Sensor and method for manufacturing same

Abstract

Provided are a sensor and a method for manufacturing the sensor, which is provided with a working electrode including a reagent layer having a property that can easily be controlled to a desired range, and a protective film. A sensor (1) comprises an insulating substrate (2), and a working electrode (10a). The working electrode (10a) comprises: an electrically conductive layer (11a) on the substrate (2); a first insulating layer (3a) which is disposed on the electrically conductive layer (11a) and which has a first opening portion (3a1) and a water-repellent top surface (3a2); a second insulating layer (4a) which is disposed on the first insulating layer (3a) and which has a second opening portion (4a1) and a top surface (4a2) having liquid repellency with respect to alcohol; a reagent layer (15a) disposed within the first opening portion (3a1) of the first insulating layer (3a); and a protective film (16a) disposed within the second opening portion (4a1) of the second insulating layer (4a).

Description

Sensor and manufacturing method thereof

[0001] The first disclosure relates to, for example, a sensor and a method for manufacturing the same.

[0002] Electrochemical sensors have been used to measure target substances in test samples such as cell culture media and blood samples. Such sensors include, for example, an insulating substrate and a working electrode disposed on the surface of the substrate, as well as 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 (e.g., an oxidoreductase and an electron carrier) involved in a redox reaction. The sensor may further include a protective film that covers only the working electrode or that covers the working electrode together with the counter electrode and / or the reference electrode to prevent leakage of the reagent from the reagent layer.

[0003] For example, the electrochemical sensor described in Patent Document 1 includes a substrate, a first electrode (working electrode) and a second electrode (reference electrode) each including a sensing layer (reagent layer) disposed on the substrate, and a flow-limiting membrane (protective membrane) containing a predetermined polymer compound that covers the entire electrodes. Patent Document 1 describes that the flow-limiting membrane (protective membrane) is formed by dip coating or casting.

[0004] U.S. Patent No. 9,014,774

[0005] However, conventional sensors having a working electrode including a reagent layer and a protective film on the conductive layer have the following problems.

[0006] In Patent Document 1, an insulating substrate having a working electrode including a conductive layer and a reagent layer is immersed in a solution containing a polymer compound and then dried to form a protective film that covers the entire insulating substrate.

[0007] However, with this method, it can be difficult to control the characteristics of the protective film on the reagent layer of the working electrode (such as the thickness of the protective film and the amount of polymer compound that makes up the protective film) within the designed range. Variations in the characteristics of the protective film on the working electrode can cause a decrease in the measurement accuracy of the sensor.

[0008] In Patent Document 1, a reagent layer containing a reagent such as an enzyme is disposed on the surface of a flat electrode.

[0009] However, in the method of forming a reagent layer on the surface of a flat electrode, it may be difficult to control the characteristics of the reagent layer (e.g., thickness of the reagent layer, amount of reagent constituting the reagent layer) within the designed range. Variation in the characteristics of the reagent layer on the working electrode may also cause a decrease in the measurement accuracy of the sensor.

[0010] An object of the first disclosure is to provide a sensor including a working electrode including a reagent layer and a protective film, the characteristics of which can be easily controlled within a desired range, and a method for manufacturing the same.

[0011] The sensor according to the first disclosure includes an insulating substrate and a working electrode disposed on the substrate.

[0012] the working electrode comprises: a conductive layer disposed on the substrate; a first insulating layer, at least a portion of which is disposed on the conductive layer, the first insulating layer having a first opening penetrating the thickness direction of the substrate at a position overlapping with a portion of the conductive layer in a plan view from the thickness direction, and a water-repellent surface; a second insulating layer disposed on the first insulating layer, the second opening penetrating the thickness direction of the substrate at a position overlapping with a portion of the first insulating layer that entirely encloses the first opening in a plan view from the thickness direction, and a surface that is liquid-repellent with respect to alcohol; a reagent layer disposed within the first opening of the first insulating layer, the reagent layer having an outer periphery defined by the inner periphery of the first opening of the first insulating layer and including a reagent participating in an oxidation-reduction reaction; and a protective film disposed within the second opening of the second insulating layer, the protective film having an outer periphery defined by the inner periphery of the second opening of the second insulating layer.

[0013] The method for manufacturing the sensor according to the first disclosure includes forming a droplet of liquid composition A containing the reagent 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 arranged, and then drying the droplet to form the reagent layer, and after forming the reagent layer, forming a droplet of liquid composition B containing a protective film component in alcohol in the second opening of the second insulating layer, and then drying the droplet to form the protective film. (Effects of the Invention)

[0014] According to the sensor and manufacturing method of the first disclosure, it is easy to control the properties of the reagent layer and protective film of the working electrode of the sensor within a desired range.

[0015] Plan view showing a substrate on which conductive layers and wirings of a first working electrode, a second working electrode, a reference electrode, and a counter electrode are disposed. Plan view showing a substrate on which conductive layers and wirings of a first working electrode, a second working electrode, a reference electrode, and a counter electrode are disposed, and a first insulating layer is further disposed thereon. Plan view showing a substrate on which conductive layers and wirings of a first working electrode, a second working electrode, a reference electrode, and a counter electrode are disposed, and a first insulating layer and a second insulating layer are further disposed thereon. 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 disposed on the substrate of FIG. 3. Plan view of a sensor according to an embodiment of the first disclosure. Cross-sectional view taken along line A-A' of a portion of the substrate of FIG. 3 corresponding to the first working electrode. 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. Cross-sectional view taken along line B-B' of a portion of the substrate of FIG. 4 corresponding to the first working electrode, obtained by drying the droplet of liquid composition A of FIG. 3. Cross-sectional view of a droplet of a 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. Cross-sectional view of a part 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 droplet of the first liquid composition B shown in FIG. 9. Cross-sectional view of a droplet of the first liquid composition B further formed in the second opening of the second insulating layer of the first working electrode, including a part of the protective film of FIG. 10. Cross-sectional view taken along line C-C' of a portion of the sensor of FIG. 5 including the first working electrode, including the first protective film obtained by drying the droplet of the first liquid composition B of FIG. 11. 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. 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 droplet of the second liquid composition B of FIG. 9. 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. 14. Cross-sectional view taken along line D-D' of a portion of the sensor of FIG. 5 including the second working electrode, including the third protective film obtained by drying the droplet of the third liquid composition B of FIG. 15. Cross-sectional view of a state in which a droplet of a 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 sensor of the comparative example, has spread by wetting.19 is a cross-sectional view of a portion of a first protective film formed on the upper surface of the second insulating layer of the first working electrode and in the second opening, obtained by drying the wetted and spread first liquid composition B of FIG. 17 ; 20 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 in the second opening, obtained by drying the wetted and spread first liquid composition B of FIG. 19 ; 21 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 in the second opening, obtained by drying the wetted and spread first liquid composition B of FIG. 19 ; 22 is a schematic diagram illustrating a method for measuring a test substance by immersing the sensor of FIG. 5 in a liquid sample; 23 is a cross-sectional view of a portion of the sensor of FIG. 5 , including a reference electrode, taken along line E-E′; 24 is a cross-sectional view of a portion of the sensor of FIG. 5 , including a counter electrode, taken along line F-F′; 25 is a control block diagram of an analytical device including a sensor according to an embodiment of the first disclosure; 25A shows the measurement results of the current value of the first working electrode (for measuring glucose) of the sensor of the example; 25B shows the measurement results of the current value of the first working electrode (for measuring glucose) of the sensor of the comparative example. FIG. 26A shows the measurement results of the current value of the second working electrode (for measuring lactate) of the sensor of the example. FIG. 26B shows the measurement results of the current value of the second working electrode (for measuring lactate) of the sensor of the comparative example. 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. 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. 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. A plan view showing the substrate of FIG. 29 on which a reagent layer of the working electrode and a silver / silver chloride layer of the reference electrode are further arranged. A plan view of a sensor according to an embodiment of the second disclosure, in which a tip opening and a flow channel are formed in the second insulating layer. 31. A plan view of a sensor according to another embodiment of the second disclosure, in which a tip opening and a flow path are formed in the first insulating layer and the second insulating layer. A cross-sectional view of a portion of the sensor of FIG. 31, including a working electrode, taken along line J-J'. A cross-sectional view of a portion of the sensor of FIG. 31, including a reference electrode, taken along line K-K'. A cross-sectional view of a portion of the sensor of FIG. 31, including a counter electrode, taken along line L-L'. A cross-sectional view of a portion of the sensor of FIG. 31, including a counter electrode and a tip opening and a flow path formed in the second insulating layer, taken along line M-M'.31 and 36 , which is immersed in a liquid sample with the tip end of the substrate at the bottom. A schematic cross-sectional view of a portion including a counter electrode, a tip end opening, and a flow channel of a sensor according to yet another embodiment, which includes a tip end opening and a flow channel formed in a portion in the thickness direction, excluding the top surface of the insulating layer, and having an enclosed structure. A cross-sectional view along line N-N' of a portion including a counter electrode, a tip end opening, and a flow channel formed in the first insulating layer and the second insulating layer of the sensor of FIG. 32 . A schematic cross-sectional view of a portion including a counter electrode, a tip end opening, and a flow channel of the sensor shown in FIGS. 31 and 36 , which is immersed in a liquid sample with the tip end of the substrate at the bottom. A schematic cross-sectional view of a portion including a counter electrode of a comparative sensor which does not include a tip end opening and a flow channel, which is immersed in a liquid sample with the tip end of the substrate at the bottom. A plan view of a sensor according to an embodiment of the second disclosure, which includes a substrate including a main body portion, a connection portion, and a base end, and a second insulating layer including a first insulating layer disposed over the entire first surface of the substrate and an insulating sheet disposed on the first surface of the substrate closer to the tip end than the bent portion. 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 a substrate. The insulating layer is omitted in FIG. 42. A schematic diagram of the sensor of FIG. 41 in which the main body of the substrate, on which the detection electrodes are arranged, is bent at the bent portion of the connection portion of the substrate and immersed in a liquid sample. The insulating layer is omitted in FIG. 43. A schematic side view of the sensor of FIG. 41 in which the main body of the substrate, on which the detection electrodes are arranged, is bent at the bent portion of the connection portion of the substrate and immersed in a liquid sample. A schematic side view of a comparative sensor in which a second insulating layer including an insulating sheet is arranged over the entire first surface of the substrate in the sensor of FIG. 41, and the main body of the substrate, on which the detection electrodes are arranged, is bent at the bent portion of the connection portion of the substrate and immersed in a liquid sample. 46 is a plan view of a sensor according to another embodiment of the second disclosure, the sensor including a substrate including a main body portion, a connection portion, and a base end portion, a first insulating layer disposed over the entire first surface of the substrate, a second insulating layer including the first insulating sheet disposed on the first surface of the substrate toward the tip end from the bent portion, and a second insulating layer including the second insulating sheet disposed on the first surface of the substrate at the base end, the second insulating layer not including an insulating sheet on the bent portion of the substrate.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. ...B shows the measurement results of the current value of the working electrode of sensor S5. 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. 50E shows the measurement results of the current value of the working electrode of sensor S5. FIG. 50F shows the measurement results of the current value of the working electrode of sensor S5. FIG. 50F shows the measurement results of the current value of the working electrode of sensor S5. FIG. 50F shows the measurement results of the current value of the working electrode of sensor S5. FIG. 50F shows the measurement results of the current value of the working electrode of sensor S5. FIG. 50F shows the measurement results of the current value of the working electrode of sensor S5. FIG. 50F shows the measurement results of the current value of the working electrode of sensor S5. FIG. 50F shows the measurement results of the current value of the working electrode of sensor S5. FIG. 50F shows 55 shows a cross-sectional view taken along line Q-Q' of a portion of the sensor shown in Fig. 54 including a first working electrode. 56 shows a cross-sectional view taken along line R-R' of a portion of the sensor shown in Fig. 54 including a second working electrode. 57 shows the results of a cyclic voltammetry test in Experiment 4 of the third disclosure, using as the working electrode either a platinum particle-free carbon electrode (Experiment 3-1), a carbon electrode containing 1% platinum particles (Experiment 3-2), or a carbon electrode containing 5% platinum particles (Experiment 3-3). 58 shows the results of measuring the current value over time by applying a potential of 0.3 V or -0.2 V in Experiment 5 of the third disclosure, using as the working electrode either a platinum particle-free carbon electrode (A) or a carbon electrode containing 1% platinum particles (B). In Experiment 6 of the third disclosure, the current value (A) corresponding to the glucose concentration and the current value (B) corresponding to the lactic acid concentration in a liquid sample were measured over time using the sensor of Experiment 2-1 (wherein the working electrode conductive layer and counter electrode were carbon conductive layers not containing platinum particles) and the sensor of Experiment 2-2 (wherein the counter electrode was a carbon conductive layer modified with platinum nanoparticles on its surface, and the working electrode conductive layer was a carbon conductive layer not containing platinum particles) (N=2).6 shows the results of time-dependent measurement of current values ​​(A) corresponding to the glucose concentration and current values ​​(B) corresponding to the lactate concentration in a liquid sample using a sensor of Experiment 3-1 (where the working electrode conductive layer and counter electrode were carbon conductive layers containing no platinum particles), a sensor of Experiment 3-2 (where the working electrode conductive layer and counter electrode were carbon conductive layers containing 1% by weight of platinum particles relative to the carbon), and a sensor of Experiment 3-3 (where the working electrode conductive layer and counter electrode were carbon conductive layers containing 5% by weight of platinum particles relative to the carbon) in Experiment 7 of the Third Disclosure (N=2).

