WO2024005025A1 - センサ及びその製造方法 - Google Patents

センサ及びその製造方法 Download PDF

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Publication number
WO2024005025A1
WO2024005025A1 PCT/JP2023/023842 JP2023023842W WO2024005025A1 WO 2024005025 A1 WO2024005025 A1 WO 2024005025A1 JP 2023023842 W JP2023023842 W JP 2023023842W WO 2024005025 A1 WO2024005025 A1 WO 2024005025A1
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Prior art keywords
sensor
insulating layer
electrode
working electrode
reagent
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Ceased
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PCT/JP2023/023842
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English (en)
French (fr)
Japanese (ja)
Inventor
哲也 則兼
圭吾 羽田
太郎 中野
健 畑山
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PHC Holdings Corp
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PHC Holdings Corp
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Application filed by PHC Holdings Corp filed Critical PHC Holdings Corp
Priority to CN202380044738.5A priority Critical patent/CN119301443A/zh
Priority to EP23831465.2A priority patent/EP4549921A4/en
Priority to US18/875,393 priority patent/US20260015647A1/en
Priority to JP2024530888A priority patent/JP7834172B2/ja
Publication of WO2024005025A1 publication Critical patent/WO2024005025A1/ja
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • 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
    • G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
    • G01N27/3272—Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue
    • A61B5/14532—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue for measuring glucose, e.g. by tissue impedance measurement
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes

Definitions

  • the first disclosure relates to, for example, a sensor and a method for manufacturing the same.
  • Electrochemical sensors have conventionally been used to measure target substances in test samples such as cell culture fluids and blood samples.
  • a sensor includes, for example, an insulating base material, a working electrode disposed on the surface of the base material, and further includes a counter electrode and/or a reference electrode.
  • the working electrode typically includes a conductive layer and a reagent layer disposed on the conductive layer and containing a reagent (for example, a redox enzyme and an electron carrier) that participates in a redox reaction.
  • the sensor may further include a protective film that covers only the working electrode or covers the working electrode together with the counter electrode and/or the reference electrode in order to prevent the reagent from flowing out from the reagent layer. .
  • the electrochemical sensor described in Patent Document 1 includes a base material, a first electrode (working electrode) and a second electrode (reference electrode) including a sensing layer (reagent layer) arranged on the base material, A flow-limiting membrane (protective membrane) containing a predetermined polymer compound is provided, covering the entirety thereof.
  • Patent Document 1 describes that the flow-limiting film (protective film) is formed by dip coating or casting.
  • an insulating base material including a working electrode including a conductive layer and a reagent layer is immersed in a solution containing a polymer compound, and dried to form a protective film that covers the entire insulating base material.
  • a reagent layer containing a reagent such as an enzyme is also arranged on the surface of a flat electrode.
  • An object of the first disclosure is to provide a sensor including a working electrode including a reagent layer and a protective film whose characteristics can be easily controlled within a desired range, and a method for manufacturing the same.
  • the sensor according to the first disclosure includes an insulating substrate and a working electrode disposed on the substrate.
  • the working electrode is a conductive layer disposed on the substrate; a first opening that penetrates through the thickness direction and is formed at a position at least partially on the conductive layer and overlaps a part of the conductive layer in a plan view from the thickness direction of the substrate; a first insulating layer having an aqueous surface; Penetration in the thickness direction is disposed on the first insulating layer and is formed at a position that overlaps with a portion of the first insulating layer that includes the entire first opening in a plan view from the thickness direction.
  • a second insulating layer having a second opening and a surface that is liquid repellent to alcohol; a reagent layer disposed within the first opening of the first insulating layer and including an outer periphery defined by an inner periphery of the first opening of the first insulating layer and a reagent involved in a redox reaction; , a protective film disposed within the second opening of the second insulating layer and including an outer periphery defined by an inner periphery of the second opening of the second insulating layer.
  • the method for manufacturing the sensor according to the first disclosure includes: Droplets of liquid composition A containing the reagent are placed in water in the first opening of the first insulating layer of the substrate on which the conductive layer, the first insulating layer, and the second insulating layer are disposed. forming and then drying to form the reagent layer; and After forming the reagent layer, forming droplets of a liquid composition B containing a protective film component in alcohol in the second opening of the second insulating layer and drying the droplets to form the protective film. including. (Effect of the invention)
  • the sensor and manufacturing method according to the first disclosure it is easy to control the characteristics of the reagent layer and protective film of the working electrode of the sensor within a desired range.
  • FIG. 2 is a plan view showing a substrate on which conductive layers and wiring of a first working electrode, a second working electrode, a reference electrode, and a counter electrode are arranged.
  • FIG. 2 is a plan view showing a substrate on which conductive layers and wiring of a first working electrode, a second working electrode, a reference electrode, and a counter electrode are arranged, and a first insulating layer is further arranged thereon.
  • FIG. 2 is a plan view showing a substrate on which conductive layers and wiring of a first working electrode, a second working electrode, a reference electrode, and a counter electrode are arranged, and a first insulating layer and a second insulating layer are further arranged thereon.
  • FIG. 4 is a plan view showing a substrate in which a reagent layer of a working electrode and a silver/silver chloride layer of a reference electrode are further arranged on the substrate of FIG. 3.
  • FIG. FIG. 1 is a plan view of a sensor according to an embodiment of the first disclosure.
  • FIG. 4 is a sectional view taken along line A-A' of a portion of the substrate in FIG. 3 corresponding to the first working electrode. 4 is a cross-sectional view of a droplet of liquid composition A containing a reagent involved in a redox reaction in water, formed in the first opening of the first insulating layer of the first working electrode of the substrate of FIG. 3.
  • FIG. 5 is a cross-sectional view taken along the line B-B' of the portion of the substrate of FIG. 4 corresponding to the first working electrode, obtained by drying the droplets of the liquid composition A of FIG. 3.
  • FIG. 5 is a cross-sectional view of a droplet of the first liquid composition B containing a protective film component in alcohol formed in the second opening of the second insulating layer of the first working electrode of the substrate of FIG. 4.
  • FIG. 9 is a cross-sectional view of a portion of the protective film in the second opening of the second insulating layer of the first working electrode of the substrate of FIG. 4, obtained by drying the droplets of the first liquid composition B shown in FIG. 9.
  • FIG. 9 is a cross-sectional view taken along the line B-B' of the portion of the substrate of FIG. 4 corresponding to the first working electrode, obtained by drying the droplets of the liquid composition A of FIG. 3.
  • FIG. 5 is a cross-sectional view of a droplet of the first liquid composition B containing a protective film
  • FIG. 11 is a cross-sectional view of a droplet of the first liquid composition B further formed within the second opening of the second insulating layer of the first working electrode, including a portion of the protective film of FIG. 10.
  • . 5 is a cross-sectional view of a droplet of a second liquid composition B containing a protective film component in alcohol formed in the second opening of the second insulating layer of the second working electrode of the substrate of FIG. 4.
  • FIG. 5 is a cross-sectional view of the second protective film in the second opening of the second insulating layer of the second working electrode of the substrate of FIG. 4, obtained by drying the droplets of the second liquid composition B of FIG. 9.
  • FIG. Cross-sectional view of a droplet of a third liquid composition B containing another protective film component in alcohol formed in the second opening of the second insulating layer of the second working electrode, including the second protective film of FIG. .
  • FIG. 7 is a cross-sectional view of a sensor of a comparative example in which a droplet of the first liquid composition B containing a protective film component in alcohol is wetted and spread, which is formed in the second opening of the second insulating layer of the first working electrode.
  • FIG. 18 is a cross-sectional view of a portion of the first protective film formed on the upper surface of the second insulating layer of the first working electrode and within the second opening, obtained by drying the spread first liquid composition B of FIG. 17; A droplet of the first liquid composition B further formed in the second opening of the second insulating layer of the first working electrode of the sensor of the comparative example, which includes a part of the first protective film in FIG. 18, wets and spreads.
  • FIG. 20 is a cross-sectional view of the first protective film formed on the upper surface of the second insulating layer and inside the second opening of the first working electrode, obtained by drying the spread first liquid composition B of FIG. 19;
  • FIG. 6 is a schematic diagram illustrating a method of measuring a test substance by immersing the sensor of FIG. 5 in a liquid sample.
  • FIG. 6 is a cross-sectional view of a portion of the sensor of FIG. 5, including a reference pole, taken along line E-E'.
  • FIG. 6 is a sectional view taken along line F-F' of a portion of the sensor of FIG. 5 including a counter electrode.
  • FIG. 1 is a control block diagram of an analysis device including a sensor according to an embodiment of the first disclosure.
  • FIG. 1 is a control block diagram of an analysis device including a sensor according to an embodiment of the first disclosure.
  • FIG. 25A shows the measurement results of the current value of the first working electrode (for glucose measurement) of the sensor of the example.
  • FIG. 25B shows the measurement results of the current value of the first working electrode (for glucose measurement) of the sensor of the comparative example.
  • FIG. 26A shows the measurement results of the current value of the second working electrode (for lactic acid measurement) of the sensor of the example.
  • FIG. 26B shows the measurement results of the current value of the second working electrode (for measuring lactic acid) of the sensor of the comparative example.
  • FIG. 2 is a plan view showing a substrate on which a working electrode conductive layer, a reference electrode conductive layer, a counter electrode, and wiring are arranged.
  • FIG. 2 is a plan view showing a substrate on which a working electrode conductive layer, a reference electrode conductive layer, a counter electrode, and wiring are arranged, and a first insulating layer is further arranged thereon.
  • FIG. 2 is a plan view showing a substrate on which a working electrode conductive layer, a reference electrode conductive layer, a counter electrode, and wiring are arranged, and a first insulating layer and a second insulating layer are further arranged thereon.
  • 30 is a plan view showing a substrate in which a reagent layer of a working electrode and a silver/silver chloride layer of a reference electrode are further arranged on the substrate of FIG. 29; FIG. FIG.
  • FIG. 7 is a plan view of a sensor according to an embodiment of the second disclosure in which a tip opening and a flow path are formed in a second insulating layer.
  • FIG. 7 is a plan view of a sensor according to another embodiment of the second disclosure, in which a distal opening and a flow path are formed in a first insulating layer and a second insulating layer.
  • 32 is a cross-sectional view of the sensor of FIG. 31, taken along line J-J', of a portion including a working electrode.
  • 32 is a sectional view taken along line K-K' of a portion of the sensor of FIG. 31 including a reference pole;
  • FIG. 32 is a sectional view taken along line L-L' of a portion of the sensor of FIG. 31 including a counter electrode.
  • FIG. 32 is a cross-sectional view taken along line MM' of a portion of the sensor of FIG. 31 including a counter electrode, a tip opening formed in the second insulating layer, and a flow path;
  • a cross-sectional view of a portion including a tract. 33 is a cross-sectional view taken along line N-N' of a portion of the sensor of FIG. 32, including a counter electrode, a tip opening formed in the first insulating layer and the second insulating layer, and a flow path.
  • FIG. 37 is a schematic cross-sectional view of a portion of the sensor shown in FIGS. 31 and 36, including a counter electrode, a tip opening, and a flow path, immersed in a liquid sample so that the tip of the substrate becomes the bottom end.
  • FIG. 3 is a schematic cross-sectional view of a portion including a counter electrode of a comparative sensor without a tip opening and a flow path, which is immersed in a liquid sample so that the tip of the substrate becomes the bottom end.
  • a substrate including a main body, a connecting portion, and a base end, a first insulating layer disposed entirely on the first surface of the substrate, and a first insulating layer disposed on the first surface of the substrate closer to the tip than the bent portion.
  • FIG. 7 is a plan view of a sensor according to an embodiment of the second disclosure, including a second insulating layer including an insulating sheet.
  • FIG. 42 is a plan view of a multiple sensor in which a plurality of the sensors of FIG. 41 are connected at the base end of the substrate. In FIG. 42, the insulating layer is omitted.
  • FIG. 42 is a schematic diagram of the sensor of FIG. 41 in which the main body portion of the substrate, on which the detection electrode is arranged, is bent at the bent portion of the connecting portion of the substrate and immersed in a liquid sample. In FIG. 43, the insulating layer is omitted.
  • FIG. 42 is a schematic side view of the sensor of FIG.
  • FIG. 41 in which the main body of the substrate, on which the detection electrode is arranged, is bent at the bend of the connecting portion of the substrate and immersed in a liquid sample.
  • a second insulating layer including an insulating sheet is disposed over the entire first surface of the substrate, and the main body of the substrate on which the detection electrode is arranged, which is bent at the bend of the connecting portion of the substrate, is A schematic diagram from the side of a comparative example sensor immersed in a sample.
  • a substrate including a main body, a connecting portion, and a proximal end, a first insulating layer disposed entirely on the first surface of the substrate, and a first insulating layer disposed on the first surface of the substrate closer to the tip than the bent portion a second insulating layer including a first insulating sheet; and a second insulating layer including a second insulating sheet disposed on the first surface of the proximal end of the substrate, and including the insulating sheet on the bent portion of the substrate.
  • FIG. 7 is a top view of a sensor according to another embodiment of the second disclosure; The sensor shown in FIG.
  • FIG. 46 further includes a plurality of third insulating sheets separated from the first insulating sheet and the second insulating sheet through the notch in the region above the bent portion of the first surface of the substrate.
  • FIG. 6 is a plan view of a sensor according to yet another embodiment of the second disclosure, further disposed with a second insulating layer;
  • FIG. 2 is a control block diagram of an analysis device including a sensor according to an embodiment of the second disclosure.
  • FIG. 3 is a plan view of a sensor S1 of a comparative example.
  • FIG. 3 is a plan view of a sensor S2 of a comparative example.
  • FIG. 3 is a plan view of the sensor S3 of the example.
  • FIG. 3 is a plan view of the sensor S4 of the example.
  • FIG. 3 is a plan view of the sensor S5 of the example.
  • FIG. 50A shows the measurement results of the current value of the working electrode of sensor S1.
  • FIG. 50B shows the measurement results of the current value of the working electrode of sensor S2.
  • FIG. 50C shows the measurement results of the current value of the working electrode of sensor S3.
  • FIG. 50D shows the measurement results of the current value of the working electrode of sensor S4.
  • FIG. 50E shows the measurement results of the current value of the working electrode of sensor S5.
  • FIG. 7 is an exploded perspective view of a sensor unit according to an embodiment of the second disclosure.
  • FIG. 3 is a cross-sectional view showing a sensor held between an upper support plate and a lower support plate.
  • FIG. 2 is an exploded perspective view of an adapter unit including a sensor unit.
  • 55 is a sectional view taken along line PP' of a portion including a counter electrode of the sensor shown in FIG. 54.
  • 55 is a cross-sectional view along the QQ' line of a portion of the sensor shown in FIG. 54 including the first working electrode.
  • 55 is a cross-sectional view taken along line RR' of a portion of the sensor shown in FIG. 54 that includes a second working electrode.
  • Experiment 4 of the third disclosure which of the carbon electrode without platinum particles (Experiment 3-1), the carbon electrode containing 1% platinum particles (Experiment 3-2), and the carbon electrode containing 5% platinum particles (Experiment 3-3) The results of a cyclic voltammetry test using this as the working electrode are shown.
  • Experiment 5 of the third disclosure either a carbon electrode without platinum particles (A) or a carbon electrode containing 1% platinum particles (B) was used as a working electrode, and a potential of 0.3 V or -0.2 V was applied. The results of measuring the current value over time are shown.
  • the sensor of Experiment 2-1 (the working electrode conductive layer and the counter electrode are carbon conductive layers containing no platinum particles) and the sensor of Experiment 2-2 (the counter electrode has platinum nanoparticles on the surface)
  • the sensor of Experiment 3-1 (the working electrode conductive layer and the counter electrode were carbon conductive layers containing no platinum particles), the sensor of Experiment 3-2 (the working electrode conductive layer and the counter electrode were platinum-free), Using a carbon conductive layer containing 1% by weight of particles based on carbon) and the sensor of Experiment 3-3 (the working electrode conductive layer and the counter electrode were carbon conductive layers containing 5% by weight of platinum particles based on carbon).
  • shows the results of measuring over time the current value (A) corresponding to the glucose concentration and the current value (B) corresponding to the lactic acid concentration in the liquid sample (N 2).
  • FIG. 7 is a plan view of a sensor according to an embodiment of the fourth disclosure.
  • FIG. 63 is a cross-sectional view of a portion of the sensor shown in FIG. 62, including a counter electrode, taken along line SS'. 63 is a cross-sectional view of a portion of the sensor shown in FIG. 62, including a working electrode, taken along line T-T'.
  • FIG. 65 shows the results of Experiment 8 of the fourth disclosure.
  • FIG. 65A shows the measurement results using the sensor of Experiment 8-1
  • FIG. 65B shows the measurement results using the sensor of Experiment 8-2.
  • FIG. 66 shows the results of Experiment 9 of the fourth disclosure.
  • FIG. 66A shows the measurement results using the sensor of Experiment 9-1
  • FIG. 66B shows the measurement results using the sensor of Experiment 9-2
  • FIG. 66C shows the measurement results using the sensor of Experiment 9-3.
  • FIG. 67 shows the results of Experiment 10 of the fourth disclosure.
  • Embodiments of a sensor and a method for manufacturing a sensor according to the first disclosure will be described below.
  • more detailed explanation than necessary may be omitted.
  • detailed explanations of well-known matters or redundant explanations of substantially the same configurations may be omitted. This is to avoid unnecessary redundancy in the following description and to facilitate understanding by those skilled in the art.
  • the material of the insulating substrate of the sensor according to the first disclosure of the present specification is not particularly limited, but includes, for example, polyethylene terephthalate, polycarbonate, polyimide, polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyoxymethylene, monomer cast nylon, Resin materials such as polybutylene terephthalate, methacrylic resin, and ABS resin, or glass materials can be used.
  • dimensions such as the thickness of the substrate are not particularly limited, a substrate having a thickness of, for example, 0.05 mm or more and 2 mm or less, preferably 0.1 mm or more and 1 mm or less can be used.
  • the conductive layer included in the working electrode of the sensor according to the first disclosure of this specification is a layer containing a conductive material such as carbon, gold, platinum, palladium, or the like.
  • the conductive layers of the working electrode, reference electrode, and counter electrode can be manufactured by forming a layer of the conductive material as described above on the surface of the substrate using a sputtering method, vapor deposition method, screen printing method, etc. .
  • the conductive layer can be processed into a predetermined pattern using a laser trimming method, if necessary.
  • the wiring of the sensor according to the embodiment described below can also be made of a similar conductive material.
  • the sensor according to the first disclosure of this specification is immersed in a liquid sample and used to detect a predetermined analyte in the liquid sample.
  • the test substance include amino acids such as glucose, lactic acid, cholesterol, bilirubin, glutamine, and glutamic acid, glycated amino acids, glycated peptides, ketone bodies (3-hydroxybutyric acid), alcohol, and the like.
  • the working electrode of the sensor according to the first disclosure of this specification includes a reagent layer containing a reagent involved in a redox reaction.
  • the reagent involved in the redox reaction may be any reagent that is involved in the redox reaction of the test substance, and can be appropriately selected depending on the test substance.
  • Reagents involved in the redox reaction can include a combination of a redox enzyme and a mediator (electron carrier), or a redox enzyme.
  • Oxidoreductases can include coenzymes.
  • oxidoreductases examples include oxidases and dehydrogenases.
  • redox enzymes include glucose oxidase, lactate oxidase, cholesterol oxidase, bilirubin oxidase, glucose dehydrogenase, lactate dehydrogenase, amino acid oxidase, amino acid dehydrogenase, glutamate oxidase, glutamate dehydrogenase, fructosyl amino acid oxidase, fructosyl peptide oxidase. , 3-hydroxybutyrate dehydrogenase, alcohol oxidase, alcohol dehydrogenase and the like. These oxidoreductases can be used to detect the test substances exemplified above.
  • Mediators include, but are not limited to, metal complexes (for example, osmium complexes, ruthenium complexes, iron complexes, etc.), quinone compounds (for example, benzoquinone, naphthoquinone, phenanthrenequinone, phenanthrolinequinone, anthraquinone, and derivatives thereof). ), phenazine compounds, viologen compounds, phenothiazine compounds, and phenol compounds.
  • metal complexes for example, osmium complexes, ruthenium complexes, iron complexes, etc.
  • quinone compounds for example, benzoquinone, naphthoquinone, phenanthrenequinone, phenanthrolinequinone, anthraquinone, and derivatives thereof.
  • phenazine compounds for example, osmium complexes, ruthenium complexes, iron complexes, etc.
  • quinone compounds for example
  • mediators include potassium ferricyanide, hexaammineruthenium, ferrocene, poly(1-vinylimidazole)-bis(bipyridine)chloroosmium, hydroquinone, 2-methyl-1,4-benzoquinone, 1,2-naphthoquinone-4 -sulfonate, 9,10-phenanthrenequinone-2-sulfonate, 9,10-phenanthrenequinone-2,7-disulfonate, 1,10-phenanthroline-5,6-dione, anthraquinone-2-sulfone acid salts, phenazine derivatives (1-methoxy-5-methylphenazinium methyl sulfate, 1-methoxy-5-ethyl phenazinium ethyl sulfate, etc.), methyl viologen, benzyl viologen, methylene blue, methylene green, 2-aminophenol, One or more selected from
  • the mediator is preferably a polymerized mediator bonded to a polymer compound from the viewpoint of sensor durability and prevention of leakage outside the sensor.
  • the polymer compound to which the mediator binds can be a homopolymer, a random copolymer, a block copolymer, or a polymer compound in which these are combined or mixed.
  • the weight average molecular weight of the polymer compound is, for example, 10,000 or more, preferably 50,000 or more, more preferably 100,000 or more, and the upper limit of the weight average molecular weight is, for example, less than 10,000,000, preferably Less than 1,000,000.
  • a polymer compound is one in which a plurality of atoms selected from at least one of carbon atoms, nitrogen atoms, oxygen atoms, and sulfur atoms are bonded in a chain to form a main chain, although there are no particular limitations thereto. can be mentioned.
  • Specific examples include natural polymers such as proteins, polypeptides, and polynucleotides, and synthetic polymers such as polyamino acids, polyimines, polyallyl compounds, poly(meth)acrylates, polyalkylene oxides, and copolymers thereof. Mention may be made of molecular compounds.
  • polyamino acids include poly(L-glutamic acid) and poly(L-lysine).
  • examples of the polyimine include polyalkylene imines such as polyethyleneimine and polypropyleneimine.
  • examples of the polyallyl compound include polyallylamine, polydiallylamine, and the like.
  • examples of polyalkylene oxide include polyethylene oxide and polypropylene oxide.
  • it is preferable that the entire polymerized mediator is hydrophilic, and furthermore, it is preferable that the polymer compound to which the mediator is bonded is hydrophilic.
  • the polymerization mediator one in which a mediator and a polymer compound are bonded via a covalent bond can be used.
  • this covalent bond for example, those described in Japanese Patent Publication No. 2003-514924 can be used, and specific examples include ether bond, thioether bond, ester bond, urethane bond, amide bond, etc. can.
  • the covalent bond may be formed from a reactive group originally possessed by a monomer forming the polymer compound, or may be formed from a reactive group possessed by a linker separately introduced into a mediator or polymer compound. .
  • polymerization mediators include those in which a phenazine compound is used as the mediator, poly(L-lysine) is used as the polymer, and the phenazine compound and poly(L-lysine) are bonded via an amide bond. can be mentioned.
  • the reagent layer included in the working electrode of the sensor according to the first disclosure of this specification can be formed by drying a liquid composition A containing a reagent involved in a redox reaction in water.
  • Liquid composition A contains the reagent in water, and may further contain components such as a buffer, a hydrophilic polymer compound, a conductive carbon filler, and a crosslinking agent, if necessary.
  • the hydrophilic polymer compound include cellulose derivatives, and examples of the cellulose derivative include one or more selected from methylcellulose, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxyethylmethylcellulose, and hydroxypropylmethylcellulose.
  • the conductive carbon filler include one or more selected from carbon black, graphite powder, porous carbon, and nanocarbon.
  • the protective film included in the working electrode of the sensor according to the first disclosure of the present specification prevents or suppresses leakage of the reagent contained in the reagent layer to the outside of the protective film, and prevents the analyte present outside the protective film from leaking out. It can be a permeable membrane.
  • the protective film having such properties preferably contains a polymer compound. Examples of the polymer compound contained in the protective film include a polymer compound containing 4-vinylpyridine as a constituent unit, a polymer compound containing a cation exchange functional group, and the like.
  • polymer compounds containing 4-vinylpyridine as a constituent unit include poly(4-vinylpyridine), a copolymer (preferably a block copolymer) of 4-vinylpyridine and alkyl methacrylate, and styrene, 4-vinylpyridine and oligomer. Copolymers (preferably random copolymers) with propylene glycol methyl ether methacrylate may be mentioned.
  • the methacrylic acid alkyl ester includes tert-butyl methacrylate.
  • examples of the oligopropylene glycol methyl ether methacrylate include tripropylene glycol methyl ether methacrylate.
  • the polymer compound containing 4-vinylpyridine as a constitutional unit is preferably crosslinked with a crosslinking agent containing two or more epoxy groups, such as polyethylene glycol diglycidyl ether (PEGDGE).
  • a crosslinking agent containing two or more epoxy groups such as polyethylene glycol diglycidyl ether (PEGDGE).
