heterocyclic skeleton; (2) A compound represented by general formula (2a): HS-(CH ２ )m-OH (2a) [In formula (2a), m represents an integer of 1 to 10.]

[0013] Compound (1) (1) An aromatic compound having an aromatic hydrocarbon ring skeleton or a nitrogen-containing heterocyclic skeleton (sometimes referred to as "compound (1)") is, in other words, an aromatic compound having at least an aromatic hydrocarbon ring skeleton or a nitrogen-containing heterocyclic skeleton.

[0014] The aromatic hydrocarbon ring may be either monocyclic or polycyclic, and examples thereof include a benzene ring and a fused polycyclic aromatic hydrocarbon ring. In the case of a polycyclic ring, the number of rings constituting the skeleton is not limited, but preferably 2 to 5 rings, and more preferably 2, 3, or 4 rings. Preferred examples of the fused polycyclic aromatic hydrocarbon ring skeleton include skeletons having a 6-membered ring. More preferred examples of the fused polycyclic aromatic hydrocarbon ring skeleton include fused bicyclic aromatic hydrocarbon ring skeletons (e.g., indene skeleton, naphthalene skeleton, azulene skeleton, etc.), fused tricyclic aromatic hydrocarbon ring skeletons (e.g., anthracene skeleton, phenanthrene skeleton, etc.), and fused tetracyclic aromatic hydrocarbon ring skeletons (e.g., pyrene skeleton, chrysene skeleton, tetracene skeleton, triphenylene skeleton, etc.). More preferred examples of the fused polycyclic aromatic hydrocarbon ring skeleton include a naphthalene skeleton, anthracene skeleton, pyrene skeleton, etc.

[0015] The nitrogen-containing heterocycle may be either monocyclic or polycyclic, with a monocyclic ring being preferred. In the case of a polycyclic ring, the number of rings constituting the skeleton is not limited, but preferably 2 or 3 rings are exemplified, and a structure in which one side of the rings is shared (fused) is preferred. Preferred examples of the nitrogen-containing heterocyclic skeleton include skeletons having 5- to 7-membered rings, more preferably skeletons having 5- or 6-membered rings, and even more preferably skeletons having 6-membered rings. The heteroatom constituting the nitrogen-containing heterocyclic skeleton is preferably a nitrogen atom, and the number of nitrogen atoms constituting the heteroatom in the heterocyclic skeleton is preferably 1 to 3, more preferably 1 or 2, and even more preferably 1. For this reason, examples of the nitrogen-containing heterocyclic skeleton include a pyrrole skeleton, an imidazole skeleton, a pyrazole skeleton, a triazole skeleton, a pyridine skeleton, a diazine skeleton (a pyrimidine skeleton, a pyrazine skeleton, a pyridazine skeleton), a triazine skeleton (a 1,2,3-triazine skeleton, a 1,2,4-triazine skeleton, a 1,3,5-triazine skeleton), a tetrazine skeleton, a pentazine skeleton, an azepine skeleton, a diazepine skeleton, and the like, and preferred examples include a pyridine skeleton, a diazine skeleton, a triazine skeleton, and the like.

[0016] The aromatic hydrocarbon ring and nitrogen-containing heterocycle constituting compound (1) may each independently have one or more substituents. When a plurality of substituents are present, they may be bonded to each other to form a ring together with the atoms to which they are bonded.

[0017] The one or more substituents each independently represent an amino group (—NH ２ ), thiol group (-SH), C １～５ alkyl group of C ２～５ an alkenyl group of C ２～５ an alkynyl group of C １～５ an alkyloxy group of C ２～５ or an alkenyloxy group of C ２～５ Preferred examples of the substituents include alkynyloxy groups represented by the following formula: In addition, each of the substituents may independently have a further substituent, and examples of the further substituents that may be had include an amino group, a thiol group, C １～５ alkyl group of C ２～５ an alkenyl group of C ２～５an alkynyl group of C １～５ Alkyloxy group, C ２～５ and an alkenyloxy group of C ２～５ Preferred examples include at least one group selected from the group consisting of alkynyloxy groups of the formula (I), an amino group, a thiol group, and C １～５ Alkyloxy group, C ２～５ and an alkenyloxy group of C ２～５ Preferred examples include at least one group selected from the group consisting of alkynyloxy groups of the following formula: The upper limit of the number of substituents is an integer representing the maximum number of substituents that can be substituted.

[0018] C １～５ Examples of the alkyl group include linear or branched alkyl groups having 1 to 5 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, and neopentyl groups. Preferred examples of the alkyl group include linear or branched alkyl groups having 1 to 4 carbon atoms, and more preferred examples include linear or branched alkyl groups having 1, 2, or 3 carbon atoms.

[0019] C ２～５ Examples of the alkenyl group include linear or branched alkenyl groups having 2 to 5 carbon atoms, such as vinyl, allyl, and 1-propenyl. Preferred examples of the alkenyl group include linear or branched alkenyl groups having 2, 3, or 4 carbon atoms, and more preferred examples include linear or branched alkenyl groups having 2 or 3 carbon atoms. In the alkenyl group, the number and position of double bonds are not limited, and examples of the number of double bonds include 1 to 3, and preferably 1 or 2.

[0020] C ２～５Examples of the alkynyl group include linear or branched alkynyl groups having 2 to 5 carbon atoms, such as ethynyl, 1-propynyl, and 2-propynyl. Preferred examples of the alkynyl group include linear or branched alkynyl groups having 2, 3, or 4 carbon atoms, and more preferred examples include linear or branched alkynyl groups having 2 or 3 carbon atoms. In the alkynyl group, the number and position of triple bonds are not limited, but examples include one or two triple bonds.

[0021] C １～５ Examples of the alkyloxy group include linear or branched alkyloxy groups having 1 to 5 carbon atoms, such as a methoxy group, an ethoxy group, and a propoxy group. Preferred examples of the alkyloxy group include linear or branched alkyloxy groups having 1 to 4 carbon atoms, more preferably linear or branched alkyloxy groups having 1, 2, or 3 carbon atoms, even more preferably alkyloxy groups having 1 or 2 carbon atoms, and particularly preferably alkyloxy groups having 1 carbon atom.

[0022] C ２～５ Examples of the alkenyloxy group include linear or branched alkenyloxy groups having 2 to 5 carbon atoms, preferably linear or branched alkenyloxy groups having 2, 3 or 4 carbon atoms, more preferably linear or branched alkenyloxy groups having 2 or 3 carbon atoms. In the alkenyloxy group, the number and positions of double bonds are not limited, and examples of the number of double bonds include 1 to 3, preferably 1 or 2.

