JP2024070325A - Method for measuring substrate in a sample - Google Patents

Abstract

[Problem] Improved method for measuring a substrate in a sample using a sensor including a working electrode comprising a nanocarbon to which a compound having an aromatic ring skeleton is attached or in close proximity, and an enzyme, and a counter electrode. [Solution] A method for measuring a substrate in a sample is disclosed, comprising step A of introducing a sample into a sensor including a working electrode comprising a nanocarbon to which a compound having an aromatic ring skeleton is attached or in close proximity, and an enzyme, and a counter electrode, step B of applying a voltage to the sensor to which the sample has been introduced, and step C of electrochemically measuring the reaction between the substrate in the sample and the enzyme using the sensor to which the voltage has been applied. [Selected Figure] None

Description

A technique is disclosed for measuring a substrate in a sample.

Nanocarbons have a high electrical conductivity, and are being increasingly used as conductive materials that transfer electrons with other substances. For example, it has been proposed to mix nanocarbons with an ink consisting of carbon, resin, and an organic solvent, print the ink on a substrate, and use it as an electrode for a biosensor (Patent Document 1). Carbon nanotubes, a type of nanocarbon, are used in sensors that measure peroxides (Patent Document 2), and are formed into a film together with an enzyme and used as an electrode for sensors and fuel cells (Patent Document 3). It has also been reported that single-walled carbon nanotubes can be used to transfer electrons directly from an enzyme to an electrode (Non-Patent Document 1). This makes it possible to use glucose dehydrogenase (FADGDH), which uses flavin adenine dinucleotide as a coenzyme, in a glucose sensor without a mediator, whereas previously a mediator was necessary. It has also been proposed to use a compound with an aromatic ring structure as an electron transfer promoter for nanocarbons (Patent Document 4).

WO2005088288 WO2011007582 WO2012002290 WO2019189808

ACS Catal. 2017, 7, 725-734

The substrate in the sample can be measured by a sensor including a working electrode containing nanocarbon and an enzyme, and a counter electrode. In this sensor, by attaching or bringing a compound having an aromatic ring skeleton to the nanocarbon or bringing it into close proximity, the electron transfer between the enzyme and the working electrode by the nanocarbon can be enhanced, thereby improving the measurement sensitivity, but it has been found that there is room for further improvement.

One challenge is to improve a method for measuring a substrate in a sample using a sensor that includes a working electrode that includes a nanocarbon to which a compound having an aromatic ring skeleton is attached or in close proximity, and an enzyme, and a counter electrode.

As a result of intensive research aimed at solving this problem, it was found that the substrate in a sample can be measured with high sensitivity by a method including step A of introducing a sample into a sensor including a working electrode containing an enzyme and nanocarbon to which a compound having an aromatic ring skeleton is attached or in close proximity, and a counter electrode, step B of applying a voltage to the sensor to which the sample has been introduced, and step C of electrochemically measuring the reaction between the substrate in the sample and the enzyme using the sensor to which the voltage has been applied. Further investigation based on this knowledge led to the completion of the inventions represented below.

[Item 1]
1. A method for measuring a substrate in a sample, comprising the steps of:
A step A of introducing a sample into a sensor including a working electrode including a nanocarbon having a compound having an aromatic ring skeleton attached thereto or adjacent thereto and an enzyme, and a counter electrode;
The method includes step B of applying a voltage to the sensor to which a sample has been introduced, and step C of electrochemically measuring a reaction between a substrate and an enzyme in the sample using the sensor to which the voltage has been applied.
[Item 2]
Item 3. The method according to item 1, wherein step B is a step of applying a voltage of 0.8 V or less for 1 second or less.
[Item 3]
3. The method according to item 1 or 2, wherein step B is a step of applying a voltage of 0.1 V to 0.4 V for 1 millisecond to 1 second.
[Item 4]
Item 3. The method according to item 2, wherein step C is a step of measuring a current based on the reaction at a voltage lower than the applied voltage in step B.
[Item 5]
Item 4. The method according to item 3, wherein step C is a step of measuring a current based on the reaction at a voltage lower than the applied voltage in step B.
[Item 6]
Item 6. The method according to any one of Items 1 to 5, wherein step C is a step of measuring a current based on the reaction at a voltage of 0.1 V or less.
[Item 7]
Item 7. The method according to any one of Items 1 to 6, wherein the nanocarbon is a carbon nanotube.
[Item 8]
Item 8. The method according to any one of Items 1 to 7, wherein the enzyme is flavin-binding glucose dehydrogenase.
[Item 9]
The compound having an aromatic ring skeleton is thymol, phenol, bis(4-hydroxyphenyl)sulfone, tyrosine disodium hydrate, sodium salicylate, toluene, 5-hydroxyindole, aniline, leucoquinizarin, carvacrol, 1,5-naphthalenediol, 4-isopropyl-3-methylphenol, 2-isopropylphenol, 4-isopropylphenol, 1-naphthol, 2-tert-butyl-5-methylphenol, 2,4,6-trimethylphenol, 2,6-diisopropylphenol ... 9. The method according to any one of Items 1 to 8, wherein the organic solvent is at least one selected from the group consisting of 2-tert-butyl-4-ethylphenol, 6-tert-butyl-2,4-xylenol, 2-tert-butyl-4-methylphenol, 2-tert-butyl-6-methylphenol, 2,4-di-tert-butylphenol, 2,4-di-tert-butyl-5-methylphenol, bis(p-hydroxyphenyl)methane, 3-tert-butylphenol, 2-isopropyl-5-methylanisole, o-cresol, m-cresol, and p-cresol.
[Item 10]
Item 10. The method according to any one of Items 1 to 9, wherein the working electrode further contains a dispersant.
[Item 11]
Item 11. The method according to any one of Items 1 to 10, wherein the counter electrode comprises at least one selected from the group consisting of porous carbon and metal oxides.
[Item 12]
Item 12. The method according to any one of items 1 to 11, wherein the sensor further comprises a reference electrode.

The substrate in the sample can be measured with high sensitivity by using a sensor including a working electrode containing a nanocarbon to which a compound having an aromatic ring skeleton is attached or in close proximity and an enzyme, and a counter electrode.

