WO2022025266A1 - Modificateur de contre-électrode - Google Patents

Modificateur de contre-électrode Download PDF

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Publication number
WO2022025266A1
WO2022025266A1 PCT/JP2021/028370 JP2021028370W WO2022025266A1 WO 2022025266 A1 WO2022025266 A1 WO 2022025266A1 JP 2021028370 W JP2021028370 W JP 2021028370W WO 2022025266 A1 WO2022025266 A1 WO 2022025266A1
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Prior art keywords
counter electrode
modifier
tert
aromatic ring
nanocarbon
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PCT/JP2021/028370
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English (en)
Japanese (ja)
Inventor
尚▲徳▼ 岩佐
勝巳 辻
圭三 米田
淳典 平塚
丈士 田中
仁志 六車
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東洋紡株式会社
国立研究開発法人産業技術総合研究所
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Priority claimed from JP2020130238A external-priority patent/JP7492707B2/ja
Priority claimed from JP2020130242A external-priority patent/JP7492708B2/ja
Application filed by 東洋紡株式会社, 国立研究開発法人産業技術総合研究所 filed Critical 東洋紡株式会社
Publication of WO2022025266A1 publication Critical patent/WO2022025266A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements

Definitions

  • nanocarbon Since nanocarbon has high electrical conductivity, its application as a conductive material for electron transfer with other substances is progressing. For example, it has been proposed to mix nanocarbon with an ink composed of carbon, a resin and an organic solvent, print it on a substrate, and use it as an electrode for a biosensor (Patent Document 1).
  • carbon nanotubes which are a type of nanocarbon, are used for sensors that measure peroxides (Patent Document 2), or they are formed into a film together with enzymes and used as electrodes for sensors and fuel cells (Patent Document 2). Patent Document 3). It has also been reported that the use of single-walled carbon nanotubes causes electron transfer from the enzyme directly to the electrode (Non-Patent Document 1).
  • FADGDH flavin adenine dinucleotide-supplemented glucose dehydrogenase
  • One issue is to provide a modifier for the counter electrode to the working electrode containing nanocarbon and an enzyme to which a compound having an aromatic ring skeleton is attached or adjacent to the compound.
  • the counter electrode to the working electrode containing nanocarbon and enzyme to which the compound having an aromatic ring skeleton is attached or adjacent is modified to contain metal oxide and / or porous carbon. It was found that it can be modified with an agent. Further studies based on such findings have led to the completion of the inventions represented by the following.
  • Item 1 A modifier containing a metal oxide and / or a porous carbon, which is a modifier for the opposite electrode to a working electrode containing nanocarbon and an enzyme to which a compound having an aromatic ring skeleton is attached or adjacent to the enzyme.
  • Item 2. Item 2. The modifier according to Item 1, wherein the metal oxide is a metal oxide insoluble in water and an organic solvent.
  • Item 3. Item 2. The modifier according to Item 1 or 2, wherein the metal oxide is at least one selected from the group consisting of Cu 2 O, CuO, Ag 2 O, MnO 2 , BaMnO 4 , OsO 4 , and PbO 2 .
  • Item 5. The modifier according to any one of Items 1 to 4, wherein the porous carbon has a particle size of 200 ⁇ m or less.
  • Item 6. The modifier according to any one of Items 1 to 5, wherein the average particle size of the porous carbon is 150 ⁇ m or less.
  • Compounds with an aromatic ring skeleton include timol, phenol, bis (4-hydroxyphenyl) sulfone, tyrosine disodium hydrate, sodium salicylate, toluene, 5-hydroxyindole, aniline, leukokinizarin, carbachlor, 1,5-naphthalene.
  • Diol 4-isopropyl-3-methylphenol, 2-isopropylphenol, 4-isopropylphenol, 1-naphthol, 2-tert-butyl-5-methylphenol, 2,4,6-trimethylphenol, 2,6-diisopropyl Phenol, 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, Item 6.
  • Item 8. Item 6. The modifier according to any one of Items 1 to 7, wherein the nanocarbon is a carbon nanotube.
  • Item 9. Item 6.
  • Item 10. A counter electrode containing a modifier according to any one of Items 1 to 9, which is a counter electrode to a working electrode containing an enzyme and nanocarbon to which a compound having an aromatic ring skeleton is attached or adjacent.