  • PEGDGE polyethylene glycol diglycidyl ether
  • a polymer compound containing 4-vinylpyridine as a constituent unit, which is crosslinked with a crosslinking agent containing two or more epoxy groups, is produced by a reaction between a pyridyl group (tertiary amine) derived from 4-vinylpyridine and an epoxy group. Contains a functional group containing a resulting quaternary ammonium cation.
  • a preferred embodiment of the polymer compound containing 4-vinylpyridine as a constituent unit, which is crosslinked with a crosslinking agent containing two or more epoxy groups, is a “second polymer compound containing a cationic functional group” described in the fourth disclosure.
  • a specific embodiment of "combined compound” is preferred.
  • Examples of the polymer compound containing a cation exchange functional group include a polymer compound containing a structural unit having a sulfonic acid group in the side chain, and preferably a perfluoro compound having a sulfonic acid group in the side chain as the structural unit.
  • a protective membrane comprising a polymeric compound containing a cation exchange functional group is preferably provided on the reagent layer to transport cations such as protons between the reagent layer and the liquid sample.
  • the protective film included in the working electrode of the sensor according to the first disclosure of this specification can be a laminate of two or more protective films, for example, a cation exchange functional layer provided on the side in contact with the reagent layer. It can be a laminate of a protective film containing a polymer compound containing a group and a protective film provided thereon containing a polymer compound containing 4-vinylpyridine as a constituent unit.
  • the protective film included in the working electrode of the sensor according to the first disclosure of this specification can be formed by drying liquid composition B containing a protective film component in alcohol.
  • the protective film component include a polymer compound, a crosslinking agent, etc. contained in the protective film.
  • the alcohol in liquid composition B include monohydric alcohols having 1 to 5 carbon atoms, with methanol, ethanol or isopropyl alcohol being particularly preferred, and ethanol being most preferred.
  • the first insulating layer included in the working electrode of the sensor according to the first disclosure of this specification includes a water-repellent surface.
  • the water-repellent surface refers to a surface having a contact angle with water of, for example, 90° or more, more preferably 100° or more, and most preferably 110° or more.
  • the upper limit of the water contact angle of the water-repellent surface of the first insulating layer is not particularly limited, but may be, for example, 160° or less (the range of the water contact angle is, for example, 90° or more and 160° or less). .
  • the second insulating layer included in the working electrode of the sensor according to the first disclosure of this specification includes a surface that is liquid repellent to alcohol (alcohol repellent).
  • the surface that is liquid repellent to alcohol refers to a surface that has a contact angle with alcohol of, for example, 45° or more, more preferably 50° or more, and most preferably 55° or more.
  • the upper limit of the contact angle to alcohol of the alcohol-repellent surface of the second insulating layer is not particularly limited, but is, for example, 100° or less (the range of the contact angle to alcohol is, for example, 45° or more and 100° or less).
  • the alcohol includes, for example, a monohydric alcohol having 1 to 5 carbon atoms, with methanol, ethanol or isopropyl alcohol being particularly preferred, and ethanol being most preferred.
  • the contact angle of the surface of the first insulating layer to water and the contact angle of the surface of the second insulating layer to alcohol values measured at 20° C. can be adopted.
  • the contact angle of the surface of the first insulating layer to water and the contact angle of the surface of the second insulating layer to alcohol can be measured using a commercially available analyzer, for example, Handy Contact Angle/Surface manufactured by KRUSS. It can be measured using a free energy analyzer MSA.
  • the contact angle of the surface with respect to water or alcohol is preferably determined by ejecting a 2 ⁇ L droplet of water or alcohol onto the surface of the object to be measured, and measuring the contact angle between the droplet and the surface 2 seconds later.
  • the water-repellent surface of the first insulating layer included in the working electrode of the sensor according to the first disclosure of this specification preferably contains a fluororesin.
  • a fluorinated hydrocarbon polymer compound can be used, such as a polymer compound containing one or more selected from vinylidene fluoride, tetrafluoroethylene, hexafluoropropylene, and perfluoro(alkyl vinyl ether).
  • the first insulating layer can be entirely made of fluororesin.
  • the alcohol-repellent surface of the second insulating layer of the working electrode of the sensor according to the first disclosure of this specification preferably contains a compound containing a perfluoroalkyl group.
  • Compounds containing perfluoroalkyl groups include fluorine-based surface modification additives.
  • Such a second insulating layer is formed by applying a composition containing an insulating matrix resin and a compound containing a perfluoroalkyl group (fluorine-based surface modification additive) in a solvent onto the first insulating layer. It can be formed by drying. At this time, a compound containing a perfluoroalkyl group is segregated on the surface, forming a surface that is liquid repellent to alcohol.
  • the second insulating layer thus formed is preferably a layer of an insulating matrix resin containing a compound containing a perfluoroalkyl group on its surface.
  • the type of insulating matrix resin is not particularly limited, but may be, for example, a polyester resin.
  • the number of carbon atoms in the perfluoroalkyl group is not particularly limited, but may range from 2 to 20 carbon atoms, for example.
  • the perfluoroalkyl group can contain a trifluoromethyl group at the end.
  • the sensor 1 of the present embodiment includes an insulating substrate 2, a first working electrode 10a, a second working electrode 10b, a reference electrode 20, a counter electrode 30, and a second working electrode 10b arranged on the substrate 2.
  • the wiring 50 is electrically connected to each of the first working electrode 10a, the second working electrode 10b, the reference electrode 20, and the counter electrode 30.
  • the sensor 1 of this embodiment includes two working electrodes, in another embodiment not shown, there may be only one working electrode, or three or more working electrodes.
  • the first working electrode 10a and the second working electrode 10b when they are not distinguished, they may be expressed as working electrodes 10a and 10b. Further, the first working electrode 10a, the second working electrode 10b, the reference electrode 20, and the counter electrode 30 may be collectively referred to as electrodes. Furthermore, although the sensor 1 of this embodiment is a three-electrode sensor that includes a working electrode, a reference electrode, and a counter electrode as electrodes, it may be a bipolar sensor that does not include a reference electrode and only includes a working electrode and a counter electrode. Although not shown, the reference electrode and/or the counter electrode may be provided on a substrate different from the substrate on which the working electrode is arranged.
  • the sensor 1 is used to detect a predetermined test substance in the liquid sample X by being immersed in the liquid sample X, as shown in FIG.
  • the liquid sample X include a culture solution containing cells C as shown in the figure, and a liquid sample prepared using blood obtained from a living body.
  • Specific examples of the test substance are as described in the ⁇ Materials> column.
  • the working electrodes 10a and 10b of the sensor 1 have reagent layers 15a and 15b containing a reagent involved in the redox reaction of the test substance in the liquid sample X on the conductive layers 11a and 11b.
  • Preferred embodiments of the reagents involved in the redox reaction are as described in the ⁇ Materials> column.
  • the reagent layers 15a and 15b of the working electrodes 10a and 10b of the sensor 1 contain a reagent that oxidizes the analyte in the liquid sample Electrons move from there to the conductive layers 11a and 11b.
  • the reagent layers 15a and 15b contain a reagent that reduces the analyte in the liquid sample X, electrons move from the conductive layers 11a and 11b to the analyte. Since the amount of moving electrons depends on the concentration of the analyte, the concentration or concentration change of the analyte in the liquid sample can be measured.
  • An example of an analysis device 100 for analyzing a test substance in a liquid sample, which includes the sensor 1, will be described with reference to FIG. 24.
  • the analysis device 100 includes a sensor 1, an analysis unit 102, and a control unit 104.
  • Working electrodes 10a, 10b, reference electrode 20, and counter electrode 30 of sensor 1 are connected to analysis unit 102 via wiring 50, respectively.
  • Analysis unit 102 is in communication with control unit 104 .
  • the analysis unit 102 includes an electrochemical measurement section 111, a control section 112, a storage section 113, and a communication section 114.
  • the electrochemical measuring section 111 is a potentiostat that measures the concentration of the analyte by applying a predetermined voltage to each electrode of the sensor 1, and includes a voltage applying section 111a and a current measuring section 111b, Preferably, it further includes a voltage measuring section (counter electrode terminal voltage measuring section) 111c.
  • the voltage application unit 111a applies a predetermined voltage to the electrodes of the sensor 1 in order to measure the concentration of the test substance contained in the liquid sample X.
  • the current measurement unit 111b measures the value of the current flowing between the working electrodes 10a, 10b and the counter electrode 30 of the sensor 1, or its change, which is measured while a voltage is applied to the electrode of the sensor 1 from the voltage application unit 111a. Detect. As described above, the current value or the change in the current value detected by the current measuring section 111b serves as an index of the concentration of the test substance in the liquid sample X or the change in concentration.
  • the voltage measurement unit 111c measures the terminal voltage of each electrode of the sensor 1.
  • the control section 112 is connected to the voltage application section 111a, the current measurement section 111b, the voltage measurement section 111c, the storage section 113, and the communication section 114.
  • the control unit 112 controls the voltage application unit 111a to apply a predetermined voltage to each electrode of the sensor 1, and transmits the measurement results in the current measurement unit 111b and voltage measurement unit 111c to the control unit 104.
  • the communication unit 114 is controlled as follows.
  • the storage unit 113 is connected to the control unit 112, and stores, for example, applied voltage values preset for each measurement target, measured values in the current measurement unit 111b and voltage measurement unit 111c, and pre-measured calibration curves. Save data.
  • the communication section 114 is controlled by the control section 112 and transmits data such as measurement results in the current measurement section 111b and the voltage measurement section 111c to the analysis section 142 of the control unit 104.
  • the control unit 104 can communicate with the analysis unit 102 via the communication section 114, and includes a display section 141 and an analysis section 142.
  • the display section 141 displays the concentration of the test substance in the liquid sample X based on the current value detected by the current measurement section 111b as a result of the analysis performed by the analysis section 142, for example.
  • the analysis unit 142 is, for example, a PC (Personal Computer), and calculates the concentration of the test substance based on the current value flowing between the working electrodes 10a, 10b and the counter electrode 30, which is measured in the current measurement unit 111b. do.
  • PC Personal Computer
  • FIG. 12 is a cross-sectional view of a portion of the sensor 1 including the first working electrode 10a, taken along line CC' in FIG.
  • FIG. 16 is a cross-sectional view of a portion of the sensor 1 including the second working electrode 10b, taken along line DD' in FIG.
  • the first working electrode 10a includes a conductive layer 11a, a first insulating layer 3a, a second insulating layer 4a, a reagent layer 15a, and a protective film 16a (FIG. 12).
  • the second working electrode 10b includes a conductive layer 11b, a first insulating layer 3b, a second insulating layer 4b, a reagent layer 15b, and a protective film 16b (FIG. 16).
  • the conductive layers 11a, 11b, the first insulating layers 3a, 3b, the second insulating layers 4a, 4b , reagent layers 15a, 15b, and protective films 16a, 16b when each element of the first working electrode 10a and the second working electrode 10b is referred to without distinction, the conductive layers 11a, 11b, the first insulating layers 3a, 3b, the second insulating layers 4a, 4b , reagent layers 15a, 15b, and protective films 16a, 16b.
  • the conductive layers 11a and 11b of the working electrodes 10a and 10b are arranged on an insulating substrate 2.
  • the main surface on the side where the conductive layers 11a and 11b are formed is referred to as a first surface 2a.
  • Preferable materials for the substrate 2 and the conductive layers 11a and 11b are as described in the ⁇ Material> column.
  • the first insulating layers 3a, 3b of the working electrodes 10a, 10b are at least partially disposed on the conductive layers 11a, 11b. As shown in FIGS. 12 and 16, part of the first insulating layers 3a, 3b may be disposed on the conductive layers 11a, 11b, or, although not shown, the entire first insulating layers 3a, 3b are conductive. It may be placed on layers 11a and 11b.
  • the first insulating layers 3a, 3b of the working electrodes 10a, 10b are the conductive layers of the working electrodes 10a, 10b of the first insulating layer 3 that covers almost the entire first surface 2a of the substrate 2. 11a and 11b.
  • the present invention is not limited to this embodiment, and the first insulating layers 3a, 3b of the working electrodes 10a, 10b may be arranged only in the vicinity of the conductive layers 11a, 11b of the working electrodes 10a, 10b.
  • the first insulating layers 3a, 3b of the working electrodes 10a, 10b are formed at positions overlapping with parts of the conductive layers 11a, 11b in a plan view from the thickness direction T of the substrate 2, and penetrated in the thickness direction T. It is characterized by having first openings 3a1 and 3b1 and water-repellent surfaces 3a2 and 3b2.
  • the shape of the inner circumferential edges 3a10, 3b10 of the first openings 3a1, 3b1 in a plan view from the thickness direction T is circular in the illustrated example, but is not limited to this, and may be a polygon (quadrangular, triangular, etc.). ) or any other shape.
  • the thickness of the portions of the first insulating layers 3a, 3b of the working electrodes 10a, 10b that cover the conductive layers 11a, 11b is not particularly limited, but can be, for example, 0.5 ⁇ m or more and 50 ⁇ m or less, preferably 2 ⁇ m or more, The thickness can be set to 20 ⁇ m or less.
  • the opening width of the first openings 3a1, 3b1 of the first insulating layers 3a, 3b of the working electrodes 10a, 10b is not particularly limited, but may be, for example, 0.5 mm or more and 5 mm or less, preferably 1 mm or more and 2 mm or less. can.
  • the first insulating layers 3a, 3b of the working electrodes 10a, 10b have water-repellent surfaces 3a2, 3b2.
  • the reagent layers 15a and 15b of the working electrodes 10a and 10b contain a reagent involved in a redox reaction in water in the first openings 3a1 and 3b1 of the first insulating layers 3a and 3b of the working electrodes 10a and 10b.
  • the liquid composition A is formed by forming droplets of the liquid composition A and then drying the droplets.
  • the surfaces 3a2 and 3b2 of the first insulating layers 3a and 3b of the working electrodes 10a and 10b are water repellent, so the droplets of liquid composition A are easily held without collapsing.
  • the characteristics of the reagent layers 15a, 15b after drying for example, the thickness of the reagent layers 15a, 15b, the amount of reagent constituting the reagent layers 15a, 15b) within a designed range.
  • Preferred embodiments of the materials constituting the first insulating layers 3a and 3b having water-repellent surfaces 3a2 and 3b2 are as described in the ⁇ Material> column.
  • the second insulating layers 4a, 4b of the working electrodes 10a, 10b are arranged on the first insulating layers 3a, 3b.
  • the second insulating layers 4a, 4b of the working electrodes 10a, 10b are the conductive layers of the working electrodes 10a, 10b of the second insulating layer 4 that covers almost the entire first surface 2a of the substrate 2. 11a and 11b.
  • the present invention is not limited to this embodiment, and the second insulating layers 4a, 4b of the working electrodes 10a, 10b may be arranged only in the vicinity of the conductive layers 11a, 11b of the working electrodes 10a, 10b.
  • the second insulating layers 4a, 4b of the working electrodes 10a, 10b are the portions of the first insulating layers 3a, 3b that enclose the entire first openings 3a1, 3b1 when viewed in plan from the thickness direction T of the substrate 2. It has second openings 4a1, 4b1 formed at positions overlapping with 3a3, 3b3 and penetrating in the thickness direction T, and surfaces 4a2, 4b2 that are liquid repellent to alcohol.
  • the shape of the inner peripheral edges 4a10, 4b10 of the second openings 4a1, 4b1 of the second insulating layers 4a, 4b of the working electrodes 10a, 10b in plan view from the thickness direction T is circular in the illustrated example;
  • the shape is not limited to this, and may be a polygon (quadrangular, triangular, etc.) or any other arbitrary shape. As shown in FIG.
  • the inner peripheral edges 3a10, 3b10 of the first openings 3a1, 3b1 of the first insulating layers 3a, 3b, the inner peripheral edges 4a10, 3b10 of the second openings 4a1, 4b1 of the second insulating layers 4a, 4b, 4b10 preferably has a parallel shape in plan view from the thickness direction T, but is not limited thereto.
  • the second openings 4a1, 4b1 of the second insulating layers 4a, 4b have, at their bottoms, the first openings 3a1, 3b1 of the first insulating layers 3a, 3b and the reagent layers 15a, 15b inside the first insulating layers 3a, 3b. This is a recess that includes a region surrounding the first openings 3a1 and 3b1 on the water-repellent surfaces 3a2 and 3b2 of the layers 3a and 3b.
  • the thickness of the second insulating layers 4a, 4b of the working electrodes 10a, 10b is not particularly limited, but can be, for example, 5 ⁇ m or more and 100 ⁇ m or less, preferably 10 ⁇ m or more and 50 ⁇ m or less.
  • the opening widths of the second openings 4a1 and 4b1 of the second insulating layers 4a and 4b of the working electrodes 10a and 10b are not particularly limited, but may be, for example, 0.5 mm or more and 5 mm or less, preferably 1 mm or more and 3 mm or less. can.
  • the second insulating layers 4a, 4b of the working electrodes 10a, 10b have surfaces 4a2, 4b2 that are liquid repellent to alcohol (alcohol repellent).
  • the protective films 16a and 16b of the working electrodes 10a and 10b are formed using a liquid containing a protective film component in alcohol in the second openings 4a1 and 4b1 of the second insulating layers 4a and 4b of the working electrodes 10a and 10b. It is formed by forming droplets of composition B and then drying them.
  • the surfaces 4a2 and 4b2 of the second insulating layers 4a and 4b of the working electrodes 10a and 10b are liquid repellent to alcohol, so the droplets of liquid composition B do not collapse. It is easy to hold without Therefore, it is easy to set the characteristics of the protective films 16a, 16b after drying (for example, the thickness of the protective films 16a, 16b, the amount of components constituting the protective films 16a, 16b) within a designed range.
  • Preferred embodiments of the materials constituting the second insulating layers 4a, 4b having surfaces 4a2, 4b2 that are liquid repellent to alcohol (alcohol repellent) are as described in the ⁇ Material> column.
  • the reagent layers 15a, 15b of the working electrodes 10a, 10b are arranged in the first openings 3a1, 3b1 of the first insulating layers 3a, 3b, and are arranged in the first openings 3a1, 3b1 of the first insulating layers 3a, 3b. It includes outer peripheral edges 15a10 and 15b10 defined by the peripheral edges 3a10 and 3b10, and a reagent involved in the redox reaction.
  • the thickness of the reagent layers 15a and 15b is not particularly limited, but as shown in the figure, the thickness of the first insulating layers 3a and 3b covering the conductive layers 11a and 11b should be approximately the same or smaller than that. is preferred.
  • Preferred embodiments of the reagents involved in the redox reaction in the reagent layers 15a and 15b are as described in the ⁇ Materials> column.
  • the protective films 16a, 16b of the working electrodes 10a, 10b are arranged in the second openings 4a1, 4b1 of the second insulating layers 4a, 4b, and are arranged in the second openings 4a1, 4b1 of the second insulating layers 4a, 4b. It includes outer peripheral edges 16a10 and 16b10 defined by peripheral edges 4a10 and 4b10. Although the thickness of the protective films 16a and 16b is not particularly limited, it is preferable that the thickness be approximately the same as or smaller than the thickness of the second insulating layers 4a and 4b as shown in the figure.
  • Preferred embodiments of the protective films 16a and 16b are as described in the ⁇ Material> column.
  • the former is referred to as the "first reagent layer 15a" of the first working electrode 10a. ”, the latter may be expressed as “second reagent layer 15b” of second working electrode 10b.
  • the reagent included in the reagent layer 15a of the first working electrode 10a may be referred to as a "first reagent”
  • the reagent included in the second reagent layer 15b of the second working electrode 10b may be referred to as a "second reagent”.
  • the senor 1 includes a plurality of working electrodes 10a and 10b, and a first reagent contained in the first reagent layer 15a of the first working electrode 10a among the plurality of working electrodes 10a and 10b;
  • the second reagents contained in the second reagent layer 15b of the second working electrode 10b, which is different from the working electrode 10a, are different from each other.
  • the first reagent is involved in the redox reaction of the first analyte in the liquid sample X
  • the second reagent is involved in the redox reaction of the first analyte in the liquid sample can be involved in the redox reaction of different second analytes, so it can be used to detect multiple analytes including the first analyte and the second analyte in liquid sample X. Can be done.
  • the first reagent preferably contains an enzyme involved in the redox reaction of glucose, more preferably glucose dehydrogenase or glucose oxidase;
  • the two reagents preferably include an enzyme involved in the redox reaction of lactic acid, more preferably lactate oxidase or lactate dehydrogenase.
  • the sensor 1 according to this aspect can detect glucose and lactic acid.
  • FIG. 22 is a cross-sectional view of a portion of the sensor 1 including the reference pole 20, taken along line EE' in FIG.
  • the reference electrode 20 includes a reference electrode conductive layer 21 disposed on the first surface 2a of the insulating substrate 2, a silver/silver chloride layer 22 disposed on the reference electrode conductive layer 21, and a silver/silver chloride layer. 22, and a reference electrode protective film 23 disposed on the reference electrode protection film 22.
  • the reference electrode conductive layer 21 can be formed of the material described for the conductive layers 11a and 11b of the working electrodes 10a and 10b.
  • the reference electrode protective film 23 can be formed of the material described in the ⁇ Material> column regarding the protective films 16a, 16b of the working electrodes 10a, 10b.
  • a part of the first insulating layer 3 disposed on the first surface 2a of the substrate 2 is disposed on the reference electrode conductive layer 21, and the reference electrode conductive layer 3 is partially disposed on the reference electrode conductive layer 21 in a plan view from the thickness direction T of the substrate 2.
  • the reference electrode includes a reference electrode first opening 301 that is formed at a position overlapping a part of the reference electrode 21 and penetrates in the thickness direction T.
  • the silver/silver chloride layer 22 of the reference electrode 20 is arranged within the reference electrode first opening 301 of the first insulating layer 3 .
  • the second insulating layer 4 disposed on the first insulating layer 3 is located at a position overlapping with a portion of the first insulating layer 3 that includes the entire reference electrode first opening 301 in a plan view from the thickness direction T.
  • a reference electrode second opening 401 penetrating in the thickness direction T is included.
  • the reference electrode protective film 23 of the reference electrode 20 is arranged within the reference electrode second opening 401 of the second insulating layer 4 .
  • FIG. 23 is a cross-sectional view of a portion of the sensor 1 including the counter electrode 30, taken along line FF' in FIG.
  • the counter electrode 30 includes a counter electrode conductive layer 31 disposed on the first surface 2a of the insulating substrate 2.
  • the counter electrode conductive layer 31 can be formed of the material described for the conductive layers 11a and 11b of the working electrodes 10a and 10b.
  • the first insulating layer 3 disposed on the first surface 2 a of the substrate 2 is partially disposed on the counter electrode conductive layer 31
  • the first insulating layer 3 disposed on the first surface 2 a of the substrate 2 is partially disposed on the counter electrode conductive layer 31 .
  • a counter electrode first opening 302 penetrating in the thickness direction T is formed at a position partially overlapping with the other electrode.
  • the second insulating layer 4 disposed on the first insulating layer 3 is formed at a position in the first insulating layer 3 that overlaps with the counter electrode first opening 302 when viewed from the thickness direction T. It includes a counter electrode second opening 402 penetrating therethrough.
  • the counter electrode conductive layer 31 of the counter electrode 30 is exposed to the outside through the counter electrode first opening 302 of the first insulating layer 3 and the counter electrode second opening 402 of the second insulating layer 4. Therefore, when the sensor 1 is immersed in the liquid sample X as shown in FIG. 21, the counter electrode conductive layer 31 of the counter electrode 30 comes into direct contact with the liquid sample X.
  • the conductive layers 11a and 11b of the working electrodes 10a and 10b, the reference electrode conductive layer 21 of the reference electrode 20, and the counter electrode conductive layer 31 of the counter electrode 30 are placed on the first surface 2a of the insulating substrate 2, the conductive layers 11a and 11b of the working electrodes 10a and 10b, the reference electrode conductive layer 21 of the reference electrode 20, and the counter electrode conductive layer 31 of the counter electrode 30 are placed. , and wiring 50 electrically connected to each of them.
  • a first layer is deposited on the first surface 2a of the substrate 2 on which the conductive layers 11a, 11b of the working electrodes 10a, 10b, the reference electrode conductive layer 21, the counter electrode conductive layer 31, and the wiring 50 are arranged.
  • An insulating layer 3 is further laminated. The portions of the first insulating layer 3 near the conductive layers 11a, 11b of the working electrodes 10a, 10b are the first insulating layers 3a, 3b of the working electrodes 10a, 10b. Parts of the first insulating layers 3a, 3b of the working electrodes 10a, 10b are arranged on the conductive layers 11a, 11b, and cover the upper surfaces of the conductive layers 11a, 11b.
  • the first insulating layers 3a, 3b of the working electrodes 10a, 10b have first openings 3a1, 3b1 of the working electrodes 10a, 10b at positions that overlap with parts of the conductive layers 11a, 11b of the working electrodes 10a, 10b.
  • the first insulating layer 3 further has a reference electrode first opening 301 at a position overlapping with a part of the reference electrode conductive layer 21 and a counter electrode first opening 302 at a position overlapping with a part of the counter electrode conductive layer 31. .
  • a second insulating layer 4 is further laminated on the first insulating layer 3.
  • the portions of the second insulating layer 4 near the conductive layers 11a, 11b of the working electrodes 10a, 10b are the second insulating layers 4a, 4b of the working electrodes 10a, 10b.
  • the second insulating layers 4a, 4b of the working electrodes 10a, 10b are located at positions overlapping portions 3a3, 3b3 of the first insulating layers 3a, 3b of the working electrodes 10a, 10b, which include the entire first openings 3a1, 3b1. It has second openings 4a1 and 4b1 formed therein.