The structure of the two-electrode closed system chip prepared in Example 1 is shown. "1" is a gold-deposited PET sheet, "2" is an adhesive sheet (0.03 mm thick), "3" is a PET sheet (0.19 mm thick), "4" is a PET sheet, "5" is a working electrode part (1.5 mm ² ), and "6" is a counter electrode part (4.5 mm ² ). In Example 1, the results of the first measurement by the linear sweep voltammetry method are shown. In Example 1, the results of the second measurement by the linear sweep voltammetry method are shown. In Example 2, the results of measurements by chronoamperometry are shown. The structure of the three-electrode closed system chip prepared in Example 3 is shown. "1" is a gold-deposited PET sheet, "2" is an adhesive sheet (0.03 mm thick), "3" is a PET sheet (0.19 mm thick), "4" is a PET sheet, "5" is a working electrode part (1.5 mm ² ), "6" is a counter electrode part (1.5 mm ² ), and "7" is a reference electrode part (1.5 mm ² ). In Example 3, the results of the first measurement by the linear sweep voltammetry method are shown. In Example 3, the results of the second measurement by the linear sweep voltammetry method are shown. In Example 3, the chronoamperometry method was used, and the measurement results at 0.1 V when the voltage application before the start of the measurement was performed under conditions of 0 V and 1 millisecond are shown. In Example 3, the chronoamperometry method was used, and the measurement results at 0.1 V when a voltage of 0.4 V was applied for 1 millisecond before the start of measurement are shown. In Example 3, among the current values obtained in FIG. 9, the current values 1 second and 5 seconds after the start of measurement are plotted. In Example 5, the current values 1 second, 2 seconds, and 5 seconds after the start of measurement are plotted. The structure of the two-electrode closed system chip prepared in the comparative example is shown in Fig. 1. "1" is a gold-deposited PET sheet, "2" is a PET sheet (0.1 mm thick) with adhesive sheets on both sides, "3" is a PET sheet, "4" is a working electrode part (2.3 ^mm2 ), and "5" is a counter electrode part (2.3 ^mm2 ). In the comparative example, the results of the first measurement by the linear sweep voltammetry method are shown. In the comparative example, the results of the second measurement by the linear sweep voltammetry method are shown.

In one embodiment, the method for measuring a substrate in a sample preferably comprises the following steps A, B, and C.
(A) a step A of introducing a sample into a sensor including a working electrode including a nanocarbon having a compound having an aromatic ring skeleton attached thereto or adjacent thereto and an enzyme, and a counter electrode;
(B) a step B of applying a voltage to the sensor into which the sample has been introduced;
(C) A step C of electrochemically measuring the reaction between the substrate and the enzyme in the sample using the sensor to which a voltage has been applied.

The sample is not particularly limited as long as it is one in which measurement of a substrate is required. The sample may be a biological sample or a non-biological sample.

Examples of living organisms in biological samples include mammals, birds, invertebrates, plants, and microorganisms. Of these, mammals are preferred. Examples of mammals include humans, dogs, cats, monkeys, cows, horses, pigs, wild boars, sheep, and rabbits. Of these, humans are preferred. The biological sample can be a sample derived from the living organism, and examples of the sample include blood, serum, plasma, saliva, cerebrospinal fluid, sweat, urine, stool, feces, lymph, tears, and cell or tissue homogenates.

Non-biological samples include any sample other than biological samples (environmental samples, etc.), such as samples derived from food, rivers, soil, etc., wastewater, etc.

Measurement includes qualitative measurement and quantitative measurement. Qualitative measurement includes, for example, detecting whether a substrate is contained in a sample, and determining whether the amount (or concentration) of the substrate in the sample is below a threshold value (or above a threshold value). Quantitative measurement includes, for example, measuring the amount (or concentration) of the substrate in the sample. Note that since the amount (or concentration) of the substrate in the sample corresponds to a current value based on the reaction between the substrate and the enzyme, the measurement of the substrate in the sample may be a measurement of that current value.

The substrate is preferably one whose reaction is catalyzed by the enzyme contained in the working electrode. Various substances can be used as the substrate depending on the type of enzyme. For example, if the enzyme is glucose dehydrogenase, the substrate can be glucose.

The sensor preferably includes a working electrode including a nanocarbon having a compound with an aromatic ring structure attached thereto or adjacent thereto and an enzyme, and a counter electrode.

The enzyme is preferably one that releases electrons in association with a catalytic reaction. Examples of such enzymes include oxidoreductases. Examples of oxidoreductases include glucose dehydrogenase, glucose oxidase, lactate oxidase, cholesterol oxidase, alcohol oxidase, sarcosine oxidase, fructosylamine oxidase, pyruvate oxidase, lactate dehydrogenase, alcohol dehydrogenase, glycerol oxidase, glycerol-3-phosphate oxidase, uricase, choline oxidase, xanthine oxidase, and hydroxybutyrate dehydrogenase.

In one embodiment, the enzyme is preferably glucose dehydrogenase, more preferably flavin-binding glucose dehydrogenase, and more preferably glucose dehydrogenase (also referred to as "FADGDH") that uses flavin adenine dinucleotide (FAD) as a coenzyme. FADGDH holds FAD in a cavity in a three-dimensional structure formed by a polypeptide, so that a substance called a mediator was conventionally required to transfer the electrons generated there to the working electrode. In contrast, by using nanocarbon, it is possible to transfer electrons to the working electrode without using a mediator. In addition, by using a compound having an aromatic ring skeleton, it is possible to transfer electrons via nanocarbon much more efficiently (or strongly).

There is no limitation on the type of FADGDH, and any type can be used. Specific examples of FADGDH include those derived from any of the following organisms: Aspergillus terreus, Aspergillus oryzae, Aspergillus niger, Aspergillus foetidus, Aspergillus aureus, Aspergillus versicolor, Aspergillus kawachi, Aspergillus awamori, Agrobacterium tumefaciens, Cytophaga marinoflava, Agaricus bisporus, Macrolepiota lacodes, Burkholderia cepacia, Mucor subtilisimus, Mucor guilliermondii, Mucor plynii, Mucor javanicus, Mucor circinelloides, Mucor circinelloides F. circinelloides, Mucor hiemalis, Mucor hiemalis F. sylvaticus, Mucor dimorphosporus, Absisia cylindrospora, Absisia hyalospora, Actinomycorum elegans, Circinella simplex, Circinella angarensis, Circinella sinensis, Circinella lacrimispora, Circinella minor, Circinella mucoroides, Circinella rigida, Circinella umbellata, Circinella muscae, Metarhizium sp. and Colletotrichum sp.

In one embodiment, preferred FADGDHs are FADGDH derived from Aspergillus oryzae, FADGDH derived from Mucor hiemalis, FADGDH derived from Mucor subtilisimus, FADGDH derived from Circinella simplex, FADGDH derived from Metarhizium sp., or FADGDH derived from Colletotrichum sp., preferably having 80% or more identity with the amino acid sequence of SEQ ID NO: 1 to 6, more preferably having 90% or more identity with the amino acid sequence of SEQ ID NO: 1 to 6, and even more preferably having 95% or more identity with the amino acid sequence of SEQ ID NO: 1 to 6, and having glucose dehydrogenation activity. The identity of the amino acid sequence can be calculated using a commercially available analysis tool or an analysis tool available via an electric communication line (Internet), for example, the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) http://www.ncbi.nlm.nih.gov/BLAST/, using the default (initial setting) parameters. The amino acid sequence of SEQ ID NO: 1 is that of FADGDH derived from Aspergillus oryzae, the amino acid sequence of SEQ ID NO: 2 is that of FADGDH derived from Mucor hiemalis, the amino acid sequence of SEQ ID NO: 3 is that of FADGDH derived from Mucor subtilisimus, the amino acid sequence of SEQ ID NO: 4 is that of FADGDH derived from Circinella simplex, the amino acid sequence of SEQ ID NO: 5 is that of FADGDH derived from Metarhizium sp., and the amino acid sequence of SEQ ID NO: 6 is that of FADGDH derived from Colletotrichum sp.