[0023] C ２～５ Examples of the alkynyloxy group include linear or branched alkenyloxy groups having 2 to 5 carbon atoms, preferably linear or branched alkynyloxy groups having 2, 3 or 4 carbon atoms, more preferably linear or branched alkynyloxy groups having 2 or 3 carbon atoms. In the alkynyloxy group, the number and positions of triple bonds are not limited, but examples include groups having 1 or 2 triple bonds.

[0024] Although not limiting the present disclosure, preferred examples of compound (1) include aromatic compounds having a single ring or two to five rings, and more preferred examples include aromatic compounds having a single ring, two rings, three rings, or four rings. Furthermore, although not limiting the present disclosure, preferred examples of compound (1) include aromatic compounds having at least one group selected from the group consisting of an amino group, a thiol group, the alkyloxy group, the alkenyloxy group, and the alkynyloxy group, and more preferred examples are aromatic compounds having at least one group selected from the group consisting of an amino group, a thiol group, and the alkyloxy group.

[0025] In the present disclosure, more preferred examples of compound (1) include aromatic compounds that are positively or neutrally charged when an electrode substrate is modified with compound (1). The charge of each compound can generally be determined from its molecular structure. For example, Examples 1, 3, and 4 used in the test examples described below can be considered to be positively charged aromatic compounds, while Example 2 can be considered to be a neutrally charged aromatic compound. In the present disclosure, the charge is referred to as a positive charge when the acid dissociation constant (pKa) of compound (1) is 6 or less in water for general chemical analysis (25°C, A2 grade water according to JIS K0557:1998) or a negative charge when the pKa is 8 or more, or a neutral charge otherwise.

[0026] Compound (1) is not limited to this, but more preferred examples include aromatic compounds represented by the following general formula (1a):

[0027]

[0028] In general formula (1a), ring A represents an aromatic hydrocarbon ring or a nitrogen-containing heterocycle, n represents an integer of 0 or more, and n R １ are the same or different and each represent an amino group, a thiol group, C １～５ alkyl group of C ２～５ an alkenyl group of C ２～５ an alkynyl group of C １～５ an alkyloxy group of C ２～５ or an alkenyloxy group of C ２～５ These groups each independently have an amino group, a thiol group, a C１～５ alkyl group of C ２～５ an alkenyl group of C ２～５ an alkynyl group of C １～５ an alkyloxy group of C ２～５ and an alkenyloxy group of C ２～５ and the alkynyloxy group may be substituted with at least one group selected from the group consisting of the alkynyloxy groups shown below.

[0029] In the general formula (1a), each ring, each group, etc., including their preferred ranges, etc., are the same as those described above.

[0030] In general formula (1a), ring A is not limited as long as it is the aforementioned aromatic hydrocarbon ring or nitrogen-containing heterocycle, but more preferred examples include a monocyclic aromatic hydrocarbon ring, a fused aromatic hydrocarbon ring having two, three, or four rings (more preferably a benzene ring skeleton, a naphthalene skeleton, an anthracene skeleton, a pyrene skeleton, etc.), or a monocyclic nitrogen-containing heterocycle (more preferably a pyridine skeleton, a diazine skeleton, a triazine skeleton, etc.).

[0031] In general formula (1a), n is an integer of 0 or more, that is, the lower limit of the integer is 0 and the upper limit is the maximum number of integers that can be substituted. Preferred examples of n include integers of 1 to 4, more preferably integers of 1 to 3, and even more preferably 1 or 2.

[0032] In general formula (1a), n R １ are the same or different (i.e., R １ When a plurality of groups are present, some or all of them may be different, or all of them may be the same), an amino group, a thiol group, C １～５ alkyl group of C ２～５ an alkenyl group of C ２～５ an alkynyl group of C １～５ an alkyloxy group of C ２～５ or an alkenyloxy group of C ２～５ n R １ are the same or different, and are preferably exemplified by an amino group, a thiol group, the alkyloxy group, the alkenyloxy group or the alkynyloxy group, more preferably an amino group, a thiol group or the alkyloxy group.

[0033] As mentioned above, these groups may each independently be substituted with at least one group selected from the group consisting of an amino group, a thiol group, the alkyl group, the alkenyl group, the alkynyl group, the alkyloxy group, the alkenyloxy group, and the alkynyloxy group.Preferably, the groups may each independently be substituted with at least one group selected from the group consisting of an amino group, a thiol group, the alkyloxy group, the alkenyloxy group, and the alkynyloxy group, more preferably at least one group selected from the group consisting of an amino group, a thiol group, and the alkyloxy group.When the group has such a substituent, a preferred example is one in which a substituent is present on a group other than an amino group, a thiol group, the alkyloxy group, the alkenyloxy group, and the alkynyloxy group.

[0034] As the aromatic compound represented by the general formula (1a), more preferably, ring A represents a monocyclic aromatic hydrocarbon ring, a fused aromatic hydrocarbon ring having two, three or four rings, or a monocyclic nitrogen-containing heterocycle, n represents an integer of 1 to 3 (more preferably, n represents 1 or 2), and n R １ are the same or different and represent an amino group, a thiol group, the alkyl group, the alkenyl group, the alkynyl group, the alkyloxy group, the alkenyloxy group, or the alkynyloxy group (more preferably an amino group, a thiol group, the alkyloxy group, the alkenyloxy group, or the alkynyloxy group, particularly preferably an amino group, a thiol group, or the alkyloxy group), wherein R １ When at least one of the above is the alkyl group, the alkenyl group, or the alkynyl group, at least one group selected from the group consisting of the alkyl group, the alkenyl group, and the alkynyl may be substituted independently with at least one group selected from the group consisting of an amino group, a thiol group, the alkynyloxy group, the alkenyloxy group, and the alkynyloxy group (more preferably at least one group selected from the group consisting of an amino group, a thiol group, and the alkynyloxy group).

[0035] As described above, preferred examples of the aromatic compound represented by general formula (1a) include aromatic compounds having at least one group selected from the group consisting of an amino group, a thiol group, the alkyloxy group, the alkenyloxy group, and the alkynyloxy group, and more preferred examples include aromatic compounds having at least one group selected from the group consisting of an amino group, a thiol group, and the alkyloxy group.