[0074] FIG. 65 shows a plan view of a sensor according to an embodiment of the Fourth Disclosure.

[0075] FIG. 65A shows a cross-sectional view of a portion of the sensor shown in FIG. 62 , including the counter electrode, taken along line S-S′.

[0076] FIG. 65B shows a cross-sectional view of a portion of the sensor shown in FIG. 62 , including the working electrode, taken along line T-T′.

[0077] FIG. 65 shows the results of Experiment 8 of the Fourth Disclosure. FIG. 65A shows the results of measurement using the sensor of Experiment 8-1, and FIG. 65B shows the results of measurement using the sensor of Experiment 8-2. Figure 66 shows the results of Experiment 9 of the fourth disclosure. Figure 66A shows the measurement results using the sensor of Experiment 9-1, Figure 66B shows the measurement results using the sensor of Experiment 9-2, and Figure 66C shows the measurement results using the sensor of Experiment 9-3. Figure 67 shows the results of Experiment 10 of the fourth disclosure.

[0016] One or more embodiments of the first to fourth disclosures in this specification will be described. In this specification, unless a temperature is specified for a method or property, the method or property refers to room temperature (25°C ± 3°C). This specification incorporates the disclosures of Japanese Patent Application Nos. 2022-107320 and 2022-116287, from which this application claims priority. Furthermore, all publications, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety.

[0017] <Embodiments of the First Disclosure> Hereinafter, embodiments of a sensor and a method for manufacturing a sensor according to the first disclosure will be described. In the embodiments, unnecessary detailed explanations may be omitted. For example, detailed explanations of well-known matters and redundant explanations of substantially identical configurations may be omitted. This is to avoid unnecessary redundancy in the following explanation and to facilitate understanding by those skilled in the art.

[0018] Furthermore, the applicant provides the accompanying drawings and the following description so that those skilled in the art can fully understand the first disclosure, and does not intend for them to limit the subject matter described in the claims.

[0019] <Materials> Examples of materials that can be used in the sensor and the manufacturing method thereof according to the first disclosure of this specification will be described.

[0020] The material of the insulating substrate of the sensor of the first disclosure of this specification is not particularly limited, and examples thereof include resin materials such as polyethylene terephthalate, polycarbonate, polyimide, polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyoxymethylene, monomer cast nylon, polybutylene terephthalate, methacrylic resin, and ABS resin, as well as glass materials. Polyethylene terephthalate, polycarbonate, and polyimide are preferred, and polyethylene terephthalate is more preferred. The dimensions of the substrate, such as its thickness, are not particularly limited, and a substrate having a thickness of, for example, 0.05 mm or more and 2 mm or less, and preferably 0.1 mm or more and 1 mm or less, can be used.

[0021] The conductive layer of the working electrode of the sensor of the first disclosure of this specification is a layer containing a conductive material such as carbon, gold, platinum, or palladium. The conductive layers of the working electrode, reference electrode, and counter electrode can be manufactured by forming a layer of such a conductive material on the surface of a substrate using a sputtering method, a vapor deposition method, a screen printing method, or the like. If necessary, the conductive layer can be processed into a predetermined pattern using a laser trimming method. The wiring of the sensor according to the embodiment described below can also be made of a similar conductive material.

[0022] The sensor disclosed in the first disclosure of this specification is used to detect a predetermined analyte in a liquid sample by immersion in the liquid sample, such as glucose, lactic acid, cholesterol, bilirubin, amino acids such as glutamine and glutamic acid, glycated amino acids, glycated peptides, ketone bodies (3-hydroxybutyric acid), and alcohol.

[0023] The working electrode of the sensor of 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 involved in the redox reaction of the test substance, and may be appropriately selected depending on the test substance. The reagent involved in the redox reaction may include a combination of an oxidoreductase and a mediator (electron carrier), or an oxidoreductase. The oxidoreductase may include a coenzyme.

[0024] Examples of oxidoreductases include oxidases and dehydrogenases. Specific examples of oxidoreductases 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, and alcohol dehydrogenase. These oxidoreductases can be used to detect the test substances exemplified above.

[0025] Furthermore, the mediator may be one or more selected from, but is not limited to, metal complexes (e.g., osmium complexes, ruthenium complexes, iron complexes, etc.), quinone compounds (e.g., benzoquinone, naphthoquinone, phenanthrenequinone, phenanthrolinequinone, anthraquinone, and derivatives thereof, etc.), phenazine compounds, viologen compounds, phenothiazine compounds, and phenol compounds. Specific examples of the mediator include one or more selected from 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-sulfonate, phenazine derivatives (1-methoxy-5-methylphenazinium methyl sulfate, 1-methoxy-5-ethylphenazinium ethyl sulfate, etc.), methyl viologen, benzyl viologen, methylene blue, methylene green, 2-aminophenol, 2-amino-4-methylphenol, and 2,4-diaminophenol. Examples of the salt include, but are not limited to, sodium salts, potassium salts, calcium salts, magnesium salts, and lithium salts.

[0026] From the viewpoint of sensor durability and suppression of leakage of the mediator from the sensor, it is desirable for the mediator to be a polymerized mediator bound to a polymer compound. The polymer compound to which the mediator is bound can be a homopolymer, random copolymer, block copolymer, or a polymer compound in which these are bound or mixed. The weight-average molecular weight of the polymer compound is, for example, 10,000 or more, preferably 50,000 or more, and more preferably 100,000 or more. The upper limit of the weight-average molecular weight is, for example, less than 10,000,000, preferably less than 1,000,000. Furthermore, the polymer compound is not particularly limited, but examples include those in which a main chain is formed by multiple atoms selected from at least one of carbon atoms, nitrogen atoms, oxygen atoms, and sulfur atoms bonded in a chain. Specific examples include natural polymer compounds such as proteins, polypeptides, and polynucleotides, as well as synthetic polymer compounds such as polyamino acids, polyimines, polyallyl compounds, poly(meth)acrylates, polyalkylene oxides, and copolymers thereof. Examples of polyamino acids include poly(L-glutamic acid) and poly(L-lysine). Examples of polyimines include polyalkyleneimines such as polyethyleneimine and polypropyleneimine. Examples of polyallyl compounds include polyallylamine and polydiallylamine. Examples of polyalkylene oxides include polyethylene oxide and polypropylene oxide. It is preferable that the polymerized mediator as a whole is hydrophilic, and it is also preferable that the polymer compound to which the mediator is bonded is hydrophilic.