The nanocarbon is not particularly limited as long as it is a substance that has an electron transfer function and is recognized as a nanocarbon. Examples of such substances include carbon materials that are mainly composed of carbon, including carbon nanotubes, carbon nanohorns, carbon nanotwists, cocoons, carbon nanocoils, graphene, and fullerenes. The carbon nanotubes may be single-walled carbon nanotubes, double-walled carbon nanotubes, or multi-walled carbon nanotubes with three or more walls. In one embodiment, the nanocarbon is preferably a carbon nanotube, and more preferably a single-walled carbon nanotube.

The compound having an aromatic ring skeleton attached to or adjacent to the nanocarbon is not particularly limited as long as it is an electron transfer promoter that promotes electron transfer between the enzyme and the working electrode by the nanocarbon. The number of ring-constituting atoms of the aromatic ring skeleton is, for example, 5 to 18, preferably 5 to 16, and more preferably 5 to 14. The aromatic ring skeleton includes a skeleton consisting of one benzene ring, a skeleton consisting of two or more (for example, 2 to 4) benzene rings (naphthalene skeleton, anthracene skeleton, etc.), a skeleton consisting of a condensed ring of a benzene ring and another aromatic ring (nitrogen-containing aromatic ring, oxygen-containing aromatic ring, sulfur-containing aromatic ring, etc.) (phenanthroline skeleton, benzofuran skeleton, benzimidazole skeleton, carbazole skeleton, etc.), and a skeleton consisting of an aromatic ring consisting of carbon and another element (nitrogen, oxygen, sulfur, etc.) (triazine skeleton, triazole skeleton, pyridine skeleton, etc.). It is preferable that the compound having an aromatic ring skeleton is a compound that does not function as a mediator by itself. Here, "does not function as a mediator by itself" means that, like benzoquinone or 1-methoxyphenazine methosulfate, it does not function by itself to transfer electrons between the electrode and the enzyme or between the electrode and the substrate.

In one embodiment, the compound having an aromatic ring skeleton preferably has an electron-donating substituent. The electron-donating substituent is a hydroxy group, an amino group, a methyl group, or the like. A preferred electron-donating substituent is a hydroxy group. Examples of compounds having an electron-donating substituent and an aromatic ring skeleton include compounds having a benzene ring substituted with a hydroxy group (e.g., thymol, phenol, bis(4-hydroxyphenyl)sulfone, tyrosine disodium hydrate, sodium salicylate, 5-hydroxyindole, leucoquinizarin, carvacrol, 1,5-naphthalenediol, 4-isopropyl-3-methylphenol, 2-isopropylphenol, 4-isopropylphenol, 1-naphthol, 2-tert-butyl-5-methylphenol, 2,4,6-trimethylphenol, 2,6-diisopropylphenol, 2-tert-butyl- 4-ethylphenol, 6-tert-butyl-2,4-xylenol, 2-tert-butyl-4-methylphenol, 2-tert-butyl-6-methylphenol, 2,4-di-tert-butylphenol, 2,4-di-tert-butyl-5-methylphenol, bis(p-hydroxyphenyl)methane, 3-tert-butylphenol, o-cresol, m-cresol, p-cresol), compounds having a benzene ring substituted with an amino group (e.g., aniline), and compounds having a benzene ring substituted with a methyl group (e.g., toluene, 2-isopropyl-5-methylanisole).

Among the above compounds, at least one selected from thymol, phenol, and carvacrol is preferred.

By attaching or bringing a compound having an aromatic ring structure to or in close proximity to the nanocarbon, it is possible to promote electron transfer between the enzyme and the working electrode via the nanocarbon. It is preferable that the compound having an aromatic ring structure and the nanocarbon are attached to or in close proximity through intermolecular interaction.

There are no particular limitations on the means for attaching or bringing the compound having an aromatic ring skeleton to or in close proximity to the nanocarbon. For example, this can be achieved by mixing the nanocarbon with the compound having an aromatic ring skeleton (including mixing in a solution), or by disposing the compound having an aromatic ring skeleton on the nanocarbon. The compound having an aromatic ring skeleton disposed in close proximity to or attached to the nanocarbon may or may not be fixed. There are no limitations on the fixation as long as it does not inhibit the functions of the nanocarbon and the compound having an aromatic ring skeleton, and any known means can be appropriately selected and used.

The amount of the compound having an aromatic ring skeleton attached to or placed in close proximity to the nanocarbon is not particularly limited. The amount of the compound having an aromatic ring skeleton is, for example, 0.001 parts by mass or more, preferably 0.01 parts by mass or more, more preferably 0.1 parts by mass or more, per 100 parts by mass of the nanocarbon. The amount of the compound having an aromatic ring skeleton is, for example, 100,000 parts by mass or less, preferably 10,000 parts by mass or less, more preferably 1,000 parts by mass or less, per 100 parts by mass of the nanocarbon. The lower limit and upper limit can be combined arbitrarily. The amount of the compound having an aromatic ring skeleton is, for example, 0.001 parts by mass or more, preferably 0.01 parts by mass or more, more preferably 0.1 parts by mass or more, per 100 parts by mass of the enzyme. The amount of the compound having an aromatic ring skeleton is, for example, 1,000,000 parts by mass or less, preferably 100,000 parts by mass or less, more preferably 10,000 parts by mass or less, per 100 parts by mass of the enzyme. The lower limit and upper limit can be combined arbitrarily.

The working electrode may further contain a dispersant. The dispersant is not particularly limited as long as it is a substance capable of suppressing aggregation of nanocarbon and dispersing it. Examples of dispersants include, but are not limited to, sodium cholate, sodium deoxycholate, sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, cetyltrimethylammonium bromide, octylphenol ethoxylate, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and sodium carboxymethylcellulose salt. Dispersants other than those described above, for example, in JP 2016-56230 A and JP 2013-230951 A, can also be used. All publications cited in this specification are incorporated herein by reference in their entirety. The dispersant may be one type alone or two or more types in combination. In one embodiment, the preferred dispersant is sodium cholate and/or sodium deoxycholate.