[0036] Although the compound (1) is not limited thereto, as one embodiment, a preferred example of the compound (1) having a thiol group and a benzene ring skeleton or a 6-membered monocyclic nitrogen-containing heterocyclic skeleton is shown as an aromatic compound represented by the following general formula (1b):

[0037]

[0038] In general formula (1b), X １ , X ２ , X ３ , X ４ and X ５ each independently represents a carbon atom or a nitrogen atom, where p is 1 in the case of a carbon atom and 0 in the case of a nitrogen atom; R １－１ , R １－２ , R １－３ , R １－４ and R １－５ are each independently a hydrogen atom, an amino group, or C １～５ alkyl group of C ２～５ an alkenyl group of C ２～５ an alkynyl group of C １～５ an alkyloxy group of C ２～５ or an alkenyloxy group of C ２～５ and the alkyl group, the alkenyl group, the alkynyl group, the alkyloxy group, the alkenyloxy group, and the alkynyloxy group each independently have an amino group and a C １～５ It may be substituted with at least one group selected from the group consisting of alkyloxy groups.

[0039] In the general formula (1b), each ring, each group, etc., including their preferred ranges, etc., are all explained in the same manner as above.

[0040] As the aromatic compound represented by the general formula (1b), X １ , X ２ , X ３ , X ４ and X ５ each independently represents a carbon atom or a nitrogen atom, where p is 1 for a carbon atom and 0 for a nitrogen atom; R １－１ , R １－２ , R １－３ , R １－４ and R １－５ each independently represents a hydrogen atom, an amino group, a methoxy group, or an ethoxy group.

[0041] As the aromatic compound represented by the general formula (1b), X １ , X ２ , X ３ , X ４ and X ５ each represents a carbon atom, p is 1, and R １－１ , R １－２ , R １－４ and R １－５ are all hydrogen atoms, and R １－３ an aromatic compound in which X is an amino group or a methoxy group; １ , X ２ , X ４ and X ５ Each represents a carbon atom, and X １ , X ２ , X ４ and X ５ p in is 1, and X ３ is a nitrogen atom, and X ３ p in is 0, and R １－１ , R １－２ , R １－４ and R １－５ are both hydrogen atoms; ２ , X ３ , X ４ and X ５ Each represents a carbon atom, and X ２ , X ３ , X ４ and X ５ p in is 1, and X １ is a nitrogen atom, and X １p in is 0, and R １－２ , R １－３ , R １－４ and R １－５ are each a hydrogen atom.

[0042] The compound (1) may be used alone or in combination of two or more.

[0043] Compound (2) (2) The compound represented by general formula (2a) (sometimes referred to as "compound (2)") is as follows: HS-(CH ２ ) ｍ —OH (2a) In formula (2a), m represents an integer of 1 to 10.

[0044] In the present disclosure, compound (2) is bonded to the electrode substrate via a sulfide bond between the electrode substrate and a sulfur atom derived from a thiol group constituting compound (2). The compound represented by general formula (2a) is not limited as long as m is an integer of 1 to 10, but preferably m is an integer of 1 to 5, more preferably m is 1, 2, or 3, and even more preferably m is 2 or 3.

[0045] The compound (2) may be used alone or in combination of two or more.

[0046] The compounds (1) and (2) may be used singly or in combination of two or more.

[0047] Formate dehydrogenase β subunit (FoDH1B) Formate dehydrogenase β subunit (FoDH1B) refers to the β subunit of formate dehydrogenase.

[0048] Formate dehydrogenase (FoDH1) is a heterodimer composed of an α subunit and a β subunit. Formate dehydrogenase, for example, oxidizes formate to carbon dioxide or converts nicotinamide adenine dinucleotide (NADH) to NAD. ＋ is an enzyme that oxidizes carbon dioxide to formic acid, or reduces carbon dioxide to formic acid. ＋to NADH. In this field, enzymes that can directly donate and accept electrons with an electrode via a redox substance present within the enzyme or on the enzyme surface are called direct electron transfer enzymes. Furthermore, a reaction in which electrons are directly transferred between an enzyme and an electrode is called a direct electron transfer reaction (DET reaction). Formate dehydrogenase is known as a type of direct electron transfer enzyme, and is capable of electron transfer via a direct electron transfer reaction. The present inventors have previously confirmed that FoDH1B, which constitutes a part of formate dehydrogenase, itself is also capable of directly donating and accepting electrons with an electrode.

[0049] FoDH1B may be a β subunit derived from wild-type formate dehydrogenase (hereinafter sometimes referred to as a "wild-type β subunit"), or may be a β subunit that has a mutation, such as a deletion, substitution, insertion, or addition of an amino acid, in the wild-type β subunit, as long as direct electron transfer between the subunit and an electrode is possible. Methanol-utilizing bacteria (e.g., the genus Methylorubrum) are known to be sources of the β subunit, and the source is not limited as long as the effects of the present disclosure can be obtained.

[0050] The amino acid sequence encoding FoDH1B can be easily determined based on databases such as UniProt (https: / / www.uniprot.org / ). For example, FoDH1B is exemplified by FoDH1B listed under Entry Number (ID) C5ATT6 in the UniProt database. While not limiting the present disclosure, the amino acid sequence represented by SEQ ID NO: 1 corresponds to the amino acid sequence encoding FoDH1B represented by C5ATT6.

[0051] As described above, the amino acid sequence encoding FoDH1B may have a mutation in the amino acid sequence to the extent that the β subunit is capable of direct electron transfer with the electrode. Here, the "extent of direct electron transfer with the electrode" means that when electrochemical measurements are performed using an electrode substrate modified with FoDH1B according to the procedures of the test examples described below, the direct electron transfer reaction is promoted compared to when an electrode substrate prepared in the same manner except that it is unmodified with FoDH1B is used. As the mutation, a mutation that promotes the direct electron transfer reaction when an applied potential of 0 V is more preferred.

[0052] The degree of promotion of the direct electron transfer reaction is not limited, but it is preferable that the current value when an electrode substrate modified with FoDH1B is used at an applied potential of 0 V is larger than the current value (which is taken as the reference value) when an electrode substrate not modified with FoDH1B is used. Here, the current value is preferably measured in the same manner as in Figures 1 and 2 (linear sweep voltammetry) of the test example described below. The degree of promotion is determined by the current value being 0.3 mA cm higher than the reference value at an applied potential of 0 V. -2 A preferred example is when the current value is 0.4 mA cm or more higher than the reference value, and more preferably when the current value is 0.4 mA cm or more higher than the reference value. -2 More preferably, 0.6 mA cm -2 The upper limit of the promotion is not limited and does not limit the present disclosure, but it is possible to increase the promotion by 10 mA cm or more. -2 Below, 5 mA cm -2 Below, 1mA cm -2 The following are examples. From this, the degree of promotion is 0.3 mA cm -2 More than 10 mA cm -2 Examples include the following:

[0053] Although not limited thereto, it is preferable that the current value when the applied potential is 0 V be larger than that when the compound AET shown in Comparative Example 3 in the test example described later is used.