[0027] The polymerized mediator may be one in which a mediator and a polymer compound are bonded via a covalent bond. Examples of such covalent bonds include those described in JP-A-2003-514924, and specific examples include ether bonds, thioether bonds, ester bonds, urethane bonds, and amide bonds. 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 the mediator or the polymer compound. A specific example of a polymerized mediator may be one in which a phenazine compound is used as the mediator and poly(L-lysine) is used as the polymer, with the phenazine compound and poly(L-lysine) bonded via an amide bond.

[0028] The reagent layer provided on the working electrode of the sensor of 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 crosslinker, as necessary. Examples of the hydrophilic polymer compound include cellulose derivatives, and examples of the cellulose derivatives include one or more selected from methyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose, and hydroxypropyl methyl cellulose. Examples of the conductive carbon filler include one or more selected from carbon black, graphite powder, porous carbon, and nanocarbon.

[0029] The protective film provided on the working electrode of the sensor of the first disclosure of this specification can be a film that prevents or suppresses leakage of the reagent contained in the reagent layer outside the protective film and is permeable to the analyte present outside the protective film. A 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 and a polymer compound containing a cation-exchange functional group.

[0030] Examples of polymer compounds containing 4-vinylpyridine as a structural unit include poly(4-vinylpyridine), copolymers (preferably block copolymers) of 4-vinylpyridine and an alkyl methacrylate, and copolymers (preferably random copolymers) of styrene, 4-vinylpyridine, and oligopropylene glycol methyl ether methacrylate. Examples of the alkyl methacrylate include tert-butyl methacrylate. Examples of the oligopropylene glycol methyl ether methacrylate include tripropylene glycol methyl ether methacrylate. Polymer compounds containing 4-vinylpyridine as a structural unit are preferably crosslinked with a crosslinking agent containing two or more epoxy groups, such as polyethylene glycol diglycidyl ether (PEG-DGE). Polymer compounds containing 4-vinylpyridine as a structural unit and crosslinked with a crosslinking agent containing two or more epoxy groups contain a quaternary ammonium cation-containing functional group generated by the reaction of the pyridyl group (tertiary amine) derived from 4-vinylpyridine with the epoxy group. A preferred embodiment of the polymer compound containing 4-vinylpyridine as a constituent unit and crosslinked with a crosslinking agent containing two or more epoxy groups is the specific embodiment of the "second polymer compound containing a cationic functional group" described in the fourth disclosure.

[0031] Examples of polymer compounds containing cation-exchange functional groups include polymer compounds containing structural units having sulfonic acid groups in their side chains. These are preferably polymer compounds containing, as structural units, perfluorocompounds having sulfonic acid groups in their side chains. More preferably, these are copolymer compounds containing, as structural units, perfluorocompounds having sulfonic acid groups in their side chains and perfluorocompounds not having ionic functional groups in their side chains. A particularly preferred example is a copolymer compound of tetrafluoroethylene and perfluoro-2-(2-fluorosulfonylethoxy)propyl vinyl ether, with Nafion® being particularly preferred. A preferred embodiment of the polymer compound containing cation-exchange functional groups is the specific embodiment of the "first polymer compound containing cation-exchange functional groups" described in the fourth disclosure. A protective membrane containing a polymer compound containing cation-exchange functional groups is preferably provided on the reagent layer to transport cations such as protons between the reagent layer and the liquid sample.

[0032] The protective film provided on the working electrode of the sensor of the first disclosure of this specification can be a laminate of two or more protective films, and can be, for example, a laminate of a protective film provided on the side in contact with the reagent layer and containing a polymer compound including a cation-exchange functional group, and a protective film provided thereon and containing a polymer compound including 4-vinylpyridine as a constituent unit.

[0033] The protective film provided on the working electrode of the sensor of the first disclosure of this specification can be formed by drying a liquid composition B containing protective film components in an alcohol. Examples of the protective film components include a polymer compound and a crosslinking agent contained in the protective film. Examples of the alcohol in liquid composition B include monohydric alcohols having 1 to 5 carbon atoms, and particularly preferably methanol, ethanol, or isopropyl alcohol, with ethanol being most preferred.

[0034] The first insulating layer of the working electrode of the sensor of the first disclosure of this specification includes a water-repellent surface. Here, 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 can be, for example, 160° or less (the range of the contact angle with water is, for example, 90° or more and 160° or less).

[0035] The second insulating layer provided in the working electrode of the sensor of the first disclosure of this specification includes a surface that is liquid-repellent (alcohol-repellent) to alcohol. Here, a surface that is liquid-repellent to alcohol refers to a surface that has a contact angle to 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 liquid-repellent surface of the second insulating layer to alcohol is not particularly limited, but can be, for example, 100° or less (the range of the contact angle to alcohol is, for example, 45° or more and 100° or less). Here, examples of alcohol include monohydric alcohols having 1 to 5 carbon atoms, with methanol, ethanol, or isopropyl alcohol being particularly preferred, and ethanol being most preferred.

[0036] The contact angle of the surface of the first insulating layer with water and the contact angle of the surface of the second insulating layer with alcohol can be measured at 20°C. The contact angle of the surface of the first insulating layer with water and the contact angle of the surface of the second insulating layer with alcohol can be measured using a commercially available analytical device, for example, a handy contact angle and surface free energy analyzer MSA manufactured by KRUSS. The contact angle of the surface with water or alcohol is preferably measured by discharging a 2 μL droplet of water or alcohol onto the surface to be measured and measuring the contact angle between the droplet and the surface after 2 seconds.

[0037] The water-repellent surface of the first insulating layer provided in the working electrode of the sensor of the first disclosure of this specification preferably contains a fluororesin. As the fluororesin, a polymer compound of a fluorohydrocarbon can be used, for example, a polymer compound containing one or more selected from vinylidene fluoride, tetrafluoroethylene, hexafluoropropylene, and perfluoro(alkyl vinyl ether), and in particular, a copolymer containing two or more selected from vinylidene fluoride, tetrafluoroethylene, hexafluoropropylene, and perfluoro(alkyl vinyl ether) is preferred, and a copolymer containing vinylidene fluoride and hexafluoropropylene is more preferred. The entire first insulating layer can be made of a fluororesin.

[0038] The second insulating layer provided in the working electrode of the sensor disclosed in the first disclosure of this specification has a liquid-repellent surface against alcohol, preferably containing a compound containing a perfluoroalkyl group. Examples of compounds containing a perfluoroalkyl group include fluorine-based surface-modifying additives. Such a second insulating layer can be formed by applying a composition containing an insulating matrix resin and a compound containing a perfluoroalkyl group (fluorine-based surface-modifying additive) in a solvent onto the first insulating layer and drying the composition. At this time, the compound containing the perfluoroalkyl group segregates on the surface, forming a liquid-repellent surface against 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-based resin. The number of carbon atoms in the perfluoroalkyl group is not particularly limited, but may be, for example, 2 to 20 carbon atoms. The perfluoroalkyl group may contain a trifluoromethyl group at its terminal.

[0039] <Overview of Sensor 1> A sensor 1 according to an embodiment of the first disclosure will be described with reference to the drawings. As shown in FIG. 5 , the sensor 1 of this embodiment includes an insulating substrate 2, a first working electrode 10a, a second working electrode 10b, a reference electrode 20, and a counter electrode 30 disposed on the substrate 2, and wiring 50 electrically connected to the first working electrode 10a, the second working electrode 10b, the reference electrode 20, and the counter electrode 30. While the sensor 1 of this embodiment includes two working electrodes, in another embodiment (not shown), the sensor may include 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 from each other, they may be referred to as the working electrodes 10a and 10b. Furthermore, 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. Although the sensor 1 of this embodiment is a three-electrode sensor including a working electrode, a reference electrode, and a counter electrode as electrodes, it may be a two-electrode sensor including only a working electrode and a counter electrode without including a reference electrode. Although not shown, the reference electrode and / or the counter electrode may be provided on a substrate separate from the substrate on which the working electrode is disposed.

[0040] As shown in Fig. 21, the sensor 1 is immersed in a liquid sample X and used to detect a predetermined test substance in the liquid sample X. Examples of the liquid sample X include a culture medium containing cells C as shown in the figure, and a liquid sample prepared using blood obtained from a living organism. Specific examples of test substances are as described in the <Materials> section.

[0041] 12 and 16, the working electrodes 10a and 10b of the sensor 1 include, on the conductive layers 11a and 11b, reagent layers 15a and 15b containing a reagent involved in the oxidation-reduction reaction of the test substance in the liquid sample X. Preferred embodiments of the reagent involved in the oxidation-reduction reaction are as described in the <Materials> section.

[0042] When the reagent layers 15a, 15b of the working electrodes 10a, 10b of the sensor 1 contain a reagent that oxidizes the analyte in the liquid sample X, electrons are transferred from the analyte to the conductive layers 11a, 11b under conditions in which a predetermined voltage is applied to the electrodes of the sensor 1. Similarly, when the reagent layers 15a, 15b contain a reagent that reduces the analyte in the liquid sample X, electrons are transferred from the conductive layers 11a, 11b to the analyte. Because the amount of transferred electrons depends on the concentration of the analyte, the concentration or change in concentration of the analyte in the liquid sample X can be measured based on the value of the current or a change in the current value flowing through the working electrodes 10a, 10b of the sensor 1.