  • Item 12. Item 12. The sensor according to Item 11, which does not include a reference electrode.
  • Item 13. Item 12. The sensor according to Item 11 or 12, wherein the surface area of the counter electrode is larger than the surface area of the working electrode.
  • the counter electrode to the working electrode containing the nanocarbon and the enzyme to which the compound having an aromatic ring skeleton is attached or adjacent is modified.
  • the structure of the electrode tip (working electrode tip) produced in the examples is shown.
  • “1” is a PET film
  • “2” is an adhesive sheet
  • "3” is a gold-deposited PET film
  • “4" is an electrode portion (working electrode portion).
  • the structure of the counter electrode chip produced in Example 1 is shown.
  • “5” is a PET film
  • “6” is an adhesive sheet
  • “7” is a gold-deposited PET film
  • “8” indicates a counter electrode portion.
  • the chronoamperogram measured by placing silver oxide on the counter electrode site is shown.
  • the chronoamperogram measured by placing manganese dioxide on the counter electrode site is shown.
  • the chronoamperogram measured by placing barium manganate on the counter electrode site is shown.
  • Comparative Example 1 the chronoamperogram measured by placing silver sulfide on the counter electrode site is shown. In Comparative Example 1, the chronoamperogram measured by placing only the carbon paste on the counter electrode portion is shown. In Comparative Example 1, the chronoamperogram measured without placing the compound and the carbon paste on the counter electrode site is shown. In Example 2, the chronoamperogram measured by placing activated carbon on the counter electrode site is shown. In Example 2, the chronoamperogram measured by placing Knobel TM on the counter electrode site is shown. In Example 2, the chronoamperogram measured without placing the porous carbon on the counter electrode site is shown.
  • the antipolar modifier is preferably a counterpolar modifier for the working electrode containing nanocarbon and an enzyme to which a compound having an aromatic ring skeleton is attached or adjacent.
  • the counter electrode is preferably the counter electrode described in 2 below, and the working electrode is preferably the working pole described in 3 below.
  • the modifier of the counter electrode preferably contains a metal oxide and / or a porous carbon.
  • the metal oxide is not particularly limited, and examples thereof include metal oxides that are insoluble in water and / or an organic solvent.
  • insoluble is used to include not only the case where it is not completely dissolved but also the case where it is slightly dissolved (for example, the solubility at room temperature is 0.0001 g / mL or less).
  • Examples of such metal oxides include copper oxides, silver oxides, manganese oxides, osmium oxides, and lead oxides.
  • Examples of the copper oxide include Cu 2 O and Cu O
  • examples of the silver oxide include Ag 2 O
  • examples of the manganese oxide include MnO 2 and BaMnO 4
  • examples of the oxide include OsO 4 .
  • Examples of the lead oxide include PbO 2 .
  • the metal oxide may be used alone or in combination of two or more.
  • As the metal oxide at least one selected from the group consisting of silver oxide and manganese oxide is preferable, and at least one selected from the group consisting of Ag 2O , MnO 2 and BaMnO 4 is more preferable.
  • the type of porous carbon is not particularly limited.
  • the porous carbon include activated carbon, carbon nanotubes, and porous carbon using an oxide as a template.
  • the carbon nanotubes may be single-walled carbon nanotubes, double-walled carbon nanotubes, or multi-walled carbon nanotubes.
  • Examples of the porous carbon using an oxide as a template include porous carbon using magnesium oxide as a template (for example, porous carbon manufactured by Toyo Carbon Co., Ltd., trade name: Knobel TM), and porous carbon using zeolite as a template. Examples thereof include quality carbon and porous carbon using alumina as a 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 arbitrarily combined.
  • the larger the specific surface area the more the reduction reaction that transfers 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, from the adsorption isotherm of nitrogen molecules at a liquid nitrogen temperature (77K) by the method of Brunar-Emmett-Teller (BET).
  • the porous carbon is a powder or granular material.
  • the particle size or average particle size of the porous carbon is not particularly limited.
  • the upper limit of the particle size or the average particle size is, for example, 200 ⁇ m, preferably 180 ⁇ m, and more preferably 150 ⁇ m.
  • the upper limit of the average particle size is preferably 100 ⁇ m, 80 ⁇ m, 50 ⁇ m, 30 ⁇ m, or 10 ⁇ m.