[0054] Such mutations include, but are not limited to, amino acid sequences in which one or more amino acids have been deleted, substituted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 1. Here, examples of the range of one or more amino acids include 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 5, 1 to 4, 1 to 3, and 1 or 2 amino acids.

[0055] The amino acid after mutation may be either a natural amino acid or an artificial amino acid. Examples of amino acids include hydrophobic amino acids, hydrophilic amino acids, basic amino acids, acidic amino acids, branched-chain amino acids, aromatic amino acids, and sulfur-containing amino acids. More specific examples of amino acids include valine, leucine, isoleucine, alanine, arginine, glutamine, lysine, aspartic acid, glutamic acid, proline, cysteine, threonine, methionine, histidine, phenylalanine, tyrosine, tryptophan, asparagine, glycine, and serine. Techniques for deleting, substituting, inserting, or adding one or more amino acids in a specific amino acid sequence are known.

[0056] Furthermore, while not limiting the present disclosure, conservative substitutions are a preferred example of substitutions. In the present disclosure, conservative substitution refers to the substitution of an amino acid residue with an amino acid residue having a side chain with similar properties. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine constitutes a conservative substitution. Other examples of conservative substitutions include substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0057] FoDH1B may be used alone or in combination of two or more.

[0058] Enzyme Electrode In the present disclosure, as described above, FoDH1B is modified (immobilized) on an electrode substrate via at least one compound selected from the group consisting of compound (1) and compound (2).

[0059] The electrode substrate is not limited as long as it is conductive, and examples thereof include metal substrates such as metals such as gold, platinum, silver, palladium, and indium tin oxide, and metal composites; and carbon substrates such as glassy carbon, graphite, and carbon black. Metal substrates are preferred examples of the electrode substrate, and gold is more preferred. These may be used alone or in combination of two or more. The shape of the electrode substrate may be linear (wire-like, etc.), flat, or thin-layer, with linear being preferred.

[0060] The surface of the electrode substrate constituting the enzyme electrode of the present disclosure may be coated with a coating. An example of the coating is a porous membrane. The enzyme electrode of the present disclosure is usually used as a microelectrode. Microelectrodes used in electrochemical techniques and methods for producing them are conventionally known in this field, and the coating can be formed on the surface of the electrode substrate according to such conventionally known methods. The coating is usually conductive.

[0061] Although not limiting the present disclosure, for example, a porous film can be formed on the surface of an electrode substrate by anodizing the electrode substrate. The anodizing method is not limited, and any method and conditions known in the art can be used. Although not limiting the present disclosure, an example of an anodizing method is a method in which the electrode substrate is immersed in an electrolyte solution and electrolyzed using the electrode substrate as the anode (positive electrode). For example, when gold is used as the electrode substrate, an anodizing process can generate a gold porous film having pores on the surface of the electrode substrate. This results in an electrode substrate with a gold porous film formed on its surface (an electrode substrate carrying a gold porous film). Although not limiting the present disclosure, examples of the electrolyte solution include conventionally known solutions such as a phosphate buffer solution at pH 7, and the solution may be appropriately selected depending on the type of coating to be formed. The components of the coating to be formed are not limited, but include gold and other metals, and a typical porous film is a metal porous film. Furthermore, it is desirable for the electrode substrate to have a linear microelectrode shape with a radius of 200 μm or less in order to perform measurements without requiring a calibration curve. From this viewpoint, the shape of the electrode substrate is preferably a linear microelectrode having a radius of 10 to 100 μm, more preferably a linear microelectrode having a radius of 20 to 70 μm, and particularly preferably a linear microelectrode having a radius of 20 to 50 μm. From this viewpoint, a preferred example of the electrode substrate is gold. The length of the electrode substrate may be determined appropriately depending on the amount of solution used during electrochemical measurement, etc.

[0062] In the enzyme electrode of the present disclosure, the content of the electrode substrate is not limited as long as the effects of the present disclosure can be obtained. Furthermore, when the electrode substrate of the present disclosure supports a coating, the content of the coating in the enzyme electrode is also not limited. Preferably, the electrode substrate supports a coating. When a coating is supported, the thickness of the coating is, for example, about 0.1 nm to 2 μm, and the thickness of the coating may be uniform or non-uniform, without limiting the present disclosure. The coating may be supported on a portion of the electrode substrate, or may be supported on the entire surface. Although not limiting the present disclosure, the coating may be supported on 50 to 100%, more preferably 90 to 100%, of the surface area of ​​the electrode substrate that comes into contact with the test sample during electrochemical measurement.

[0063] The electrode substrate constituting the enzyme electrode of the present disclosure may be further covered with a non-conductive packaging material, as long as at least a portion of its surface is exposed. Examples of non-conductive packaging materials include packaging materials (e.g., resins) such as heat-shrink tubing conventionally known in the art. The packaging material may be selected appropriately as long as it does not impede the effects of the present disclosure. While not limiting the present disclosure, a preferred example is when a gold wire is covered with a packaging material as the electrode substrate. For example, by covering at least the sides of the gold wire with a packaging material and then cutting the wire together with the packaging material, an electrode can be easily produced in which the gold (electrode substrate) is exposed in the cross section and the sides are covered with the packaging material. Furthermore, for example, by covering a gold wire with a packaging material to form a two-layered wire, and then cutting the packaging material at the tip of the two-layered wire, an electrode can be easily produced in which the gold (electrode substrate) is exposed at the tip and the uncut portions of the sides are covered with the packaging material. Furthermore, when the electrode substrate has the coating (e.g., a porous film), it is sufficient that the coating is formed on at least a part or the entire surface of the exposed electrode substrate portion (preferably, the coating is supported on 50 to 100%, more preferably 90 to 100% of the surface area of ​​the exposed electrode substrate portion), and preferably, the coating is not formed on the surface of the packaging material.

[0064] As described above, in the present disclosure, the electrode substrate is modified (immobilized) with FoDH1B via at least one compound selected from the group consisting of compound (1) and compound (2).