[0043] An example of an analytical device 100 equipped with the sensor 1 for analyzing a test substance in a liquid sample will be described with reference to FIG.

[0044] 24 , the analysis device 100 includes a sensor 1, an analysis unit 102, and a control unit 104. The working electrodes 10a and 10b, the reference electrode 20, and the counter electrode 30 of the sensor 1 are each connected to the analysis unit 102 via wiring 50. The analysis unit 102 is capable of communicating with the control unit 104.

[0045] The analysis unit 102 includes an electrochemical measurement section 111 , a control section 112 , a storage section 113 , and a communication section 114 .

[0046] The electrochemical measurement unit 111 is a potentiostat that applies a predetermined voltage to each electrode of the sensor 1 to measure the concentration of the test substance, and includes a voltage application unit 111a and a current measurement unit 111b, and preferably further includes a voltage measurement unit (counter electrode terminal voltage measurement unit) 111c.

[0047] 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.

[0048] The current measuring unit 111b detects the value or change in the current flowing between the working electrodes 10a, 10b and the counter electrode 30 of the sensor 1, measured while a voltage is applied from the voltage applying unit 111a to the electrodes of the sensor 1. As described above, the current value or change in the current value detected by the current measuring unit 111b is an index of the concentration or change in concentration of the test substance in the liquid sample X.

[0049] The voltage measurement unit 111 c measures the terminal voltage of each electrode of the sensor 1 .

[0050] The control unit 112 is connected to the voltage application unit 111a, the current measurement unit 111b, the voltage measurement unit 111c, the storage unit 113, and the communication unit 114. The control unit 112 controls the voltage application unit 111a to apply a predetermined voltage to each electrode of the sensor 1, and controls the communication unit 114 to transmit the measurement results of the current measurement unit 111b and the voltage measurement unit 111c to the control unit 104.

[0051] The memory unit 113 is connected to the control unit 112 and stores data such as the value of the applied voltage that is preset for each measurement object, the measurement values ​​in the current measurement unit 111b and the voltage measurement unit 111c, and a calibration curve that has been measured in advance.

[0052] The communication section 114 is controlled by the control section 112 and transmits data such as the measurement results of the current measurement section 111 b and the voltage measurement section 111 c to the analysis section 142 of the control unit 104 .

[0053] The control unit 104 is capable of communicating with the analysis unit 102 via a communication unit 114 , and includes a display unit 141 and an analysis unit 142 .

[0054] The display unit 141 displays the results of the analysis in the analysis unit 142, such as the concentration of the test substance in the liquid sample X from the current value detected by the current measurement unit 111b.

[0055] The analysis unit 142 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 electrodes 10a, 10b and the counter electrode 30, which is measured by the current measurement unit 111b.

[0056] <Working Electrode> The features of the working electrodes 10a and 10b of the sensor 1 will be described with reference to the drawings, particularly Fig. 12 and Fig. 16. 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. 5. 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. 5.

[0057] 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 ). In the following description, when the elements of the first working electrode 10a and the second working electrode 10b are referred to without distinction, they will be referred to as conductive layers 11a and 11b, first insulating layers 3a and 3b, second insulating layers 4a and 4b, reagent layers 15a and 15b, and protective films 16a and 16b.

[0058] The conductive layers 11a and 11b of the working electrodes 10a and 10b are disposed on an insulating substrate 2. The main surface on which the conductive layers 11a and 11b are formed is referred to as a first surface 2a.

[0059] Preferred embodiments of the materials for the substrate 2 and the conductive layers 11a and 11b are as described in the <Materials> section.

[0060] At least a portion of the first insulating layers 3a and 3b of the working electrodes 10a and 10b is disposed on the conductive layers 11a and 11b. As shown in Figures 12 and 16, a portion of the first insulating layers 3a and 3b may be disposed on the conductive layers 11a and 11b, or, although not shown, the entire first insulating layers 3a and 3b may be disposed on the conductive layers 11a and 11b.

[0061] In the illustrated embodiment, the first insulating layers 3a and 3b of the working electrodes 10a and 10b refer to the portions of the first insulating layer 3 that cover almost the entire first surface 2a of the substrate 2 and are located near the conductive layers 11a and 11b of the working electrodes 10a and 10b. However, the present invention is not limited to this embodiment, and the first insulating layers 3a and 3b of the working electrodes 10a and 10b may be located only in the portions near the conductive layers 11a and 11b of the working electrodes 10a and 10b.

[0062] The first insulating layers 3a, 3b of the working electrodes 10a, 10b are characterized by having first openings 3a1, 3b1 penetrating in the thickness direction T and water-repellent surfaces 3a2, 3b2 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. The shape of the inner peripheral 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 thereto and may be a polygon (such as a square or a triangle) or any other shape.

[0063] The thickness of the portions of the first insulating layers 3a and 3b of the working electrodes 10a and 10b that cover the conductive layers 11a and 11b is not particularly limited, but can be, for example, 0.5 μm or more and 50 μm or less, and preferably 2 μm or more and 20 μm or less.

[0064] 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 can be, for example, 0.5 mm or more and 5 mm or less, preferably 1 mm or more and 2 mm or less.

[0065] The first insulating layers 3a, 3b of the working electrodes 10a, 10b have water-repellent surfaces 3a2, 3b2. As described below, the reagent layers 15a, 15b of the working electrodes 10a, 10b are formed by forming droplets of a liquid composition A containing a reagent involved in a redox reaction in water within the first openings 3a1, 3b1 of the first insulating layers 3a, 3b of the working electrodes 10a, 10b, and then drying the droplets. Because the surfaces 3a2, 3b2 of the first insulating layers 3a, 3b of the working electrodes 10a, 10b are water-repellent during droplet formation, the droplets of the liquid composition A are easily retained without collapsing. This makes it easy to maintain the properties of the reagent layers 15a, 15b after drying (e.g., the thickness of the reagent layers 15a, 15b, the amount of reagent constituting the reagent layers 15a, 15b) within the designed ranges.

[0066] Preferred embodiments of the material constituting the first insulating layers 3a, 3b having the water-repellent surfaces 3a2, 3b2 are as described in the <Material> column.

[0067] The second insulating layers 4a, 4b of the working electrodes 10a, 10b are disposed on the first insulating layers 3a, 3b. In the illustrated embodiment, the second insulating layers 4a, 4b of the working electrodes 10a, 10b refer to the portions of the second insulating layer 4 that cover almost the entire first surface 2a of the substrate 2 and are located near the conductive layers 11a, 11b of the working electrodes 10a, 10b. However, this embodiment is not limiting, and the second insulating layers 4a, 4b of the working electrodes 10a, 10b may be disposed only in the portions near the conductive layers 11a, 11b of the working electrodes 10a, 10b.

[0068] The second insulating layers 4a, 4b of the working electrodes 10a, 10b have second openings 4a1, 4b1 that penetrate in the thickness direction T and are formed at positions that overlap with portions 3a3, 3b3 of the first insulating layers 3a, 3b that entirely encompass the first openings 3a1, 3b1 when viewed in a plane from the thickness direction T of the substrate 2, and have surfaces 4a2, 4b2 that are liquid-repellent with respect to alcohol.

[0069] 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 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 (rectangle, triangle, etc.) or any other shape. As shown in Figure 3, the inner peripheral edges 3a10, 3b10 of the first openings 3a1, 3b1 of the first insulating layers 3a, 3b and the inner peripheral edges 4a10, 4b10 of the second openings 4a1, 4b1 of the second insulating layers 4a, 4b are preferably parallel to each other in a plan view from the thickness direction T, but is not limited to this. The second openings 4a1, 4b1 of the second insulating layers 4a, 4b are recesses at their bottoms that include the first openings 3a1, 3b1 of the first insulating layers 3a, 3b and the reagent layers 15a, 15b therein, as well as the areas of the water-repellent surfaces 3a2, 3b2 of the first insulating layers 3a, 3b that surround the first openings 3a1, 3b1.

[0070] The thickness of the second insulating layers 4a and 4b of the working electrodes 10a and 10b is not particularly limited, but can be, for example, 5 μm or more and 100 μm or less, and preferably 10 μm or more and 50 μm or less.

[0071] The opening width of the second openings 4a1, 4b1 of the second insulating layers 4a, 4b of the working electrodes 10a, 10b is not particularly limited, but can be, for example, 0.5 mm or more and 5 mm or less, preferably 1 mm or more and 3 mm or less.

[0072] The second insulating layers 4a, 4b of the working electrodes 10a, 10b have surfaces 4a2, 4b2 that are liquid-repellent (alcohol-repellent) to alcohol. As described below, the protective films 16a, 16b of the working electrodes 10a, 10b are formed by forming droplets of a liquid composition B containing protective film components in alcohol within the second openings 4a1, 4b1 of the second insulating layers 4a, 4b of the working electrodes 10a, 10b, and then drying the droplets. Because the surfaces 4a2, 4b2 of the second insulating layers 4a, 4b of the working electrodes 10a, 10b are liquid-repellent to alcohol during the formation of the droplets of liquid composition B, the droplets of liquid composition B are easily retained without collapsing. This makes it easy to maintain the properties of the protective films 16a, 16b after drying (e.g., the thickness of the protective films 16a, 16b, the amounts of the components constituting the protective films 16a, 16b) within the designed ranges.

[0073] Preferred embodiments of the material constituting the second insulating layers 4a, 4b having surfaces 4a2, 4b2 that are liquid-repellent (alcohol-repellent) to alcohol are as described in the <Material> column.