  • the lower limit of the particle size or the 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 arbitrarily combined.
  • the particle size or the average particle size can be measured by, for example, a mesh method or a laser diffraction / scattering method.
  • the counter electrode is preferably the counter electrode to the working electrode containing the enzyme and nanocarbon to which the compound having an aromatic ring skeleton is attached or adjacent.
  • the counter electrode preferably contains a modifier for the counter electrode, and the modifier for the counter electrode is preferably the modifier according to 1 above.
  • the working electrode is preferably the working pole described in 3 below.
  • the counter electrode may further contain a dispersant.
  • the dispersant is preferably combined with porous carbon, and the dispersant may be a substance capable of suppressing and dispersing the aggregation of the porous carbon.
  • the dispersant is not particularly limited, and examples thereof include sodium cholic acid, sodium deoxycholate, sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, cetyltrimethylammonium bromide, and octylphenol ethoxylate.
  • the dispersant may be used alone or in combination of two or more. In one embodiment, preferred dispersants are sodium cholic acid, 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 linear shape, a coil shape, a net shape, a rod shape, a film shape, or a plate shape.
  • the counter electrode preferably has a substrate suitable for an electrode, and the substrate is preferably one in which a metal film (for example, a metal thin film) is formed on an insulating substrate.
  • a metal film for example, a metal thin film
  • the insulating substrate for example, a glass substrate or a plastic substrate (for example, a PET substrate) can be used.
  • the type of metal forming the metal film is not particularly limited as long as it is used for the electrode, and examples thereof include gold, platinum, and titanium.
  • the counter electrode is formed with a carbon film (for example, a carbon film made of carbon paste, preferably a carbon thin film) formed in place of the metal film or at least a part thereof on the metal film.
  • a carbon film for example, a carbon film made of carbon paste, preferably a carbon thin film
  • the carbon paste preferably contains a counter electrode modifier (eg, metal oxide) in addition to the components used for the electrodes, preferably a carbon, a binder, and a counter electrode modifier (eg, metal oxide). Is preferably contained.
  • Examples of carbon include graphite and carbon black.
  • Examples of the binder include a hydrocarbon binder, an alcohol binder, and an ester binder.
  • Examples of the hydrocarbon binder include heptane, octane, nonane, decane, undecane, dodecane, tridecane, liquid paraffin, and squalane.
  • Examples of the alcohol binder include oleyl alcohol and stearyl alcohol.
  • Examples of the ester binder include phthalates such as dioctyl phthalate. The carbon and the binder may be used alone or in combination of two or more.
  • the content of the binder 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 further preferably 20 parts by mass with respect to 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 more preferably 200 parts by mass with respect to 100 parts by mass of carbon. The lower limit and the upper limit can be arbitrarily combined.
  • the content of the counter electrode modifier in the carbon paste is also 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 further preferably 20 parts by mass with respect to 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 more preferably 200 parts by mass with respect to 100 parts by mass of carbon. The lower limit and the upper limit can be arbitrarily combined.
  • the content (or loading capacity) of the counter electrode modifier per 100 parts by mass of carbon in the counter electrode may 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. can.
  • the counter electrode is a substrate on which a modifier for the counter electrode (for example, porous carbon) is loaded.
  • a modifier for the counter electrode for example, porous carbon
  • the load capacity of the modifier of the counter electrode is not particularly limited.
  • the loading method of the modifier of the opposite electrode is not particularly limited.
  • a solution in which a modifier for counter electrode (for example, porous carbon) is dispersed or dissolved is prepared and a predetermined portion on the substrate (the substrate is a metal thin film formed on an insulating substrate), It can be loaded by dropping it on a place where a metal thin film is formed) and drying it.
  • the dispersion medium or solvent is not particularly limited, and examples thereof include water, an alcohol-based solvent (for example, ethanol), a ketone-based solvent (for example, acetone), and a combination thereof.
  • the dispersant it is preferable to add the dispersant to the dispersion liquid in which the modifier of the opposite electrode (for example, porous carbon) is dispersed.
  • the mixing ratio of the dispersant is arbitrary, but for example, it is preferably 0.2 to 2% (w / v).
  • the mixing ratio of the porous carbon is also arbitrary, but it is preferable to add 0.05 to 0.5% (w / v), for example.