  • the second insulating layer 4 further has a reference electrode second opening 401 at a position overlapping with the reference electrode first opening 301 of the first insulating layer 3 , and a reference electrode second opening 401 overlapping with the counter electrode first opening 302 of the first insulating layer 3 A counter electrode second opening 402 is provided at the position.
  • the second insulating layer 4 is formed by coating an ink containing an insulating matrix resin and a compound containing a perfluoroalkyl group (fluorine-based surface modification additive) in a solvent on the first insulating layer. It can be formed by drying.
  • the substrate 2 shown in FIG. 3 On which the conductive layers 11a, 11b of the working electrodes 10a, 10b, the reference electrode conductive layer 21, the counter electrode conductive layer 31, the wiring 50, the first insulating layer 3, and the second insulating layer 4 are arranged.
  • a cross section along line AA' of a portion corresponding to the first working electrode 10a is shown in FIG.
  • the first opening 3a1 of the first insulating layer 3a of the working electrode 10a is a recess that includes the conductive layer 11a of the working electrode 10a at its bottom.
  • the first insulating layer 3a of the working electrode 10a has a water-repellent surface 3a2.
  • droplets of liquid composition A containing a reagent involved in a redox reaction are formed in water in the first opening 3a1 of the first insulating layer 3a of the working electrode 10a.
  • the droplet of the liquid composition A is formed at an internal angle ⁇ a (as shown in FIG. 7) at the contact point with the surface 3a2 of the first insulating layer 3a. ) is maintained without collapsing until it reaches the water contact angle of the surface 3a2 of the first insulating layer 3a.
  • droplets of the liquid composition A in the first opening 3a1 of the first insulating layer 3a of the working electrode 10a in this way they are dried to form a reagent layer 15a as shown in FIG.
  • droplets of the liquid composition A containing a reagent involved in a redox reaction in water are held without collapsing within the first opening 3a1 of the first insulating layer 3a having the water-repellent surface 3a2. Therefore, it is easy to set the characteristics of the reagent layer 15a after drying (for example, the thickness of the reagent layer 15a, the amount of reagent constituting the reagent layer 15a) within a designed range.
  • the surface of the first insulating layer 3a does not have water repellency, when a droplet of the liquid composition A is formed in the first opening 3a1 of the first insulating layer 3a, the droplet may collapse.
  • the liquid composition A is likely to wet the outside of the first opening 3a1 of the first insulating layer 3a, and it is not easy to adjust the properties of the reagent layer obtained by drying to a designed range.
  • composition of liquid composition A containing a reagent involved in a redox reaction in water is as described in the ⁇ Materials> column.
  • droplets of liquid composition B containing a protective film component in alcohol are formed in the second opening 4a1 of the second insulating layer 4a of the working electrode 10a. .
  • the droplet of the liquid composition B forms an internal angle ⁇ b (FIG. 9) at the point of contact with the surface 4a2 of the second insulating layer 4a. ) is maintained without collapsing until it reaches the contact angle with the alcohol on the surface 4a2 of the second insulating layer 4a.
  • the droplets of the liquid composition B in the second opening 4a1 of the second insulating layer 4a of the working electrode 10a in this manner are dried to form a protective film 16a as shown in FIG.
  • the droplets of the liquid composition B containing the protective film component in alcohol do not collapse within the second opening 4a1 of the second insulating layer 4a having the surface 4a2 that is liquid repellent to alcohol. Since it is easily retained, it is easy to set the characteristics of the protective film 16a after drying (for example, the thickness of the protective film 16a, the amount of components constituting the protective film 16a) within a designed range.
  • the surface of the second insulating layer 4a does not have liquid repellency to alcohol, when a droplet of the liquid composition B is formed in the second opening 4a1 of the second insulating layer 4a, the droplet There is a high possibility that the liquid composition B will wet the outside of the second opening 4a1 of the second insulating layer 4a, and it is not easy to adjust the properties of the protective film obtained by drying to the designed range. (See Comparative Examples described below with reference to FIGS. 17 to 20).
  • a preferred embodiment of the composition of liquid composition B containing a protective film component in alcohol is as described in the ⁇ Materials> column.
  • FIGS. 9 to 12 show an example in which the protective film 16a is formed in the second opening 4a1 of the second insulating layer 4a of the working electrode 10a in two steps.
  • a liquid composition B containing a protective film component in alcohol is poured into the second opening 4a1 of the second insulating layer 4a. After forming the droplets, they are dried to form a part of the protective film 16a in the thickness direction, as shown in FIG. Subsequently, as shown in FIG.
  • droplets of the liquid composition B are further formed in the second opening 4a1 of the second insulating layer 4a, and then dried to form a protective film 16a, as shown in FIG. form the whole.
  • the present invention is not limited to this example, and the protective film may be formed in one step, or may be formed in three or more steps.
  • the senor 1 includes a plurality of working electrodes 10a, 10b, and a first reagent contained in the first reagent layer 15a of the first working electrode 10a among the plurality of working electrodes 10a, 10b. and the second reagent contained in the second reagent layer 15b of the second working electrode 10b, which is different from the first working electrode 10a, are different from each other.
  • the method for manufacturing the sensor 1 includes, as a step of forming a reagent layer,
  • the first reagent layer 15a of the first working electrode 10a included in the plurality of working electrodes 10a, 10b is placed in the first opening 3a1 of the first insulating layer 3a of the first working electrode 10a, and the first reagent is contained in water.
  • forming droplets of the first liquid composition A and then drying them see FIGS. 6 to 8
  • a second action different from the first working electrode 10a included in the plurality of working electrodes 10a, 10b are examples of the first reagent layer.
  • the second reagent layer 15b of the pole 10b is placed in the first opening 3b1 of the first insulating layer 3b of the second working electrode 10b with a second liquid composition A containing a second reagent different from the first reagent in water. Forming a droplet and then drying it (not shown, but the same process as in FIGS. 6 to 8) It is preferable to include.
  • the reagent layers 15a and 15b of the plurality of working electrodes 10a and 10b can be formed individually and independently, so that the composition of the liquid composition A containing the reagent in water can be changed. By adjusting, it is easy to form reagent layers 15a and 15b containing different reagents on the plurality of working electrodes 10a and 10b.
  • the first reagent layer 15a of the first working electrode 10a contains, for example, glucose dehydrogenase or glucose oxidase. a first reagent containing the first reagent;
  • the first working electrode 10a includes a first protective film 16a containing a polymer compound containing 4-vinylpyridine as a constituent unit, as shown in FIG.
  • first protective film 16a Formation of such a first protective film 16a is as follows: After forming the first reagent layer 15a of the first working electrode 10a, a polymer compound containing 4-vinylpyridine as a constituent unit in alcohol is added to the second opening 4a1 of the second insulating layer 4a of the first working electrode 10a.
  • the first protective film 16a may be formed by forming droplets of the first liquid composition B containing and then drying the droplets.
  • the first protective film 16a may be formed in two steps as shown in FIGS. 9 to 12, or may be formed in one step or three or more steps (not shown).
  • the second reagent layer 15b of the second working electrode 10b contains, for example, lactate oxidase or lactate dehydrogenase. and a second reagent containing the second reagent.
  • the protective film 16b of the second working electrode 10b is, as shown in FIG.
  • a third protective film 16bb containing a polymer compound containing 4-vinylpyridine as a constituent unit is disposed on the second protective film 16ba.
  • the formation of the protective film 16b including the second protective film 16ba and the third protective film 16bb is as follows: After forming the second reagent layer 15b of the second working electrode 10b, a polymer compound containing the cation exchange functional group in alcohol is added to the second opening 4b1 of the second insulating layer 4b of the second working electrode 10b. forming droplets of the second liquid composition B containing and then drying to form a second protective film 16ba (see FIGS. 13 and 14); and after forming the second protective film 16ba, performing a second action.
  • Droplets of a third liquid composition B containing a polymer compound containing 4-vinylpyridine as a constituent unit in alcohol are formed in the second opening 4b1 of the second insulating layer 4b of the pole 10b, and then dried. to form a third protective film 16bb (see FIGS. 15 and 16).
  • a third protective film 16bb can include.
  • the protective films 16a and 16b of the plurality of working electrodes 10a and 10b can be formed individually and independently, so the composition of the liquid composition B containing the protective film component in alcohol can be adjusted. By doing so, it is easy to form protective films 16a and 16b having different compositions optimized according to the reagent layers 15a and 15b on the reagent layers 15a and 15b containing different reagents of the plurality of working electrodes 10a and 10b. It is.
  • the method according to this embodiment can include a step of forming the reference electrode 20.
  • the step of forming the reference electrode 20 is as follows: In the substrate 2 shown in FIG. 3 on which the reference electrode conductive layer 21, the first insulating layer 3, and the second insulating layer 4 are arranged, the reference electrode conductive layer 21 in the reference electrode first opening 301 of the first insulating layer 3 placing a silver/silver chloride layer 22 on top (FIG. 4); and after placing the silver/silver chloride layer 22, the silver/silver chloride in the reference electrode second opening 401 of the second insulating layer 4 of the substrate 2; Arranging the reference electrode protective film 23 on the layer 22 (FIG. 5, FIG. 22) can include.
  • Example of the first disclosure As an example of the first disclosure, a sensor 1 having the configuration shown in FIG. 5 was manufactured. An outline of the materials and manufacturing method used for the sensor 1 having the configuration shown in FIG. 5 is shown below.
  • substrate As the insulating substrate 2, a 188 ⁇ m thick substrate made of polyethylene terephthalate and having the shape shown in FIG. 1 etc. was used.
  • the conductive layers 11a and 11b of the working electrodes 10a and 10b and the reference electrode are formed in the shape shown in FIG.
  • the conductive layer 21, the counter conductive layer 31, and the wiring 50 electrically connected to each of them were formed of a carbon conductive layer with a thickness of 5 ⁇ m.
  • first insulating layer (first insulating layer) Subsequently, as shown in FIG. 2, a first layer of the substrate 2 made of a fluororesin containing a copolymer containing vinylidene fluoride and hexafluoropropylene is placed on the first surface 2a of the substrate 2 on which the carbon conductive layer is disposed. A first insulating layer 3 covering the surface 2a and the carbon conductive layer and having a thickness of 5 ⁇ m on the carbon conductive layer was laminated. The first insulating layer 3 was formed by applying a composition that forms the fluororesin by curing onto the first surface 2a of the substrate 2 on which the carbon conductive layer is disposed, and heating the composition at 140° C. for 1 hour.
  • the first opening 3a1 of the first insulating layer 3a of the first working electrode 10a was circular with a diameter of 1.2 mm.
  • the first opening 3b1 of the first insulating layer 3b of the second working electrode 10b was circular with a diameter of 1.4 mm.
  • the reference electrode first opening 301 was a circle with a diameter of 1.1 mm, and the counter electrode first opening 302 was a rectangle with a size of 1.8 mm x 2.1 mm.
  • the water contact angle of the surface of the first insulating layer 3 was 134.2°.
  • To measure the water contact angle use a handy contact angle/surface free energy analyzer MSA manufactured by KRUSS Co., Ltd. at 20°C. This was done by measuring the contact angle between the drop and the surface.
  • the second opening 4a1 of the second insulating layer 4a of the first working electrode 10a and the second opening 4b1 of the second insulating layer 4b of the second working electrode 10b were each circular with a diameter of 2 mm.
  • the second opening 401 of the reference electrode was a circle with a diameter of 2 mm, and the second opening 402 of the counter electrode was a rectangle with a size of 1.8 mm x 2.1 mm.
  • the contact angle of the surface of the second insulating layer 4 (including the surfaces 4a2 and 4b2 of the second insulating layers 4a and 4b of the working electrodes 10a and 10b) with respect to ethanol was 60.7°.
  • the contact angle for ethanol was measured using a handy contact angle/surface free energy analyzer MSA manufactured by KRUSS, at 20°C, a droplet of 2 ⁇ L of ethanol was discharged onto the surface of the object to be measured, and after 2 seconds, This was done by measuring the contact angle between the droplet and the surface.
  • the first opening 3a1 of the first insulating layer 3a of the first working electrode 10a contains a sodium phosphate buffer (pH 7.4), a carbon black dispersion, a polymer-bonded mediator, glucose oxidase, and a crosslinking agent in water.
  • a 0.4 ⁇ L droplet of the first liquid composition A was formed and dried to form a first reagent layer 15a containing glucose oxidase and a mediator.
  • a carbon black dispersion, hydroxypropyl cellulose, a polymer-bonded mediator, lactic acid oxidase, polyimidazole, poly-L-lysine and a crosslinking agent are placed in water.
  • a 0.6 ⁇ L droplet of the second liquid composition A was formed and dried to form a second reagent layer 15b containing lactate oxidase and a mediator.
  • a first liquid composition B (P4VP-tBuMA polymer dispersion) was prepared by mixing the following reagents in ethanol to the following final concentrations and reacting for about 1 hour.
  • ⁇ P4VP-tBuMA Mn of poly-4-vinylpyridine: 74,000, Mn of poly-tert-butyl methacrylate: 87,000, Mw/Mn: 1.16, manufactured by NARD
  • final concentration 3.55 wt% ⁇ Random copolymer of tripropylene glycol methyl ether methacrylate-styrene-4-vinylpyridine manufactured by Nard
  • final concentration 4.45 wt% ⁇ PEGDGE poly(ethylene glycol) diglycidyl ether, Mn: ⁇ 1,000, manufactured by Sigma-Aldrich
  • final concentration 7mM A droplet of 0.65 ⁇ L of the first liquid composition B is formed in the second opening 4a1 of the second insulating layer 4a of
  • a dispersion liquid was prepared.
  • the resulting Nafion (registered trademark) dispersion having a concentration of 16.12 wt % was designated as a second liquid composition B.
  • This second liquid composition B includes Nafion® in a solvent containing a lower alcohol.
  • a droplet of 0.60 ⁇ L of the second liquid composition B was formed in the second opening 4b1 of the second insulating layer 4b of the second working electrode 10b and dried to form a second protective film 16ba. (See Figure 14).
  • the same composition as the first liquid composition B (P4VP-tBuMA polymer dispersion) was used as a third liquid composition B in the following treatment. That is, a droplet of 0.65 ⁇ L of the third liquid composition B is formed in the second opening 4b1 of the second insulating layer 4b of the second working electrode 10b on which the second protective film 16ba is formed, and then dried. Thus, a third protective film 16bb was formed (see FIG. 16).
  • a reference electrode protective film 23 was placed on the silver/silver chloride layer 22 in the reference electrode second opening 401 of the second insulating layer 4 to form the reference electrode 20 (FIG. 22).
  • first insulating layer 3 On the first insulating layer 3, a composition containing the same polyester resin as in the example in a solvent but not containing a fluorine-based surface modification additive was coated and heated at 140° C. for 1 hour to form a thickness of 38 ⁇ m. A second insulating layer was formed.
  • the contact angle of the surface of the second insulating layer of the sensor of the comparative example thus obtained with respect to ethanol was 11.2°.
  • the first liquid composition B P4VP-tBuMA polymer dispersion
  • a droplet of .65 ⁇ L was formed, as shown in FIG. 17, the droplet did not remain in the second opening 4a1, but the first liquid composition B wetted and spread to the upper surface of the second insulating layer 4a.
  • this droplet was dried, as shown in FIG. 18, a part of the first protective film 16a was formed not only inside the second opening 4a1 but also on the upper surface of the second insulating layer 4a.
  • the first liquid composition B After drying, a droplet of 0.65 ⁇ L of the first liquid composition B was formed, and as shown in FIG. 19, the first liquid composition B wetted the upper surface of the second insulating layer 4a. It expanded. When it was dried, a part of the first protective film 16a was formed not only inside the second opening 4a1 but also on the upper surface of the second insulating layer 4a, as shown in FIG.
  • the second liquid composition B (Nafion (registered trademark)), which is the same as that used in the example, was placed in the second opening 4b1 of the second insulating layer 4b of the second working electrode 10b of the sensor of the comparative example.
  • the second protective film 16ba was irregularly formed not only within the second opening 4b1 but also on the upper surface of the second insulating layer 4b.
  • the third protective film 16bb was formed only in the second opening 4b1. Instead, they were also irregularly formed on the upper surface of the second insulating layer 4b.
  • ⁇ Glucose and lactic acid measurement experiment> (Culture solution) In a solution of RPMI-1640 Medium (R1383, manufactured by Sigma-Aldrich), MES (2-Morpholinoethanesulfonic acid monohydrate) (manufactured by Dojindo Chemical Co., Ltd.) and MOPS (3-Morpholinopropanesul) were added as buffer components. fonic acid) (manufactured by Dojin Chemical Co., Ltd.) A culture solution was prepared by adding each to a final concentration of 25mM, and adjusting the glucose concentration to 30mM, the lactic acid concentration to 15mM, and the pH to 7.4.
  • the sensor 1 of the above example and the sensor of the comparative example were immersed in the above culture solution, and a voltage of 100 mV was applied to the first working electrode and the second working electrode with respect to the reference electrode.
  • FIG. 25A shows the measurement results of the current value of the first working electrode (for glucose measurement) of the sensor 1 of the example.
  • the measurement results of the current value of the first working electrode (for glucose measurement) of the sensor of the comparative example are shown in FIG. 25B.
  • the current value in the sensor 1 of the example was stable, whereas the current value in the sensor of the comparative example decreased over time.
  • the glucose permeability was high from the beginning and that the reagent containing glucose oxidase leaked.
  • FIG. 26A shows the measurement results of the current value of the second working electrode (for lactic acid measurement) of the sensor 1 of the example.
  • FIG. 26B shows the measurement results of the current value of the second working electrode (for measuring lactic acid) of the sensor of the comparative example.
  • the current value in the sensor 1 of the example was stable, whereas the current value in the sensor of the comparative example decreased over time.
  • the material of the insulating substrate of the sensor according to the second disclosure of this specification is not particularly limited, but, for example, the same material as the material of the insulating substrate of the sensor of the first disclosure of this specification can be used.
  • the conductive layer included in the working electrode, reference electrode, and counter electrode of the sensor according to the second disclosure of this specification is a layer containing a conductive material such as carbon, gold, platinum, palladium, or the like.
  • the conductive layer can be manufactured by forming a layer of the conductive material as described above on the surface of the substrate using a sputtering method, a vapor deposition method, a screen printing method, or the like.
  • the conductive layer can be processed into a predetermined pattern using a laser trimming method, if necessary.
  • the wiring of the sensor according to the second disclosure of this specification can also be made of the same conductive material as the conductive layer.
  • the sensor according to the second disclosure of this specification is used to detect a predetermined analyte in the liquid sample by being immersed in the liquid sample.
  • the liquid sample contains water as a solvent.
  • the liquid sample include a cell culture solution and a liquid sample prepared using blood obtained from a living body.
  • the analyte are as described with respect to the analyte by the sensor of the first disclosure herein.
  • the working electrode of the sensor according to the second disclosure of this specification includes a reagent layer containing a reagent involved in a redox reaction.
  • the reagent involved in the redox reaction may be any reagent that is involved in the redox reaction of the test substance, and can be appropriately selected depending on the test substance.
  • Reagents involved in the redox reaction can include a combination of a redox enzyme and a mediator (electron carrier), or a redox enzyme.
  • Oxidoreductases can include coenzymes.
  • oxidoreductases examples include oxidases and dehydrogenases. Specific examples of oxidoreductases are as described with respect to the sensor of the first disclosure herein.
  • the mediator is not particularly limited, and examples thereof are as described with respect to the sensor of the first disclosure of this specification.
  • the reagent layer included in the working electrode of the sensor according to the second disclosure of the present specification can further contain components such as a buffer, a hydrophilic polymer compound, a conductive carbon filler, and a crosslinking agent.
  • a buffer such as a buffer, a hydrophilic polymer compound, a conductive carbon filler, and a crosslinking agent.
  • hydrophilic polymer compounds and conductive carbon fillers are as described with respect to the sensor of the first disclosure herein.
  • the protective film included in the working electrode of the sensor according to the second disclosure of this specification prevents or suppresses the reagent contained in the reagent layer from leaking out of the protective film, and prevents the analyte present outside the protective film from leaking out. It can be a permeable membrane.
  • the protective film having such properties preferably contains a polymer compound. Examples of the polymer compound contained in the protective film include a polymer compound containing 4-vinylpyridine as a constituent unit, a polymer compound containing a cation exchange functional group, and the like.
  • Examples of the polymer compound containing 4-vinylpyridine as a constitutional unit and the polymer compound containing a cation exchange functional group are as described with respect to the sensor disclosed in the first disclosure of this specification.
  • the protective film included in the working electrode of the sensor of the second disclosure herein can be a laminate of two or more protective films, an example of which is as described with respect to the sensor of the first disclosure herein. That's right.
  • Another suitable example of the protective film included in the working electrode of the sensor according to the second disclosure of this specification is a protective film containing a polymer compound containing 4-vinylpyridine as a constituent unit.
  • the reference electrode of the sensor according to the second disclosure of this specification can be provided with a protective film.
  • the protective film of the reference electrode can be made of the material described above for the protective film of the working electrode.
  • a sensor 1101 according to an embodiment of the second disclosure will be described with reference to the drawings.
  • the sensor 1101 of this embodiment as shown in FIG. 31 and its cross-sectional views, FIGS. 33, 34, 35, and 36, an insulating substrate 1200 including a tip 1201 immersed in a liquid sample; a detection electrode 1300 that is disposed near the tip 1201 on the first surface 1202 of the substrate 1200 and includes a working electrode 1310, a reference electrode 1320, and a counter electrode 1330; Wiring 1005 arranged on the first surface 1202 of the substrate 1200 and connected to the detection electrode 1300; an insulating layer 1050 disposed on the first surface 1202 of the substrate 1200 and formed to cover at least a portion of the detection electrode 1300 and the wiring 1005; a tip opening 1061 formed in the insulating layer 1050 and communicating with the upper surface 1331 of the counter electrode 1330 and opening toward the tip 1201 of the substrate 1200;
  • the insulating layer 1050 includes a channel
  • the sensor 1101 of this embodiment includes two working electrodes 1310, in another embodiment not shown, there may be only one working electrode, or there may be three or more working electrodes.
  • the sensor 1101 is used to detect a predetermined test substance in the liquid sample X1 by being immersed in the liquid sample X1, as shown in FIGS. 39 and 43. Specific examples of the liquid sample and test substance are as described in the ⁇ Materials> column.
  • the working electrode 1310 of the sensor 1101 includes a working electrode conductive layer 1311 and a reagent disposed on the working electrode conductive layer 1311 that participates in the redox reaction of the test substance in the liquid sample X1.
  • a reagent layer 1315 containing the reagent layer 1315 is included. Preferred embodiments of the reagents involved in the redox reaction are as described in the ⁇ Materials> column.
  • the reagent layer 1315 of the working electrode 1310 contains a reagent that oxidizes the analyte in the liquid sample Electrons move. Similarly, when the reagent layer 1315 includes a reagent that reduces the analyte in the liquid sample X1, electrons move from the working electrode 1310 to the analyte. Since the amount of moving electrons depends on the concentration of the test substance, the concentration or change in concentration of the test substance in the liquid sample X1 is measured based on the current value or change in the current value flowing through the working electrode 1310 of the sensor 1101. be able to.
  • the analysis device 1100 includes a sensor 1101, an analysis unit 1102, and a control unit 1104.
  • Detection electrodes 1300 including working electrode 1310, reference electrode 1320, and counter electrode 1330 of sensor 1101 are each connected to analysis unit 1102 via wiring 1005.
  • Analysis unit 1102 is in communication with control unit 1104.
  • the analysis unit 1102 includes an electrochemical measurement section 1111, a control section 1112, a storage section 1113, and a communication section 1114.
  • the electrochemical measurement section 1111 is a potentiostat that measures the concentration of a test substance by applying a predetermined voltage to the detection electrode 1300 of the sensor 1101, and includes a voltage application section 1111a and a current measurement section 1111b. , preferably further includes a voltage measuring section (counter electrode terminal voltage measuring section) 1111c.
  • the voltage application unit 1111a applies a predetermined voltage to the detection electrode 1300 of the sensor 1101 in order to measure the concentration of the test substance contained in the liquid sample X1.
  • the current measurement unit 1111b measures the value of the current flowing between the working electrode 1310 and the counter electrode 1330 of the sensor 1101, or its change, which is measured while a voltage is applied from the voltage application unit 1111a to the detection electrode 1300 of the sensor 1101. Detect. As described above, the current value or the change in the current value detected by the current measurement unit 1111b serves as an index of the concentration or concentration change of the test substance in the liquid sample X1.
  • the voltage measurement unit 1111c measures the terminal voltage of the counter electrode 1330 of the sensor 1101.
  • the control section 1112 is connected to the voltage application section 1111a, the current measurement section 1111b, the voltage measurement section 1111c, the storage section 1113, and the communication section 1114.
  • the control unit 1112 controls the voltage application unit 1111a to apply a predetermined voltage to the detection electrode 1300 of the sensor 1101, and transmits the measurement results in the current measurement unit 1111b and voltage measurement unit 1111c to the control unit 1104.
  • the communication unit 1114 is controlled so as to do so.
  • the storage unit 1113 is connected to the control unit 1112, and stores, for example, applied voltage values preset for each measurement target, measured values in the current measurement unit 1111b and voltage measurement unit 1111c, and pre-measured calibration curves. Save data.
  • the communication section 1114 is controlled by the control section 1112 and transmits data such as measurement results in the current measurement section 1111b and the voltage measurement section 1111c to the analysis section 1142 of the control unit 1104.