[0074] The reagent layers 15a, 15b of the working electrodes 10a, 10b are disposed within the first openings 3a1, 3b1 of the first insulating layers 3a, 3b, and include outer peripheral edges 15a10, 15b10 defined by the inner peripheral edges 3a10, 3b10 of the first openings 3a1, 3b1 of the first insulating layers 3a, 3b, and a reagent involved in an oxidation-reduction reaction. The thickness of the reagent layers 15a, 15b is not particularly limited, but is preferably approximately the same as or smaller than the thickness of the portions of the first insulating layers 3a, 3b that cover the conductive layers 11a, 11b, as shown in the figure.

[0075] Preferred embodiments of the reagents involved in the oxidation-reduction reaction in the reagent layers 15a and 15b are as described in the <Materials> section.

[0076] The protective films 16a and 16b of the working electrodes 10a and 10b are disposed within the second openings 4a1 and 4b1 of the second insulating layers 4a and 4b, and include outer peripheral edges 16a10 and 16b10 defined by the inner peripheral edges 4a10 and 4b10 of the second openings 4a1 and 4b1 of the second insulating layers 4a and 4b. The thickness of the protective films 16a and 16b is not particularly limited, but is preferably approximately the same as or smaller than the thickness of the second insulating layers 4a and 4b, as shown in the figure.

[0077] The preferred embodiments of the protective films 16a and 16b are as described in the <Material> section.

[0078] In the following description, 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 may be referred to as the "first reagent layer 15a" of the first working electrode 10a and the latter as the "second reagent layer 15b" of the second working electrode 10b. Furthermore, the reagent contained in the reagent layer 15a of the first working electrode 10a may be referred to as the "first reagent," and the reagent contained in the second reagent layer 15b of the second working electrode 10b may be referred to as the "second reagent."

[0079] In a preferred embodiment, sensor 1 includes multiple working electrodes 10 a, 10 b, and a first reagent contained in first reagent layer 15 a of first working electrode 10 a and a second reagent contained in second reagent layer 15 b of second working electrode 10 b, which is different from first working electrode 10 a, are different from each other. Sensor 1 according to this preferred embodiment can be used to detect multiple analytes, including a first analyte and a second analyte, in liquid sample X, because the first reagent is involved in an oxidation-reduction reaction of a first analyte in liquid sample X and the second reagent is involved in an oxidation-reduction reaction of a second analyte different from the first analyte in liquid sample X.

[0080] In the sensor 1 according to the preferred embodiment, which includes the plurality of working electrodes 10 a, 10 b, the first reagent preferably contains an enzyme involved in the oxidation-reduction reaction of glucose, more preferably glucose dehydrogenase or glucose oxidase, and the second reagent preferably contains an enzyme involved in the oxidation-reduction reaction of lactic acid, more preferably lactate oxidase or lactate dehydrogenase. The sensor 1 according to this embodiment can detect glucose and lactic acid.

[0081] <Reference Electrode> The structure of the reference electrode 20 of the sensor 1 will be described with reference to the drawings, particularly Fig. 22. Fig. 22 is a cross-sectional view of a portion of the sensor 1 including the reference electrode 20, taken along line EE' in Fig. 5.

[0082] The reference electrode 20 includes a reference electrode conductive layer 21 disposed on the first surface 2 a of the insulating substrate 2, a silver / silver chloride layer 22 disposed on the reference electrode conductive layer 21, and a reference electrode protective film 23 disposed on the silver / silver chloride layer 22.

[0083] The reference electrode conductive layer 21 can be formed from the materials described for the conductive layers 11a and 11b of the working electrodes 10a and 10b.

[0084] The reference electrode protective film 23 can be formed from the materials listed in the <Materials> section for the protective films 16a and 16b of the working electrodes 10a and 10b.

[0085] The first insulating layer 3 disposed on the first surface 2 a of the substrate 2 has a portion disposed on the reference electrode conductive layer 21 and includes a reference electrode first opening 301 penetrating in the thickness direction T and formed at a position overlapping with a portion of the reference electrode conductive layer 21 in a plan view from the thickness direction T of the substrate 2. The silver / silver chloride layer 22 of the reference electrode 20 is disposed in the reference electrode first opening 301 of the first insulating layer 3.

[0086] The second insulating layer 4 disposed on the first insulating layer 3 includes a reference electrode second opening 401 penetrating in the thickness direction T and formed at a position overlapping with a portion of the first insulating layer 3 that entirely encompasses the reference electrode first opening 301 in a plan view from the thickness direction T. The reference electrode protective film 23 of the reference electrode 20 is disposed in the reference electrode second opening 401 of the second insulating layer 4.

[0087] <Counter Electrode> The structure of the counter electrode 30 of the sensor 1 will be described with reference to the drawings, particularly Fig. 23. 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. 5.

[0088] The counter electrode 30 includes a counter electrode conductive layer 31 disposed on the first surface 2 a of the insulating substrate 2 .

[0089] The counter electrode conductive layer 31 can be formed from the materials described for the conductive layers 11a and 11b of the working electrodes 10a and 10b.

[0090] The first insulating layer 3 disposed on the first surface 2 a of the substrate 2 has a portion disposed on the counter electrode conductive layer 31 and includes a counter electrode first opening 302 penetrating in the thickness direction T and formed at a position overlapping with a portion of the counter electrode conductive layer 31 in a plan view from the thickness direction T of the substrate 2.

[0091] The second insulating layer 4 arranged on the first insulating layer 3 includes a counter electrode second opening 402 that penetrates in the thickness direction T and is formed at a position in the first insulating layer 3 that overlaps with the counter electrode first opening 302 when viewed in a plane from the thickness direction T.

[0092] Counter electrode conductive layer 31 of counter electrode 30 is exposed to the outside through counter electrode first opening 302 in first insulating layer 3 and counter electrode second opening 402 in second insulating layer 4. Therefore, when sensor 1 is immersed in liquid sample X as shown in FIG. 21 , counter electrode conductive layer 31 of counter electrode 30 comes into direct contact with liquid sample X.

[0093] <Method for Manufacturing Sensor 1> A preferred embodiment of a method for manufacturing the sensor 1 will be described with reference to the drawings, particularly FIGS.

[0094] First, as shown in FIG. 1 , the conductive layers 11 a and 11 b of the working electrodes 10 a and 10 b, the reference electrode conductive layer 21 of the reference electrode 20, the counter electrode conductive layer 31 of the counter electrode 30, and wiring 50 electrically connected to each of them are arranged on a first surface 2 a of an insulating substrate 2.

[0095] Next, as shown in FIG. 2 , a first insulating layer 3 is further laminated on the first surface 2a of the substrate 2 on which the conductive layers 11a and 11b of the working electrodes 10a and 10b, the reference electrode conductive layer 21, the counter electrode conductive layer 31, and the wiring 50 are arranged. The portions of the first insulating layer 3 near the conductive layers 11a and 11b of the working electrodes 10a and 10b are the first insulating layers 3a and 3b of the working electrodes 10a and 10b. Portions of the first insulating layers 3a and 3b of the working electrodes 10a and 10b are disposed on the conductive layers 11a and 11b, covering the upper surfaces of the conductive layers 11a and 11b. The first insulating layers 3a and 3b of the working electrodes 10a and 10b have first openings 3a1 and 3b1 of the working electrodes 10a and 10b at positions overlapping portions of the conductive layers 11a and 11b of the working electrodes 10a and 10b. The first insulating layer 3 further has a reference electrode first opening 301 at a position overlapping a part of the reference electrode conductive layer 21 , and a counter electrode first opening 302 at a position overlapping a part of the counter electrode conductive layer 31 .

[0096] Next, as shown in FIG. 3 , 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 and 11b of the working electrodes 10a and 10b constitute the second insulating layers 4a and 4b of the working electrodes 10a and 10b. The second insulating layers 4a and 4b of the working electrodes 10a and 10b have second openings 4a1 and 4b1 formed at positions overlapping with portions 3a3 and 3b3 of the first insulating layers 3a and 3b of the working electrodes 10a and 10b that entirely encompass the first openings 3a1 and 3b1. The second insulating layer 4 further has a reference electrode second opening 401 at a position overlapping the reference electrode first opening 301 of the first insulating layer 3, and a counter electrode second opening 402 at a position overlapping the counter electrode first opening 302 of the first insulating layer 3. Preferably, the second insulating layer 4 can be formed by applying an ink containing an insulating matrix resin and a compound containing a perfluoroalkyl group (a fluorine-based surface modification additive) in a solvent onto the first insulating layer and drying it.

[0097] 6 shows a cross section along line A-A' of a portion of the substrate 2 on which the conductive layers 11a and 11b of the working electrodes 10a and 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, the portion corresponding to the first working electrode 10a, as shown in FIG. 6. As shown in FIG. 6, 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.

[0098] Next, as shown in Fig. 7, a droplet of liquid composition A containing a reagent involved in a redox reaction in water is formed in the first opening 3a1 of the first insulating layer 3a of the working electrode 10a. At this time, because the surface 3a2 of the first insulating layer 3a of the working electrode 10a is water-repellent, the droplet of liquid composition A is held without collapsing until the interior angle θa (shown in Fig. 7) at the point of contact with the surface 3a2 of the first insulating layer 3a reaches the water contact angle of the surface 3a2 of the first insulating layer 3a.