  • the counter electrode modifier (eg, porous carbon) may be immobilized on the substrate. Immobilization can be carried out by appropriately selecting a known method. For example, a liquid in which a substance suitable for immobilization such as a tetrafluoroethylene / perfluoro [2- (fluorosulfonylethoxy) polyvinyl ether] copolymer (eg, Nafion TM) and carboxylmethyl cellulose is dissolved is placed on the substrate. It can be immobilized by dropping the modifier of the opposite electrode on the loaded portion and drying it. In one embodiment, it is preferable that the modifier for the counter electrode is loaded on the substrate and then treated with a polymer substance such as carboxylmethyl cellulose so as to cover these substances.
  • a substance suitable for immobilization such as a tetrafluoroethylene / perfluoro [2- (fluorosulfonylethoxy) polyvinyl ether] copolymer (eg, Nafion TM) and carb
  • the surface area of the counter electrode is not particularly limited. In one embodiment, the surface area of the counter electrode is preferably larger than the surface area of the working electrode from the viewpoint of amplifying the current value due to electron transfer between the enzyme and the working electrode by nanocarbon.
  • the lower limit of the surface area of the counter electrode is preferably 1.5 times, 2 times, 2.5 times, 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 times, 150 times, or 100 times the surface area of the working electrode. The lower limit and the upper limit can be arbitrarily combined.
  • the senor preferably comprises a working electrode containing an enzyme and a nanocarbon to which a compound having an aromatic ring skeleton is attached or adjacent to the sensor, and a counter electrode.
  • the counter electrode is preferably the counter electrode according to 2 above.
  • the enzyme preferably releases electrons by a catalytic reaction.
  • enzymes include redox enzymes.
  • the oxidoreductase include glucose dehydrogenase, glucose oxidase, lactic acid oxidase, cholesterol oxidase, alcohol oxidase, sarcosine oxidase, fructosylamine oxidase, pyruvate oxidase, lactic acid dehydrogenase, alcohol dehydrogenase, glycerol oxidase, and glycerol-3-lin.
  • acid oxidase uricase, choline oxidase, xanthin oxidase, and hydroxybutyric acid dehydrogenase.
  • the enzyme is preferably glucose dehydrogenase, preferably flavin-bound glucose dehydrogenase, and preferably glucose dehydrogenase (also referred to as "FADGDH") with flavin adenine dinucleotide (FAD) as a coenzyme. .. Since FADGDH holds FAD in the depression of the three-dimensional structure formed by the polypeptide, a substance called a mediator is conventionally required to transfer the electrons generated there to the working electrode. On the other hand, by using nanocarbon, it is possible to transfer electrons to the working electrode without using a mediator. Further, by using a compound having an aromatic ring skeleton, electron transfer via nanocarbon can be performed remarkably efficiently (or strongly).
  • FADGDH The type of FADGDH is not limited, and any one can be used. Specific examples of FADGDH include those derived from one of the following organisms: Aspergillus teres, Aspergillus oryzae, Alpergillus niger, Aspergillus foetidas, Alpergillus aureus, Aspergillus bargecolor, Aspergillus.
  • Preferred FADGDHs in one embodiment are FADGDH from Aspergillus oryzae, FADGDH from Mucor Hiemaris, FADGDH from Mucor subtilisimas, FADGDH from silcinella simplex, FADGDH from Metallydium sp. Yes, preferably it has 80% or more identity with the amino acid sequences of SEQ ID NOs: 1-6, more preferably 90% or more identity with the amino acid sequences of SEQ ID NOs: 1-6, and even more preferably. , which has 95% or more identity with the amino acid sequences of SEQ ID NOs: 1 to 6 and has glucose dehydrogenating activity.
  • the identity of the amino acid sequence can be calculated using commercially available or available analysis tools over the telecommunications line (Internet), for example, the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignmentment). searchtool) It can be calculated using the default (initial setting) parameters at http://www.ncbi.nlm.nih.gov/BLAST/.
  • 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 Hiemaris
  • the amino acid sequence of SEQ ID NO: 3 is that of Mucor.
  • the amino acid sequence of SEQ ID NO: 4 is that of FADGDH derived from silcinella simplex
  • the amino acid sequence of SEQ ID NO: 5 is that of FADGDH derived from Metallydium SP.
  • the amino acid sequence of is that of FADGDH derived from collettricham SP.