  • the control unit 1104 can communicate with the analysis unit 1102 via the communication section 1114, and includes a display section 1141 and an analysis section 1142.
  • the display unit 1141 displays the concentration of the test substance in the liquid sample X1 based on the current value detected by the current measurement unit 1111b as a result of the analysis in the analysis unit 1142, for example.
  • the analysis unit 1142 is, for example, a PC (Personal Computer), and calculates the concentration of the test substance based on the value of the current flowing between the working electrode 1310 and the counter electrode 1330, which is measured by the current measurement unit 1111b.
  • PC Personal Computer
  • the substrate 1200 of the sensor 1101 of this embodiment includes a tip 1201 that is immersed in a liquid sample.
  • the substrate 1200 may be a plate-shaped body.
  • the tip 1201 is a portion of the peripheral edge of the substrate 1200 that becomes the tip when the sensor 1101 is immersed in the liquid sample X1 (see FIG. 43).
  • Preferred embodiments of the substrate 1200 are as described in the ⁇ Material> column.
  • a detection electrode 1300 including a working electrode 1310, a reference electrode 1320, and a counter electrode 1330 is arranged near the tip 1201 on the first surface 1202 of the substrate 1200.
  • the wiring 1005 of the sensor 1101 is arranged on the first surface 1202 of the substrate 1200 and is electrically connected to each electrode of the detection electrode 1300 (working electrode 1310, reference electrode 1320, and counter electrode 1330). Preferred embodiments of the material of the wiring 1005 are as described in the ⁇ Material> column.
  • the insulating layer 1050 of the sensor 1101 is placed on the first surface 1202 of the substrate 1200 and is formed to cover at least a portion of the detection electrode 1300 and the wiring 1005.
  • the insulating layer 1050 includes a first insulating layer 1051 disposed on the first surface 1202 of the substrate 1200 and a second insulating layer 1052 disposed on the first insulating layer 1051.
  • the insulating layer 1050 may consist of only one layer, or may include three or more layers.
  • the insulating layer 1050 may be any insulating layer, and the material thereof is not particularly limited.
  • the insulating layer 1050 may be an insulating layer containing an insulating cured resin, an insulating layer containing an insulating sheet, or a plurality of insulating layers. It may be a combination of
  • An insulating layer containing an insulating cured resin can be formed by curing a curable resin composition.
  • a curable resin composition a composition that can be polymerized and/or crosslinked by irradiation with active energy rays to produce an insulating cured resin can be used.
  • active energy rays include ultraviolet rays, electron beams, X-rays, infrared rays, and visible rays, with ultraviolet rays or electron beams being preferred.
  • a negative resist composition can be used as the curable resin composition.
  • fluororesin is preferable.
  • the fluororesin may be any resin containing a polymer containing fluorine atoms on the main chain carbon and/or side chain, such as poly(meth)acrylate resin containing fluorine atoms on the main chain carbon and/or side chain, Examples include polyester resin.
  • the thickness of the insulating layer containing the insulating cured resin is not particularly limited, but may be, for example, 1 ⁇ m or more and 50 ⁇ m or less.
  • the insulating layer including the insulating sheet can include the insulating sheet and a bonding layer including an adhesive or adhesive disposed on one side of the insulating sheet.
  • An insulating layer including an insulating sheet can be formed by attaching an insulating sheet including a bonding layer onto the first surface 1202 of the substrate 1200 directly or via another insulating layer.
  • the insulating sheet can be an insulating resin sheet, such as a polyethylene terephthalate sheet.
  • a bonding layer for example, a double-sided tape or a layer of adhesive or adhesive can be used.
  • the outer surface of the insulating sheet is preferably a water-repellent surface, and may be a water-repellent surface imparted with water repellency by, for example, water-repellent coating or surface roughening treatment.
  • the thickness of the insulating layer including the insulating sheet is not particularly limited, but the thickness of the insulating sheet can be, for example, 10 ⁇ m or more and 200 ⁇ m or less, preferably 20 ⁇ m or more and 100 ⁇ m or less, and one of the insulating sheets
  • the thickness of the bonding layer disposed on the surface can be, for example, 1 ⁇ m or more and 50 ⁇ m or less, preferably 5 ⁇ m or more and 20 ⁇ m or less.
  • a preferred embodiment of the insulating layer 1050 includes a first insulating layer 1051 containing an insulating cured resin disposed on the first surface 1202 of the substrate 1200, and a first insulating layer 1051 containing an insulating sheet disposed on the first insulating layer 1051. 2 insulating layers 1052.
  • the second insulating layer 1052 can include an insulating sheet and a bonding layer disposed on the surface of the insulating sheet on the first insulating layer 1051 side, and the insulating sheet is connected to the first insulating layer through the bonding layer. Preferably, it is pasted on layer 1051.
  • the thickness of the first insulating layer 1051 containing an insulating cured resin is not particularly limited; )
  • the thickness of the portion covering the surface can be, for example, 0.5 ⁇ m or more and 50 ⁇ m or less, preferably 2 ⁇ m or more and 20 ⁇ m or less.
  • Examples of the thickness of the second insulating layer 1052 including the insulating sheet are as described in the previous paragraph.
  • FIG. 33 is a cross-sectional view of a portion including the working electrode 1310 of the sensor 1101 shown in FIG. 31, taken along line JJ' in FIG.
  • the working electrode 1310 includes a working electrode conductive layer 1311 disposed on the first surface 1202 of the substrate 1200 and a reagent layer 1315 disposed on the working electrode conductive layer 1311.
  • Working electrode 1310 preferably further includes a working electrode protective film 1316 disposed on reagent layer 1315 as shown.
  • Preferred materials for the working electrode conductive layer 1311, the reagent layer 1315, and the working electrode protective film 1316 are as described in the ⁇ Material> column.
  • a wiring 1005 is connected to the working electrode conductive layer 1311 of the working electrode 1310 (see FIGS. 27 and 31).
  • the working electrode 1310 includes the working electrode protective film 1316
  • the working electrode protective film 1316 suppresses leakage of reagent from the reagent layer 1315. Further, since the working electrode protective film 1316 is porous, the liquid sample X1 can permeate therein. Therefore, when the sensor 1101 is immersed in the liquid sample X1, the liquid sample X1 can easily reach the working electrode 1310, and immersion of the liquid sample X1 into the working electrode 1310 is less likely to be inhibited.
  • the insulating layer 1050 includes a working electrode opening 1501 that penetrates in the thickness direction T1 and is formed at a position overlapping with at least a portion of the working electrode 1310 when viewed in plan from the thickness direction T1 of the substrate 1200.
  • the working electrode opening 1501 is located on the working electrode conductive layer 1311, and the reagent layer 1315 and the working electrode protective film 1316 are arranged within the working electrode opening 1501.
  • the outer periphery of each of the reagent layer 1315 and the working electrode protective film 1316 is defined by the inner periphery of the working electrode opening 1501.
  • the shape of the inner peripheral edge of the working electrode opening 1501 of the insulating layer 1050 when viewed from the thickness direction T1 is circular in the illustrated example, but is not limited to this, and may be polygonal (quadrangular, triangular, etc.). or any other arbitrary shape.
  • the opening width (the width of the widest part) of the working electrode opening 1501 of the insulating layer 1050 is not particularly limited, but may be, for example, 0.5 mm or more and 10 mm or less, preferably 1 mm or more and 5 mm or less.
  • the working electrode opening 1501 of the insulating layer 1050 is constituted by a working electrode first opening 1511 of the first insulating layer 1051 and a working electrode second opening 1521 of the second insulating layer 1052.
  • the working electrode first opening 1511 is formed in the first insulating layer 1051 at a position that overlaps with a part of the working electrode conductive layer 1311 when viewed from the thickness direction T1, and penetrates in the thickness direction T1.
  • the second working electrode opening 1521 is formed at a position that overlaps a portion of the first insulating layer 1051 that includes the entire first working electrode opening 1511 in the second insulating layer 1052 when viewed in plan from the thickness direction T1. It penetrates in the thickness direction T1.
  • the reagent layer 1315 is disposed within the working electrode first opening 1511 of the first insulating layer 1051 and includes an outer peripheral edge defined by an inner peripheral edge of the working electrode first opening 1511.
  • the working electrode protective film 1316 is disposed within the second working electrode opening 1521 of the second insulating layer 1052 and includes an outer peripheral edge defined by an inner peripheral edge of the second working electrode opening 1521.
  • the shape of the inner periphery of the working electrode first opening 1511 of the first insulating layer 1051 and the working electrode second opening 1521 of the second insulating layer 1052 in plan view from the thickness direction T1 is circular in the illustrated example. However, the shape is not limited to this, and each can be a polygon (quadrangular, triangular, etc.) or any other arbitrary shape.
  • the opening width (the width of the widest part) of the working electrode first opening 1511 of the first insulating layer 1051 is not particularly limited, but may be, for example, 0.5 mm or more and 5 mm or less, preferably 1 mm or more and 2 mm or less.
  • the opening width (the width of the widest part) of the working electrode second opening 1521 of the second insulating layer 1052 is not particularly limited, but may be, for example, 0.5 mm or more and 10 mm or less, preferably 1 mm or more and 5 mm or less. .
  • FIG. 34 is a cross-sectional view of a portion of the sensor 1101 in FIG. 31 including the reference pole 1320, taken along the line K-K' in FIG.
  • the reference electrode 1320 includes a reference electrode conductive layer 1321 disposed on the first surface 1202 of the substrate 1200 and a silver/silver chloride layer 1322 disposed on the reference electrode conductive layer 1321.
  • Reference electrode 1320 preferably further includes a reference electrode overcoat 1326 disposed over silver/silver chloride layer 1322 as shown.
  • Preferred materials for the reference electrode conductive layer 1321 and the reference electrode protective film 1326 are as described in the ⁇ Material> column.
  • a wiring 1005 is connected to the reference electrode conductive layer 1321 of the reference electrode 1320 (see FIGS. 27 and 31).
  • Reference electrode protective film 1326 suppresses leakage of silver from silver/silver chloride layer 1322. Further, since the reference electrode protective film 1326 is porous, the liquid sample X1 can permeate therein. Therefore, when the sensor 1101 is immersed in the liquid sample X1, the liquid sample X1 can easily reach the reference electrode 1320, and immersion of the liquid sample X1 into the reference electrode 1320 is less likely to be inhibited.
  • the insulating layer 1050 includes a reference electrode opening 1502 that penetrates in the thickness direction T1 and is formed at a position overlapping at least a portion of the reference electrode 1320 when viewed in plan from the thickness direction T1 of the substrate 1200.
  • a part of the reference electrode conductive layer 1321, a silver/silver chloride layer 1322, and a reference electrode protective film 1326 are arranged within the reference electrode opening 1502. By disposing the reference electrode protective film 1326 within the reference electrode opening 1502, leakage of silver from the silver/silver chloride layer 1322 is more effectively suppressed.
  • the outer periphery of each of the silver/silver chloride layer 1322 and the reference electrode protective film 1326 is defined by the inner periphery of the reference electrode opening 1502.
  • the shape of the inner periphery of the reference electrode opening 1502 of the insulating layer 1050 in a plan view from the thickness direction T1 is circular in the illustrated example, but is not limited to this, and may be a polygon (quadrangular, triangular, etc.) or any other arbitrary shape.
  • the opening width (the width of the widest part) of the reference electrode opening 1502 of the insulating layer 1050 is not particularly limited, but can be, for example, 0.5 mm or more and 10 mm or less, preferably 1 mm or more and 5 mm or less.
  • the reference electrode opening 1502 in the insulating layer 1050 is constituted by a reference electrode first opening 1512 in the first insulating layer 1051 and a reference electrode second opening 1522 in the second insulating layer 1052.
  • the reference electrode first opening 1512 is formed in the first insulating layer 1051 at a position that overlaps with a part of the reference electrode conductive layer 1321 when viewed from the thickness direction T1, and penetrates in the thickness direction T1.
  • the reference electrode second opening 1522 is formed at a position that overlaps a portion of the first insulating layer 1051 that includes the entire reference electrode first opening 1512 in the second insulating layer 1052 when viewed in plan from the thickness direction T1. It penetrates in the thickness direction T1.
  • a portion of the reference electrode conductive layer 1321 and the silver/silver chloride layer 1322 are arranged in the reference electrode first opening 1512 of the first insulating layer 1051 and are defined by the inner peripheral edge of the reference electrode first opening 1512. Including the outer periphery.
  • the reference electrode protective film 1326 is disposed in the reference electrode second opening 1522 of the second insulating layer 1052 and includes an outer peripheral edge defined by an inner peripheral edge of the reference electrode second opening 1522.
  • the shapes of the inner peripheral edges of the reference electrode first opening 1512 of the first insulating layer 1051 and the reference electrode second opening 1522 of the second insulating layer 1052 in a plan view from the thickness direction T1 are each circular in the illustrated example.
  • the shape is not limited to this, and each can be a polygon (quadrangular, triangular, etc.) or any other arbitrary shape.
  • the opening width (the width of the widest part) of the reference electrode first opening 1512 of the first insulating layer 1051 is not particularly limited, but may be, for example, 0.5 mm or more and 5 mm or less, preferably 1 mm or more and 2 mm or less.
  • the opening width (the width of the widest part) of the reference electrode second opening 1522 of the second insulating layer 1052 is not particularly limited, but may be, for example, 0.5 mm or more and 10 mm or less, preferably 1 mm or more and 5 mm or less. .
  • FIG. 35 is a cross-sectional view of a portion of the sensor 1101 in FIG. 31 including the counter electrode 1330, taken along the line L-L' in FIG.
  • the counter electrode 1330 is placed on the first surface 1202 of the substrate 1200.
  • counter electrode 1330 consists entirely of a conductive layer. In order to specifically refer to the conductive layer portion of the counter electrode, it may be referred to as a "counter electrode conductive layer” or "counter electrode conductive layer.”
  • a preferred embodiment of the material of the counter electrode 1330 is as described in the ⁇ Material> column as the material of the conductive layer of the counter electrode.
  • a wiring 1005 is connected to the counter electrode 1330 (see FIGS. 27 and 31).
  • the insulating layer 1050 includes a counter electrode opening 1503 that penetrates in the thickness direction T1 and is formed at a position overlapping at least a part of the counter electrode 1330 when viewed from the thickness direction T1 of the substrate 1200.
  • the upper surface 1331 of the counter electrode 1330 is exposed through the counter electrode opening 1503.
  • the shape of the inner periphery of the counter electrode opening 1503 of the insulating layer 1050 in a plan view from the thickness direction T1 is a quadrilateral in the illustrated example, but is not limited to this, and may be a circle, a polygon other than a quadrangle (a triangle) etc.) or any other shape.
  • the opening width (the width of the widest part) of the counter electrode opening 1503 of the insulating layer 1050 is not particularly limited, but may be, for example, 0.5 mm or more and 10 mm or less, preferably 1 mm or more and 5 mm or less.
  • a portion of the upper surface 1331 of the counter electrode 1330 is exposed through the counter electrode opening 1503, but the present invention is not limited thereto.
  • the counter electrode opening 1503 may have a shape that includes the entire counter electrode 1330, and the entire upper surface 1331 of the counter electrode 1330 may be exposed through the counter electrode opening 1503.
  • the counter electrode opening 1503 of the insulating layer 1050 is constituted by a counter electrode first opening 1513 of the first insulating layer 1051 and a counter electrode second opening 1523 of the second insulating layer 1052.
  • the counter electrode first opening 1513 is formed in the first insulating layer 1051 at a position overlapping a part of the counter electrode 1330 when viewed in plan from the thickness direction T1, and penetrates in the thickness direction T1.
  • the counter electrode second opening 1523 is formed in the second insulating layer 1052 at a position that coincides with the counter electrode first opening 1513 of the first insulating layer 1051 in a plan view from the thickness direction T1. penetrate through.
  • the counter-electrode first opening 1513 of the first insulating layer 1051 and the counter-electrode second opening 1523 of the second insulating layer 1052 match, and it is sufficient that they at least partially overlap.
  • the inner peripheral shape and opening width of the counter electrode first opening 1513 of the first insulating layer 1051 and the counter electrode second opening 1523 of the second insulating layer 1052 are not particularly limited. may be within the ranges stated above.
  • FIG. 36 is a cross-sectional view of a portion of the sensor 1101 in FIG. 31 including the counter electrode 1330, the tip opening 1061, and the flow path 1062, taken along the line M-M' in FIG.
  • the tip opening 1061 is formed in the insulating layer 1050, communicates with the upper surface 1331 of the counter electrode 1330, and opens toward the tip 1201 of the substrate 1200.
  • the flow path 1062 is formed in the insulating layer 1050 and connects the tip opening 1061 and the upper surface 1331 of the counter electrode 1330.
  • Channel 1062 guides liquid sample X1 from tip opening 1061 to upper surface 1331 of counter electrode 1330.
  • the channel 1062 can guide the liquid sample X1 from the tip opening 1061 to the upper surface 1331 of the counter electrode 1330 by capillary action.
  • FIG. 39 is a schematic cross-sectional view corresponding to FIG. 36 of the sensor 1101 of this embodiment, which is immersed in the liquid sample X1 from the tip 1201 of the substrate 1200 and includes a tip opening 1061 and a flow path 1062.
  • FIG. 40 is a schematic cross-sectional view of a sensor 1150 of a comparative example that is immersed in liquid sample X1 and has the same structure as the sensor 1101 of this embodiment except that it does not include the tip opening 1061 and the flow path 1062.
  • the liquid sample X1 flows from the tip opening 1061 through the channel 1062 to the counter electrode 1330. It is guided to the upper surface 1331.
  • the upper surface 1331 of the counter electrode 1330 of the sensor 1101 can sufficiently contact the liquid sample X1.
  • this is preferable because the upper surface 1331 of the counter electrode 1330 of the sensor 1101 can sufficiently contact the liquid sample X1 even if the depth of the liquid sample X1 is not sufficient. Therefore, by using the sensor 1101 of this embodiment, the measurement accuracy of the analyte in the liquid sample X1 is increased.
  • the counter electrode 1330 and the tip 1201 on the first surface 1202 of the substrate 1200 are The region in between is covered with an insulating layer 1050.
  • the sensor 1150 of the comparative example with this structure is immersed in the liquid sample X1 so that the tip 1201 of the substrate 1200 becomes the tip, the liquid sample It is easy to keep away from the counter electrode 1330 due to liquid repellency.
  • the sensor 1150 of the comparative example contact between the counter electrode 1330 and the liquid sample X1 is likely to be inhibited, and the test substance in the liquid sample X1 may not be normally measured.
  • the counter electrode 1330 is a carbon conductive layer
  • the upper surface 1331 of the counter electrode 1330 has high water repellency, so that contact of the liquid sample X1 containing water as a solvent with the upper surface 1331 of the counter electrode 1330 is particularly likely to be inhibited.
  • This problem of the sensor 1150 of the comparative example can be solved by the sensor 1101 of this embodiment in which the tip opening 1061 and the flow path 1062 are formed in the insulating layer 1050.
  • the sensor 1101 of this embodiment shown in FIGS. 31 and 36 has a portion of the insulating layer 1050 between the counter electrode 1330 and the tip portion 1201 of the substrate 1200 when viewed in plan from the thickness direction T1 of the substrate 1200.
  • a tip opening 1061 and a flow path 1062 are formed.
  • the flow path 1062 is connected to the counter electrode opening 1503 of the insulating layer 1050, and the upper surface 1331 of the counter electrode 1330 is exposed through the counter electrode opening 1503, so that it cannot be immersed in the liquid sample X1.
  • the effect of the upper surface 1331 of the counter electrode 1330 coming into contact with the liquid sample X1 is particularly high.
  • the tip opening 1061 and flow path 1062 of the sensor 1101 shown in FIGS. 31 and 36 are recesses formed in the insulating layer 1050, which are closed on the side where the substrate 1200 is present and open on the top surface 1053 side of the insulating layer 1050.
  • it is not limited to this embodiment.
  • the tip opening 1061 and the flow path 1062 may be surrounded.
  • the flow path 1062 included in the sensor 1101 shown in FIGS. 31 and 36 has an expanded portion 1062A that expands the flow path width W (see FIG. 31) toward the tip opening 1061.
  • the passage 1062 having the expanded portion 1062A facilitates the movement of the liquid sample X1 from the tip opening 1061 to the upper surface 1331 of the counter electrode 1330 when the sensor 1101 is immersed in the liquid sample X1.
  • the channel width W is perpendicular to the direction from the counter electrode 1330 to the tip 1201 of the substrate 1200 (MM' direction in FIG. 31) when the sensor 1101 is viewed from the thickness direction T1 of the substrate 1200. It refers to the width of the flow path 1062 in the direction (LL' direction in FIG. 31).
  • the insulating layer 1050 includes a first insulating layer 1051 disposed on the first surface 1202 of the substrate 1200, and a second insulating layer 1052 disposed on the first insulating layer 1051.
  • a tip opening 1061 and a flow path 1062 are formed in the second insulating layer 1052.
  • the first insulating layer 1051 includes an insulating cured resin
  • the second insulating layer 1052 includes an insulating sheet.
  • the first insulating layer 1051 containing an insulating cured resin can be formed by applying a curable resin composition to the first surface 1202 of the substrate 1200 and curing the coating by irradiating the coating with active energy rays.
  • the second insulating layer 1052 including an insulating sheet can be formed by attaching an insulating sheet to the first insulating layer 1051 including an insulating cured resin via a bonding layer. After forming the second insulating layer 1052 including an insulating sheet, a portion of the insulating sheet between the counter electrode 1330 and the tip 1201 of the substrate 1200 is peeled off to form a tip opening 1061 and a flow path in the second insulating layer 1052. 1062 can be formed.
  • a second insulating layer 1052 including an insulating sheet in which a tip opening 1061 and a flow path 1062 are formed can be formed.
  • the tip opening 1061 and the flow path 1062 are further formed in the first insulating layer 1051 in the sensor 1101 shown in FIGS. 31 and 36. ing.
  • the sensor 1101'' shown in FIGS. 32 and 38 has a portion 1515 of the first insulating layer 1051 between the counter electrode 1330 and the tip 1201 of the substrate 1200 (see FIG. 36). ) in which a tip opening 1061 and a flow path 1062 are further formed. Since the sensor 1101'' shown in FIGS.
  • the first insulating layer 1051 is a layer containing an insulating cured resin
  • the second insulating layer 1052 is a layer containing an insulating sheet.
  • the sensor 1101'' has an insulating layer 1050 including a first insulating layer 1051 including an insulating cured resin and a second insulating layer 1052 including an insulating sheet by the procedure described in the previous paragraph.
  • the portion 1515 (see FIG. 36) of the first insulating layer 1051 containing an insulating cured resin is peeled off.
  • the first insulating layer 1051 lacking the portion 1515 may be formed.
  • the sensor 1101'' can also be manufactured by forming the second insulating layer 1052 thereon.
  • a first insulating layer 1051 is laminated on the first surface 1202 of the substrate 1200 on which the working electrode conductive layers 1311, 1311, the reference electrode conductive layer 1321, the counter electrode 1330, and the wiring 1005 are arranged.
  • the first insulating layer 1051 has working electrode first openings 1511, 1511 at a position overlapping with a part of the working electrode conductive layer 1311, 1311, and a reference electrode first opening 1512 at a position overlapping with a part of the reference electrode conductive layer 1321.
  • the first insulating layer 1051 includes an insulating cured resin.
  • the first insulating layer 1051 containing an insulating cured resin can be formed by applying a curable resin composition to the first surface 1202 of the substrate 1200 and curing the coating by irradiating the coating with active energy rays. can.
  • a second insulating layer 1052 is further laminated on the first insulating layer 1051 of the substrate 1200 in FIG. 28.
  • the second insulating layer 1052 includes a second working electrode opening 1521 having a shape that completely encloses the first working electrode opening 1511 , 1511 at a position overlapping with the first working electrode opening 1511 , 1511 of the first insulating layer 1051 .
  • a reference electrode second opening 1522 having a shape that encloses the entire reference electrode first opening 1512 at a position overlapping with the reference electrode first opening 1512, and a counter electrode first opening 1522 at a position overlapping with the counter electrode first opening 1513.
  • the second insulating layer 1052 further has a tip opening 1061 and a flow path 1062 in a portion between the counter electrode 1330 and the tip 1201 of the substrate 1200 when viewed in plan from the thickness direction T1 of the substrate 1200.
  • the second insulating layer 1052 includes an insulating sheet.
  • the second insulating layer 1052 including the insulating sheet can further include a bonding layer including an adhesive or adhesive disposed on one side of the insulating sheet.
  • the second insulating layer 1052 including an insulating sheet can be formed by attaching an insulating sheet onto the first insulating layer 1051 via a bonding layer.
  • a reagent layer 1315 is placed in the working electrode first openings 1511, 1511 of the first insulating layer 1051, and a reagent layer 1315 is placed in the reference electrode first opening 1512 of the first insulating layer 1051.
  • a silver/silver chloride layer 1322 is disposed.
  • working electrode protective films 1316, 1316 are placed in the working electrode second openings 1521, 1521 of the second insulating layer 1052 to form working electrodes 1310, 1310, and the second insulating layer A reference electrode protective film 1326 is disposed within the reference electrode second opening 1522 of the layer 1052 to form the reference electrode 1320.
  • the order of formation of each layer in FIGS. 30 and 31 is not particularly limited, and may be performed in a different order.