[0099] Droplets of liquid composition A are formed in the first opening 3a1 of the first insulating layer 3a of the working electrode 10a in this manner, and then dried to form a reagent layer 15a as shown in FIG. 8 . As described above, droplets of liquid composition A containing a reagent involved in a redox reaction in water are likely to be retained without collapsing within the first opening 3a1 of the first insulating layer 3a, which has a water-repellent surface 3a2. This makes it easy to maintain the properties of the reagent layer 15a after drying (e.g., the thickness of the reagent layer 15a, the amount of reagent constituting the reagent layer 15a) within the designed range. If the surface of the first insulating layer 3a were not water-repellent, when droplets of liquid composition A were formed in the first opening 3a1 of the first insulating layer 3a, the droplets would likely collapse and the liquid composition A would likely wet the outside of the first opening 3a1 of the first insulating layer 3a. This makes it difficult to adjust the properties of the reagent layer obtained by drying within the designed range.

[0100] A preferred embodiment of the composition of the liquid composition A containing a reagent involved in an oxidation-reduction reaction in water is as described in the <Materials> section.

[0101] After the reagent layer 15a is formed, a droplet of liquid composition B containing a protective film component in alcohol is formed in the second opening 4a1 of the second insulating layer 4a of the working electrode 10a, as shown in Fig. 9. At this time, because the surface 4a2 of the second insulating layer 4a is liquid-repellent to alcohol, the droplet of liquid composition B is held without collapsing until the interior angle θb (shown in Fig. 9) at the point of contact with the surface 4a2 of the second insulating layer 4a reaches the contact angle of the surface 4a2 of the second insulating layer 4a with the alcohol.

[0102] After droplets of liquid composition B are formed in the second opening 4a1 of the second insulating layer 4a of the working electrode 10a in this manner, they are dried to form a protective film 16a, as shown in FIG. 10 . As described above, droplets of liquid composition B containing protective film components in alcohol are likely to be retained without collapsing within the second opening 4a1 of the second insulating layer 4a, which has a surface 4a2 that is liquid-repellent to alcohol. This makes it easy to maintain the properties of the protective film 16a after drying (e.g., the thickness of the protective film 16a and the amount of components constituting the protective film 16a) within the designed range. If the surface of the second insulating layer 4a were not liquid-repellent to alcohol, when droplets of liquid composition B were formed within the second opening 4a1 of the second insulating layer 4a, the droplets would likely collapse and the liquid composition B would likely wet the outside of the second opening 4a1 of the second insulating layer 4a, making it difficult to adjust the properties of the protective film obtained upon drying within the designed range (see the comparative example described below with reference to FIGS. 17 to 20 ).

[0103] A preferred embodiment of the composition of the liquid composition B containing the protective film component in alcohol is as described in the <Materials> section.

[0104] 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 stages. In this embodiment of the method for manufacturing the sensor 1, after the reagent layer 15a is formed, as shown in FIG. 9, droplets of a liquid composition B containing protective film components in alcohol are formed in the second opening 4a1 of the second insulating layer 4a and then dried to form a portion of the protective film 16a in the thickness direction as shown in FIG. 10. Subsequently, as shown in FIG. 11, further droplets of the liquid composition B are formed in the second opening 4a1 of the second insulating layer 4a and then dried to form the entire protective film 16a as shown in FIG. 12. However, the present invention is not limited to this example, and the protective film may be formed in one stage or in three or more stages.

[0105] As described above, in a preferred embodiment, the sensor 1 includes a plurality of working electrodes 10a, 10b, and the first reagent contained in the first reagent layer 15a of the first working electrode 10a among the plurality of working electrodes 10a, 10b is different from the second reagent contained in the second reagent layer 15b of the second working electrode 10b different from the first working electrode 10a. The manufacturing method of sensor 1 according to this preferred embodiment preferably includes, as a step of forming a reagent layer, the step of forming first reagent layer 15a of first working electrode 10a included in the plurality of working electrodes 10a, 10b by forming droplets of a first liquid composition A containing a first reagent in water within first opening 3a1 of first insulating layer 3a of first working electrode 10a, followed by drying (see FIGS. 6 to 8 ), and forming second reagent layer 15b of second working electrode 10b, different from first working electrode 10a included in the plurality of working electrodes 10a, 10b, by forming droplets of a second liquid composition A containing a second reagent different from the first reagent in water within first opening 3b1 of first insulating layer 3b of second working electrode 10b, followed by drying (steps similar to those shown in FIGS. 6 to 8 , although not shown).

[0106] In the manufacturing method of the sensor 1 according to this embodiment, the reagent layers 15a, 15b of the multiple working electrodes 10a, 10b can be formed individually and independently. Therefore, by adjusting the composition of the liquid composition A containing a reagent in water, it is easy to form the reagent layers 15a, 15b containing different reagents on the multiple working electrodes 10a, 10b.

[0107] In a sensor 1 containing different reagents on multiple working electrodes 10a, 10b manufactured by the method according to the preferred embodiment described above, the first reagent layer 15a on the first working electrode 10a contains a first reagent containing, for example, glucose dehydrogenase or glucose oxidase. In this embodiment, the first working electrode 10a includes a first protective film 16a containing a polymer compound containing 4-vinylpyridine as a structural unit, as shown in FIG. 12 . Formation of such a first protective film 16a may include, after forming the first reagent layer 15a on the first working electrode 10a, forming droplets of a first liquid composition B containing a polymer compound containing 4-vinylpyridine as a structural unit in alcohol within the second opening 4a1 of the second insulating layer 4a of the first working electrode 10a, followed by drying to form the first protective film 16a. Formation of the first protective film 16a may be performed in two stages, as shown in FIGS. 9 to 12 , or in one stage or three or more stages (not shown).

[0108] In the sensor 1 manufactured by the method according to the preferred embodiment described above, in which the working electrodes 10a, 10b contain different reagents, the second reagent layer 15b of the second working electrode 10b contains a second reagent containing, for example, lactate oxidase or lactate dehydrogenase. In this embodiment, the protective film 16b of the second working electrode 10b includes, as shown in FIG. 16 , a second protective film 16ba disposed on the second reagent layer 15b and containing a polymer compound having a cation exchange functional group, and a third protective film 16bb disposed on the second protective film 16ba and containing a polymer compound having 4-vinylpyridine as a structural unit. Formation of protective film 16b including second protective film 16ba and third protective film 16bb can include the following steps: after forming second reagent layer 15b on second working electrode 10b, forming droplets of second liquid composition B containing a polymer compound including the cation exchange functional group in alcohol in second opening 4b1 of second insulating layer 4b of second working electrode 10b, and then drying to form second protective film 16ba (see FIGS. 13 and 14); and after forming second protective film 16ba, forming droplets of third liquid composition B containing a polymer compound including 4-vinylpyridine as a structural unit in alcohol in second opening 4b1 of second insulating layer 4b of second working electrode 10b, and then drying to form third protective film 16bb (see FIGS. 15 and 16).

[0109] In the method according to the present embodiment, the protective films 16a, 16b for the multiple working electrodes 10a, 10b can be formed individually and independently. Therefore, by adjusting the composition of the liquid composition B containing protective film components in alcohol, it is easy to form protective films 16a, 16b of different compositions optimized according to the reagent layers 15a, 15b on the multiple working electrodes 10a, 10b, each containing a different reagent.

[0110] The method according to this embodiment may include a step of forming the reference electrode 20. The step of forming the reference electrode 20 may include: in the substrate 2 on which the reference electrode conductive layer 21, the first insulating layer 3, and the second insulating layer 4 are arranged as shown in Fig. 3 , arranging a silver / silver chloride layer 22 on the reference electrode conductive layer 21 in the reference electrode first opening 301 of the first insulating layer 3 (Fig. 4); and, after arranging the silver / silver chloride layer 22, arranging a reference electrode protective film 23 on the silver / silver chloride layer 22 in the reference electrode second opening 401 of the second insulating layer 4 of the substrate 2 (Figs. 5 and 22).

[0111] <Example of the First Disclosure> As an example of the first disclosure, a sensor 1 having the configuration shown in Fig. 5 was manufactured. The materials used for the sensor 1 having the configuration shown in Fig. 5 and the manufacturing method thereof will be outlined below.

[0112] (Substrate) As the insulating substrate 2, a substrate made of polyethylene terephthalate and having a thickness of 188 μm, having the shape shown in FIG. 1 etc., was used.

[0113] (Conductive Layer) A carbon paste was applied to the first surface 2 a of the insulating substrate 2 and heated at 140° C. for 1 hour, thereby forming the conductive layers 11 a and 11 b of the working electrodes 10 a and 10 b, the reference electrode conductive layer 21, and the counter electrode conductive layer 31, as well as the wiring 50 electrically connected to each of them, each having the shape illustrated in FIG. 1 , from a 5 μm-thick carbon conductive layer.

[0114] (First insulating layer) Subsequently, a first insulating layer 3 was laminated on the first surface 2a of the substrate 2 on which the carbon conductive layer was disposed, the first insulating layer 3 being made of a fluororesin containing a copolymer containing vinylidene fluoride and hexafluoropropylene, the first insulating layer 3 covering the first surface 2a of the substrate 2 and the carbon conductive layer, and having a thickness of 5 μm on the carbon conductive layer, as shown in Fig. 2. The first insulating layer 3 was formed by applying a composition that forms the fluororesin upon curing to the first surface 2a of the substrate 2 on which the carbon conductive layer was disposed, and heating it at 140°C for 1 hour.

[0115] The first opening 3a1 of the first insulating layer 3a of the first working electrode 10a was a circle with a diameter of 1.2 mm. The first opening 3b1 of the first insulating layer 3b of the second working electrode 10b was a circle 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 dimensions of 1.8 mm x 2.1 mm.

[0116] The water contact angle of the surface of the first insulating layer 3 (including surfaces 3a2 and 3b2 of the first insulating layers 3a and 3b of the working electrodes 10a and 10b) was 134.2°. The water contact angle was measured using a handy contact angle and surface free energy analyzer MSA manufactured by KRUSS GmbH by discharging a 2 μL droplet of water onto the surface to be measured at 20° C. and measuring the contact angle between the droplet and the surface after 2 seconds.