  • Nanocarbon is not particularly limited as long as it has an electron transfer function and is recognized as nanocarbon.
  • Such substances mean carbon materials composed primarily of carbon, including, for example, carbon nanotubes, carbon nanohorns, carbon nanotwists, cocoons, carbon nanocoils, graphene, fullerenes and the like.
  • the carbon nanotubes may be single-walled carbon nanotubes, double-walled carbon nanotubes, or multi-walled carbon nanotubes.
  • the nanocarbon is preferably a carbon nanotube, 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 in the aromatic ring skeleton is, for example, 5 to 18, preferably 5 to 16, and more preferably 5 to 14.
  • Aromatic ring skeletons include 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 benzene ring and another aromatic ring (nitrogen-containing aromatic ring).
  • Oxygen-containing aromatic ring, sulfur-containing aromatic ring, etc. and skeleton phenanthroline skeleton, benzofuran skeleton, benzoimidazole skeleton, carbazole skeleton, etc.), carbon and other elements (nitrogen, oxygen, sulfur, etc.)
  • skeleton consisting of an aromatic ring (triazine skeleton, triazole skeleton, pyridine skeleton, etc.) are included.
  • the compound having an aromatic ring skeleton is preferably a compound which does not have a function as a mediator by itself.
  • it does not function as a mediator by itself means that electron transfer is performed independently between an electrode and an enzyme or between an electrode and a substrate, such as benzoquinone and 1-methoxyphenazinemethsulfate. Means not have.
  • 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 the compound having an electron-donating substituent and an aromatic ring skeleton include a compound having a benzene ring substituted with a hydroxy group (eg, timol, phenol, bis (4-hydroxyphenyl) sulfone, tyrosine disodium hydrate, etc.
  • thymol phenol and carvacrol are preferable.
  • the means for adhering or bringing the compound having an aromatic ring skeleton to or close to the nanocarbon is not particularly limited.
  • it can be carried out by mixing the nanocarbon and the compound having an aromatic ring skeleton (including mixing in a solution), or by arranging the compound having an aromatic ring skeleton on the nanocarbon.
  • the compound having an aromatic ring skeleton arranged in close proximity to or attached to the nanocarbon may or may not be fixed. Immobilization is not limited as long as it does not inhibit the functions of the compound having a nanocarbon and an aromatic ring skeleton, and can be appropriately selected from known means and used.
  • the amount of the compound having an aromatic ring skeleton attached to or close to the nanocarbon in order to promote electron transfer is not particularly limited.
  • the amount of the compound having an aromatic ring skeleton is not particularly limited.
  • the amount of the compound having an aromatic ring skeleton is, for example, 0.001 part by mass or more, preferably 0.01 part by mass or more, and more preferably 0.1 part by mass or more with respect to 100 parts by mass of 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, and more preferably 1000 parts by mass or less with respect to 100 parts by mass of nanocarbon.
  • the lower limit and the upper limit can be arbitrarily combined.
  • the amount of the compound having an aromatic ring skeleton is, for example, 0.001 part by mass or more, preferably 0.01 part by mass or more, and more preferably 0.1 part by mass or more with respect to 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, and more preferably 10,000 parts by mass or less with respect to 100 parts by mass of the enzyme.
  • the lower limit and the upper limit can be arbitrarily combined.
  • the working electrode may further contain a dispersant.
  • the dispersant is not particularly limited as long as it is a substance capable of suppressing the aggregation of nanocarbons and dispersing them.
  • examples of the dispersant include sodium cholic acid, sodium deoxycholate, sodium dodecylsulfate, sodium dodecylbenzenesulfonate, cetyltrimethylammonium bromide, octylphenol ethoxylate and the like.
  • the dispersant may be used alone or in combination of two or more.
  • preferred dispersants are sodium cholic acid, 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 linear shape, a coil shape, a net shape, a rod shape, a film shape, or a plate shape.
  • the working electrode preferably has a substrate suitable for an electrode on which an enzyme used in a biosensor is immobilized, and the substrate has a metal film (for example, a metal thin film) formed on an insulating substrate.
  • the product is a product.
  • the insulating substrate for example, a glass substrate or a plastic substrate (for example, a PET substrate) can be used.
  • the type of metal forming the metal film is not particularly limited as long as it is used for the electrode, and examples thereof include gold, platinum, and titanium.