The shape of the working electrode is not particularly limited as long as it can be used as an electrode, and may be, for example, a wire, coil, net, rod, film, or plate. In one embodiment, the working electrode preferably has a substrate suitable for an electrode on which an enzyme used in a biosensor is immobilized, and the substrate is preferably an insulating substrate on which a metal film (e.g., a metal thin film) is formed. Examples of materials for the insulating substrate include plastic, photosensitive materials, paper, glass, ceramics, and biodegradable materials. The insulating substrate is preferably a glass substrate or a plastic substrate (e.g., a PET substrate). The type of metal that can form the metal film is not particularly limited as long as it is used for an electrode, and examples of the metal that can form the metal film include precious metals such as gold, platinum, and palladium, copper, aluminum, nickel, titanium, indium tin oxide (ITO), and zinc oxide (ZnO). The substrate may also be an insulating substrate on which a carbon film (e.g., a carbon thin film made of carbon paste) is formed instead of a metal film.

In one embodiment, the working electrode is preferably a substrate on which nanocarbon, a compound having an aromatic ring skeleton, and an enzyme are loaded (particularly immobilized), and preferably has a substrate and a layer (which may also be referred to as a "reagent layer") containing nanocarbon, a compound having an aromatic ring skeleton, and an enzyme formed (or laminated) on at least one surface of the substrate. In one embodiment, the working electrode is preferably immersed in a solvent. This embodiment may be referred to as an "open system". In an open system, the working electrode may be immersed in a solvent to which a sample has been added, or a sample may be added to a solvent in which the working electrode is immersed. In another embodiment, it is preferable to have an electrode including a working electrode and a counter electrode, and a cover laminated on the electrode via a spacer having a slit. In this embodiment, a cavity for supplying a sample can be formed by the electrode, the slit, and the cover. This embodiment may be referred to as a "closed system". For the configuration of the closed system, for example, WO 2018/043050 (or U.S. Patent Application Publication No. 2019/194714) or the like can be referred to. In a closed system, the solvent containing the sample can be injected into the cavity by capillary action by contacting the cavity (particularly the tip or inlet). The solvent is typically a buffer solution, such as an acetate buffer solution, a citrate buffer solution, a phosphate buffer solution, or a borate buffer solution. The solvent may or may not contain a compound having an aromatic ring structure.

The method of loading the nanocarbon, the compound having an aromatic ring skeleton, and the enzyme is not particularly limited. For example, the loading can be performed by repeatedly preparing a solution in which each of these substances is dispersed or dissolved, dropping the solution onto a predetermined portion of the substrate (if the substrate is an insulating substrate on which a metal thin film is formed, the portion where the metal thin film is formed), and drying. Alternatively, the loading can be performed by preparing a solution in which each of these substances is dispersed or dissolved, mixing the solutions together, dropping the solutions onto a predetermined portion of the substrate, and drying. The dispersion medium or solvent is not particularly limited, and examples thereof include water, alcohol-based solvents (e.g., ethanol), ketone-based solvents (e.g., acetone), and combinations of two or more of these.

In one embodiment, it is preferable to add a dispersant to the dispersion liquid in which the nanocarbon is dispersed. The mixing ratio of the dispersant is arbitrary, but it is preferable to mix it at 0.2 to 2% (w/v), for example. The mixing ratio of the nanocarbon is also arbitrary, but it is preferable to mix it at 0.05 to 0.5% (w/v), for example.

The order of loading is arbitrary, but in one embodiment, it is preferable to load them in the order of nanocarbon → enzyme → compound having an aromatic ring skeleton, or nanocarbon → compound having an aromatic ring skeleton → enzyme, and it is also preferable to load them simultaneously.

The amounts of nanocarbon, compound having an aromatic ring skeleton, and enzyme used are not particularly limited.

In one embodiment, the nanocarbon, the compound having an aromatic ring skeleton, and the enzyme may be immobilized on a substrate. The immobilization can be performed by appropriately selecting a known method. For example, the immobilization can be performed by dropping a liquid in which a substance suitable for immobilization, such as tetrafluoroethylene/perfluoro[2-(fluorosulfonylethoxy)polyvinyl ether] copolymer (e.g., Nafion (trademark)) and carboxymethyl cellulose, is dissolved, onto the portion of the substrate on which the above substances are loaded, and then drying. In one embodiment, after loading the nanocarbon, the compound having an aromatic ring skeleton, and the enzyme onto the substrate, it is preferable to treat these substances with a polymer substance such as carboxymethyl cellulose so as to cover them.

In one embodiment, the working electrode is preferably one in which nanocarbon and an enzyme are loaded (particularly immobilized) on a substrate, and preferably has a substrate and a layer (which may also be referred to as a "reagent layer") containing nanocarbon and an enzyme formed (or laminated) on at least one surface of the substrate. The method of loading (or immobilizing) nanocarbon and enzyme on a substrate can be the same as the method of loading (or immobilizing) nanocarbon, a compound having an aromatic ring skeleton, and an enzyme on a substrate. It is preferable that the working electrode (or the layer) does not contain (is not immobilized) a compound having an aromatic ring skeleton. In an open system, the working electrode may be immersed in a solvent containing a sample and a compound having an aromatic ring skeleton, or the sample may be added to the solvent containing a compound having an aromatic ring skeleton in which the working electrode is immersed. In a closed system, the solvent containing the sample and the compound having an aromatic ring skeleton can be injected into the cavity by capillary action by contacting the cavity (particularly the tip portion or inlet thereof). The solvent is typically a buffer solution, such as an acetate buffer solution, a citrate buffer solution, a phosphate buffer solution, or a borate buffer solution.

The concentration of the compound having an aromatic ring skeleton in the solvent is not particularly limited. The lower limit of the concentration is, for example, 0.000001% (w/v), preferably 0.000005% (w/v), more preferably 0.00001% (w/v), more preferably 0.00005% (w/v), more preferably 0.0001% (w/v), more preferably 0.0005% (w/v), more preferably 0.001% (w/v), more preferably 0.005% (w/v), more preferably 0.01% (w/v). The upper limit of the concentration is, for example, 2% (w/v), preferably 1.5% (w/v), more preferably 1% (w/v). The lower limit and upper limit can be combined arbitrarily.

The counter electrode is not particularly limited as long as it is a counter electrode that includes a nanocarbon and an enzyme to which a compound having an aromatic ring skeleton is attached or in close proximity. In one embodiment, the counter electrode preferably includes a counter electrode modifier. In one embodiment, the counter electrode modifier preferably includes a metal oxide and/or porous carbon.