  • the substrate 1200 is A main body 1210 including a tip 1201 and a first end 1211 opposite to the tip 1201, in which a detection electrode 1300 is arranged; A second end 1222 connected to the first end 1211 of the main body 1210 and a third end 1223 opposite to the second end 1222, from the second end 1222 to the third end 1223.
  • the connecting portion 1220 of the substrate 1200 includes a bending portion 1225 near the third end 1223 that bends the substrate 1200 so that the first surface 1202 side of the substrate 1200 protrudes.
  • FIGS. 41, 42, 46, and 47 show plan views of the sensor 1101 before the substrate 1200 is bent at the bending portion 1225.
  • the wiring 1005 is preferably arranged on the first surface 1202 of the substrate 1200 from the main body part 1210 to the base end part 1230 via the connecting part 1220.
  • the terminal 1006 is arranged on the first surface 1202 of the proximal end portion 1230, and the wiring 1005 is connected to one of the detection electrodes 1300 on the first surface 1202 of the main body portion 1210 and the proximal end.
  • the portion 1230 is electrically connected to one of the terminals 1006 on the first surface 1202.
  • the substrate 1200 of the sensor 1101 shown in FIG. 41 is bent at the bending portion 1225 so that the first surface 1202 side on which the detection electrode 1300 and the like are arranged protrudes.
  • the substrate 1200 bent at the bending portion 1225 has a shape in which the main body portion 1210 and a portion of the connecting portion 1220 closer to the second end portion 1222 than the bending portion 1225 protrude from the base end portion 1230.
  • the main body 1210 of the substrate 1200 and the portion of the connecting portion 1220 closer to the second end 1222 than the bent portion 2125 has a substantially vertically protruding shape.
  • the sensor 1101 shown in FIG. 41 which includes the substrate 1200 bent at the bending portion 1225 of the connecting portion 1220, does not require tilting the proximal end portion 1230 of the substrate 1200 (for example, FIG. 43), as shown in FIGS. 44), the main body 1210 of the substrate 1200 on which the detection electrode 1300 is arranged is oriented so that the distal end 1201 is the lower end. , it is possible to immerse it in the liquid sample X1 for the measurement of the test substance.
  • a multiple sensor 1105 shown in FIG. 42 is one in which the base end portions 1230 of the substrate 1200 of the four sensors 1101 are integrated.
  • the insulating layer 1050 is disposed in a region of the first surface 1202 of the substrate 1200 that is closer to the second end 1222 than the bent portion 1225 of the main body 1210 and the connecting portion 1220.
  • the second insulating layer 1052 includes a first insulating sheet.
  • the portion of the connecting portion 1220 closer to the second end 1222 than the bent portion 1225 refers to the portion of the connecting portion 1220 from the end of the connecting portion 1220 on the second end 1222 side to the second end 1222. It is a part.
  • the insulating layer 1050 further includes a first insulating layer 1051 containing an insulating cured resin formed over the entire first surface 1202 of the substrate 1200.
  • the second insulating layer 1052 including the first insulating sheet includes, in addition to the first insulating sheet, a bonding layer (not shown) including a pressure-sensitive adhesive or an adhesive. and is attached to the above region of the first surface 1202 of the substrate 1200.
  • the part immersed in the liquid sample is achieved, and the tip opening 1061 and the flow path 1062 can be easily formed in the first insulating sheet of the second insulating layer 1052, which is preferable.
  • the insulating layer 1050 of the sensor 1101 shown in FIG. 41 includes a non-insulating region 1055 that does not include an insulating sheet on the first surface 1202 of the substrate 1200 in the region of the bent portion 1225 of the connecting portion 1220. The effects produced by this configuration will be explained with reference to FIGS. 44 and 45.
  • FIG. 44 is a schematic side view of the sensor 1101 shown in FIG. 41 in which the main body 1210 including the tip 1201 of the substrate 1200 is immersed in the liquid sample X1.
  • the first surface 1202 side of the substrate 1200 protrudes at the bent portion 1225 of the connecting portion 1220, and the bent portion of the main body portion 1210 and the connecting portion 1220 The portion closer to the second end portion 1222 than 1225 is bent so as to be approximately vertical.
  • the insulating layer 1050 includes a first insulating layer 1051 containing an insulating cured resin that covers the entire first surface 1202 of the substrate 1200, and A second insulating layer 1052 including a first insulating sheet covers a region of the first surface 1202 of the substrate 1200 that is closer to the second end 1222 than the bent portion 1225 of the main body 1210 and the connecting portion 1220; including.
  • the insulating layer 1050 has a non-insulating region on the first surface 1202 of the substrate 1200, which does not include an insulating sheet but includes a first insulating layer 1051 containing an insulating cured resin, on the area of the bent portion 1225 of the connecting portion 1220. Contains 1055.
  • the second insulating layer 1052 including the first insulating sheet is flat and not deformed. Therefore, the substrate 1200 is not affected by contraction or expansion that occurs after deforming the first insulating sheet, and the portions of the main body portion 1210 and the connecting portion 1220 closer to the second end portion 1222 than the bent portion 1225 have a flat shape. Retained. As a result, as shown in FIG. 44, the main body 1210 of the substrate 1200 of the sensor 1101 is held in a vertical position with the tip 1201 as the lower end in the liquid sample X1, and the detection electrode 1300 measures the analyte. can be performed successfully.
  • FIG. 45 shows the same structure as FIGS. 41 and 44 except that the second insulating layer 1052 including the first insulating sheet is formed on the entire first surface 1202 of the substrate 1200, including the area above the bent portion 1225.
  • a comparative example sensor 1151 having the same structure as sensor 1101 is shown.
  • the second insulating layer 1052 including the insulating sheet on the first surface 1202 of the substrate 1200 is stretched by bending the substrate 1200 at the bending portion 1225 . Since the second insulating layer 1052 including the stretched insulating sheet tends to contract, warping as shown in FIG. 45 occurs in the main body portion 1210 and the connecting portion 1220 of the substrate 1200.
  • the main body portion 1210 of the substrate 1200 that has been warped is inclined with the first surface 1202 side on which the detection electrode 1300 is disposed facing upward. For this reason, as shown in FIG. 45, contact between the liquid sample X1 and the detection electrode 1300 on the first surface 1202 of the main body 1210 is inhibited, and the accuracy of measurement of the analyte by the detection electrode 1300 is reduced.
  • the sensor 1101 shown in FIGS. 41 and 44 overcomes this problem of the sensor 1151 of the comparative example because the insulating layer 1050 includes a non-insulating region 1055 in the region above the bent portion 1225 of the first surface 1202 of the substrate 1200. Eliminate.
  • an insulating layer 1050 is formed on a region of the first surface 1202 of the substrate 1200 that is closer to the second end portion 1222 than the bent portion 1225 of the main body portion 1210 and the connecting portion 1220.
  • a second insulating layer 1052a including a first insulating sheet is disposed, and a second insulating layer 1052b including a second insulating sheet is disposed on a region of the proximal end portion 1230 of the first surface 1202 of the substrate 1200. including.
  • the insulating layer 1050 further includes a first insulating layer 1051 containing an insulating cured resin formed over the entire first surface 1202 of the substrate 1200.
  • the insulating layer 1050 includes a non-insulating region 1055 that does not include an insulating sheet but includes the first insulating layer 1051 on the first surface 1202 of the substrate 1200 in a region of the bent portion 1225 of the connecting portion 1220 .
  • the second insulating layer 1052a including the first insulating sheet and the second insulating layer 1052b including the second insulating sheet are respectively arranged in the above region of the first surface 1202 of the substrate 1200 from above the first insulating layer 1051. According to this embodiment, a decrease in detection accuracy due to warping of the substrate 1200 of the sensor 1101 can be suppressed, and the insulation between the wiring 1005 and the terminal 1006 on the base end portion 1230 of the substrate 1200 can be improved.
  • the first insulating sheet of the second insulating layer 1052a and the second insulating sheet of the second insulating layer 1052b are connected to the bent portion 1225 of the connecting portion 1220 on the first surface 1202 of the substrate 1200.
  • the regions are separated via a notch 1056 formed in a direction intersecting the direction in which the connecting portion 1220 extends.
  • a plurality of insulating layers 1050 are provided on the first surface 1202 of the substrate 1200 in the region of the bent portion 1225 of the connecting portion 1220, and are separated from the second insulating layers 1052a and 1052b via the notch 1056.
  • a second insulating layer 1052c including a third insulating sheet According to this embodiment, a decrease in detection accuracy due to warping of the substrate 1200 of the sensor 1101 can be suppressed, and the insulation between the wirings 1005 on the bent portion 1225 of the connection portion 1220 of the substrate 1200 can be improved.
  • ⁇ Sensor unit 900> The sensor unit 900 will be described with reference to FIGS. 51, 52, and 53.
  • the illustrated sensor unit 900 is used to position the main body 1210 of the substrate 1200 including the detection electrode 1300 of the sensor 1101 with respect to each well of the 24-well plate 925.
  • the sensor unit 900 includes a lower support plate 957, six sets of multiple sensors 1105 (see FIG. 42), and a port 961 for supplying additives, which are arranged in order from the bottom.
  • An upper support plate 959 and a gasket sheet 960 are provided.
  • the multi-sensor 1105 is formed by connecting four sensors 1101 shown in FIG. 42 at the base end 1230 of the substrate 1200 and integrating them.
  • the first surface 1202 side of the substrate 1200 protrudes at the bent portion 1225 of the connecting portion 1220 of the substrate 1200, and the side of the first surface 1202 of the substrate 1200 protrudes from the bent portion 1225 of the main body portion 1210 and the connecting portion 1220 with respect to the base end portion 1230.
  • the portion on the second end 1222 side is bent vertically.
  • six sets of multiple sensors 1105 including four sensors 1101 are attached to the lower support plate 957.
  • the lower support plate 957 is provided with through holes 957b at positions corresponding to the main body portion 1210 and the connection portion 1220 of the substrate 1200 of each of the 24 sensors 1101 included in the 6 sets of multiple sensors 1105.
  • the main body portion 1210 and the connecting portion 1220 of the substrate 1200 of each of the 24 sensors 1101 are inserted into each of the through holes 957b of the lower support plate 957.
  • the sensor 1101 is inserted into the through hole 957b of the lower support plate 957, as shown in FIG. becomes.
  • bent portion 1225 of the connecting portion 1220 of the substrate 1200 is inserted downward into the portion of the inner peripheral wall of the through hole 957b of the lower support plate 957 that engages with the inside of the bent portion 1225 of the connecting portion 1220 of the substrate 1200 of the sensor 1101.
  • a first engaging portion 957d is provided to support the first engaging portion 957d.
  • the lower side of the upper support plate 959 is provided with pin-shaped second engaging portions 959a that are inserted into each of the 24 through holes 957b of the lower support plate 957.
  • the first engaging portion 957d of the lower support plate 957 has a curved upper surface shape including a curved upper surface.
  • the second engaging portion 959a of the upper support plate 959 has a lower curved portion shape including a curved lower surface.
  • the sensor 1101 is supported such that the distal end 1201 of the substrate 1200 is the lower end, and the portion of the substrate 1200 on the distal side of the main body 1210 and the bent portion 1225 of the connecting portion 1220 is vertical.
  • the posture of the main body portion 1210 of the substrate 1200 of the sensor 1101 with respect to the liquid sample contained in each well of the well plate can be stabilized, and the accuracy of detection by the sensor 1101 can be improved.
  • a gasket sheet 960 having through holes is stacked on the upper surface of the upper support plate 959 to complete the sensor unit 900.
  • the sensor unit 900 can be combined with other members to configure an adapter unit 920. As shown in FIG. 53, in the adapter unit 920, an adapter bottom (culture container installation part) 924, a well plate (culture container) 925, an adapter top 926, and a sensor unit 900 are placed in this order from the bottom. In this embodiment, the well plate 925 has 24 4 ⁇ 6 wells.
  • the adapter top 926 is provided to adjust the height of the well plate 925, and different adapter tops 926 are used depending on the height of the well plate 925. This is to adjust the height relationship between the sensor unit 900 and the well plate 925 when the sensor unit 900 is placed on the adapter top 926.
  • the sensor unit 900 placed on the adapter top 926 has four legs (support bodies) 940 provided on its lower surface that pass through the through holes 941 of the lower adapter top 926 and serve as a culture container installation part. is inserted into the positioning hole 942 provided in the adapter bottom 924 of the adapter.
  • the main body 1210 of the substrate 1200 including the detection electrode 1300 of the sensor 1101 is held in a stable posture within each well of the well plate 925. ing.
  • the terminal 1006 on the base end 1230 of the substrate 1200 of the sensor 1101 included in the sensor unit 900 is connected to the external analysis unit 1102 through the through hole of the gasket sheet 960. It can be electrically connected to the chemical measurement section 1111 (see FIG. 48). That is, the adapter unit 920 provided with the sensor unit 900 can configure the analysis apparatus 1100 shown in FIG. 48 by combining with the external analysis unit 1102 and control unit 1104.
  • Example of second disclosure The following five sensors were created as examples or comparative examples of the second disclosure. Note that the sensors S1 to S5 used in the following experiments were not provided with the reagent layers 1315, 1315 and the working electrode protective films 1316, 1316 of the working electrodes 1310, 1310, and the reference electrode protective film 1326 of the reference electrode 1320.
  • Sensor S1 is a comparative example of the structure shown in FIG. 49A.
  • a first insulating layer 1051 and a second insulating layer 1052 including an insulating sheet are laminated over the entire first surface 1202 of the substrate 1200.
  • the tip opening 1061 and the flow path 1062 are not formed, and the portion of the first surface 1202 of the substrate 1200 between the counter electrode 1330 and the tip 1201 is also formed with the first insulating layer 1051 and the second insulating layer. It is covered with an insulating layer 1050 including 1052.
  • the senor S1 does not have the non-insulated region 1055 of the insulating layer 1050 to prevent warping of the substrate 1200, and the tip opening 1061 and flow path 1062 for guiding the liquid sample to the upper surface 1331 of the counter electrode 1330. .
  • the sensor S2 includes a second insulating layer 1052a that includes a first insulating sheet disposed at a portion of the substrate 1200 closer to the tip than the bent portion 1225 of the connecting portion 1220, instead of the second insulating layer 1052 that covers the entire sensor S1. and a second insulating layer 1052b including a second insulating sheet disposed on the base end portion 1230 of the substrate 1200, and an insulating sheet including the first insulating layer 1051 on the bent portion 1225 of the connecting portion 1220 of the substrate 1200.
  • a non-insulating region 1055 that does not include is arranged.
  • the senor S2 has a non-insulated region 1055 of the insulating layer 1050 to prevent warping of the substrate 1200, but also has a tip opening 1061 and a channel 1062 for guiding the liquid sample to the upper surface 1331 of the counter electrode 1330. do not have.
  • sensor S3 Sensor S3 is an example of sensor 1101 having the structure shown in FIG. 49C. Similar to the sensor 1101 shown in FIG. 46, the sensor S3 includes a non-insulating region 1055 of the insulating layer 1050 to prevent warping of the substrate 1200, and a tip opening 1061 and a flow path 1062 formed only in the second insulating layer 1052. (See FIG. 36).
  • sensor S4 Sensor S4 is an example of sensor 1101 having the structure shown in FIG. 49D.
  • the second insulating layer 1052a including the first insulating sheet of the insulating layer 1050 and the second insulating layer 1052b including the second insulating sheet are integrated into an integrated structure that covers the entire first surface 1202 of the substrate 1200.
  • a second insulating layer 1052 including an insulating sheet is substituted, and a portion 1515 (see FIGS. 36 and 49C) of the first insulating layer 1051 between the counter electrode 1330 and the tip 1201 of the substrate 1200 is peeled off to form a tip. It has an opening 1061 and a flow path 1062.
  • the sensor S4 does not have the non-insulating region 1055 of the insulating layer 1050 for preventing the substrate 1200 from warping, and the tip opening 1061 and the flow path formed in the first insulating layer 1051 and the second insulating layer 1052 1062 (see FIG. 38).
  • sensor S5 Sensor S5 is an example of sensor 1101 having the structure shown in FIG. 49E.
  • the sensor S5 in the sensor S3, a portion 1515 (see FIGS. 36 and 49C) of the first insulating layer 1051 between the counter electrode 1330 and the tip 1201 of the substrate 1200 is peeled off to form the tip opening 1061 and the flow path 1062. That is.
  • the sensor S5 includes a non-insulating region 1055 of the insulating layer 1050 for preventing warping of the substrate 1200, a tip opening 1061 and a flow path 1062 formed in the first insulating layer 1051 and the second insulating layer 1052 (see FIG. 38).
  • substrate As an insulating substrate, a 188 ⁇ m thick substrate 1200 made of polyethylene terephthalate and including a main body portion 1210, a connecting portion 1220, and a base end portion 1230 and having a shape shown in FIGS. 49A to 49E was used.
  • the main body 1210 of the substrate 1200 was a rectangle with a width of 11 mm and a width of 4.2 mm.
  • the bent portion 1225 of the connecting portion 1220 of the substrate 1200 protrudes toward the first surface 1202, and the portion of the main body portion 1210 and the connecting portion 1220 that is closer to the tip than the bent portion 1225 is formed. , and bent so as to be perpendicular to the proximal end 1230.
  • a conductive layer 1321, a counter electrode (counter electrode conductive layer) 1330, a wiring 1005, and a terminal 1006 were formed.
  • first insulating layer 1051 On the first surface 1202 of the substrate 1200 on which the carbon conductive layer is disposed, as shown in FIG. A first insulating layer 1051 having a length of 5 ⁇ m was laminated.
  • the first insulating layer 1051 is formed by applying a curable resin composition (negative resist composition) that is cured by active energy ray irradiation to produce an insulating cured resin, and irradiating the coating film with active energy rays. did.
  • Appropriate masking was performed to form working electrode first openings 1511, 1511, reference electrode first opening 1512, and counter electrode first opening 1513 in the first insulating layer 1051.
  • a silver/silver chloride layer 1322 was placed inside the reference electrode first opening 1512 of the first insulating layer 1051.
  • a second insulating layer 1052 was disposed in a predetermined region on the first surface 1202 of the substrate 1200 on which the carbon conductive layer and the first insulating layer 1051 were disposed.
  • a second insulating layer 1052 which is a laminate of a 50 ⁇ m thick polyethylene terephthalate sheet (insulating sheet) whose surface has been water-repellent treated and a 10 ⁇ m thick bonding layer containing an adhesive, is attached to the substrate via the bonding layer.
  • 1200 was attached to a predetermined area on the first insulating layer 1051 disposed on the first surface 1202.
  • the working electrode second openings 1521, 1521 and the reference electrode second opening 1522 were each circular with a diameter of 2.0 mm.
  • the second opening 1523 of the counter electrode was a rectangle with a width of 2.1 mm in the horizontal direction of the main body 1210 and a width of 1.8 mm in the vertical direction.
  • sensors S1 to S5 were used in which the reagent layers 1315, 1315 and the working electrode protective films 1316, 1316 of the working electrodes 1310, 1310, and the reference electrode protective film 1326 of the reference electrode 1320 were not provided.
  • a 24-well plate was prepared, each having cylindrical wells with a depth of 17.40 mm, a well bottom diameter of 16.26 mm, and a well diameter of 15.62 mm. 0.85 mL, 0.94 mL, 1.0 mL, or 1.2 mL of RPMI medium supplemented with 500 ⁇ M ascorbic acid (hereinafter referred to as "liquid sample X1") was added to each well of the 24-well plate.
  • the sensors S1 to S5 are held horizontally with the base end 1230 of the substrate 1200 facing upward, and the main body 1210 of the substrate 1200 is held with the distal end 1201 facing upward.
  • the samples were immersed in each volume of liquid sample X1 in the wells so that
  • the voltage difference between the working electrodes 1310 and 1310 (not including the reagent layer and protective film) of each of the sensors S1 to S5 and the reference electrode 1320 (not including the protective film) was controlled by a potentiostat to be 500 mV.
  • the current values between each of the two working electrodes 1310, 1310 and the counter electrode 1330 were measured over time until 0.7 hours later. Further, in order to confirm whether the liquid sample X1 was in contact with the counter electrode 1330, the voltage of the counter electrode 1330 was measured over time.
  • the measurement was performed while vibrating the 24-well plate after a certain period of time had passed after the start of the measurement.
  • FIG. 50 shows the measurement results of the current values of the two working electrodes 1310 and 1310 when each of the four volumes of liquid samples is used for each of the sensors S1 to S5.
  • FIG. 50A shows the results for sensor S1
  • FIG. 50B shows the results for sensor S2
  • FIG. 50C shows the results for sensor S3
  • FIG. 50D shows the results for sensor S4,
  • FIG. 50E shows the results for sensor S5. .
  • Patent Document 2 discloses a cell culture analysis device having a cartridge that fits into a plate provided with a plurality of cell culture vessels.
  • the cell culture analyzer of Patent Document 2 has sensors that measure the inside of each culture container, and a plurality of openings into which these sensors are inserted are provided in the cartridge, and the sensors and fiber cables are connected in each opening. is connected. These plurality of fiber cables are connected to an external control unit.
  • the sensor of the cell culture analyzer described in Patent Document 2 has the following problems.
  • a second object of the disclosure is to provide a sensor and a sensor unit including the same, in which interference with contact of a liquid sample to a detection electrode of the sensor is suppressed when the sensor is immersed in a liquid sample.
  • a sensor used while immersed in a liquid sample an insulating substrate including a tip portion immersed in the liquid sample; a detection electrode disposed near the tip on the first surface of the substrate and including a working electrode, a counter electrode, and a reference electrode; Wiring arranged on the first surface of the substrate and connected to the detection electrode; an insulating layer disposed on the first surface of the substrate and formed to cover at least a portion of the detection electrode and the wiring; a tip opening formed in the insulating layer that communicates with the upper surface of the counter electrode and opens toward the tip of the substrate; A sensor comprising: a channel formed in the insulating layer, connecting the tip opening and the upper surface of the counter electrode, and guiding the liquid sample from the tip opening to the upper surface of the counter electrode.
  • the insulating layer includes a counter electrode opening that penetrates in the thickness direction and is formed at a position overlapping with the counter electrode in a plan view from the thickness direction of the substrate, the upper surface of the counter electrode is exposed through the counter electrode opening;
  • the sensor described in Appendix 1. (Additional note 3)
  • the insulating layer includes: a working electrode opening penetrating in the thickness direction formed at a position overlapping with the working electrode in a plan view from the thickness direction of the substrate; a reference electrode opening formed in a position overlapping with the reference electrode and penetrating in the thickness direction;
  • the working electrode includes a working electrode protective film disposed within the working electrode opening,
  • the reference electrode includes a reference electrode protective film disposed within the reference electrode opening.
  • the insulating layer is a first insulating layer containing an insulating cured resin, disposed on the first surface of the substrate; a second insulating layer including an insulating sheet, disposed on the first insulating layer, the tip opening and the flow path are formed in the second insulating layer;
  • the sensor described in any one of Supplementary Notes 1 to 3. (Appendix 5) The tip opening and the flow path are further formed in the first insulating layer.
  • the sensor described in Appendix 4. (Appendix 6) The sensor according to any one of Supplementary Notes 1 to 5, wherein the flow path has an expanded portion that expands the flow path width toward the tip opening.
  • the substrate is a main body portion including the tip portion and a first end portion opposite to the tip portion, and in which the detection electrode is disposed;
  • the main body includes a second end connected to the first end, and a third end opposite to the second end, and extends from the second end to the third end.
  • the connecting portion of the substrate includes a bending portion near the third end portion that bends the substrate so that the first surface side protrudes.
  • the insulating layer is a first insulating layer disposed on a region of the main body portion and a portion of the connecting portion closer to the second end than the bent portion on the first surface of the substrate. including sheets, The sensor described in Appendix 7. (Appendix 9) The insulating layer has a non-insulating area that does not include an insulating sheet on the first surface of the substrate, on the area of the bending part of the connecting part. The sensor described in Appendix 8.
  • the insulating layer further includes a second insulating sheet disposed on the base end region of the first surface of the substrate, The first insulating sheet and the second insulating sheet are separated on the first surface of the substrate in the region of the bent portion of the connecting portion,
  • the sensor according to appendix 8 or 9. (Appendix 11) The sensor according to any one of Supplementary Notes 1 to 10, and A sensor unit including a support member including an engaging part that engages with the substrate of the sensor and supports the sensor so that the tip end of the substrate becomes a lower end.
  • the material thereof is not particularly limited, but for example, the same material as the insulating substrate of the sensor according to the first disclosure of this specification may be used. Available.
  • the conductive layer included in the working electrode, reference electrode, and counter electrode of the sensor according to the third disclosure of this specification is a layer containing a conductive material such as carbon or gold.
  • the conductive layer can be manufactured by forming a layer of the conductive material as described above on the surface of the substrate using a sputtering method, a vapor deposition method, a screen printing method, or the like.
  • the conductive layer can be processed into a predetermined pattern using a laser trimming method, if necessary.
  • the conductive layer of the working electrode may be referred to as the working electrode conductive layer
  • the conductive layer of the reference electrode may be referred to as the reference electrode conductive layer.
  • the conductive layer of the counter electrode is simply referred to as a counter electrode because the conductive layer itself constitutes the counter electrode. More preferred embodiments of the conductive materials constituting the working electrode conductive layer and the counter electrode are as described below.
  • the wiring of the sensor according to the third disclosure of this specification can also be made of the same conductive material as the conductive layer.
  • the sensor according to the third disclosure of this specification is used to detect a predetermined analyte (analyte) in the liquid sample by being immersed in the liquid sample.
  • the liquid sample contains water as a solvent.