  • the substrate may be one in which a carbon film (for example, a carbon thin film made of carbon paste) is formed on the insulating substrate instead of the metal film.
  • the working electrode is preferably one in which nanocarbon, a compound having an aromatic ring skeleton, and an enzyme are loaded on a substrate.
  • the loading method of these substances is not particularly limited.
  • a solution in which each of these substances was dispersed or dissolved was prepared, and a metal thin film was sequentially formed at a predetermined portion on the substrate (when the substrate was a metal thin film formed on an insulating substrate). It can be loaded by repeating the operation of dropping it on a place) and drying it.
  • these can be mixed, dropped onto a predetermined portion on the substrate, and dried to be loaded.
  • the dispersion medium or solvent is not particularly limited, and examples thereof include water, an alcohol-based solvent (for example, ethanol), a ketone-based solvent (for example, acetone), and a combination thereof.
  • a dispersant to the dispersion liquid in which nanocarbon is dispersed.
  • the mixing ratio of the dispersant is arbitrary, but for example, it is preferably 0.2 to 2% (w / v).
  • the blending ratio of nanocarbon is also arbitrary, but it is preferable to blend 0.05 to 0.5% (w / v), for example.
  • the loading order is arbitrary, but in one embodiment, it is preferable to load in the order of nanocarbon ⁇ enzyme ⁇ compound having an aromatic ring skeleton or nanocarbon ⁇ compound having an aromatic ring skeleton ⁇ enzyme, and these are loaded at the same time. It is also preferable to load it.
  • the amount of nanocarbon, a compound having an aromatic ring skeleton, and an enzyme is not particularly limited.
  • the nanocarbon, the compound having an aromatic ring skeleton, and the enzyme may be immobilized on the substrate. Immobilization can be carried out by appropriately selecting a known method. For example, a liquid in which a substance suitable for immobilization such as a tetrafluoroethylene / perfluoro [2- (fluorosulfonylethoxy) polyvinyl ether] copolymer (eg, Nafion TM) and carboxylmethyl cellulose is dissolved is placed on the substrate.
  • a substance suitable for immobilization such as a tetrafluoroethylene / perfluoro [2- (fluorosulfonylethoxy) polyvinyl ether] copolymer (eg, Nafion TM) and carboxylmethyl cellulose is dissolved is placed on the substrate.
  • a substance suitable for immobilization such as a tetrafluoroethylene / perfluoro [2- (fluorosulfonylethoxy) polyvinyl
  • the senor further contains a solvent for immersing the working electrode and the counter electrode.
  • the working electrode is a substrate on which nanocarbon and an enzyme are loaded
  • the solvent (including the substance or substrate to be measured) in which the working electrode is immersed may contain a compound having an aromatic ring skeleton.
  • the solvent include a buffer solution, and examples thereof include an acetate buffer solution, a citrate buffer solution, a phosphate buffer solution, and a borolate buffer solution.
  • the concentration of the substance or substrate to be measured in the solvent is not particularly limited and can be set to any concentration required for measurement.
  • 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), and more preferably 0.00005%.
  • the upper limit of the concentration is, for example, 2% (w / v), preferably 1.5% (w / v), and more preferably 1% (w / v). The lower limit and the upper limit can be arbitrarily combined.
  • the sensor may include a reference electrode, but even if the sensor does not include a reference electrode, electron transfer can be detected or measured by the two electrodes of the working electrode and the counter electrode. It is preferable that the reference electrode is not included.
  • the sensor can further be equipped with a configuration normally provided by a biosensor such as a potentiometer and a current detection circuit.
  • a biosensor such as a potentiometer and a current detection circuit.
  • the specific configuration of the counter electrode, potentiometer, current detection circuit, etc. is arbitrary as long as the desired measurement can be performed by the sensor, and it is possible to appropriately select and design from means known in the art. can.
  • Example 1 Using a sheet in which gold was vapor-deposited on a PET substrate, an electrode chip having an electrode portion (working electrode portion) of 2.25 mm 2 was produced (FIG. 1).
  • FIG. 1 “1" is a PET film
  • “2” is an adhesive sheet
  • "3” is a gold-deposited PET film
  • "4" is an electrode portion (working electrode portion).