[0065] The modification (immobilization) of an electrode substrate with compound (1) and / or compound (2) can be carried out by known modification methods, such as thiol modification, chemical surface modification using amine electrolytic oxidation, physical surface modification using π-π interactions, and physical surface modification using hydrophobic interactions. As mentioned above, the electrode substrate may carry a coating. Therefore, in the present disclosure, the electrode substrate and the electrode substrate carrying the coating may be collectively referred to as the "electrode substrate." (Thus, the present disclosure can be said to encompass electrode substrates modified with compound (1) and / or compound (2) via a coating.) The thiol modification method allows various compounds having a thiol group to be modified onto the surface of the electrode substrate through sulfide bonds. The chemical surface modification method using amine electrolytic oxidation allows various compounds having an amino group to be oxidized on the electrode substrate, thereby modifying the compound onto the surface of the electrode substrate. The physical surface modification method using π-π interactions allows various compounds having a pyrene skeleton to be modified onto a carbon electrode substrate, such as carbon nanotubes, through π-π interactions of aromatic rings. According to the physical surface modification method using hydrophobic interaction, for example, a highly hydrophobic compound can be applied to the surface of the electrode substrate to modify the surface of the electrode substrate with the compound. Such modifications can be carried out according to conventionally known procedures.

[0066] Although not limiting the present disclosure, the thiol modification method will be further described as an example of a modification method. When compound (1) and compound (2) have a thiol group, the compound can be bonded to an electrode substrate via a sulfide bond using a sulfur atom derived from the thiol group constituting compound (1) or compound (2) according to a conventionally known thiol modification method. Thiol modification methods are conventionally known, as exemplified by the methods for forming self-assembled monolayers (SAMs) described in Non-Patent Documents 5 and 6. Although not limiting the present disclosure, the bonding shown in the test examples described below follows a procedure according to a conventionally known technique, and a person skilled in the art can bond compound (1) and / or compound (2) having a thiol group to an electrode substrate according to this procedure.

[0067] By further contacting the electrode substrate modified with compound (1) and / or compound (2) with FoDH1B, the electrode substrate can be modified (immobilized) with FoDH1B via compound (1) and / or compound (2). FoDH1B may be present on the surface of the packaging material, but as described above, the electrode substrate is modified with FoDH1B via at least compound (1) and / or compound (2) that modify (immobilize) the electrode substrate.

[0068] The contact of FoDH1B with an electrode substrate modified with compound (1) and / or compound (2) is not limited as long as contact is possible, and examples include dripping or coating a FoDH1B-containing solution onto the electrode substrate, or immersing the electrode substrate in the solution. Examples of FoDH1B-containing solutions include phosphate buffers (potassium phosphate buffer, etc.) and Tris buffers containing FoDH1B. The content of FoDH1B in the solution may be determined appropriately. The pH of the FoDH1B-containing solution is 3 to 11, preferably 5 to 9. Examples of contact conditions include 4 to 25°C and 5 minutes to 4 hours, more preferably 30 minutes to 2 hours. The pH can be measured at a solution temperature of 25°C using a commonly available pH meter.

[0069] In this manner, an electrode substrate modified with FoDH1B via compound (1) and / or compound (2) can be produced. The enzyme electrode of the present disclosure includes an electrode substrate modified (immobilized) with FoDH1B via compound (1) and / or compound (2) in this manner.

[0070] In the enzyme electrode of the present disclosure, the content of compound (1) and / or compound (2) is not limited as long as the effects of the present disclosure can be obtained. For example, the content of compound (1) and / or compound (2) is preferably about 0.0000001 to 1 part by mass, more preferably about 0.00001 to 0.1 part by mass, per 100 parts by mass of the coating.

[0071] In the enzyme electrode of the present disclosure, the content of FoDH1B is not limited as long as the effects of the present disclosure are obtained. For example, the content of FoDH1B is preferably about 0.0000001 to 1 part by mass, and more preferably about 0.00001 to 0.1 part by mass, per 100 parts by mass of the coating.

[0072] The enzyme electrode of the present disclosure may or may not contain an electron mediator (water-soluble iron porphyrin ([5,10,15,20-tetrakis(2,6-dichloro-3-sulfonate)-popyrinato]Fe(HO)(OH-)), ABTS (2,2'-azinobis-(3-ethylbenzthiozoline-6-sulfonic acid), etc.). Since the enzyme electrode of the present disclosure contains FoDH1B, which is capable of directly donating and receiving electrons to and from the electrode, it is preferable that the enzyme electrode does not contain an electron mediator.

[0073] Biosensor The present disclosure encompasses a biosensor including the enzyme electrode as a working electrode. In the present disclosure, the sensor may be referred to as the "biosensor of the present disclosure."

[0074] The biosensor of the present disclosure includes the enzyme electrode as a working electrode, and may further include a counter electrode, or may include a counter electrode and a reference electrode. In other words, the biosensor of the present disclosure may be a two-electrode type or a three-electrode type. A preferred example of the biosensor of the present disclosure is a three-electrode type including a counter electrode and a reference electrode.

[0075] The material of the counter electrode is not particularly limited, but examples thereof include platinum, gold, diamond, palladium, and carbon, and more preferably platinum.

[0076] The material of the reference electrode is not particularly limited, but examples thereof include silver / silver chloride, silver, platinum, palladium, and carbon, and more preferably silver / silver chloride.

[0077] The biosensor of the present disclosure may or may not contain an electron mediator. A preferred example of the biosensor of the present disclosure is one that does not contain an electron mediator.

[0078] The enzyme electrode and biosensor of the present disclosure can be used in various electrochemical measurement techniques by using an electrochemical analyzer such as a potentiostat, a galvanostat, etc. Electrochemical analyzers are commercially available.

[0079] Electrochemical Measurement Method Therefore, the present disclosure encompasses an electrochemical measurement method using the enzyme electrode or the biosensor. In the present disclosure, this method may be referred to as the "measurement method of the present disclosure."

[0080] In the present disclosure, the coenzyme to be measured is a coenzyme. Examples of the coenzyme include NADH, FAD, and NADPH, and a preferred example is NADH. The coenzyme to be measured may be used alone or in combination of two or more.

[0081] The sample containing a coenzyme is not limited as long as it contains a coenzyme, and examples thereof include body fluids (whole blood, serum, plasma, saliva, cerebrospinal fluid, synovial fluid, urine, interstitial fluid, sweat, tears, saliva, etc.), various tissues and cells derived from living organisms, samples derived from such body fluids, tissues, cells, etc. (including concentrates and dried products thereof), pharmaceuticals, chemicals, or fermented products (including fermented foods), production process solutions thereof, and samples derived from such production process solutions. The sample containing a coenzyme is preferably liquid at room temperature (20°C). The content of the coenzyme in the sample is not particularly limited as long as the measurement is performed appropriately. These may be used alone or in combination of two or more.