  • the liquid sample include a cell culture solution and a liquid sample prepared using blood obtained from a living body.
  • the test substance are as described with respect to the test substance by the sensor of the first disclosure of this specification, and particularly preferably one or more selected from glucose and lactic acid.
  • the liquid sample is preferably a liquid sample containing cells, especially living cells, and particularly preferably a cell culture solution.
  • the working electrode of the sensor according to the third disclosure of this specification includes a reagent layer disposed on the working electrode conductive layer and containing a reagent involved in the redox reaction of the analyte.
  • the reagent can be appropriately selected depending on the test substance.
  • the reagent may include a combination of an oxidoreductase and a mediator (electron carrier), or an oxidoreductase. Oxidoreductases can include coenzymes.
  • oxidoreductases examples include oxidases and dehydrogenases. Specific examples of oxidoreductases are as described with respect to the sensor of the first disclosure herein, and may particularly preferably be glucose oxidase, lactate oxidase, glucose dehydrogenase or lactate dehydrogenase.
  • the mediator is not particularly limited, and examples thereof are as described with respect to the sensor of the first disclosure of this specification.
  • the reagent layer included in the working electrode of the sensor according to the third disclosure of the present specification can further contain components such as a buffer, a hydrophilic polymer compound, a conductive carbon filler, and a crosslinking agent.
  • a buffer such as a buffer, a hydrophilic polymer compound, a conductive carbon filler, and a crosslinking agent.
  • hydrophilic polymer compounds and conductive carbon fillers are as described with respect to the sensor of the first disclosure herein.
  • the working electrode of the sensor according to the third disclosure of this specification can further include a protective film.
  • the protective film on the working electrode can be a film that prevents or suppresses leakage of the reagent contained in the reagent layer to the outside of the protective film and is permeable to the test substance present outside the protective film.
  • the protective film having such properties preferably contains a polymer compound.
  • the polymer compound contained in the protective film include a polymer compound containing 4-vinylpyridine as a constituent unit and a polymer compound containing a cation exchange functional group.
  • Examples of the polymer compound containing a cation exchange functional group include a polymer compound having proton conductivity.
  • Examples of cation exchange functional groups include anionic functional groups.
  • Examples of the polymer compound containing 4-vinylpyridine as a constituent unit and the polymer compound having proton conductivity are as described with respect to the sensor disclosed in the first disclosure of this specification.
  • the protective film included in the working electrode of the sensor of the third disclosure herein can be a laminate of two or more protective films, an example of which is as described with respect to the sensor of the first disclosure herein. That's right.
  • Another suitable example of the protective film included in the working electrode of the sensor according to the third disclosure of this specification is a protective film containing a polymer compound containing 4-vinylpyridine as a constituent unit.
  • the reference electrode of the sensor according to the third disclosure of this specification can be provided with a protective film.
  • the protective film of the reference electrode can be made of the material described above for the protective film of the working electrode.
  • the counter electrode of the sensor according to the third disclosure of this specification may be covered with a protective film. Preferred embodiments of the protective film covering the counter electrode will be described later.
  • a portion of the electrode including a working electrode and a counter electrode disposed on an insulating substrate other than the portion that contacts the liquid sample may be covered with an insulating layer.
  • the insulating layer can contain an insulating resin.
  • the insulating layer can have a multilayer structure of two or more layers.
  • the multilayer insulating layer can include, for example, a first insulating layer disposed on an insulating substrate and a conductive layer, and a second insulating layer disposed on the first insulating layer. Preferred embodiments of the materials constituting the first insulating layer and the second insulating layer are as described with respect to the sensor according to the first disclosure.
  • the sensor 300 of this embodiment includes an insulating substrate 2, a first working electrode 10a, a second working electrode 10b, and a reference electrode arranged on the first surface 2a of the substrate 2. 20 and a counter electrode 60, and a wiring 50 electrically connected to each of the first working electrode 10a, the second working electrode 10b, the reference electrode 20, and the counter electrode 60.
  • the sensor 300 of this embodiment includes two working electrodes, in another embodiment not shown, there may be only one working electrode, or three or more working electrodes.
  • the first working electrode 10a and the second working electrode 10b are not distinguished, they may be expressed as working electrodes 10a and 10b.
  • the first working electrode 10a, the second working electrode 10b, the reference electrode 20, and the counter electrode 60 may be collectively referred to as an electrode.
  • the sensor 300 of this embodiment is a three-electrode sensor that includes a working electrode, a reference electrode, and a counter electrode as electrodes, it may be a bipolar sensor that does not include a reference electrode but only includes a working electrode and a counter electrode.
  • the reference electrode and/or the counter electrode may be provided on a substrate different from the substrate on which the working electrode is arranged.
  • the sensor 300 is used to detect a predetermined analyte in the liquid sample X by being immersed in the liquid sample X, in the same way as shown in FIG. 21 regarding the sensor 1 of the first disclosure.
  • Specific examples of the liquid sample and test substance are as described in the ⁇ Materials> column.
  • the working electrodes 10a and 10b of the sensor 300 include working electrode conductive layers 11a and 11b, and reagent layers 15a and 15b containing reagents involved in the redox reaction of the test substance in the liquid sample.
  • reagent layers 15a and 15b containing reagents involved in the redox reaction of the test substance in the liquid sample.
  • Preferred embodiments of the reagent are as described in the ⁇ Materials> column.
  • the reagent layers 15a and 15b of the working electrodes 10a and 10b of the sensor 300 contain a reagent that oxidizes the analyte in the liquid sample
  • the analyte is removed from the analyte under the condition that a predetermined voltage is applied to the electrodes of the sensor 300. Electrons move to the working electrode conductive layers 11a and 11b.
  • the reagent layers 15a and 15b contain a reagent that reduces the analyte in the liquid sample
  • electrons move from the working electrode conductive layers 11a and 11b to the analyte. Since the amount of moving electrons depends on the concentration of the test substance, the concentration or change in concentration of the test substance in the liquid sample is measured based on the current value or change in the current value flowing through the working electrodes 10a and 10b of the sensor 300. can do.
  • the sensor 300 can constitute an analysis device for analyzing a test substance in a liquid sample.
  • the analysis device includes a sensor 300, an analysis unit 102, and a control unit 104 in which the sensor 1 according to the first disclosure is replaced with the sensor 300 according to the third disclosure in the analysis device 100 shown in FIG.
  • An example of this is an analyzer equipped with the following. The functions of this analyzer are as described with respect to the analyzer 100 shown in FIG. 24.
  • FIG. 54 and FIGS. 55, 56, and 57 are cross-sectional views thereof.
  • the structure of the reference pole 20 of the sensor 300 according to the third disclosure is the same as the structure of the reference pole 20 of the sensor 1 according to the first disclosure.
  • the description of the reference pole 20 of the sensor 1 according to the first disclosure with reference to FIG. 22 is cited as the description of the reference pole 20 of the sensor 300 according to the third disclosure.
  • the sensor 300 includes an insulating substrate 2, working electrode conductive layers 11a and 11b, a counter electrode 60, a reference electrode conductive layer 21, and a wiring 50, which are arranged on the first surface 2a of the substrate 2.
  • the counter electrode 60 is entirely made of a conductive layer. To specifically refer to the conductive layer portion of the counter electrode, it may also be referred to as a "counter electrode conductive layer” or "counter electrode conductive layer.”
  • a first insulating layer 3 is further arranged on the first surface 2a of the substrate 2, and a second insulating layer 4 is further arranged on the first insulating layer 3.
  • the height of the upper surface of the first insulating layer 3 from the first surface 2a of the substrate 2 is the height of the working electrode conductive layers 11a, 11b, the counter electrode 60, the reference electrode conductive layer 21, and the wiring 50 from the first surface 2a of the substrate 2. greater than the height from. Therefore, a part of the first insulating layer 3 is arranged on the working electrode conductive layers 11a, 11b, the counter electrode 60, the reference electrode conductive layer 21, and the wiring 50.
  • the first insulating layer 3 includes a counter electrode first opening 302 formed at a position overlapping a part of the counter electrode 60 and penetrating through the substrate 2 in the thickness direction T.
  • the second insulating layer 4 includes a second counter-electrode opening 402 that is formed in a position overlapping the entire counter-electrode first opening 302 of the first insulating layer 3 and that penetrates through the substrate 2 in the thickness direction T.
  • the counter electrode 60 is exposed to the outside through the first counter electrode opening 302 and the second counter electrode opening 402 .
  • a portion 60a of the counter electrode 60 that comes into contact with the liquid sample when the sensor 300 is immersed in the liquid sample is located at the bottom of the recess formed by the first opening 302 of the counter electrode.
  • the working electrodes 10a and 10b include working electrode conductive layers 11a and 11b, and a reagent that is disposed on the working electrode conductive layers 11a and 11b and is involved in the redox reaction of the test substance. It includes reagent layers 15a and 15b. Preferred embodiments of the reagent in the reagent layers 15a and 15b are as described in the ⁇ Materials> column.
  • the former in order to distinguish between the "reagent layer 15a" of the first working electrode 10a and the “reagent layer 15b" of the second working electrode 10b, the former is The latter may be referred to as the "second reagent layer 15b" of the second working electrode 10b.
  • the reagent included in the reagent layer 15a of the first working electrode 10a may be referred to as a "first reagent”
  • the reagent included in the second reagent layer 15b of the second working electrode 10b may be referred to as a "second reagent”.
  • the sensor 300 includes a plurality of working electrodes 10a, 10b, and a first reagent contained in the first reagent layer 15a of the first working electrode 10a among the plurality of working electrodes 10a, 10b;
  • the second reagents contained in the second reagent layer 15b of the second working electrode 10b, which is different from the working electrode 10a, are different from each other.
  • the first reagent participates in the redox reaction of the first analyte in the liquid sample
  • the second reagent is different from the first analyte in the liquid sample. Since it can participate in the redox reaction of the second analyte, it can be used to detect a plurality of analytes including the first analyte and the second analyte in a liquid sample.
  • the working electrodes 10a and 10b can include protective films 16a and 16b disposed on the reagent layers 15a and 15b.
  • Preferred embodiments of the protective films 16a and 16b are as described in the ⁇ Material> column.
  • a first protective film 16a consisting of a single layer is disposed on the first reagent layer 15a
  • a protective film 16b consisting of a second protective film 16ba and a third protective film 16bb is disposed on the second reagent layer 15b.
  • the protective films 16a and 16b are not limited to this example, and may have the same structure, or may have another structure not shown.
  • the first insulating layer 3 has a first working electrode formed in a position overlapping with a portion of each of the working electrode conductive layers 11a and 11b and extending through the substrate 2 in the thickness direction T. It includes openings 303a and 303b. Further, the second insulating layer 4 has a working electrode hole formed in the first insulating layer 3 at a position that overlaps with a portion that includes the whole of the first working electrode openings 303a and 303b, and which penetrates through the thickness direction T of the substrate 2. Two openings 403a and 403b are provided.
  • the working electrodes 10a, 10b are exposed to the outside through the working electrode first openings 303a, 303b and the working electrode second openings 403a, 403b. Portions 11a1 and 11b1 of the working electrode conductive layers 11a and 11b that come into contact with the liquid sample when the sensor 300 is immersed in the liquid sample are located at the bottom of the recess formed by the working electrode first openings 303a and 303b. . Further, the reagent layers 15a and 15b are contained in the first working electrode openings 303a and 303b, and the outer periphery of the reagent layers 15a and 15b is defined by the inner periphery of the first working electrode openings 303a and 303b. The protective films 16a, 16b are enclosed in the working electrode second openings 403a, 403b, and the outer peripheral edges of the protective films 16a, 16b are defined by the inner peripheral edges of the working electrode second openings 403a, 403b.
  • ⁇ Suppression of hydrogen peroxide generation by sensor 300> When the sensor 300 according to the third disclosure is immersed in a liquid sample and electrochemically measures an analyte in the liquid sample, the sensor 300 suppresses and/or generates hydrogen peroxide during measurement. By reducing the concentration of hydrogen peroxide, the concentration of hydrogen peroxide in the liquid sample is maintained at less than 15 ⁇ M during the measurement. Hydrogen peroxide is known to be toxic to cells. For this reason, when using an electrochemical sensor to continuously measure a test substance in a liquid sample containing cells, such as a cell culture medium, over a long period of several days or more, the concentration of hydrogen peroxide during measurement must be It is desirable to suppress the increase.
  • the sensor 300 it is possible to maintain the hydrogen peroxide concentration in the liquid sample at a low concentration of less than 15 ⁇ M during the measurement without causing a significant decrease in the detection sensitivity of the analyte.
  • measuring the test substance in a liquid sample electrochemically means, for example, connecting the sensor 300 immersed in the liquid sample to a potentiostat, and using the reference electrode 20 as a reference, the working electrodes 10a, 10b. It refers to detecting the current between the working electrodes 10a, 10b and the counter electrode 60 when a predetermined potential is applied to the electrodes, and measuring the analyte based on the detected current. Electrochemical measurements using the sensor 300 are typically carried out over a period of several hours or days, such as one hour or more, preferably 72 hours (3 days) or more, and more preferably 96 hours (4 days) or more.
  • Sensor 300 maintains the hydrogen peroxide concentration in the liquid sample below 15 ⁇ M during this period.
  • the hydrogen peroxide concentration of the liquid sample during said period is more preferably maintained below 10 ⁇ M.
  • the counter electrode 60 includes a catalyst for decomposing hydrogen peroxide in the portion 60a that contacts the liquid sample.
  • the generation of hydrogen peroxide is thought to be mainly caused by a reaction on the counter electrode 60 in which oxygen (O 2 ) in the liquid sample is reduced to hydrogen peroxide (H 2 O 2 ).
  • oxygen (O 2 ) in the liquid sample is reduced to hydrogen peroxide (H 2 O 2 ).
  • the generation of hydrogen peroxide on the counter electrode 60 can be a problem.
  • the working electrodes 10a, 10b are used, which are provided with reagent layers 15a, 15b containing oxidase, dehydrogenase, etc. involved in the oxidation of the test substance.
  • reagent layers 15a, 15b containing oxidase, dehydrogenase, etc. involved in the oxidation of the test substance.
  • the sensor 300 which includes the counter electrode 60 containing a catalyst that decomposes hydrogen peroxide in the portion 60a that contacts the liquid sample, oxygen (O 2 ) in the liquid sample is converted to water (H 2 O) on the counter electrode 60 during measurement. ), the generation of hydrogen peroxide is suppressed.
  • the reagents contained in the reagent layers 15a and 15b of the working electrodes 10a and 10b there are reagents that involve a reaction that generates hydrogen peroxide during measurement (for example, oxidase and dehydrogenase).
  • the hydrogen peroxide generated on the working electrodes 10a and 10b is also decomposed into water by the catalyst included in the counter electrode 60, so that the hydrogen peroxide concentration in the liquid sample being measured is reduced. increase may be suppressed.
  • the catalyst can be, for example, a catalyst containing a metal.
  • Catalysts containing metals are preferred because they are more stable than organic catalysts such as catalase.
  • a catalyst containing metal a catalyst containing one or more selected from platinum, nickel, palladium, iron, manganese, and tungsten is preferable, and a catalyst containing platinum is particularly preferable.
  • Catalysts containing these metals are preferred because they have a high activity for decomposing hydrogen peroxide, and catalysts containing platinum are particularly preferred from the viewpoint of catalytic activity and stability.
  • the catalyst may be included in the portion of the counter electrode that contacts the liquid sample, may be contained only in the portion that contacts the liquid sample, or may be contained in the entire counter electrode. It's okay. Further, the catalyst may be included in the counter electrode as a film that coats the surface of at least the part that contacts the liquid sample, or it may be contained in the counter electrode as a film that covers the surface of at least the part that contacts the liquid sample, or it may be dispersed in a conductive material that constitutes the entire counter electrode or at least the part that contacts the liquid sample. It may be included in the state.
  • the counter electrode in which the catalyst is included as a film that covers the surface of the part that contacts the liquid sample for example, a metal that can be used as the catalyst is applied to the liquid by sputtering, plating, etc.
  • a counter electrode obtained using a dispersion containing particles of the catalyst in a liquid medium is preferred because it can be produced by a simple method such as coating or printing.
  • the catalyst particles include particles of the metals described above, with platinum particles being particularly preferred.
  • the platinum particles are preferably platinum nanoparticles having an average particle diameter of less than 1 ⁇ m, for example, 800 nm or less, preferably 600 nm or less.
  • examples of the counter electrode in which the catalyst is contained in a dispersed state in a conductive material that constitutes the entire counter electrode or at least a portion that contacts a liquid sample include particles of the catalyst and the conductive material.
  • examples include counter electrodes obtained by forming the whole or at least the part that contacts the liquid sample using a pasty or liquid composition containing particles of . Since the counter electrode of this embodiment can contain the catalyst at the same time as forming the counter electrode on the substrate, it can be produced with a small number of steps.
  • examples of the catalyst particles include particles of the metals described above, with platinum particles being particularly preferred. Preferred embodiments of the platinum particles are as described above.
  • the counter electrode can contain carbon, gold, etc. as a conductive material, and preferably contains carbon as a conductive material.
  • carbon conductive carbon materials such as glassy carbon, carbon black, graphite, diamond-like carbon, graphene, carbon nanotubes, and fullerene can be used.
  • the content of the catalyst in the counter electrode can be appropriately set depending on the type and activity of the catalyst.
  • the platinum particles are contained in an amount of, for example, 0.5% by weight or more based on the carbon. , preferably 1% by weight or more, more preferably 3% by weight, particularly preferably 5% by weight or more, and the upper limit is not particularly limited, but from a cost perspective it is preferably 50% by weight or less (the ratio of the platinum particles to the carbon is, for example, 0%). 5% by weight or more and 50% by weight or less), more preferably 30% by weight or less, and most preferably 10% by weight or less.
  • the ratio of carbon to platinum particles within this range, it is possible to suppress an increase in the hydrogen peroxide concentration in the liquid sample during the measurement period.
  • the surface of the counter electrode may be further covered with a protective film.
  • the protective film covering the counter electrode preferably contains a polymer compound having a cation exchange functional group, particularly a polymer compound having a cation exchange functional group in a side chain.
  • examples of the polymer compound having a cation exchange functional group include a polymer compound containing a structural unit having a sulfonic acid group, preferably a perfluoro compound having a sulfonic acid group in the side chain.
  • a polymeric compound particularly preferably a copolymer of tetrafluoroethylene and perfluoro-2-(2-fluorosulfonylethoxy)propyl vinyl ether, most preferably Nafion®.
  • Anions such as chloride ions, organic acid ions, etc. that may be included in the liquid sample can adversely affect the activity of catalysts that decompose hydrogen peroxide, such as platinum catalysts.
  • a protective film containing a polymer compound having a cation exchange functional group it is possible to prevent anions from coming into contact with the counter electrode, thereby suppressing a decrease in the activity of the catalyst.
  • the working electrode conductive layers 11a and 11b of the working electrodes 10a and 10b have at least the portions 11a1 and 11b1 that come into contact with the liquid sample, and preferably the entire portion contains a catalyst that decomposes hydrogen peroxide. It is preferable not to include it.
  • the working electrode conductive layers 11a and 11b of the working electrodes 10a and 10b of the sensor 300 contain the catalyst, particularly a catalyst containing platinum, there is a possibility that background noise during measurement may increase and the current response value may decrease. .
  • the working electrode conductive layer 11a, 11b or the portion 11a1, 11b1 that does not contain a catalyst that decomposes hydrogen peroxide is a layer of a conductive material such as carbon, gold, etc. that does not contain a catalyst that decomposes hydrogen peroxide, or a portion thereof. Can be mentioned.
  • the working electrode conductive layers 11a and 11b of the working electrodes 10a and 10b are three electrodes including the working electrode conductive layers 11a and 11b as a working electrode, a silver silver chloride (saturated KCl) electrode as a reference electrode, and a platinum electrode as a counter electrode. It was measured when a potential in the range of -0.2V to 0.3V (vs. the silver-silver chloride (saturated KCl) electrode) was applied to the working electrode conductive layer in phosphate buffered saline using a formula electrolytic cell. satisfies the condition that the current value is ⁇ 5.5 nA/mm 2 or less.
  • the sensor 300 including the working electrode conductive layers 11a and 11b that satisfies this condition is preferable because the background current when measuring the analyte is sufficiently small and the responsiveness of the sensor is high.
  • working electrode conductive layers 11a and 11b include a carbon conductive layer containing carbon, and more preferably a carbon conductive layer that does not contain a catalyst that decomposes hydrogen peroxide, such as a catalyst containing platinum.
  • the carbon those exemplified for the conductive material of the counter electrode 60 can be used.
  • the current value of ⁇ 5.5 nA/mm 2 or less means that the vertical projected area of the portion of the working electrode conductive layers 11a and 11b used as the working electrode that comes into contact with the phosphate buffered saline is 1 mm 2 This means that the absolute value of the current value per unit is 5.5 nA or less.
  • the measurement of the current value using the above three-electrode electrolytic cell can be performed by the procedure described in ⁇ Third Disclosure/Experiment 5> mentioned above.
  • the present inventors further found that in the sensor 300 according to the present disclosure, in an embodiment in which the portion 60a of the counter electrode 60 that contacts the liquid sample includes a catalyst containing platinum, a negative current with a large absolute value can be caused to flow at a low potential.
  • the counter electrode 60 containing a platinum-containing catalyst in the portion 60a that contacts the liquid sample can efficiently function as a counter electrode with a smaller area compared to a counter electrode that does not contain a platinum-containing catalyst, thereby reducing the sensor size.
  • the projected area of the counter electrode 60 is preferably 250% or less, more preferably 100% or less, and preferably 3% or more of the projected area of the working electrode conductive layers 11a and 11b.
  • the projected area of the counter electrode can be, for example, 3% or more and 250% or less of the projected area of the working electrode conductive layer), more preferably 10% or more.
  • the sensor 300 including the counter electrode 60 containing a platinum-containing catalyst in the portion 60a that contacts the liquid sample is also suitable for a case where a plurality of working electrodes 10a and 10b are provided as shown in the figure.
  • the projected area of the counter electrode 60 refers to the vertical projected area of the portion 60a of the counter electrode 60 that contacts the liquid sample (corresponding to the bottom surface of the recess formed by the first counter electrode opening 302).
  • the projected area of the working electrode conductive layers 11a, 11b is the area of the portions 11a1, 11b1 of the working electrode conductive layers 11a, 11b that come into contact with the liquid sample (corresponding to the bottom surfaces of the recesses formed by the working electrode first openings 303a, 303b). , refers to the projected area in the vertical direction, and if a plurality of working electrodes 10a, 10b are present, refers to the total area thereof.
  • Example of third disclosure> In experiments 1 to 7 of the sensor according to the third disclosure, a sensor 300 having the configuration shown in FIG. 54 was used as an example and a comparative example of the sensor of the third disclosure.
  • substrate Similar to the first disclosed example, a 188 ⁇ m thick substrate made of polyethylene terephthalate and having the shape shown in FIG. 54 was used as the insulating substrate 2.
  • the electrode conductive layers 11a, 11b, the reference electrode conductive layer 21, the counter electrode (counter electrode conductive layer) 60, and the wiring 50 electrically connected to each of them were formed of a carbon conductive layer with a thickness of 5 ⁇ m.
  • first insulating layer (first insulating layer) Subsequently, the first surface 2a of the substrate 2 and the carbon conductive layer made of a fluororesin containing a copolymer containing vinylidene fluoride and hexafluoropropylene are placed on the first surface 2a of the substrate 2 on which the carbon conductive layer is disposed. A covering first insulating layer 3 having a thickness of 5 ⁇ m on the carbon conductive layer was laminated. The method for manufacturing the first insulating layer 3 is as described in the first disclosed embodiment.
  • the first working electrode opening 303a on the first working electrode conductive layer 11a of the first insulating layer 3 has a circular diameter of 1.2 mm
  • the working electrode first opening 303b on the second working electrode conductive layer 11b has a circular shape with a diameter of 1.2 mm.
  • the reference electrode first opening 301 was a circle with a diameter of 1.1 mm
  • the counter electrode first opening 302 was a rectangle with a size of 1.8 mm x 2.1 mm.
  • the working electrode second openings 403a and 403b of the second insulating layer 4 on the working electrode conductive layers 11a and 11b were each circular with a diameter of 2 mm.
  • the second opening 401 of the reference electrode was a circle with a diameter of 2 mm, and the second opening 402 of the counter electrode was a rectangle with a size of 1.8 mm x 2.1 mm.
  • First working electrode (for glucose measurement) reagent layer) A sodium phosphate buffer (pH 7.4), a carbon black dispersion, a polymer-bonded mediator, and glucose oxidase are placed in water in the working electrode first opening 303a of the first insulating layer 3 on the first working electrode conductive layer 11a. A droplet of 0.4 ⁇ L of the first liquid composition A containing a crosslinking agent and a crosslinking agent was formed and dried to form a first reagent layer 15a containing glucose oxidase and a mediator.
  • a first liquid composition B (P4VP-tBuMA polymer dispersion) was prepared by mixing the following reagents in ethanol to the following final concentrations and reacting for about 1 hour.
  • ⁇ P4VP-tBuMA Mn of poly-4-vinylpyridine: 74,000, Mn of poly-tert-butyl methacrylate: 87,000, Mw/Mn: 1.16, manufactured by NARD
  • final concentration 5.72 wt% ⁇ PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ⁇ 1,000, manufactured by Sigma-Aldrich), final concentration 0.9 wt%
  • a droplet of 0.65 ⁇ L of the first liquid composition B is formed in the second working electrode opening 403a of the second insulating layer 4 on the first working electrode 10a, dried, and then the first liquid composition B is dried. 1 A droplet of 0.65 ⁇ L of liquid composition B was formed and dried to form a first protective film 16a.