  • FIG. 2 Using a sheet in which gold was vapor-deposited on a PET substrate, an electrode chip having a counter electrode portion of 9 mm 2 was produced (FIG. 2).
  • "5" is a PET film
  • "6” is an adhesive sheet
  • "7” is a gold-deposited PET film
  • "8” is a counter electrode portion.
  • Graphite powder and liquid paraffin were mixed at a mass ratio of 1: 1 to prepare a carbon paste.
  • the prepared carbon paste and the following compounds (1) to (3) were mixed at a mass ratio of 2: 1 to prepare a counter electrode tip coated on the counter electrode site.
  • Silver oxide (2)
  • Manganese dioxide (3) Barium manganate
  • the working electrode tip and the counter electrode tip prepared above were set on the working electrode and the counter electrode of the electrochemical analyzer (ALS / CHI 660B, BAS Co., Ltd.), respectively. These two electrodes were immersed in 40 mM sodium phosphate buffer (pH 7.4). When glucose was not added (0 mM) to this buffer solution, or when glucose was added to 5 mM or 14 mM, measurement was performed by chronoamperometry, and an overvoltage of + 0.4 V was applied between the two electrodes. The current value was measured.
  • FIGS. 3 Chronoamperograms measured at glucose concentrations of 0 mM, 5 mM, and 14 mM are shown in FIGS. 3 (silver oxide), 4 (manganese dioxide), and 5 (barium manganate), respectively.
  • the current values 5 seconds after the start of measurement are as shown in Table 1 below.
  • FIGS. 6 Chronoamperograms measured at glucose concentrations of 0 mM, 5 mM, and 14 mM are shown in FIGS. 6 (silver sulfide), 7 (carbon paste only), and 8 (no carbon paste), respectively.
  • FIGS. 6 to 8 the current values 5 seconds after the start of measurement are shown in Table 2 below.
  • Example 1 From the results of Example 1 and Comparative Example 1, even under the condition that the glucose response current cannot be measured only by applying the carbon paste to the counter electrode portion, the glucose response current can be increased by adding silver oxide, manganese dioxide, or barium manganate. It turned out to be measured. On the other hand, it was found that the glucose response current was not measured when silver sulfide was added.
  • Example 2 Similar to Example 1, an electrode chip having an electrode portion of 2.25 mm 2 was produced using a sheet in which gold was vapor-deposited on a PET substrate (FIG. 1).
  • aqueous dispersion containing 15% (w / v) of the following porous carbon of (1) and an aqueous dispersion containing 1.5% (w / v) of the following porous carbon of (2) were prepared. .. (1) Activated carbon powder (Fuji Film Wako Pure Chemical Industries, Ltd., 031-18103, particle size 100 mesh passing) (2) Knobel TM powder (Toyo Tanso Co., Ltd., MJ (4) 010, specific surface area 1100 m 2 / g, particle size 100 mesh passage)
  • the working electrode tip and the counter electrode tip prepared above were set on the working electrode and the counter electrode of the electrochemical analyzer (ALS / CHI 660B, BAS Co., Ltd.), respectively. These two electrodes were immersed in 40 mM sodium phosphate buffer (pH 7.4). When glucose was not added (0 mM) to this buffer solution, or when glucose was added to 5 mM or 14 mM, measurement was performed by chronoamperometry, and an overvoltage of + 0.4 V was applied between the two electrodes. The current value was measured.
  • FIG. 9 activated carbon
  • FIG. 10 Kerbel TM
  • FIG. 11 without porous carbon
  • Example 3 Examples of the case where activated carbon powders having different particle sizes are used are shown below. 37.5 ⁇ L of aqueous dispersion containing 2% (w / v) sodium cholic acid and 0.15% (w / v) single-walled carbon nanotubes (SuperPureTubes, NanoIntegras, outer diameter 1.1-1.7 nm). And 5.4 ⁇ L of FADGDH (having the amino acid sequence of SEQ ID NO: 2; 60 U / ⁇ L) dissolved in ultrapure water was mixed.
  • a 1% (w / v) Timor solution dissolved in 50% (v / v) ethanol was diluted 10-fold with ultrapure water to prepare a 0.1% (w / v) Timor solution, and 32.4 ⁇ L of the solution was prepared.