The metal oxide is not particularly limited, and examples thereof include metal oxides that are insoluble in water and/or organic solvents. Here, the term "insoluble" is used to mean not only the case where the metal oxide is completely insoluble, but also the case where the metal oxide is slightly soluble (for example, the solubility at room temperature is 0.0001 g/mL or less). Examples of such metal oxides include copper oxide, silver oxide, manganese oxide, osmium oxide, and lead oxide. Examples of copper oxides include Cu ₂ O and CuO, examples of silver oxides include Ag ₂ O, examples of manganese oxides include MnO ₂ and BaMnO ₄ , and examples of osmium oxides include OsO _4. Examples of lead oxides include PbO _2. The metal oxide may be one type alone or a combination of two or more types. The metal oxide is preferably at least one type selected from the group consisting of silver oxide and manganese oxide, and more preferably at least one type selected from the group consisting of Ag ₂ O, MnO ₂ , and BaMnO ₄ .

The type of porous carbon is not particularly limited. Examples of porous carbon include activated carbon, carbon nanotubes, and porous carbon with an oxide template. Carbon nanotubes may be single-walled carbon nanotubes, double-walled carbon nanotubes, or multi-walled carbon nanotubes with three or more layers. Examples of porous carbon with an oxide template include porous carbon with a magnesium oxide template (for example, porous carbon manufactured by Toyo Tanso Co., Ltd., product name: Knobel (trademark)), porous carbon with a zeolite template, and porous carbon with an alumina template.

The specific surface area of the porous carbon is not particularly limited. The lower limit of the specific surface area is, for example, 500 m ² /g, preferably 1000 m ² /g. The upper limit of the specific surface area is, for example, 5000 m ² /g, preferably 4000 m ² /g, and more preferably 3000 m ² /g. The lower limit and the upper limit can be combined arbitrarily. The larger the specific surface area, the more the reduction reaction of transferring electrons from the counter electrode to the substance on the counter electrode surface can be promoted. The specific surface area can be determined, for example, by the Brunauer-Emmett-Teller (BET) method from the adsorption isotherm of nitrogen molecules at liquid nitrogen temperature (77K).

The porous carbon is preferably a powder. The particle size or average particle size of the porous carbon is not particularly limited. The upper limit of the particle size or average particle size is, for example, 200 μm, preferably 180 μm, and more preferably 150 μm. It is also preferable that the upper limit of the average particle size is 100 μm, 80 μm, 50 μm, 30 μm, or 10 μm. The lower limit of the particle size or average particle size is, for example, 10 nm, preferably 15 nm, and more preferably 20 nm. The lower limit and the upper limit can be combined arbitrarily. The smaller the particle size or average particle size, the higher the dispersibility in the solution, and the more likely it is that clogging will be prevented during handling with a micropipette, dispenser, or the like, and the more likely it is that handling accuracy will be improved. The particle size or average particle size can be measured, for example, by a mesh method or a laser diffraction scattering method.

The counter electrode may further contain a dispersant. In particular, when the counter electrode contains porous carbon as a counter electrode modifier, a dispersant is preferable because it can suppress the aggregation of the porous carbon and disperse it. Examples of dispersants include, but are not limited to, sodium cholate, sodium deoxycholate, sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, cetyltrimethylammonium bromide, octylphenol ethoxylate, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and carboxymethylcellulose sodium salt. For example, dispersants other than those described in JP 2016-56230 A and JP 2013-230951 A can also be used. The dispersant may be one type alone or a combination of two or more types. In one embodiment, the preferred dispersant is sodium cholate and/or sodium deoxycholate.

The shape of the counter electrode is not particularly limited as long as it can be used as an electrode, and may be, for example, a wire, coil, mesh, rod, film, or plate. In one embodiment, the counter electrode preferably has a substrate suitable for an electrode, and the substrate is preferably an insulating substrate on which a metal film (e.g., a metal thin film) is formed. Examples of materials for the insulating substrate include plastic, photosensitive materials, paper, glass, ceramics, and biodegradable materials. The insulating substrate is preferably a glass substrate or a plastic substrate (e.g., a PET substrate). The type of metal that forms the metal film is not particularly limited as long as it is used for an electrode, and examples of the metal that can be used include precious metals such as gold, platinum, and palladium, copper, aluminum, nickel, titanium, indium tin oxide (ITO), and zinc oxide (ZnO).

In one embodiment, the counter electrode is preferably one in which a carbon film (e.g., a carbon film made of carbon paste, preferably a thin carbon film) is formed in place of the metal film or on at least a portion of the metal film. The carbon paste preferably contains a counter electrode modifier in addition to the components used in the electrode, and preferably contains carbon, a binder, and a counter electrode modifier.

Examples of carbon include graphite and carbon black. Examples of binders include hydrocarbon binders, alcohol binders, and ester binders. Examples of hydrocarbon binders include heptane, octane, nonane, decane, undecane, dodecane, tridecane, liquid paraffin, and squalane. Examples of alcohol binders include oleyl alcohol and stearyl alcohol. Examples of ester binders include phthalate esters such as dioctyl phthalate. Carbon and binders may each be used alone or in combination of two or more types.

The binder content in the carbon paste is not particularly limited. The lower limit of the content is, for example, 1 part by mass, preferably 5 parts by mass, more preferably 10 parts by mass, and even more preferably 20 parts by mass, per 100 parts by mass of carbon. The upper limit of the content is, for example, 1000 parts by mass, preferably 500 parts by mass, and even more preferably 200 parts by mass, per 100 parts by mass of carbon. The lower limit and upper limit can be combined in any manner.

The content of the counter electrode modifier in the carbon paste is not particularly limited. The lower limit of the content is, for example, 1 part by mass, preferably 5 parts by mass, more preferably 10 parts by mass, and even more preferably 20 parts by mass, per 100 parts by mass of carbon. The upper limit of the content is, for example, 1000 parts by mass, preferably 500 parts by mass, and even more preferably 200 parts by mass, per 100 parts by mass of carbon. The lower limit and upper limit can be combined in any manner.

The content (or loading) of the counter electrode modifier per 100 parts by mass of carbon in the counter electrode can be selected from the same range as the content of the counter electrode modifier per 100 parts by mass of carbon in the carbon paste.

In one embodiment, the counter electrode is preferably a substrate on which a counter electrode modifier is loaded, and preferably has a substrate and a layer (which may also be referred to as a "reagent layer") containing the counter electrode modifier formed (or laminated) on at least one surface of the substrate. There are no particular limitations on the amount of counter electrode modifier loaded.

The method of loading the counter electrode modifier is not particularly limited. For example, a solution in which the counter electrode modifier is dispersed or dissolved is prepared, and the solution is dropped onto a predetermined portion of the substrate (where the metal thin film is formed, if the substrate is an insulating substrate on which a metal thin film is formed), and then dried, thereby loading the modifier. The dispersion medium or solvent is not particularly limited, and examples thereof include water, alcohol-based solvents (e.g., ethanol), ketone-based solvents (e.g., acetone), and combinations thereof.