  • a dispersion liquid was prepared.
  • the resulting Nafion (registered trademark) dispersion having a concentration of 16.12 wt % was designated as a second liquid composition B.
  • This second liquid composition B includes Nafion® in a solvent containing a lower alcohol.
  • a droplet of 0.60 ⁇ L of the second liquid composition B is formed in the second working electrode opening 403b of the second insulating layer 4 on the second working electrode 10b, and dried to form a second protective film 16ba. was formed.
  • the same composition as the first liquid composition B (P4VP-tBuMA polymer dispersion) was used as a third liquid composition B in the following treatment. That is, a droplet of 0.65 ⁇ L of the third liquid composition B is formed in the second working electrode opening 403b of the second insulating layer 4 on which the second protective film 16ba is formed, and dried. 3 protective film 16bb was formed. In this way, a protective film 16b, in which the third protective film 16bb was laminated on the second protective film 16ba, was formed on the second working electrode 10b.
  • Reference pole Since the cross-sectional structure of the reference electrode 20 is the same as that shown in FIG. 22 regarding the sensor of the first disclosure, a method for manufacturing the reference electrode 20 will be outlined below with reference to FIG. 22.
  • a silver-silver chloride paste was applied on the reference electrode conductive layer 21 in the reference electrode first opening 301 of the first insulating layer 3 and heated at 140° C. for 1 hour to form a silver-silver chloride layer 22 .
  • the reference electrode protective film 23 was placed on the silver-silver chloride layer 22 in the reference electrode second opening 401 of the second insulating layer 4, thereby forming the reference electrode 20.
  • Experiment 1-1 In the above method, a carbon conductive layer was formed using an ordinary carbon paste that did not contain a catalyst such as platinum, and the sensor 300 of Experiment 1-1 was manufactured. In the sensor of Experiment 1-1, the working electrode conductive layers 11a and 11b, the reference electrode conductive layer 21, and the counter electrode (counter electrode conductive layer) 60 were all made of carbon conductive layers that did not contain a catalyst that decomposed hydrogen peroxide.
  • Experiment 1-2 After forming a carbon conductive layer using a normal carbon paste that does not contain a catalyst such as platinum, platinum was deposited only on the counter electrode 60 before forming the first insulating layer 3.
  • the sensor 300 of Experiment 1-2 was manufactured using the above procedure.
  • the counter electrode 60 has a portion 60a exposed through the counter electrode first opening 302 of the first insulating layer 3 and the counter electrode second opening 402 of the second insulating layer 4 that is in contact with the liquid sample.
  • the working electrode conductive layers 11a, 11b and the reference electrode conductive layer 21 include a conductive layer and a platinum vapor deposited film laminated thereon, and the working electrode conductive layers 11a, 11b and the reference electrode conductive layer 21 are made of carbon conductive layers that do not contain a catalyst such as platinum.
  • Liquid samples containing glucose and lactic acid as test substances were phosphate buffered saline (PBS) (Takara Bio PBS Tablets T9181), glucose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and sodium lactate (Sigma-Aldrich).
  • PBS containing 30mM glucose and 15.6mM lactic acid and having a pH adjusted to 7.0 was prepared using PBS (manufactured by Nippon Steel & Co., Ltd.).
  • the sensor of Experiment 1-1 or Experiment 1-2 was immersed in the liquid sample, and a voltage of 100 mV was applied to the first working electrode 10a and the second working electrode 10b with respect to the reference electrode 20 (Ag/AgCl).
  • a hydrogen peroxide concentration measurement sample was prepared using the liquid sample after 7 days of measurement according to the formulation shown in the table below.
  • the absorbance at 666 nm of the hydrogen peroxide concentration measurement sample was measured for 5 minutes using a plate reader (at 37° C.).
  • a hydrogen peroxide concentration measurement sample was prepared by mixing a solution containing hydrogen peroxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) with a known concentration instead of the liquid sample in the above table, and the absorbance at 666 nm was similarly measured. By doing so, a calibration curve showing the relationship between hydrogen peroxide concentration and absorbance was created.
  • the hydrogen peroxide concentration in the liquid sample was determined from the calibration curve.
  • the determined hydrogen peroxide concentration is a value corrected for the amount of water evaporated from the liquid sample during continuous measurement for 7 days. The results are shown in the table below.
  • the sensor of Experiment 1-2 which has a counter electrode made of a carbon conductive layer with a platinum vapor-deposited film laminated on the part that contacts the liquid sample, is different from the sensor of Experiment 1-1, which has a counter electrode made of only a carbon conductive layer. It was confirmed that the generation of hydrogen peroxide in the liquid sample could be suppressed or reduced during the measurement of the test substance in the liquid sample.
  • Experiment 2-1 A sensor 300 in which all conductive layers including the counter electrode 60 were made of carbon conductive layers, which was the same as the sensor in Experiment 1-1, was used as the sensor in Experiment 2-1.
  • Experiment 2-2 In the sensor of Experiment 2-1, a platinum nanoparticle dispersion liquid (manufactured by Sigma-Aldrich, platinum particle diameter: 70 ⁇ 6 nm) was dropped and dried, and then a 21.5 wt % Nafion (registered trademark) dispersion (manufactured by Sigma-Aldrich) was dropped and dried to form a protective film (not shown).
  • a sensor 300 for Experiment 2-2 was manufactured.
  • the portion 60a of the counter electrode 60 that comes into contact with the liquid sample is a carbon conductive layer whose surface is modified with platinum nanoparticles, and the surface of the portion 60a is coated with cations. It is coated with a protective film (not shown) containing Nafion®, a polymeric compound with exchangeable functional groups.
  • a hydrogen peroxide concentration measurement sample was prepared using the liquid sample after 7 days of measurement according to the formulation shown in the table below.
  • the absorbance at 666 nm of the hydrogen peroxide concentration measurement sample was measured for 5 minutes using a plate reader (at 37° C.).
  • a hydrogen peroxide concentration measurement sample is prepared by mixing a solution containing hydrogen peroxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) with a known concentration, and the absorbance at 666 nm is similarly measured. By this, a calibration curve between hydrogen peroxide concentration and absorbance was prepared.
  • the hydrogen peroxide concentration in the liquid sample was determined from the calibration curve.
  • the determined hydrogen peroxide concentration is a value corrected for the amount of water evaporated from the liquid sample during continuous measurement for 7 days. The results are shown in the table below.
  • the sensor 300 of Experiment 2-2 which has the counter electrode 60 made of a carbon conductive layer modified with platinum nanoparticles in the portion 60a that contacts the liquid sample, is different from the sensor 300 of Experiment 2-2, which has the counter electrode 60 made of only a carbon conductive layer. It was confirmed that the generation of hydrogen peroxide in the liquid sample could be suppressed or reduced during the measurement of the test substance in the liquid sample, compared to the sensor 300 of No.-1.
  • Experiment 2-1 The sensor 300 in which all the conductive layers including the counter electrode 60 were made of carbon conductive layers, which was the same as the sensor in Experiments 1-1 and 2-1, was used as the sensor in Experiment 3-1.
  • Experiment 3-2 Using a carbon paste containing carbon (containing graphite and carbon black) and 1% by weight of platinum particles (average particle size 390 nm) based on the carbon, the working electrode conductive layer 11a was , 11b, the reference electrode conductive layer 21, the counter electrode (counter electrode conductive layer) 30, and the wiring 50 were formed, but the sensor 300 of Experiment 3-2 was manufactured by the above procedure.
  • Experiment 3-3 was carried out using the sensor and procedure of Experiment 3-2, except that a carbon paste containing the carbon and platinum particles of 5% by weight relative to the carbon was used as the carbon paste.
  • a sensor 300 was manufactured.
  • a hydrogen peroxide concentration measurement sample was prepared using the liquid sample after 7 days of measurement according to the formulation shown in the table below.
  • the absorbance at 666 nm of the hydrogen peroxide concentration measurement sample was measured for 5 minutes using a plate reader (at 37° C.).
  • a hydrogen peroxide concentration measurement sample is prepared by mixing a solution containing hydrogen peroxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) with a known concentration, and the absorbance at 666 nm is similarly measured. By this, a calibration curve between hydrogen peroxide concentration and absorbance was prepared.
  • the hydrogen peroxide concentration in the liquid sample was determined from the calibration curve.
  • the determined hydrogen peroxide concentration is a value corrected for the amount of water evaporated from the liquid sample during continuous measurement for 7 days. The results are shown in the table below.
  • the sensor 300 of Experiment 3-2 and Experiment 3-3 which has the counter electrode 60 made of a carbon conductive layer containing platinum particles, is different from the sensor 300 of Experiment 3-1, which has the counter electrode 60 made of only a carbon conductive layer.
  • the generation of hydrogen peroxide in the liquid sample could be suppressed or reduced during the measurement of the test substance in the liquid sample.
  • a carbon conductive layer containing no platinum (hereinafter referred to as “carbon electrode without platinum particles”) used as the counter electrode 60 in Experiments 3-1, 3-2, and 3-3, a carbon conductive layer containing 1% by weight of platinum particles based on carbon. (hereinafter referred to as “carbon electrode containing 1% platinum particles”) and a carbon conductive layer containing 5% by weight of platinum particles relative to carbon (hereinafter referred to as “carbon electrode containing 5% platinum particles”).
  • carbon electrode containing 1% platinum particles a carbon conductive layer containing 5% by weight of platinum particles relative to carbon
  • a silver chloride (saturated KCl) electrode manufactured by BAS
  • a platinum electrode is used as a counter electrode.
  • a three-electrode electrolytic cell was constructed, and each electrode was connected to a potentiostat.
  • the working electrode, counter electrode, and reference electrode were prepared using phosphate buffered saline (PBS) (Takara Bio PBS Tablets T9181; 0.14 M NaCl, 0.0027 M KCl, 0.010 M PO4 3+ ), and cyclic voltammetry was performed by applying a potential in the range of ⁇ 0.5 V to the reference electrode to the working electrode at a sweep rate of 10 mV/s.
  • PBS phosphate buffered saline
  • the electrode with a higher platinum particle content ratio has a larger absolute value of positive current value on the high potential side and a larger absolute value of negative current value on the lower potential side.
  • the negative current value on the low potential side is considered to be due to reduction of oxygen, etc. in PBS. This means that when an electrode containing platinum particles is used as a working electrode, the background noise increases, the current response value decreases, and the influence of environment-dependent disturbances such as oxygen concentration increases, which may reduce sensitivity. suggested that there is a sex.
  • an electrode containing platinum particles when using an electrode containing platinum particles as a counter electrode, due to the increase in current due to reduction of oxygen, etc., in the negative potential region, a negative current with a large absolute value occurs at a low potential (potential with a small absolute value). can flow. Therefore, an electrode containing platinum particles can function as a counter electrode more efficiently with a smaller area than a carbon electrode that does not contain platinum, and thus can contribute to reducing the sensor size. Furthermore, by driving the sensor of the present disclosure using an electrode containing platinum particles as a counter electrode at a low potential in a negative potential region, it is also possible to reduce the burden on the potentiostat that is the measuring device.
  • the working electrode, counter electrode, and reference electrode were prepared using phosphate buffered saline (PBS) (Takara Bio PBS Tablets T9181; 0.14 M NaCl, 0.0027 M KCl, Amperometric measurement was performed in which the working electrode was immersed in 0.010 M PO 4 3+ ), a potential of 0.3 V or -0.2 V was applied to the working electrode and the current value was measured over time with respect to the reference electrode. .
  • FIG. 59 shows the measurement results of the current value per area of the working electrode.
  • FIG. 59A shows the measurement results when a carbon electrode without platinum particles was used as a working electrode.
  • FIG. 59B shows measurement results when a carbon electrode containing 1% platinum particles was used as a working electrode.
  • the current value (absolute value) when a carbon electrode without platinum particles is used as a working electrode is significantly smaller than the current value (absolute value) when a carbon electrode containing 1% platinum particles is used as a working electrode. was confirmed (A and B have different vertical axis scales). This result shows that conductive layers that do not contain platinum particles are suitable for the working electrode conductive layers 11a and 11b because of their low background current.
  • the part 60a of the counter electrode 60 in the sensor of Experiment 2-1 that contacts the liquid sample to be measured is a carbon conductive layer whose surface is modified with platinum nanoparticles
  • the working electrode conductive layers 11a and 11b are carbon conductive layers that do not contain platinum particles.
  • the measurement results of the current values of the first working electrode 10a and the second working electrode 10b are shown in FIGS. 60A and 60B, respectively.
  • the current value of the first working electrode 10a shown in FIG. 60A corresponds to the glucose concentration.
  • the current value of the second working electrode 10b shown in FIG. 60B corresponds to the lactic acid concentration.
  • all conductive layers including the working electrode conductive layers 11a and 11b and the counter electrode 60 were carbon conductive layers containing 1% by weight of platinum particles based on carbon.
  • all conductive layers including the working electrode conductive layers 11a and 11b and the counter electrode 60 were carbon conductive layers containing 5% by weight of platinum particles based on carbon.
  • the measurement results of the current values of the first working electrode 10a and the second working electrode 10b are shown in FIGS. 61A and 61B, respectively.
  • the current value of the first working electrode 10a shown in FIG. 61A corresponds to the glucose concentration.
  • the current value of the second working electrode 10b shown in FIG. 61B corresponds to the lactic acid concentration.
  • the sensor of Experiment 3-2 in which the working electrode conductive layers 11a, 11b and the counter electrode 60 contained 1% by weight of platinum particles based on carbon was the sensor of Experiment 3-2 in which the working electrode conductive layers 11a, 11b and the counter electrode 60 contained no platinum particles. No significant decrease in current response value was observed compared to sensor No. 1.
  • the sensor of Experiment 3-3 in which the working electrode conductive layers 11a, 11b and the counter electrode 60 contain platinum particles in an amount of 5% by weight relative to carbon is the sensor in which the working electrode conductive layers 11a, 11b and the counter electrode 60 do not contain platinum particles.
  • the current response value was clearly decreased. This suggests that the presence of platinum in the working electrode conductive layers 11a and 11b reduces the responsiveness of the sensor.
  • Such a sensor is sometimes used for continuous monitoring, in which the test substance is continuously measured over a long period of several days or more while immersed in a liquid sample.
  • the sensor is desired not only to have high detection sensitivity for the test substance but also to have less negative impact (eg, cytotoxicity) on cultured cells contained in the liquid sample.
  • a sensor that includes a working electrode that includes a reagent layer containing a reagent involved in the redox reaction of the test substance, such as an oxidoreductase or a mediator, and a counter electrode is used to electrochemically detect the test substance in a liquid sample. It has been found that when measuring, hydrogen peroxide can be generated during long-term measurements. If the hydrogen peroxide concentration in a liquid sample exceeds a certain level, it may be toxic to cells.
  • the third disclosure provides a sensor including a working electrode and a counter electrode that can suppress the generation of hydrogen peroxide during measurement or reduce the generated hydrogen peroxide without causing a noticeable decrease in sensitivity.
  • the challenge is to provide.
  • the sensor according to the third disclosure suppresses the generation of hydrogen peroxide during measurement without causing a significant decrease in sensitivity when used for measuring a test substance in a liquid sample such as a cell culture solution. Since the generated hydrogen peroxide can be reduced, an increase in the hydrogen peroxide concentration in the liquid sample can be suppressed. Therefore, the sensor according to the third disclosure has little negative influence on cells in a liquid sample, and can be suitably used for continuous monitoring over a long period of time.
  • a sensor for measuring an analyte in a liquid sample comprising: a working electrode including a working electrode conductive layer; and a reagent layer disposed on the working electrode conductive layer and containing a reagent involved in a redox reaction of the analyte; The opposite and Equipped with When the sensor is immersed in the liquid sample and the analyte in the liquid sample is electrochemically measured, the hydrogen peroxide concentration in the liquid sample is maintained at less than 15 ⁇ M during the measurement. , sensor. (Appendix 3-2) The sensor according to appendix 3-1, wherein the counter electrode includes a catalyst that decomposes hydrogen peroxide in a portion that contacts the liquid sample.
  • the working electrode conductive layer is a working electrode, a silver silver chloride (saturated KCl) electrode as a reference electrode, and a platinum electrode as a counter electrode
  • the current value measured when a potential in the range of -0.2V to 0.3V (vs. the above-mentioned silver-silver chloride (saturated KCl) electrode) is applied to the working electrode conductive layer is ⁇ 5.5 nA/mm 2 or less.
  • the sensor according to any one of Supplementary Notes 3-1 to 3-9, which satisfies the following conditions.
  • the material of the insulating substrate used in the sensor according to the fourth disclosure of this specification is not particularly limited, but, for example, the same material as the material of the insulating substrate of the sensor according to the first disclosure of this specification can be used.
  • the conductive layer included in the working electrode, reference electrode, and counter electrode of the sensor according to the fourth disclosure of this specification is a layer containing a conductive material such as carbon, gold, platinum, palladium, or the like.
  • the conductive layer can be manufactured by forming a layer of the conductive material as described above on the surface of the substrate using a sputtering method, a vapor deposition method, a screen printing method, or the like.
  • the conductive layer can be processed into a predetermined pattern using a laser trimming method, if necessary.
  • the conductive layer of the working electrode may be referred to as the working electrode conductive layer
  • the conductive layer of the reference electrode may be referred to as the reference electrode conductive layer.
  • the conductive layer of the counter electrode is simply referred to as a counter electrode because the conductive layer itself constitutes the counter electrode.
  • the wiring of the sensor according to the fourth disclosure of this specification can also be made of the same conductive material as the conductive layer.
  • the sensor according to the fourth disclosure of this specification is immersed in a liquid sample and used to detect a predetermined analyte in the liquid sample.
  • the liquid sample contains water as a solvent.
  • the liquid sample include a cell culture solution and a liquid sample prepared using blood obtained from a living body.
  • the test substance are as described with respect to the test substance by the sensor of the first disclosure of this specification, and particularly preferably one or more selected from glucose and lactic acid.
  • the working electrode of the sensor according to the fourth disclosure of this specification includes a reagent layer disposed on the working electrode conductive layer and containing a reagent involved in the redox reaction of the analyte.
  • the reagent can be appropriately selected depending on the test substance.
  • the reagent may include a combination of an oxidoreductase and a mediator (electron carrier), or an oxidoreductase. Oxidoreductases can include coenzymes.
  • oxidoreductases examples include oxidases and dehydrogenases. Specific examples of oxidoreductases are as described with respect to the sensor of the first disclosure herein, and may particularly preferably be glucose oxidase, lactate oxidase, glucose dehydrogenase or lactate dehydrogenase.
  • the mediator is not particularly limited, and examples thereof are as described with respect to the sensor of the first disclosure of this specification.
  • the reagent layer included in the working electrode of the sensor according to the fourth disclosure of the present specification can further contain components such as a buffer, a hydrophilic polymer compound, a conductive carbon filler, and a crosslinking agent.
  • a buffer such as a buffer, a hydrophilic polymer compound, a conductive carbon filler, and a crosslinking agent.
  • hydrophilic polymer compounds and conductive carbon fillers are as described with respect to the sensor of the first disclosure herein.
  • the working electrode of the sensor according to the fourth disclosure of this specification further includes a first protective film disposed on the reagent layer and a second protective film disposed on the first protective film.
  • the first protective film and the second protective film may be collectively referred to as a protective film.
  • the protective film on the reagent layer can be a film that prevents or suppresses leakage of the reagent contained in the reagent layer to the outside of the protective film, and is permeable to the test substance present outside the protective film. Preferred embodiments of the materials constituting the first protective film and the second protective film on the reagent layer having such properties will be described later.
  • the reference electrode of the sensor according to the fourth disclosure of this specification can be provided with a protective film.
  • the protective film of the reference electrode can contain a polymer compound. Examples of such polymer compounds include the polymer compounds described below contained in the first protective film or the second protective film disposed on the reagent layer of the working electrode, and the polymer compounds contained in the second protective film are particularly preferable.
  • a portion of the electrode including a working electrode and a counter electrode disposed on an insulating substrate other than the portion that contacts the liquid sample may be covered with an insulating layer.
  • the insulating layer can contain an insulating resin.
  • the insulating layer can have a multilayer structure of two or more layers.
  • the multilayer insulating layer can include, for example, a first insulating layer disposed on an insulating substrate and a conductive layer, and a second insulating layer disposed on the first insulating layer. Preferred embodiments of the materials constituting the first insulating layer and the second insulating layer are as described with respect to the sensor according to the first disclosure.
  • the sensor 400 of this embodiment includes an insulating substrate 2, a first working electrode 10a, a second working electrode 10b, and a reference electrode arranged on the first surface 2a of the substrate 2. 20 and a counter electrode 30, and a wiring 50 electrically connected to each of the first working electrode 10a, the second working electrode 10b, the reference electrode 20, and the counter electrode 30.
  • the sensor 400 of this embodiment includes two working electrodes, in another embodiment not shown, there may be only one working electrode, or three or more working electrodes. In the following description, when the first working electrode 10a and the second working electrode 10b are not distinguished, they may be expressed as working electrodes 10a and 10b.
  • the first working electrode 10a, the second working electrode 10b, the reference electrode 20, and the counter electrode 30 may be collectively referred to as electrodes.
  • the sensor 400 of this embodiment is a three-electrode sensor that includes a working electrode, a reference electrode, and a counter electrode as electrodes, it may be a bipolar sensor that does not include a reference electrode and only includes a working electrode and a counter electrode.
  • the reference electrode and/or the counter electrode may be provided on a substrate different from the substrate on which the working electrode is arranged.
  • the sensor 400 is used to detect a predetermined analyte in the liquid sample X by being immersed in the liquid sample X, in the same way as shown in FIG. 21 regarding the sensor 1 of the first disclosure.
  • Specific examples of the liquid sample and test substance are as described in the ⁇ Materials> column.
  • the first working electrode 10a of the sensor 400 includes a working electrode conductive layer 11a, a reagent layer 15a containing a reagent involved in the redox reaction of the analyte in the liquid sample, and a layer on the reagent layer 15a.
  • the first protective film 18aa is disposed on the first protective film 18aa
  • the second protective film 18ab is disposed on the first protective film 18aa.
  • Preferred embodiments of the reagent are as described in the ⁇ Materials> column.
  • the cross-sectional structure of the second working electrode 10b of the sensor 400 is substantially the same as the cross-sectional structure of the first working electrode 10a shown in FIG.
  • the working electrode conductive layer 11a is connected to the working electrode conductive layer 11, the reagent layer 15a is connected to the reagent layer 15b, the first protective film 18aa is connected to the first protective film 18ba, and the second protective film 18ab is connected to the working electrode 10b. 18bb, the protective film 18a is replaced with a protective film 18b.
  • the reagent layers 15a and 15b of the working electrodes 10a and 10b of the sensor 400 contain a reagent that oxidizes the analyte in the liquid sample
  • the analyte is removed from the analyte under the condition that a predetermined voltage is applied to the electrodes of the sensor 400. Electrons move to the working electrode conductive layers 11a and 11b.
  • the reagent layers 15a and 15b contain a reagent that reduces the analyte in the liquid sample, electrons move from the working electrode conductive layers 11a and 11b to the analyte.
  • the concentration or concentration change of the test substance in the liquid sample is measured based on the value of the current flowing through the working electrodes 10a, 10b of the sensor 400, or the change in the current value. can do.
  • the sensor 400 can constitute an analysis device for analyzing a test substance in a liquid sample.
  • the analysis device includes a sensor 400, an analysis unit 102, and a control unit 104 in which the sensor 1 according to the first disclosure is replaced with the sensor 400 according to the fourth disclosure in the analysis device 100 shown in FIG.
  • An example of this is an analyzer equipped with the following. The functions of this analyzer are as described with respect to the analyzer 100 shown in FIG. 24.
  • FIG. 62 and FIGS. 63 and 64 are cross-sectional views thereof.
  • the structure of the reference pole 20 of the sensor 400 according to the fourth disclosure is the same as the structure of the reference pole 20 of the sensor 1 according to the first disclosure. Therefore, the description of the reference pole 20 of the sensor 1 according to the first disclosure with reference to FIG. 22 is cited as the description of the reference pole 20 of the sensor 400 according to the fourth disclosure.
  • the sensor 400 includes an insulating substrate 2, working electrode conductive layers 11a and 11b, a counter electrode 30, a reference electrode conductive layer 21, and a wiring 50, which are arranged on the first surface 2a of the substrate 2.
  • the counter electrode 30 is entirely made of a conductive layer. To specifically refer to the conductive layer portion of the counter electrode, it may also be referred to as a "counter electrode conductive layer” or "counter electrode conductive layer.”
  • a first insulating layer 3 is further arranged on the first surface 2a of the substrate 2, and a second insulating layer 4 is further arranged on the first insulating layer 3.
  • the height of the upper surface of the first insulating layer 3 from the first surface 2a of the substrate 2 is the height of the working electrode conductive layers 11a, 11b, the counter electrode 30, the reference electrode conductive layer 21, and the wiring 50, greater than the height from. Therefore, a part of the first insulating layer 3 is arranged on the working electrode conductive layers 11a, 11b, the counter electrode 30, the reference electrode conductive layer 21, and the wiring 50.
  • the first insulating layer 3 includes a counter electrode first opening 302 that is formed at a position overlapping a part of the counter electrode 30 and penetrates through the substrate 2 in the thickness direction T.