[0082] The coenzyme and / or the sample may be mixed with any liquid or the like to the extent that it does not affect the measurement results. Examples of liquids include solutions containing inorganic salts, phosphate buffers (potassium phosphate buffer, etc.), McIlvain's buffer, Tris buffer, citrate buffer, acetate buffer, water, etc. Such a mixed solution with a liquid or the like may be used as a sample to be contacted with the enzyme electrode or biosensor of the present disclosure. The sample may also be pretreated as necessary. The content of the coenzyme in the mixed solution is not particularly limited as long as the measurement is performed appropriately. These may be used alone or in combination of two or more.

[0083] The measurement method of the present disclosure can be carried out by contacting the enzyme electrode or biosensor of the present disclosure with a coenzyme and measuring the current value. Therefore, the measurement method of the present disclosure preferably includes the steps of (i) contacting the enzyme electrode or biosensor of the present disclosure with (ii) a sample containing the coenzyme and measuring the current value.

[0084] Examples of electrochemical measurement methods (methods for measuring current values) include amperometry such as chronoamperometry and potential step chronoamperometry; and voltammetry such as cyclic voltammetry, linear sweep voltammetry, and differential pulse voltammetry.

[0085] The contact between (i) the enzyme electrode or biosensor of the present disclosure and (ii) a sample containing a coenzyme is not limited as long as they can be brought into contact with each other, but a preferred example is a method in which the enzyme electrode or biosensor of the present disclosure is immersed in a sample containing a coenzyme.

[0086] The contact conditions between (i) the enzyme electrode or biosensor of the present disclosure and (ii) a sample containing a coenzyme are, for example, about 4 to 80°C, preferably about 15 to 40°C. The contact time is, for example, about 1 second to 2 minutes, preferably about 5 seconds to 1 minute, and more preferably about 10 to 30 seconds. The pH of the sample is not limited as long as the measurement of the present disclosure can be carried out. However, taking as an example the sample as a liquid such as a solution, an example of the pH at room temperature (25°C) is about 2 to 8.5, preferably about 4 to 8. During contact, stirring may or may not be performed.

[0087] The measurement method of the present disclosure can be carried out under voltage application conditions. Therefore, the measurement method of the present disclosure preferably includes the steps of (i) contacting the enzyme electrode or biosensor of the present disclosure with (ii) a sample containing a coenzyme under voltage application conditions and measuring a current value.

[0088] The applied potential is, for example, about −0.5 to 0.7 V, preferably about −0.2 to 0.2 V, and more preferably 0 V. The application of the potential is preferably initiated after (i) the enzyme electrode or biosensor of the present disclosure is brought into contact with (ii) a sample containing a coenzyme.

[0089] The measurement method of the present disclosure can measure the catalytic current accompanying the oxidation of a coenzyme by FoDH1B as a current value. For example, when the coenzyme is NADH, the NAD of NADH by FoDH1B can be measured. ＋The catalytic current accompanying the oxidation reaction to coenzyme is measured as a current value. The current measurement time is, for example, about 1 to 60 seconds, preferably about 5 to 30 seconds. Since the present disclosure enables real-time measurement, the measurement is preferably initiated between immediately (0 seconds) and 60 seconds after contact of (i) the enzyme electrode or biosensor of the present disclosure with (ii) a sample containing a coenzyme, and more preferably between immediately after contact and 30 seconds.

[0090] Thus, according to the present disclosure, the catalytic current can be measured as a current value. According to the present disclosure, as can be seen from the test examples described below, even when the same sample is used, the current value can be measured at a higher value by using compound (1) and / or compound (2). That is, according to the present disclosure, the combined use of compound (1) and / or compound (2) can promote the DET-type reaction by FoDH1B, allowing the current value to be measured with high sensitivity. High-sensitivity measurement is important for more easily determining the presence or content of a coenzyme in a sample. Therefore, the measurement method of the present disclosure, which uses an electrode substrate modified with FoDH1B via compound (1) and / or compound (2), is excellent in terms of more efficient electrochemical measurement of coenzymes. In the present disclosure, measurement includes detection (presence or absence of a coenzyme), measurement (measurement of a current value), and quantification (determination of the concentration of a coenzyme in a sample, etc.), and detection, measurement, and / or quantification are possible by appropriately selecting the electrochemical measurement method (a current measurement method such as chronoamperometry) depending on the purpose.

[0091] Furthermore, the measurement method of the present disclosure can measure current values ​​with high sensitivity even at an applied potential of 0 V. Generally, application of a high potential (e.g., 0.4 V or higher) can cause electrochemical decomposition of substances other than coenzymes in a sample, and when substances other than coenzymes are electrochemically decomposed, a change in current (an increase or decrease in current value) due to the decomposition can occur. In such cases, the influence of current changes due to substances other than coenzymes increases the likelihood that current measurement based on the coenzyme (i.e., coenzyme-specific current measurement) will be hindered. The measurement method of the present disclosure is also advantageous in that it can effectively measure current values ​​even at an applied potential of 0 V.

[0092] Furthermore, while the measurement method of the present disclosure enables highly sensitive measurement by contacting the enzyme electrode or biosensor of the present disclosure with a coenzyme, the sample provided for contact with the enzyme electrode or biosensor in the measurement method of the present disclosure does not necessarily contain a coenzyme. That is, the measurement method of the present disclosure does not preclude contacting a sample that may contain a coenzyme (which may also be referred to as a sample whose coenzyme content is unknown) with the enzyme electrode or biosensor of the present disclosure and measuring the current value. Therefore, the measurement method of the present disclosure can also be said to include a step of contacting the enzyme electrode or biosensor of the present disclosure with a sample that may contain a coenzyme and measuring the current value. Therefore, the coenzyme, the sample containing the coenzyme, and the sample that may contain the coenzyme that are contacted with the enzyme electrode or biosensor of the present disclosure can be collectively referred to as the test sample. Therefore, the measurement method of the present disclosure can also be said to include a step of contacting (I) the enzyme electrode or biosensor of the present disclosure with (II) the test sample and measuring the current value. In the above explanation, the various procedures, conditions, etc. are the same as those described above, except that the coenzyme and / or a sample containing the coenzyme is replaced with the test sample.