In one embodiment, it is preferable to add a dispersant to the dispersion liquid in which the counter electrode modifier (particularly porous carbon) is dispersed. The mixing ratio of the dispersant is arbitrary, but it is preferable to mix it at 0.2 to 2% (w/v), for example. The mixing ratio of the porous carbon is also arbitrary, but it is preferable to mix it at 0.05 to 0.5% (w/v), for example.

In one embodiment, the counter electrode modifier may be immobilized on the substrate. The immobilization can be performed by appropriately selecting a known method. For example, the counter electrode modifier can be immobilized by dropping a liquid in which a substance suitable for immobilization, such as tetrafluoroethylene/perfluoro[2-(fluorosulfonylethoxy)polyvinyl ether] copolymer (e.g., Nafion (trademark)) and carboxymethyl cellulose, is dissolved, onto the portion of the substrate on which the counter electrode modifier is loaded, and then drying. In one embodiment, after the counter electrode modifier is loaded onto the substrate, it is preferable to treat the substrate with a polymer substance such as carboxymethyl cellulose so as to cover the substances.

The surface area of the counter electrode is not particularly limited. In one embodiment, in order to amplify the current value due to electron transfer between the enzyme and the working electrode by the nanocarbon, the surface area of the counter electrode is preferably equal to or greater than the surface area of the working electrode. The lower limit of the surface area of the counter electrode is preferably 1, 1.5, 2, 2.5, or 3 times the surface area of the working electrode. The upper limit of the surface area of the counter electrode is not particularly limited, but is, for example, 200, 150, or 100 times the surface area of the working electrode. The lower and upper limits can be combined in any manner.

In an open system, the counter electrode may be immersed in a solvent. Examples of the solvent include the same solvents as those exemplified for immersing the working electrode. The counter electrode and working electrode may be immersed in the same solvent.

The sensor (particularly the electrode layer) may further include a reference electrode. The reference electrode can be used as a reference for the potential when measuring the electrode potential. In a closed system, a detection electrode for detecting that a sample has been supplied to the cavity may be included. The reference electrode and the detection electrode may be made of the same material as the working electrode and the counter electrode (precious metals such as platinum, gold, and palladium, copper, aluminum, nickel, titanium, ITO, ZnO, carbon, etc.). In one embodiment, the reference electrode may be a silver-silver chloride electrode. Even if the sensor does not include a reference electrode, electron transfer can be detected or measured using two electrodes, the working electrode and the counter electrode, so it is preferable that the sensor does not include a reference electrode in terms of simplifying the configuration.

The sensor may further include components that are typically included in biosensors, such as a potentiostat and a current detection circuit. The specific components of the working electrode, counter electrode, potentiostat, current detection circuit, etc. may be any as long as the sensor is capable of performing the intended measurement, and may be appropriately selected and designed from means known in the art.

In step A, the method of introducing the sample into the sensor can be appropriately selected depending on the configuration of the sensor. In an open system, for example, the sample is added to a solvent in which the electrode is immersed, and in a closed system, for example, the sample is brought into contact with the cavity (particularly the tip or inlet).

In step B, applying a voltage to the sensor may be referred to as "conditioning". The voltage applied to the sensor is not particularly limited and may be, for example, 0.8 V or more, but even if it is 0.8 V or less, a sufficient effect may be obtained. The applied voltage is preferably 0.7 V or less, 0.6 V or less, 0.5 V or less, or 0.4 V or less. The applied voltage is, for example, more than 0 V, 0.01 V or more, 0.05 V or more, 0.08 V or more, 0.1 V or more, or 0.15 V or more. The lower limit and upper limit can be combined arbitrarily. In addition, when the sensor includes a reference electrode, the applied voltage is, for example, 0.15 V or more, preferably 0.2 V or more, more preferably 0.3 V or more, and even more preferably 0.4 V or more.

In step B, the time for which the voltage is applied to the sensor is not particularly limited and may be, for example, 1 second or more, but a sufficient effect can be achieved with a time of 1 second or less. The application time is preferably 1 millisecond or more, and any application time can be selected within the range of 1 millisecond to 1 second.

In step C, the electrochemical measurement of the reaction between the substrate and the enzyme in the sample is preferably a measurement of the current based on the reaction when a predetermined voltage is applied. The applied voltage in step C is preferably lower than the applied voltage in step B, and is preferably 0.4 V or less, 0.3 V or less, 0.2 V or less, or 0.1 V or less. The applied voltage in step C is preferably -0.8 V or more, -0.6 V or more, or -0.4 V or more. Examples of voltage-current measurement methods include, but are not limited to, the step method (chronoamperometry, etc.) and the sweep method (cyclic voltammetry, etc.).

The method for measuring a substrate in a sample may include any further step in addition to steps A, B, and C. For example, when measuring a substrate in a sample two or more times, step B can be omitted from the second and subsequent measurements, and therefore the method may include step D in which only step C is repeated.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these.

[Example 1]
An electrode chip with two electrode sites was designed using a gold-deposited sheet on a PET substrate (Figure 1). In Figure 1, "1" is the gold-deposited PET sheet, "2" is the adhesive sheet (0.03 mm thick), "3" is the PET sheet (0.19 mm thick), "4" is the PET sheet, "5" is the working electrode site (1.5 mm ² ), and "6" is the counter electrode site (4.5 mm ² ).

The working electrode and counter electrode were created by bonding together the components "1" to "3" in Figure 1.

0.21 μL (0.31 μg) of an aqueous dispersion containing 2% (w/v) sodium cholate and 0.15% (w/v) single-walled carbon nanotubes (Meijo eDIPS EC1.5, Meijo Nano Carbon Co., Ltd., central diameter 1-3 nm) was mixed with 0.03 μL (0.5 U) of FADGDH (having the amino acid sequence of SEQ ID NO: 2; 16.6 U/μL) dissolved in ultrapure water. 20 mg of thymol was dissolved in 2000 μL of 50% (v/v) ethanol to prepare a 1% (w/v) thymol solution, and then 100 μL of the thymol solution was added to 900 μL of pure water and mixed to prepare a 0.1% (w/v) thymol solution. By adding and mixing 0.18 μL (0.18 μg) of 0.1% (w/v) thymol solution to the above single-walled carbon nanotube and FADGDH mixture, a working electrode reagent (0.42 μL per chip) containing a mixture of single-walled carbon nanotubes, FADGDH, and thymol was prepared, which was then dropped onto the working electrode portion "5" in Figure 1 and dried, thereby immobilizing the single-walled carbon nanotubes, FADGDH, and thymol on the working electrode portion.