[0117] (Second insulating layer) Subsequently, as shown in FIG. 3, a composition containing a polyester resin and a fluorine-based surface-modifying additive containing a perfluoroalkyl group in a solvent was applied onto the first insulating layer 3, and the composition was heated at 140° C. for 1 hour to laminate a second insulating layer 4 having a thickness of 40 μm.

[0118] The second opening 4a1 in the second insulating layer 4a of the first working electrode 10a and the second opening 4b1 in the second insulating layer 4b of the second working electrode 10b were each a circle with a diameter of 2 mm. The reference electrode second opening 401 was a circle with a diameter of 2 mm, and the counter electrode second opening 402 was a rectangle measuring 1.8 mm × 2.1 mm.

[0119] The contact angle of ethanol on the surface of the second insulating layer 4 (including surfaces 4a2 and 4b2 of the second insulating layers 4a and 4b of the working electrodes 10a and 10b) was 60.7°. The contact angle with ethanol was measured using a handy contact angle and surface free energy analyzer MSA manufactured by KRUSS GmbH by discharging a 2 μL droplet of ethanol onto the surface to be measured at 20° C. and measuring the contact angle between the droplet and the surface after 2 seconds.

[0120] (First Working Electrode (for Glucose Measurement) Reagent Layer) A 0.4 μL droplet of a first liquid composition A containing a sodium phosphate buffer solution (pH 7.4), a carbon black dispersion, a polymer-bound mediator, glucose oxidase, and a crosslinking agent in water was formed in the first opening 3 a 1 of the first insulating layer 3 a of the first working electrode 10 a, and then dried to form a first reagent layer 15 a containing glucose oxidase and the mediator.

[0121] At this time, the droplets of the first liquid composition A did not overflow from the first opening 3a1, and the first reagent layer 15a after drying was formed only within the first opening 3a1 (see FIGS. 7 and 8).

[0122] (Second Working Electrode (for Lactic Acid Measurement) Reagent Layer) A 0.6 μL droplet of a second liquid composition A containing a carbon black dispersion, hydroxypropyl cellulose, a polymer-bound mediator, lactate oxidase, polyimidazole, poly-L-lysine, and a crosslinking agent in water was formed in the first opening 3 b 1 of the first insulating layer 3 b of the second working electrode 10 b, and dried to form a second reagent layer 15 b containing lactate oxidase and the mediator.

[0123] At this time, the droplets of second liquid composition A did not overflow from first opening 3b1, and second reagent layer 15b after drying was formed only within first opening 3b1 (see FIGS. 7 and 8).

[0124] (Protective Film for First Working Electrode (for Glucose Measurement)) The following reagents were mixed with ethanol to the following final concentrations and reacted for about 1 hour to prepare a first liquid composition B (P4VP-tBuMA polymer dispersion). P4VP-tBuMA (poly-4-vinylpyridine Mn: 74,000, poly-tert-butyl methacrylate Mn: 87,000, Mw / Mn: 1.16, manufactured by NARD), final concentration 3.55 wt % Tripropylene glycol methyl ether methacrylate-styrene-4-vinylpyridine random copolymer (manufactured by NARD), final concentration 4.45 wt % PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ∼1,000, manufactured by Sigma-Aldrich), final concentration 7 mM A 0.65 μL droplet of the first liquid composition B was formed in the second opening 4 a 1 of the second insulating layer 4 a of the first working electrode 10 a and dried, and then another 0.65 μL droplet of the first liquid composition B was formed and dried to form a first protective film 16 a (see FIG. 12 ).

[0125] At this time, the droplets of the first liquid composition B did not overflow from the second opening 4a1, and after drying, the first protective film 16a was formed only within the second opening 4a1 (see FIGS. 9, 10, 11 and 12).

[0126] (Second Working Electrode (for Lactic Acid Measurement) Protective Film) 23,093.67 mg of a 21.5 wt% Nafion® dispersion (Sigma-Aldrich Corporation) was mixed with 6,472.79 mg of ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) and 1,226.46 mg of a 5 mol / L aqueous sodium hydroxide solution (Fujifilm Wako Pure Chemical Industries, Ltd.) to adjust the pH of the dispersion (neutralization of the cation exchange groups). The precipitate was dissolved using a vortex mixer to prepare 30,792.92 mg of a 16.12 wt% Nafion® dispersion. The resulting 16.12 wt% Nafion® dispersion was designated as second liquid composition B. This second liquid composition B contains Nafion® in a solvent containing a lower alcohol.

[0127] A 0.60 μL droplet 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 FIG. 14).

[0128] At this time, the droplets of the second liquid composition B did not overflow from the second opening 4b1, and after drying, the second protective film 16ba was formed only within the second opening 4b1 (see FIGS. 13 and 14).

[0129] Subsequently, the same composition as the first liquid composition B (P4VP-tBuMA polymer dispersion) was used in the following process as a third liquid composition B. That is, a 0.65 μL droplet of the third liquid composition B was formed in the second opening 4 b 1 of the second insulating layer 4 b of the second working electrode 10 b on which the second protective film 16 ba was formed, and then dried to form a third protective film 16 bb (see FIG. 16 ).

[0130] At this time, the droplets of the third liquid composition B did not overflow from the second opening 4b1, and after drying, the third protective film 16bb was formed only within the second opening 4b1 (see FIGS. 15 and 16).

[0131] (Reference Electrode) A silver / silver chloride paste was applied to the reference electrode conductive layer 21 in the first reference electrode opening 301 of the first insulating layer 3 and heated at 140° C. for 1 hour to form a silver / silver chloride layer 22 .

[0132] Subsequently, a reference electrode protective film 23 was disposed on the silver / silver chloride layer 22 in the reference electrode second opening 401 of the second insulating layer 4 to form a reference electrode 20 (FIG. 22).

[0133] Comparative Example A comparative sensor was fabricated in the same manner as in Sensor 1 of the above-described Example, except that the second insulating layer was fabricated in the following manner.

[0134] A composition containing the same polyester resin as in the example in a solvent but not containing a fluorine-based surface-modifying additive was applied onto the first insulating layer 3, and heated at 140°C for 1 hour to form a second insulating layer having a thickness of 38 μm.

[0135] The contact angle of the surface of the second insulating layer of the comparative sensor thus obtained with ethanol was 11.2°.

[0136] The following problem occurred when forming the protective films on the first and second working electrodes of the sensor of the comparative example.

[0137] A 0.65 μL droplet of the first liquid composition B (P4VP-tBuMA polymer dispersion) used in the Examples was formed in the second opening 4a1 of the second insulating layer 4a of the first working electrode 10a of the comparative sensor. As shown in FIG. 17, the droplet did not remain in the second opening 4a1, but rather spread to the upper surface of the second insulating layer 4a. When this droplet was dried, a portion of the first protective film 16a was formed not only in the second opening 4a1 but also on the upper surface of the second insulating layer 4a, as shown in FIG. 18. After drying, another 0.65 μL droplet of the first liquid composition B was formed. As shown in FIG. 19, the first liquid composition B spread to the upper surface of the second insulating layer 4a. When this was dried, a portion of the first protective film 16a was formed not only in the second opening 4a1 but also on the upper surface of the second insulating layer 4a.

[0138] Although not shown, when droplets of the second liquid composition B (Nafion (registered trademark) dispersion) used in the Examples were formed in the second opening 4b1 of the second insulating layer 4b of the second working electrode 10b of the sensor of the Comparative Example and then dried, a second protective film 16ba was formed irregularly not only in the second opening 4b1 but also on the upper surface of the second insulating layer 4b. Furthermore, when droplets of the third liquid composition B (P4VP-tBuMA polymer dispersion) used in the Examples were formed and then dried, a third protective film 16bb was formed irregularly not only in the second opening 4b1 but also on the upper surface of the second insulating layer 4b.

[0139] <Glucose and Lactic Acid Measurement Experiment> (Culture Solution) To a solution of RPMI-1640 Medium (R1383, manufactured by Sigma-Aldrich), MES (2-morpholinoethanesulfonic acid monohydrate) (manufactured by Dojindo Laboratories) and MOPS (3-morpholinopropanesulfonic acid) (manufactured by Dojindo Laboratories) were added as buffer components to a final concentration of 25 mM, and the glucose concentration was further adjusted to 30 mM, the lactic acid concentration to 15 mM, and the pH to 7.4 to prepare a culture solution.

[0140] (Current Measurement) The sensor 1 of the example and the sensor of the comparative example were immersed in the culture solution, and a voltage of 100 mV was applied to the first working electrode and the second working electrode relative to the reference electrode, and the current between the first working electrode and the counter electrode and the current between the second working electrode and the counter electrode were measured for about 11 days. The measurement was performed with N=2.

[0141] The measurement results of the current value at the first working electrode (for measuring glucose) of sensor 1 of the example are shown in Figure 25A. The measurement results of the current value at the first working electrode (for measuring glucose) of the sensor of the comparative example are shown in Figure 25B. The current value at sensor 1 of the example was stable, whereas the current value at the sensor of the comparative example decreased over time. It was presumed that the sensor of the comparative example had high glucose permeability from the beginning and that the reagent containing glucose oxidase leaked out because it was difficult to control the thickness of the first protective film.

[0142] Figure 26A shows the measurement results of the current value at the second working electrode (for measuring lactate) of sensor 1 of the example. Figure 26B shows the measurement results of the current value at the second working electrode (for measuring lactate) of the sensor of the comparative example. The current value at sensor 1 of the example was stable, whereas the current value at the sensor of the comparative example decreased over time. It was presumed that the sensor of the comparative example had high lactate permeability from the beginning and that the reagent containing lactate oxidase leaked because it was difficult to control the thickness of the second protective film and the third protective film.