  • the second insulating layer 4 includes a second counter-electrode opening 402 that is formed in a position overlapping the entire counter-electrode first opening 302 of the first insulating layer 3 and that penetrates through the substrate 2 in the thickness direction T.
  • the counter electrode 30 is exposed to the outside through the first counter electrode opening 302 and the second counter electrode opening 402 .
  • the working electrodes 10a and 10b include working electrode conductive layers 11a and 11b, and a reagent layer disposed on the working electrode conductive layers 11a and 11b and containing a reagent involved in the redox reaction of the test substance. 15a and 15b.
  • Preferred embodiments of the reagent in the reagent layers 15a and 15b are as described in the ⁇ Materials> column.
  • the former in order to distinguish between the "reagent layer 15a" of the first working electrode 10a and the “reagent layer 15b" of the second working electrode 10b, the former is The latter may be referred to as the "second reagent layer 15b" of the second working electrode 10b.
  • the reagent included in the reagent layer 15a of the first working electrode 10a may be referred to as a "first reagent”
  • the reagent included in the second reagent layer 15b of the second working electrode 10b may be referred to as a "second reagent”.
  • the sensor 400 includes a plurality of working electrodes 10a, 10b, and a first reagent contained in the first reagent layer 15a of the first working electrode 10a among the plurality of working electrodes 10a, 10b;
  • the second reagents contained in the second reagent layer 15b of the second working electrode 10b, which is different from the working electrode 10a, are different from each other.
  • the first reagent participates in the redox reaction of the first analyte in the liquid sample
  • the second reagent is different from the first analyte in the liquid sample. Since it can participate in the redox reaction of the second analyte, it can be used to detect a plurality of analytes including the first analyte and the second analyte in a liquid sample.
  • the working electrodes 10a, 10b have first protective films 18aa, 18ba placed on the reagent layers 15a, 15b, and second protective films 18aa, 18ba placed on the first protective films 18aa, 18ba.
  • Protective films 18a and 18b are provided, each consisting of films 18ab and 18bb.
  • the first insulating layer 3 has a working electrode first opening penetrating in the thickness direction T of the substrate 2, which is formed at a position overlapping a part of each of the working electrode conductive layers 11a and 11b. 303a and 303b.
  • the second insulating layer 4 has a working electrode hole formed in the first insulating layer 3 at a position that overlaps with a portion that includes the whole of the first working electrode openings 303a and 303b, and which penetrates through the thickness direction T of the substrate 2. Two openings 403a and 403b are provided.
  • the reagent layers 15a, 15b are contained in the working electrode first openings 303a, 303b, and the outer peripheral edges of the reagent layers 15a, 15b are defined by the inner peripheral edges of the working electrode first openings 303a, 303b.
  • the first protective films 18aa, 18ba and the second protective films 18ab, 18bb are included in the working electrode second openings 403a, 403b, and the outer peripheral edges of the first protective films 18aa, 18ba and the second protective films 18ab, 18bb are It is defined by the inner periphery of the working electrode second openings 403a and 403b.
  • the first protective films 18aa and 18ba contain a first polymer compound containing a cation-exchanging functional group
  • the second protective films 18ab and 18bb contain a cationic functional group. It is characterized by containing a second polymer compound containing.
  • a sensor with this feature can be used for continuous monitoring applications where the test substance is continuously measured over a long period of several days while immersed in a liquid sample. Stable measurements are possible without peeling.
  • polymeric compounds containing cation-exchange functional groups e.g., tetrafluoroethylene and perfluoro-2-(2-fluorosulfonyl)
  • cation-exchange functional groups e.g., tetrafluoroethylene and perfluoro-2-(2-fluorosulfonyl)
  • a first protective film containing a polymer compound containing 4-vinylpyridine as a constituent unit for example, P4VP-tBuMA (a copolymer compound of poly-4-vinylpyridine and polyvinyl pyridine) is provided on the reagent layer.
  • P4VP-tBuMA a copolymer compound of poly-4-vinylpyridine and polyvinyl pyridine
  • a sensor that includes a two-layered protective film in which a second protective film containing a block copolymer compound of -tert-butyl methacrylate) is further provided on the first protective film.
  • the present inventors found that when performing continuous electrochemical measurements of test substances by immersing this sensor in a liquid sample, the detected current value changes significantly after a long period of time exceeding 3 days. It has been found that an event may occur in which an accurate measurement cannot be made due to the detection of In the sensor that showed an abnormal value, it was confirmed that peeling had occurred between the first protective film and the second protective film.
  • the present inventors have found that the above problem can be solved by the sensor according to the fourth disclosure, which includes a first protective film and a second protective film having the above characteristics.
  • the first polymer compound of the first overcoat includes a cation exchange functional group (i.e., an anionic functional group), while the second polymer compound of the second overcoat includes a cationic functional group. It is considered that the first protective film and the second protective film are in close contact with each other at the interface due to electrostatic bonding, and peeling is suppressed.
  • the first polymer compound contained in the first protective film may have a cation-exchangeable functional group, and preferably has a cation-exchangeable functional group in its side chain.
  • a sulfonic acid group is preferable.
  • the first polymer compound having a sulfonic acid group in the side chain is preferably a polymer compound containing a perfluoro compound having a sulfonic acid group in the side chain as a constitutional unit, and more preferably has a sulfonic acid group in the side chain.
  • a copolymer compound containing a perfluoro compound and a perfluoro compound that does not have an ionic functional group in its side chain as a constituent unit is particularly preferably a copolymer compound containing tetrafluoroethylene and perfluoro-2-(2-fluorosulfonyl). ethoxy)propyl vinyl ether, most preferably Nafion(R).
  • the second polymer compound contained in the second protective film may contain a cationic functional group, and preferably has a cationic functional group in its side chain. Since the pH of the liquid sample to be analyzed may vary during long-term measurements, the cationic functional group in the second polymer compound is preferably a pH-independent cationic functional group. In particular, a functional group containing a quaternary ammonium cation is preferred. The quaternary ammonium cation preferably originates from the reaction of a tertiary amine with an epoxy group. Tertiary amines include pyridyl groups, particularly 4-pyridyl groups.
  • the second polymer compound includes a first unit containing a cationic functional group and a second unit containing a hydrophobic functional group, and the ratio of the second unit to the total amount of constitutional units of the second polymer compound is 50. It is preferable that it is mol% or more. Since such a second polymer compound has high hydrophobicity, even when the sensor according to the fourth disclosure is immersed in an aqueous liquid sample such as a cell culture medium and measured for a long time, the second protective film is not exposed to water. Because of this, it does not swell, and peeling at the interface with the first protective film is easily suppressed.
  • the upper limit of the content of the second unit containing a hydrophobic functional group in the second polymer compound is not particularly limited, but for example, if the ratio of the second unit to the total amount of constituent units of the second polymer compound is 70 mol% or less, Something can happen.
  • the hydrophobic functional group include saturated hydrocarbon groups having 1 to 10 carbon atoms.
  • the second polymer compound that includes a first unit that includes a cationic functional group and a second unit that includes a hydrophobic functional group is, for example, a third unit that includes a tertiary amine and a second unit that includes a hydrophobic functional group.
  • the epoxy group of the crosslinking agent compound is preferably 6.8 mol% or more, more preferably 23 mol% or less (the tertiary amine in the third unit), based on the tertiary amine in the third unit.
  • the resulting second polymer compound is a high-density quaternary polymer compound. This is preferable because the electrostatic bond between the first protective film and the second protective film becomes strong.
  • the number of moles of the tertiary amine is It can be calculated based on the molecular weight of the monomer of the third unit, and the number of moles of epoxy groups is the molecular weight of the crosslinking agent compound (if the crosslinking agent compound is a polymer compound, its number average molecular weight is taken as the uniform molecular weight). It can be calculated based on
  • the third unit containing a tertiary amine in the uncrosslinked polymer compound is preferably a structural unit derived from 4-vinylpyridine.
  • a preferred example of the uncrosslinked polymer compound is a copolymer compound of 4-vinylpyridine and tert-butyl methacrylate.
  • the copolymer compound more preferably has a second unit derived from tert-butyl methacrylate relative to the total amount of the third unit derived from 4-vinylpyridine and the second unit derived from tert-butyl methacrylate. contains 50 mol% or more, more preferably 70 mol% or less (the ratio of the second unit to the total amount of the third unit and the second unit is, for example, 50 mol% or more and 70 mol% or less).
  • the copolymer compound of 4-vinylpyridine and tert-butyl methacrylate is particularly preferably a block copolymer compound.
  • the molecular weight of the uncrosslinked polymer compound is not particularly limited, but may have a number average molecular weight (Mn) of 150,000 to 500,000, for example.
  • the crosslinking agent compound containing two or more epoxy groups is, for example, a linear compound having epoxy groups at both ends, preferably a polyalkylene glycol compound whose both ends are each modified with a glycidyl group.
  • the molecular weight of poly(ethylene glycol) diglycidyl ether is not particularly limited, but may have a number average molecular weight (Mn) of 200 to 2,000, for example.
  • the second protective films 18ab and 18bb have a second protective film including a first unit containing a quaternary ammonium cation derived from a reaction between a tertiary amine and an epoxy group, and a second unit containing a hydrophobic functional group.
  • a preferred method of manufacturing the sensor 400 according to the third disclosure includes: A mixture containing an uncrosslinked polymer compound containing a third unit containing a tertiary amine and a second unit containing a hydrophobic functional group, and a crosslinking agent compound containing two or more epoxy groups in an alcohol, By maintaining the temperature at a temperature of °C or higher for 6 hours or more, the tertiary amine of the third unit and the epoxy group of the crosslinking agent compound are reacted, and the third unit is A first step of obtaining a second protective film-forming composition containing the second polymer compound containing the first unit and the second unit in alcohol by converting it into the first unit containing an ammonium cation.
  • the second protective film forming composition substrate 2; Working electrode conductive layers 11a and 11b arranged on the substrate 2; reagent layers 15a, 15b disposed on the working electrode conductive layers 11a, 11b; first protective films 18aa, 18ba disposed on reagent layers 15a, 15b;
  • a sensor component comprising: a second step of coating and drying the first protective films 18aa, 18ba to form second protective films 18ab, 18bb containing the second polymer compound; including.
  • the crosslinking reaction between the tertiary amine of the third unit in the uncrosslinked polymer compound and the epoxy group of the crosslinker compound sufficiently progresses, and the density of the quaternary ammonium cation increases.
  • a second protective film-forming composition containing a high second polymer compound can be obtained.
  • the temperature conditions in the first step are more preferably 30°C or higher, more preferably 40°C or higher, and the upper limit is preferably lower than the boiling point of alcohol, for example, when using ethanol as the alcohol, it is preferably lower than 78°C. preferable.
  • the reaction time in the first step is preferably 12 hours or more, and the upper limit is not particularly limited, but is, for example, 48 hours or less (the reaction time in the first step is, for example, 12 hours or more and 48 hours or less).
  • the alcohol ethanol, methanol, and propanol are preferred, and ethanol is particularly preferred.
  • Preferred embodiments of the beautiful fragrance copolymer compound and the crosslinking agent compound are as described above.
  • the composition for forming the second protective film is applied or dropped on the first protective films 18aa and 18ba to coat them, and then dried by volatilizing the alcohol as a solvent, and then Protective films 18ab and 18bb are formed.
  • substrate As in the first disclosed embodiment, a 188 ⁇ m thick substrate made of polyethylene terephthalate and having the shape shown in FIG. 62 was used as the insulating substrate 2.
  • conductive layer By applying carbon paste on the first surface 2a of the insulating substrate 2 and heating it at 140° C. for 1 hour, a working electrode conductive layer 11a and a reference electrode conductive layer having the same shape as in FIG. 1 related to the first disclosure are formed. 21 and the counter electrode (counter electrode conductive layer) 30, and the wiring 50 electrically connected to each of them were formed of a carbon conductive layer with a thickness of 5 ⁇ m.
  • first insulating layer (first insulating layer) Subsequently, the first surface 2a of the substrate 2 and the carbon conductive layer made of a fluororesin containing a copolymer containing vinylidene fluoride and hexafluoropropylene are placed on the first surface 2a of the substrate 2 on which the carbon conductive layer is disposed. A covering first insulating layer 3 having a thickness of 5 ⁇ m on the carbon conductive layer was laminated. The method for manufacturing the first insulating layer 3 is as described in the first disclosed embodiment.
  • the working electrode first opening 303a on the working electrode conductive layer 11a of the first insulating layer 3 was circular with a diameter of 1.2 mm.
  • the reference electrode first opening 301 was a circle with a diameter of 1.1 mm, and the counter electrode first opening 302 was a rectangle with a size of 1.8 mm x 2.1 mm.
  • the working electrode second opening 403a of the second insulating layer 4 on the working electrode conductive layer 11a was circular with a diameter of 2 mm.
  • the second opening 401 of the reference electrode was a circle with a diameter of 2 mm, and the second opening 402 of the counter electrode was a rectangle with a size of 1.8 mm x 2.1 mm.
  • a sodium phosphate buffer solution (pH 7.4) with a final concentration of 3 mM and a final concentration of 14 mg/mL (carbon black concentration) are applied on the working electrode conductive layer 11a.
  • a reagent layer 15a containing glucose dehydrogenase and a mediator was formed.
  • a first protective film 18aa is formed on the reagent layer 15a
  • a second protective film 18ab is formed on the first protective film 18aa
  • a first protective film 18ab is formed on the first protective film 18aa.
  • a protective film 18a having a two-layer structure consisting of a protective film 18aa and a second protective film 18ab was formed. A specific method for manufacturing the first protective film 18aa and the second protective film 18ab will be explained in Experiments 8 to 10.
  • Reference pole Since the cross-sectional structure of the reference electrode 20 is the same as that shown in FIG. 22 regarding the sensor of the first disclosure, a method for manufacturing the reference electrode 20 will be outlined below with reference to FIG. 22.
  • a silver-silver chloride paste was applied on the reference electrode conductive layer 21 in the reference electrode first opening 301 of the first insulating layer 3 and heated at 140° C. for 1 hour to form a silver-silver chloride layer 22 .
  • the reference electrode protective film 23 was placed on the silver-silver chloride layer 22 in the reference electrode second opening 401 of the second insulating layer 4, thereby forming the reference electrode 20.
  • Nafion® dispersion 30,792.92 mg of Nafion® dispersion was prepared.
  • 0.6 mg of the obtained Nafion (registered trademark) dispersion having a concentration of 16.12 wt% is applied onto the reagent layer 15a in the second opening 403a of the working electrode of the second insulating layer 4 and dried to form cations.
  • a first protective film 18aa containing Nafion (registered trademark) as a polymer compound containing an exchangeable functional group was formed.
  • P4VP-tBuMA is a block copolymer of poly-4-vinylpyridine (P4VP) with a predetermined molecular weight and poly-tert-butyl methacrylate (tBuMA) with a predetermined molecular weight.
  • Second protective film forming composition of Experiment 8-1 ⁇ P4VP-tBuMA (Mn of poly-4-vinylpyridine: 74,000, Mn of poly-tert-butyl methacrylate: 87,000, Mw/Mn: 1.16, manufactured by Polymer Source), final concentration 5.72wt % ⁇ PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ⁇ 1,000, manufactured by Sigma-Aldrich), final concentration 0.91 wt%
  • Second protective film forming composition of Experiment 8-2 ⁇ P4VP-tBuMA (Mn of poly-4-vinylpyridine: 120,000, Mn of poly-tert-butyl methacrylate: 270,000, Mw/Mn: 1.15, manufactured by Polymer Source), final concentration 5.72wt % ⁇ PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ⁇ 1,000, manufactured by Sigma-Aldrich), final concentration 2.0 wt%
  • the sensor 400 provided with the second protective film 18ab of Experiment 8-1 was used as the sensor of Experiment 8-1.
  • the sensor 400 provided with the second protective film 18ab of Experiment 8-2 was used as the sensor of Experiment 8-2.
  • RPMI-1640 Medium manufactured by Sigma-Aldrich Co., Ltd. R1383
  • glucose manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
  • sodium lactate manufactured by Sigma-Aldrich Co., Ltd.
  • FIG. 65A shows the measurement results using the sensor of Experiment 8-1
  • FIG. 65B shows the measurement results using the sensor of Experiment 8-2.
  • P4VP-tBuMA containing P4VP of Mn: 74,000 and tBuMA of Mn: 87,000, and containing PEGDGE at a final concentration of 0.91 wt%.
  • the sensor of Experiment 8-2 provided with the protective film 18ab no abnormally high current value was detected even after 4 days or more had elapsed from the start of measurement.
  • compositions were designed for the composition for forming the second protective film, each containing P4VP-tBuMA and PEGDGE at the following final concentrations in ethanol.
  • the ratio (mol%) of epoxy groups derived from PEGDGE to pyridine derived from P4VP the number of moles of pyridine derived from P4VP calculated based on the molecular weight of the monomer of P4VP and , and the number of moles of epoxy groups derived from PEGDGE calculated based on the number average molecular weight of PEGDGE (considered as a uniform molecular weight of PEGDGE).
  • Composition A (10.17 mol% of epoxy group derived from PEGDGE with respect to pyridine derived from 4-vinylpyridine): ⁇ P4VP-tBuMA (Mn of poly-4-vinylpyridine: 120,000, Mn of poly-tert-butyl methacrylate: 270,000, Mw/Mn: 1.15, manufactured by Polymer Source), final concentration 5.72wt % ⁇ PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ⁇ 1,000, manufactured by Sigma-Aldrich), final concentration 0.9 wt%
  • Composition B (6.77 mol% of epoxy groups derived from PEGDGE with respect to pyridine derived from 4-vinylpyridine): ⁇ P4VP-tBuMA (Mn of poly-4-vinylpyridine: 120,000, Mn of poly-tert-butyl methacrylate: 270,000, Mw/Mn: 1.15, manufactured by Polymer Source), final concentration 5.72wt % ⁇ PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ⁇ 1,000, manufactured by Sigma-Aldrich), final concentration 0.6 wt%
  • a mixture containing the components of Composition A in ethanol was pre-crosslinked by holding it at 40°C for 16 hours to prepare a composition for forming a second protective film in Experiment 9-1.
  • a mixture containing the components of Composition B in ethanol was pre-crosslinked by holding it at 40°C for 16 hours to prepare a composition for forming a second protective film in Experiment 9-2.
  • a mixture containing the components of Composition B in ethanol was pre-crosslinked by holding it at 53° C. for 16 hours to prepare a composition for forming a second protective film in Experiment 9-3.
  • the sensor from Experiment 9-1, Experiment 9-2, or Experiment 9-3 was immersed in the same liquid sample used in Experiment 8, and a voltage of 100 mV was applied to the working electrode 10a with respect to the reference electrode 20 (Ag/AgCl). A voltage was applied, and the current value between the working electrode 10a and the counter electrode 30 was measured continuously for about 14 days.
  • FIG. 66A shows the measurement results using the sensor of Experiment 9-1
  • FIG. 66B shows the measurement results using the sensor of Experiment 9-2
  • FIG. 66C shows the measurement results using the sensor of Experiment 9-3. .
  • the second protective film 18ab is formed using a second protective film forming composition that has been pre-crosslinked by mixing P4VP-tBuMA and PEGDGE and then reacting them for a certain period of time. ) It was suggested that the sensor provided on the first protective film 18aa can suppress the occurrence of an abnormally high current value during long-term measurement. It was also suggested that by increasing the temperature during pre-crosslinking, it is possible to further suppress the occurrence of abnormally high current values during long-term measurements.
  • compositions for forming the second protective film were designed for the composition for forming the second protective film, each containing P4VP-tBuMA and PEGDGE in ethanol at the following final concentrations.
  • Composition C (22.67 mol% of epoxy groups derived from PEGDGE with respect to pyridine derived from 4-vinylpyridine): ⁇ P4VP-tBuMA (Mn of poly-4-vinylpyridine: 120,000, Mn of poly-tert-butyl methacrylate: 270,000, Mw/Mn: 1.15, manufactured by Polymer Source), final concentration 5.72wt % ⁇ PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ⁇ 1,000, manufactured by Sigma-Aldrich), final concentration 2.0 wt%
  • Composition D (10.17 mol% of epoxy group derived from PEGDGE with respect to pyridine derived from 4-vinylpyridine): ⁇ P4VP-tBuMA (Mn of poly-4-vinylpyridine: 120,000, Mn of poly-tert-butyl methacrylate: 270,000, Mw/Mn: 1.15, manufactured by Polymer Source), final concentration 5.72wt % ⁇ PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ⁇ 1,000, manufactured by Sigma-Aldrich), final concentration 0.9 wt%
  • Composition E (6.77 mol% of epoxy groups derived from PEGDGE with respect to pyridine derived from 4-vinylpyridine): ⁇ P4VP-tBuMA (Mn of poly-4-vinylpyridine: 120,000, Mn of poly-tert-butyl methacrylate: 270,000, Mw/Mn: 1.15, manufactured by Polymer Source), final concentration 5.72wt % ⁇ PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ⁇ 1,000, manufactured by Sigma-Aldrich), final concentration 0.6 wt%
  • a mixture containing the components of Composition C in ethanol was pre-crosslinked by holding it at room temperature (25°C ⁇ 3°C) for about 1 hour to prepare the second protective film forming composition of Experiment 10-1.
  • a mixture containing the components of Composition D in ethanol was pre-crosslinked by holding it at 40° C. for 16 hours to prepare a composition for forming a second protective film in Experiment 10-2.
  • a mixture containing the components of Composition E in ethanol was pre-crosslinked by holding it at 53° C. for 16 hours to prepare a composition for forming a second protective film in Experiment 10-3.
  • Each sensor after being stored at 60°C for 14 days was immersed in the same liquid sample used in Experiment 8, and a voltage of 100 mV was applied to the working electrode 10a with respect to the reference electrode 20 (Ag/AgCl).
  • the current value between the electrode 10a and the counter electrode 30 was measured continuously for 10 days.
  • the second protective film 18ab is formed using a second protective film forming composition that has been pre-crosslinked by reacting P4VP-tBuMA and PEGDGE for 6 hours or more after mixing, with Nafion containing a cation exchange functional group.
  • the sensors of Experiments 10-2 and 10-3 provided on the first protective film 18aa containing (registered trademark) had smaller fluctuations in current value after the temperature accelerated test compared to the sensor of Experiment 10-1. confirmed. Furthermore, it was confirmed that by increasing the temperature during pre-crosslinking, it was possible to further reduce the fluctuation in current value after the temperature accelerated test.
  • Such a sensor is sometimes used for continuous monitoring, in which a test substance is continuously measured over a long period of several days or more while immersed in a liquid sample.
  • the sensor is required to have small changes in responsiveness in order to enable stable measurements during the measurement period.
  • the fourth disclosure of the present specification to provide a sensor that can perform stable measurements with little change in response even when measurements are performed for a long period of time while immersed in a liquid sample, and a method for manufacturing the same. shall be.
  • the sensor according to the fourth disclosure can be suitably used for continuous monitoring over a long period of time.
  • a sensor comprising an insulating substrate and a working electrode disposed on the substrate,
  • the working electrode is a working electrode conductive layer disposed on the substrate;
  • a reagent layer containing a reagent involved in a redox reaction, disposed on the working electrode conductive layer;
  • a first protective film containing a first polymer compound containing a cation exchange functional group, disposed on the reagent layer;
  • a sensor comprising: a second protective film that is disposed on the first protective film and includes a second polymer compound that includes a cationic functional group.
  • the second polymer compound includes the first unit containing the cationic functional group and the second unit containing the hydrophobic functional group, and the second unit is based on the total amount of the constituent units of the second polymer compound.
  • the sensor according to appendix 4-1 wherein the ratio of is 50 mol% or more.
  • the sensor according to appendix 4-1 or 4-2, wherein the cationic functional group is pH independent.
  • the sensor according to appendix 4-3, wherein the cationic functional group includes a quaternary ammonium cation derived from a reaction between a tertiary amine and an epoxy group.
  • the working electrode is a working electrode conductive layer disposed on the substrate; a reagent layer containing a reagent involved in a redox reaction, disposed on the working electrode conductive layer; a first protective film containing a first polymer compound containing a cation exchange functional group, disposed on the reagent layer; A first unit disposed on the first protective film that includes a quaternary ammonium cation derived from a reaction between a tertiary amine and an epoxy group, and a second unit that includes a hydrophobic functional group.
  • a method for manufacturing a sensor comprising: a second protective film containing a dipolymer compound; A mixture comprising an uncrosslinked polymer compound containing a third unit containing a tertiary amine and the second unit containing the hydrophobic functional group, and a crosslinking agent compound containing two or more epoxy groups in alcohol. , by holding the tertiary amine of the third unit and the epoxy group of the crosslinking agent compound at a temperature of 20° C. or higher for 6 hours or more, thereby converting the third unit into the third unit.
  • the uncrosslinked polymer compound includes the third unit and the second unit in a ratio of 50 mol% or more to the total amount of the third unit and the second unit.
  • Appendix 4-7 The method described in Appendix 4-7, including as follows.
  • Appendix 4-9) The uncrosslinked polymer compound and the crosslinking agent compound are contained in the mixture such that the epoxy group of the crosslinking agent compound is 6.8 mol% or more with respect to the tertiary amine of the third unit.
  • the method described in Appendix 4-7 or 4-8. (Appendix 4-10) The method according to any one of Supplementary Notes 4-7 to 4-9, wherein the uncrosslinked polymer compound is a copolymer compound of 4-vinylpyridine and tert-butyl methacrylate.

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