An aqueous dispersion containing 10% (w/v) activated carbon powder (UNP, Universal Engineering, Inc., average particle size 0.3 μm measured by laser diffraction scattering method, specific surface area 1355 ^m2 /g) having a particle size of 0.3 μm was prepared, and 2 μL (200 μg) of the aqueous dispersion was mixed with 0.5 μL (25 μg) of 5% (w/v) Nafion™ solution (equivalent amount per chip) to produce a counter electrode reagent (2.5 μL per chip) containing the activated carbon powder and Nafion™. This was dropped onto the counter electrode site "6" in Figure 1 and dried to immobilize the activated carbon powder on the counter electrode site.

By attaching a PET sheet ("4" in Figure 1) to the above electrode chip, a 2.25 μL cavity was formed on the working electrode and counter electrode, and a two-electrode closed system chip was created in which a specified amount of sample could be injected by capillary action by contacting a droplet of sample.

The working and counter electrodes of the electrode chip prepared above were set on the working and counter electrodes of an electrochemical analyzer (ALS/CHI 660B, BAS Corporation), respectively. A 40 mM sodium phosphate buffer solution (pH 7.4) containing no glucose (0 mg/dL) or 100 mg/dL glucose was prepared, and a droplet of this sample was placed in contact with the electrode chip and injected into the cavity. Measurements were then performed using the linear sweep voltammetry method to examine the current-voltage characteristics. Two consecutive measurements were performed for each glucose sample.

The results of the first measurement of each glucose sample are shown in Figure 2. Compared to the case of 0 mg/dL glucose (Figure 2(a)), a glucose response current was detected in the presence of 100 mg/dL glucose, and the minimum applied voltage at which the glucose response current was detected was -0.2 V (Figure 2(b)). In the results of the second measurement of each glucose sample (Figure 3), compared to the case of 0 mg/dL glucose (Figure 3(a)), a glucose response current was detected in the presence of 100 mg/dL glucose, and the minimum applied voltage at which the glucose response current was detected was -0.4 V, which was 0.2 V smaller than the minimum applied voltage in the first measurement (Figure 3(b)). This shift in the minimum applied voltage is thought to be due to the rearrangement of the substances involved in electron transfer, namely FADGDH, thymol, single-walled carbon nanotubes, and interactions between substances at the electrode, caused by the voltage application in the first measurement.

[Example 2]
As in Example 1, a two-electrode closed system chip and a glucose sample (40 mM sodium phosphate buffer (pH 7.4) containing 100 mg/dL glucose) were prepared, and measurements were performed by the chronoamperometry method. In order to reduce the reaction of substances that affect the accuracy of glucose measurements, such as ascorbic acid and 2-pyridinealdoxime methiodide (PAM) in blood, a low measurement voltage is preferable. From the results of FIG. 3, measurements at a constant voltage by chronoamperometry were performed at −0.1 V. The number of measurements was limited to one per electrode chip. When measuring at −0.1 V, when the voltage application conditions before the start of measurement were 0 V and 1 millisecond, no glucose response current was detected (FIG. 4(a)). On the other hand, when the voltage application conditions before the start of measurement were 0.8 V and 1 second, a glucose response current was detected (FIG. 4(b)). From these results, it is considered that the application of voltage under specific conditions before the start of measurement also caused rearrangement of interactions between substances involved in electron transfer in the chronoamperometry method.

Voltage was applied under multiple conditions before the start of the measurement. Measurements were performed at -0.1 V. The current values 10 seconds after the start of the measurement were as shown in Table 1.

From the results in Table 1, it was found that conditioning in the chronoamperometry method is effective at an applied voltage of 0.1 V to 0.8 V, preferably 0.2 V to 0.8 V, and more preferably 0.3 V to 0.8 V, regardless of whether the voltage application time is 1 millisecond, 0.1 second, or 1 second, and a glucose response current is detected. Furthermore, since a glucose response current is detected under conditioning conditions of an applied voltage of 0.1 V or more and a voltage application time of 1 millisecond or more, it can be said that there is a conditioning effect even under conditions of an applied voltage of 0.8 V or more and a voltage application time of 1 second or more.

[Example 3]
An electrode chip with three electrode sites was designed using a gold-deposited sheet on a PET substrate (Figure 5). In Figure 5, "1" is the gold-deposited PET sheet, "2" is the adhesive sheet (0.03 mm thick), "3" is the PET sheet (0.19 mm thick), "4" is the PET sheet, "5" is the working electrode site (1.5 mm ² ), "6" is the counter electrode site (1.5 mm ² ), and "7" is the reference electrode site (1.5 mm ² ).

The working electrode, counter electrode, and reference electrode were created by bonding together the components "1" to "3" in Figure 5.

The reagent used on the working electrode was prepared in the same manner as in Example 1, dropped onto the working electrode, and dried to immobilize single-walled carbon nanotubes, FADGDH, and thymol. No reagent was used on the counter electrode or reference electrode.

By attaching a PET sheet ("4" in Figure 5) to the above electrode chip, a 2.25 μL cavity was formed on the working electrode, counter electrode, and reference electrode, and a three-electrode closed system chip was created that can inject a specified amount of sample by capillary action by contacting a droplet of sample.

The working electrode, counter electrode, and reference electrode of the electrode chip prepared above were set on the working electrode, counter electrode, and reference electrode of an electrochemical analyzer (ALS/CHI 660B, BAS Corporation), respectively. Droplets of 0, 100, 300, or 600 mg/dL glucose sample dissolved in 50 mM sodium phosphate buffer (pH 7.4) were brought into contact with the electrode chip and injected into the cavity, and measurements were performed using the linear sweep voltammetry method. Two consecutive measurements were performed for each glucose sample.

The results of the first measurement of each glucose sample are shown in Figure 6. Compared to the case of 0 mg/dL glucose (Figure 6(a)), a glucose response current was detected in the presence of 100 to 600 mg/dL glucose (Figures 6(b) to (d)), and the minimum applied voltage at which the glucose response current was detected was 0.15 V. In the results of the second measurement of each glucose sample (Figure 7), a response current was detected in the presence of glucose compared to the case of 0 mg/dL glucose (Figure 7(a)), and the minimum applied voltage at which the glucose response current was detected was -0.2 V, which was 0.35 V lower than the minimum applied voltage in the first measurement. As with the two-electrode closed system chip of Example 1, it is thought that the voltage application in the first measurement also caused rearrangement of the substances involved in electron transfer, i.e., FADGDH, thymol, single-walled carbon nanotubes, and interactions between substances in the electrodes in the three-electrode closed system chip.

[Example 4]
A three-electrode closed chip was prepared in the same manner as in Example 3, and measurements were performed by the chronoamperometry method. From the results of FIG. 7, measurements at a constant voltage by chronoamperometry were performed at 0.1 V. The number of measurements was limited to one per electrode chip. The glucose concentration dissolved in 50 mM sodium phosphate buffer (pH 7.4) was prepared at 0 to 750 mg/dL. When measuring at 0.1 V, when the voltage application conditions before the start of the measurement were 0 V and 1 millisecond, no glucose response current was detected (FIG. 8). On the other hand, when the voltage application conditions before the start of the measurement were 0.4 V and 1 millisecond, a glucose concentration-dependent response current was detected (FIG. 9). As a result of plotting the current values 1 second and 5 seconds after the start of the measurement, a current value corresponding to the glucose concentration was obtained at 1 second or more (FIG. 10).