[0093] When a test sample is contacted, if the current value after contact with the test sample is higher than the current value before contact with the test sample, it can be determined that a coenzyme is present in the test sample. If the current value before contact with the test sample and the current value after contact with the test sample are the same or lower, it can be determined that a coenzyme is not present in the test sample. Furthermore, if the current value after contact with the test sample is higher than the current value before contact with the test sample, the concentration of the coenzyme in the test sample can be determined based on the current value (measured value). According to the measurement method of the present disclosure, the presence or absence of a coenzyme can also be known in real time.

[0094] Thus, the enzyme electrode and biosensor of the present disclosure are useful as means for electrochemically measuring coenzymes in real time. According to the present disclosure, coenzymes can be measured with high sensitivity without calibration or an incubation step. Therefore, the enzyme electrode, biosensor, and measurement method of the present disclosure are also useful for real-time monitoring of coenzymes. Furthermore, while coenzymes are generally components that can be present in trace amounts in a sample, the present disclosure achieves a lower detection limit down to a concentration range of several μM, as shown in the test examples described below, thereby achieving high sensitivity in coenzyme measurement. Therefore, the enzyme electrode and the like of the present disclosure are useful as tools for coenzyme measurement in a variety of fields, including the medical field, the chemical field, the bioindustry, and the food industry.

[0095] The present disclosure encompasses any and all combinations of the constituent features described herein. Furthermore, the various characteristics (properties, structures, functions, etc.) described for each embodiment of the present disclosure may be combined in any way to identify the subject matter encompassed by the present disclosure. In other words, the present disclosure encompasses all subject matter consisting of any and all combinations of the combinable characteristics described herein.

[0096] Hereinafter, the embodiments of the present disclosure will be described more specifically with reference to examples, but the embodiments of the present disclosure are not limited to the following examples.

[0097] Test Example 1. Test Procedure - Preparation of Enzyme Electrode The formate dehydrogenase β subunit (FoDH1B) was expressed in the host Escherichia coli ArcticExpress (DE3) strain and purified prior to use. In this test example, the amino acid sequence represented by SEQ ID NO: 1 was used as the amino acid sequence encoding FoDH1B. The expression plasmid used was a pET21a in which the FoDH1B gene had been recombined. Specifically, FoDH1B was expressed in Escherichia coli DE3 according to a conventional method and then purified according to the following procedure. The cultured cells were harvested by centrifugation (20°C, 3,000 × g, 5 minutes) and then suspended in 5 mL of protein extraction reagent BugBuster® (Merck) per gram of wet weight of cells and mixed for 20 minutes. The resulting suspension was centrifuged (4°C, 16,500 × g, 20 minutes) to obtain a cell extract. The supernatant of the bacterial extract was added dropwise to a column support, TALON® metal affinity resin (Clontech Laboratories), to adsorb FoDH1B. After washing with a mixture of 7 mL of HisTALON Equilibration buffer and 0.5 mL of HisTALON Elution buffer, the FoDH1B was eluted with 5 mL of HisTALON Elution buffer. To remove imidazole from the eluate, buffer exchange was performed using a centrifugal ultrafiltration filter, and the resulting FoDH1B solution was stored at -80°C.

[0098] A gold microplate electrode (gold wire with a diameter of 100 μm) was used as the electrode substrate. The gold wire was insulated by encasing it in a non-conductive packaging material (heat-shrink tubing) to form a two-layered wire. The heat-shrink tubing was removed from the tip of this two-layered wire to expose the tip of the gold wire. The tip of the gold wire was then anodized to form a porous oxide coating layer on the surface of the exposed tip of the gold wire. Specifically, the anodization method involved applying a potential of 1.1 V to the electrode substrate in a 0.1 M potassium phosphate buffer solution at pH 7.0 for 120 seconds, followed by a potential sweep from 1.3 to 0 V, to form a porous coating approximately 1 μm thick on the surface of the electrode substrate. According to this procedure, gold present on the surface of the tip of the gold wire was dissolved as gold ions in the buffer solution and then re-deposited on the electrode surface, forming a porous coating on the surface of the electrode substrate. In this way, an electrode substrate carrying a porous film was obtained.

[0099] Electrode substrates with porous coatings were modified with the nine compounds described below, and the results were designated Examples 1 to 6 and Comparative Examples 1 to 3. In Examples 1 to 6 and Comparative Examples 2 and 3, the electrode substrates were modified with the compounds described below by thiol modification according to Non-Patent Document 6. Specifically, each electrode substrate was left overnight at room temperature (20°C) in a solution in which the corresponding compound was dissolved in 100% ethanol to a concentration of 10 mM, thereby producing electrode substrates whose surfaces were modified with the compounds (electrode substrates with each compound immobilized on the surface). In Comparative Example 1, the surface of the electrode substrate was modified using a compound not containing a thiol group according to a conventionally known physical surface modification method based on hydrophobic interaction. After the modification, 30 μL of the FoDH1B solution (100 mM Tris buffer, pH 8.0, protein concentration 0.1 mg / mL) was dropped onto the surface of each electrode substrate and allowed to stand at 4°C for 2 hours to prepare an electrode substrate with FoDH1B supported on a porous coating, which was used as an enzyme electrode (solid lines in Figures 1 and 2 described below). That is, the surface portion of the enzyme electrode in each Example and Comparative Example included a configuration in which a compound was interposed between the surface of the electrode substrate and the FoDH1B supported thereon. In addition, in each Example and Comparative Example, a comparison electrode was used that was prepared in the same manner as the corresponding enzyme electrode except that it did not support FoDH1B (dashed lines in Figures 1 and 2 described below). In each Reference Example, FoDH1B was directly supported on an electrode substrate that had not undergone surface modification with the compounds in the corresponding Examples and Comparative Examples, and was used as the enzyme electrode (the former is the solid line in the Reference Example in Figures 1 and 2 described below, and the latter is the dashed line in the same Reference Example).