  • 0.6 ⁇ L of the mixed solution was dropped onto the 2.25 mm 2 electrode site (working electrode site) shown in FIG. 1 and dried. After drying, 0.6 ⁇ L of 1% (w / v) Nafion solution was added dropwise and dried to prepare a working electrode chip on which carbon nanotubes, FADGDH, and thymol were immobilized.
  • An aqueous dispersion containing 10% (w / v) of the following activated carbon powders (1), (3) and (4) having different particle sizes was prepared, and an electrode portion (counter electrode portion) of 2.25 mm 2 shown in FIG. 1 was prepared. ) was dropped with 1.0 ⁇ L and dried. After drying, 0.6 ⁇ L of 1% (w / v) Nafion solution was added dropwise and dried to prepare a counter electrode tip in which activated carbon powder was immobilized on an electrode site.
  • the working electrode tip and the counter electrode tip prepared above were set on the working electrode and the counter electrode of the electrochemical analyzer (ALS / CHI 660B, BAS Co., Ltd.), respectively. These two electrodes were immersed in 40 mM sodium phosphate buffer (pH 7.4). Measurement by chronoamperometry was performed when glucose was not added (0 mM) or glucose was added to 14 mM in this buffer solution, and the current value when an overvoltage of + 0.4 V was applied between the two electrodes. Was measured.

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Abstract

L'invention concerne un modificateur de contre-électrode. Un modificateur qui contient un oxyde métallique et/ou un carbone poreux est utilisé, en tant que modificateur de contre-électrode, pour la réalisation d'une électrode de travail contenant une enzyme et des nanocarbones auxquels adhère ou à proximité desquels se trouve un composé ayant un squelette de cycle aromatique.
PCT/JP2021/028370 2020-07-31 2021-07-30 Modificateur de contre-électrode WO2022025266A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024101215A1 (fr) * 2022-11-11 2024-05-16 東洋紡株式会社 Procédé de mesure de matrice dans un échantillon

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JP2001318071A (ja) * 2000-03-02 2001-11-16 Fumiyo Kusunoki 潰瘍性大腸疾患測定電極、潰瘍性大腸疾患測定装置及び潰瘍性大腸疾患判定方法
WO2005088288A1 (fr) * 2004-03-10 2005-09-22 National Institute Of Advanced Industrial Science And Technology Biocapteur à nanotube de carbone
JP2013011495A (ja) * 2011-06-28 2013-01-17 Mitsui Eng & Shipbuild Co Ltd 生化学反応用炭素電極
JP2013190212A (ja) * 2012-03-12 2013-09-26 Dainippon Printing Co Ltd バイオセンサ及びその製造方法
JP2016188794A (ja) * 2015-03-30 2016-11-04 シーシーアイ株式会社 バイオセンサ
WO2018088391A1 (fr) * 2016-11-08 2018-05-17 Jsr株式会社 Capteur d'enzyme et système de capteur d'enzyme
JP2019138718A (ja) * 2018-02-08 2019-08-22 日東電工株式会社 センサ用電極材、センサ用電極、センサ、及びバイオセンサ

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Publication number Priority date Publication date Assignee Title
JP2001318071A (ja) * 2000-03-02 2001-11-16 Fumiyo Kusunoki 潰瘍性大腸疾患測定電極、潰瘍性大腸疾患測定装置及び潰瘍性大腸疾患判定方法
WO2005088288A1 (fr) * 2004-03-10 2005-09-22 National Institute Of Advanced Industrial Science And Technology Biocapteur à nanotube de carbone
JP2013011495A (ja) * 2011-06-28 2013-01-17 Mitsui Eng & Shipbuild Co Ltd 生化学反応用炭素電極
JP2013190212A (ja) * 2012-03-12 2013-09-26 Dainippon Printing Co Ltd バイオセンサ及びその製造方法
JP2016188794A (ja) * 2015-03-30 2016-11-04 シーシーアイ株式会社 バイオセンサ
WO2018088391A1 (fr) * 2016-11-08 2018-05-17 Jsr株式会社 Capteur d'enzyme et système de capteur d'enzyme
JP2019138718A (ja) * 2018-02-08 2019-08-22 日東電工株式会社 センサ用電極材、センサ用電極、センサ、及びバイオセンサ

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024101215A1 (fr) * 2022-11-11 2024-05-16 東洋紡株式会社 Procédé de mesure de matrice dans un échantillon

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