[Example 5]
A three-electrode closed system chip was prepared in the same manner as in Example 3, except that silver-silver chloride ink (BAS) was applied to the reference electrode and dried, and measurements were performed by the chronoamperometry method. The voltage application conditions before the start of the measurement were 0.4 V, 1 millisecond, and the measurement at a constant voltage was 0.05 V. The number of measurements was only one per electrode chip. The glucose concentration dissolved in 50 mM sodium phosphate buffer (pH 7.4) was adjusted to 0 to 750 mg/dL. The current values 1 second, 2 seconds, and 5 seconds after the start of the measurement were plotted, and a current value corresponding to the glucose concentration was obtained at 1 second or more (FIG. 11).

The results of Examples 4 and 5 demonstrated that, similar to the two-electrode closed-system chip of Example 2, a conditioning effect can be obtained by applying a voltage before starting measurement in the chronoamperometry method using a three-electrode closed-system chip.

[Comparative Example]
An electrode chip with two electrode sites was fabricated using a gold-deposited sheet on a PET substrate (Figure 12). In Figure 12, "1" is the gold-deposited PET sheet, "2" is a PET sheet (0.1 mm thick) with adhesive sheets on both sides, "3" is the PET sheet, "4" is the working electrode site (2.3 ^mm2 ), and "5" is the counter electrode site (2.3 ^mm2 ).

The working electrode and counter electrode were created by bonding together the components "1" and "2" in Figure 12.

FADGDH (having the amino acid sequence of SEQ ID NO:2; 5.4 U/μL) was prepared by dissolving in ultrapure water, and 0.5 μL (2.7 U) was dropped onto the working electrode site "4" in Figure 12 and allowed to dry. Potassium ferricyanide, an oxidation-reduction mediator, was dissolved in ultrapure water to prepare a 134 mM solution, and 0.5 μL was dropped onto this working electrode site and allowed to dry. No reagent was used on the counter electrode site.

By attaching a PET sheet ("3" in Figure 12) to the above electrode chip, a 0.9 μL cavity was formed on the working electrode and counter electrode, and a two-electrode closed system chip was created that can inject a specified amount of sample by capillary action by contacting a droplet of sample.

As in Example 1, two consecutive measurements were performed for each glucose sample using the linear sweep voltammetry method. The results of the first measurement for each glucose sample are shown in Figure 13. Compared to the case of 0 mg/dL glucose (Figure 13(a)), a glucose response current was detected in the presence of 100 mg/dL glucose, and the minimum applied voltage at which the glucose response current was detected was -0.1 V (Figure 13(b)). In the results of the second measurement for each glucose sample (Figure 14), a response current was detected in the presence of 100 mg/dL glucose, and the minimum applied voltage at which the glucose response current was detected was -0.03 V (Figure 14(b)), compared to the case of 0 mg/dL glucose (Figure 14(a)).

In FIG. 14(b), a negative current value was measured at -0.2 to -0.04 V, but this is not considered to be a response current associated with the oxidation reaction of glucose. That is, in the first measurement in the presence of 100 mg/dL glucose, glucose is oxidized by the enzyme reaction of FADGDH at the working electrode site and reduced potassium ferricyanide is generated, which is oxidized by the working electrode site and electrons are transferred to the electrode, resulting in a positive current value being measured as the response current of glucose. On the other hand, a reduction reaction occurs at the counter electrode site, and reduced potassium ferricyanide is generated. In the second measurement, a reverse reaction occurs in the low applied voltage range of -0.2 to -0.04 V, that is, the reduced mediator generated by the first measurement is oxidized at the counter electrode site, and reduced potassium ferricyanide is generated at the other working electrode site by transferring electrons from the electrode to potassium ferricyanide, which is considered to be the reason why the reduction current is measured as a negative current value.

Therefore, when potassium ferricyanide, a redox mediator, was used, the minimum applied voltage at which a glucose-responsive current was detected was not lower in the second measurement than in the first measurement, and the conditioning effect seen in Examples 1 to 5 was not observed.

Claims (12)

1. A method for measuring a substrate in a sample, comprising the steps of:
A step A of introducing a sample into a sensor including a working electrode including a nanocarbon having a compound having an aromatic ring skeleton attached thereto or adjacent thereto and an enzyme, and a counter electrode;
The method includes step B of applying a voltage to the sensor to which a sample has been introduced, and step C of electrochemically measuring a reaction between a substrate and an enzyme in the sample using the sensor to which the voltage has been applied. The method according to claim 1, wherein step B is a step of applying a voltage of 0.8 V or less for 1 second or less. The method according to claim 1, wherein step B is a step of applying a voltage of 0.1 V to 0.4 V for 1 millisecond to 1 second. The method according to claim 2, wherein step C is a step of measuring the current based on the reaction at a voltage lower than the applied voltage of step B. The method according to claim 3, wherein step C is a step of measuring a current based on the reaction at a voltage lower than the applied voltage of step B. The method of claim 1, wherein step C is a step of measuring a current based on the reaction at a voltage of 0.1 V or less. The method of claim 1, wherein the nanocarbon is a carbon nanotube. The method of claim 1, wherein the enzyme is flavin-binding glucose dehydrogenase. Compounds having an aromatic ring skeleton include thymol, phenol, bis(4-hydroxyphenyl)sulfone, tyrosine disodium hydrate, sodium salicylate, toluene, 5-hydroxyindole, aniline, leucoquinizarin, carvacrol, 1,5-naphthalenediol, 4-isopropyl-3-methylphenol, 2-isopropylphenol, 4-isopropylphenol, 1-naphthol, 2-tert-butyl-5-methylphenol, 2,4,6-trimethylphenol, 2,6-diisopropylphenol, 2-tert The method according to claim 1, wherein the phenol is at least one selected from the group consisting of 2-tert-butyl-4-ethylphenol, 6-tert-butyl-2,4-xylenol, 2-tert-butyl-4-methylphenol, 2-tert-butyl-6-methylphenol, 2,4-di-tert-butylphenol, 2,4-di-tert-butyl-5-methylphenol, bis(p-hydroxyphenyl)methane, 3-tert-butylphenol, 2-isopropyl-5-methylanisole, o-cresol, m-cresol, and p-cresol. The method of claim 1, wherein the working electrode further comprises a dispersant. The method of claim 1, wherein the counter electrode comprises at least one material selected from the group consisting of porous carbon and metal oxides. The method of claim 1, wherein the sensor further includes a reference electrode.

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