[0100] The compounds used in Examples 1 to 6 are as follows: Compound used in Example 1 (ATP; aminothiophenol) The compound (ATP) used in Example 1 is a compound represented by the general formula (1b) in which X １ , X ２ , X ３ , X ４ and X ５ each represents a carbon atom, p is 1, and R １－１ , R １－２ , R １－４ and R １－５ are all hydrogen atoms, and R１－３ The compound (MBT; methoxybenzenethiol) used in Example 2 is an aromatic compound represented by the general formula (1b) in which X １ , X ２ , X ３ , X ４ and X ５ each represents a carbon atom, p is 1, and R １－１ , R １－２ , R １－４ and R １－５ are all hydrogen atoms, and R １－３ The compound (4Mpy) used in Example 3 is an aromatic compound in which X is a methoxy group. １ , X ２ , X ４ and X ５ Each represents a carbon atom, and X １ , X ２ , X ４ and X ５ p in is 1, and X ３ is a nitrogen atom, and X ３ p in is 0, and R １－１ , R １－２ , R １－４ and R １－５ In the general formula (1b), X is an aromatic compound in which X is a hydrogen atom. ２ , X ３ , X ４ and X ５ Each represents a carbon atom, and X ２ , X ３ , X ４ and X ５ p in is 1, and X １ is a nitrogen atom, and X １ p in is 0, and R １－２ , R １－３ , R １－４ and R １－５are all hydrogen atoms. Compound (2-ME; mercaptoethanol) used in Example 5 Compound (2-ME) used in Example 5 is a compound in which m is 2 in general formula (2a). Compound (MP; mercaptopropanol) used in Example 6 Compound (MP) used in Example 6 is a compound in which m is 3 in general formula (2a).

[0101] Electrochemical measurements using a biosensor were performed using an electrochemical analyzer (ALD1040C; BAS, Inc., Japan). The FoDH1B-modified enzyme electrode, silver / silver chloride electrode, and platinum wire were used as the working, reference, and counter electrodes, respectively. An NADH-containing solution (NADH dissolved in potassium phosphate buffer, final NADH concentration: 10 mM) was added to 0.4 M potassium phosphate buffer (pH 8.0) at 25°C. Measurements were initiated 15 seconds after addition by linear sweep voltammetry (-0.5 to 0 V). Current measurements were performed for a total of 50 seconds, and current-potential curves were obtained. The ATP-modified electrode, which showed the greatest improvement in current, was used to measure the concentration response of the current at an applied voltage of 0 V by amperometry. Specifically, an NADH-containing solution was continuously added dropwise to the buffer solution, and the solution was stirred, and current values ​​were recorded in the low concentration range (2 to 300 μM) and the high concentration range (100 to 5000 μM).

[0102] 2. Results The measurement results of linear sweep voltammetry (-0.5 to 0 V) ​​are shown in Figures 1 and 2, the measurement results of the current-time curve (0 V) are shown in Figure 3, and the current response results at each NADH concentration are shown in Figure 4.

[0103] 1 and 2, when the enzyme (FoDH1B) was supported on the electrode substrate via the compounds used in Examples 1 to 6 (i.e., compounds belonging to compound (1) or (2)) (solid lines in each table), a significant improvement in the current value (the value of the catalytic current generated by contact with the coenzyme) was observed compared to when FoDH1B was supported on the electrode substrate via the compounds used in Comparative Examples 1 to 3 (solid lines in each table). In particular, when the compounds shown in Examples 1 to 6 were used, a significant improvement in the current value was observed even at an applied potential of 0 V.

[0104] This demonstrates that coenzymes can be measured efficiently by using an electrode substrate on which FoDH1B is supported via the compounds used in Examples 1 to 6 above.

[0105] Figure 3 shows the concentration dependence of the current-time curve (0 V), with the arrow in the figure indicating the time point at which the coenzyme (NADH) was added. As shown in Figure 3, it was found that the intermittent addition of the coenzyme gradually increased the current value in a manner dependent on the coenzyme concentration. Furthermore, in this test example, an increase in the current value was measured just 15 seconds after the addition of the coenzyme. Thus, in this test example, a current value reflecting the addition of the coenzyme could be measured in real time without incubation. This demonstrates that the method of the present disclosure can detect coenzymes in a very short time.

[0106] Furthermore, as shown in Figure 4, a correlation was observed between the added coenzyme concentration and the measured current value. In particular, a linear response was observed in the low coenzyme concentration range up to 600 μM in the sample. In this case, the detection limit was 5.2 μM.

[0107] These findings demonstrate that this method allows for real-time monitoring of coenzymes without the need for calibration or incubation.

[0108] Although not limiting the present disclosure, a comparison of the results obtained by the above procedure and the results obtained using an existing sensor is shown in FIG. 5. In FIG. 5, from the left, TM The results of measurements were performed according to Non-Patent Document 3 for the "NADH assay," Non-Patent Document 7 for the "EnzyFluo NAD+ / NADH Assay Kit2," and Non-Patent Document 4 for the "non-enzymatic NADH sensor." As shown in FIG. 5, compared to the use of existing sensors, this biosensor does not require incubation, allowing for rapid coenzyme measurement, can measure coenzymes over a wide concentration range, and does not require the creation of a calibration curve. From these results, it can be said that this biosensor has sensor performance suitable for real-time monitoring that does not require calibration.

Claims

1. An enzyme electrode comprising an electrode substrate and a formate dehydrogenase β subunit (FoDH1B) immobilized on the electrode substrate via at least one compound selected from the group consisting of the following (1) and (2): (1) an aromatic compound having an aromatic hydrocarbon ring skeleton or a nitrogen-containing heterocyclic skeleton; (2) a compound represented by the general formula (2a): HS-(CH ２ )m-OH (2a) [In formula (2a), m represents an integer of 1 to 10.] 2. The compound (1) is an aromatic compound represented by the following general formula (1a): [In formula (1a), ring A represents an aromatic hydrocarbon ring or a nitrogen-containing heterocycle, n represents an integer of 0 or more, and n R １ are the same or different and each represent an amino group, a thiol group, C １～５ alkyl group of C ２～５ an alkenyl group of C ２～５ an alkynyl group of C １～５ an alkyloxy group of C ２～５ or an alkenyloxy group of C ２～５ These groups each independently have an amino group, a thiol group, a C １～５ alkyl group of C ２～５ an alkenyl group of C ２～５ an alkynyl group of C １～５ an alkyloxy group of C ２～５ and an alkenyloxy group of C ２～５ and / or the compound (2) is represented by the general formula (2a), wherein m is an integer of 1 to 3.

3. The enzyme electrode according to claim 2, wherein in the general formula (1a), n is an integer of 1 to 3, the aromatic hydrocarbon ring is a benzene ring or a fused polycyclic aromatic hydrocarbon ring having two, three or four rings, and the nitrogen-containing heterocycle is a monocyclic nitrogen-containing heterocycle.

4. A biosensor comprising the enzyme electrode according to claim 1 as a working electrode.

5. A method for electrochemically measuring a coenzyme, using the enzyme electrode according to any one of claims 1 to 3 or the biosensor according to claim 4.

6. The electrochemical measurement method according to claim 5, wherein the coenzyme is NADH.

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