[0143] <Embodiments of the Second Disclosure> Hereinafter, embodiments of a sensor and a sensor unit according to the second disclosure will be described. In the present embodiments, unnecessary detailed explanations may be omitted. For example, detailed explanations of already well-known matters and redundant explanations of substantially identical configurations may be omitted. This is to avoid unnecessary redundancy in the following explanation and to facilitate understanding by those skilled in the art.

[0144] Furthermore, the applicant provides the accompanying drawings and the following description so that those skilled in the art can fully understand the second disclosure, and does not intend for them to limit the matters described in the "Additional Notes Regarding the Second Disclosure" below.

[0145] <Materials> Examples of materials that can be used for the sensor according to the second disclosure of this specification will be described. 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 insulating substrate of the sensor according to the first disclosure of this specification can be used.

[0146] The conductive layers provided on the working electrode, reference electrode, and counter electrode of the sensor of the second disclosure of this specification are layers containing a conductive material such as carbon, gold, platinum, or palladium. The conductive layers can be manufactured by forming a layer of such a conductive material on the surface of a substrate using a sputtering method, a vapor deposition method, a screen printing method, or the like. If necessary, the conductive layer can be processed into a predetermined pattern using a laser trimming method.

[0147] The wiring of the sensor disclosed in the second specification can also be made of the same conductive material as the conductive layer.

[0148] The sensor of the second disclosure of this specification is used to detect a predetermined analyte in a liquid sample by immersion in the liquid sample. The liquid sample preferably contains water as a solvent. Examples of the liquid sample include a cell culture medium and a liquid sample prepared using blood obtained from a living body. Examples of the analyte are as described with respect to the analyte detected by the sensor of the first disclosure of this specification.

[0149] The working electrode of the sensor of 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 involved in the redox reaction of the test substance, and may be appropriately selected depending on the test substance. The reagent involved in the redox reaction may include a combination of an oxidoreductase and a mediator (electron carrier), or an oxidoreductase. The oxidoreductase may include a coenzyme.

[0150] The oxidoreductase may be an oxidase or a dehydrogenase. Specific examples of the oxidoreductase are as described in relation to the sensor of the first disclosure of this specification.

[0151] Furthermore, the mediator is not particularly limited, and examples thereof are as described in relation to the sensor of the first disclosure of this specification.

[0152] The reagent layer provided on the working electrode of the sensor disclosed in the second disclosure of this specification may further contain, in addition to the reagent, components such as a buffer, a hydrophilic polymer compound, a conductive carbon filler, a crosslinking agent, etc. Examples of the hydrophilic polymer compound and the conductive carbon filler are as described for the sensor disclosed in the first disclosure of this specification.

[0153] The protective film provided on the working electrode of the sensor of the second disclosure of this specification can be a film that prevents or suppresses leakage of the reagent contained in the reagent layer outside the protective film and is permeable to the analyte present outside the protective film. A 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 and a polymer compound containing a cation-exchange functional group.

[0154] 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 in relation to the sensor disclosed in the first aspect of this specification.

[0155] The protective film provided on the working electrode of the sensor of the second disclosure of this specification can be a laminate of two or more protective films, examples of which are as described for the sensor of the first disclosure of this specification.

[0156] Another suitable example of the protective film provided on the working electrode of the sensor of the second disclosure of this specification is a protective film containing a polymer compound containing 4-vinylpyridine as a constituent unit.

[0157] The reference electrode of the sensor disclosed in the second specification may be provided with a protective film. The protective film of the reference electrode may be made of the materials described above for the protective film of the working electrode.

[0158] <Outline of Sensor 1101> A sensor 1101 according to an embodiment of the second disclosure will be described with reference to the drawings. As shown in FIG. 31 and its cross-sectional views in FIGS. 33, 34, 35, and 36, the sensor 1101 of this embodiment comprises: an insulating substrate 1200 including a tip portion 1201 to be immersed in a liquid sample; a detection electrode 1300 disposed near the tip portion 1201 on a first surface 1202 of the substrate 1200 and including a working electrode 1310, a reference electrode 1320, and a counter electrode 1330; a wiring 1005 disposed 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 so as to cover at least a part of the detection electrode 1300 and the wiring 1005; a tip opening 1061 formed on the insulating layer 1050, communicating with an upper surface 1331 of the counter electrode 1330 and opening to the tip portion 1201 side of the substrate 1200; A channel 1062 formed in the insulating layer 1050 connects the tip opening 1061 and the upper surface 1331 of the counter electrode 1330 and guides the liquid sample from the tip opening 1061 to the upper surface 1331 of the counter electrode 1330 .

[0159] The sensor 1101 of this embodiment includes two working electrodes 1310, but in another embodiment not shown, the number of working electrodes may be only one, or may be three or more.

[0160] 39 and 43, the sensor 1101 is immersed in a liquid sample X1 and used to detect a predetermined test substance in the liquid sample X1. Specific examples of the liquid sample and the test substance are as described in the <Materials> section.

[0161] 33 , the working electrode 1310 of the sensor 1101 includes a working electrode conductive layer 1311 and a reagent layer 1315 that contains a reagent involved in the redox reaction of the test substance in the liquid sample X1 and is disposed on the working electrode conductive layer 1311. Preferred embodiments of the reagent involved in the redox reaction are as described in the <Materials> section.

[0162] When the reagent layer 1315 of the working electrode 1310 contains a reagent that oxidizes the analyte in the liquid sample X1, electrons are transferred from the analyte to the working electrode 1310 under conditions in which a predetermined voltage is applied to the detection electrode 1300 of the sensor 1101. Similarly, when the reagent layer 1315 contains a reagent that reduces the analyte in the liquid sample X1, electrons are transferred from the working electrode 1310 to the analyte. Because the amount of transferred electrons depends on the concentration of the analyte, the concentration or change in concentration of the analyte in the liquid sample X1 can be measured based on the value of the current or a change in the current flowing through the working electrode 1310 of the sensor 1101.

[0163] An example of an analytical device 1100 for analyzing a test substance in a liquid sample, which includes a sensor 1101, will be described with reference to FIG.

[0164] 48 , the analysis device 1100 includes a sensor 1101, an analysis unit 1102, and a control unit 1104. The detection electrodes 1300, including a working electrode 1310, a reference electrode 1320, and a counter electrode 1330 of the sensor 1101, are each connected to the analysis unit 1102 via wiring 1005. The analysis unit 1102 is capable of communicating with the control unit 1104.

[0165] The analysis unit 1102 includes an electrochemical measurement unit 1111 , a control unit 1112 , a storage unit 1113 , and a communication unit 1114 .

[0166] The electrochemical measurement unit 1111 is a potentiostat that applies a predetermined voltage to the detection electrode 1300 of the sensor 1101 to measure the concentration of the test substance, and includes a voltage application unit 1111a and a current measurement unit 1111b, and preferably further includes a voltage measurement unit (counter electrode terminal voltage measurement unit) 1111c.

[0167] 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.

[0168] The current measuring unit 1111b detects the value or change in the current flowing between the working electrode 1310 and the counter electrode 1330 of the sensor 1101, measured while a voltage is applied from the voltage applying unit 1111a to the detection electrode 1300 of the sensor 1101. As described above, the current value or change in the current value detected by the current measuring unit 1111b is an index of the concentration or change in concentration of the test substance in the liquid sample X1.

[0169] The voltage measurement unit 1111 c measures the terminal voltage of the counter electrode 1330 of the sensor 1101 .

[0170] The control unit 1112 is connected to the voltage application unit 1111a, the current measurement unit 1111b, the voltage measurement unit 1111c, the storage unit 1113, and the communication unit 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 controls the communication unit 1114 to transmit the measurement results of the current measurement unit 1111b and the voltage measurement unit 1111c to the control unit 1104.

[0171] The memory unit 1113 is connected to the control unit 1112 and stores data such as the value of the applied voltage that is preset for each measurement object, the measurement values ​​in the current measurement unit 1111b and the voltage measurement unit 1111c, and a calibration curve that has been measured in advance.

[0172] The communication section 1114 is controlled by the control section 1112 and transmits data such as the measurement results of the current measurement section 1111b and the voltage measurement section 1111c to the analysis section 1142 of the control unit 1104.

[0173] The control unit 1104 is capable of communicating with the analysis unit 1102 via a communication unit 1114 , and includes a display unit 1141 and an analysis unit 1142 .

[0174] The display unit 1141 displays the results of the analysis in the analysis unit 1142, such as the concentration of the test substance in the liquid sample X1 from the current value detected in the current measurement unit 1111b.

[0175] 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.

[0176] <Structure of Sensor 1101> Next, the structure of the sensor 1101 of this embodiment will be described mainly with reference to FIG. 31 and its cross-sectional views of FIGS.

[0177] The substrate 1200 of the sensor 1101 of this embodiment includes a tip 1201 that is immersed in the liquid sample. The substrate 1200 can be a plate-like body. The tip 1201 is a portion of the periphery of the substrate 1200 that becomes the tip when the sensor 1101 is immersed in the liquid sample X1 (see FIG. 43). Preferred aspects of the substrate 1200 are as described in the <Material> section.

[0178] In the sensor 1101 , a detection electrode 1300 including a working electrode 1310 , a reference electrode 1320 and a counter electrode 1330 is disposed near a tip 1201 on a first surface 1202 of a substrate 1200 .

[0179] The wiring 1005 of the sensor 1101 is disposed on the first surface 1202 of the substrate 1200, and is electrically connected to each electrode (working electrode 1310, reference electrode 1320, and counter electrode 1330) of the detection electrode 1300. Preferred embodiments of the material of the wiring 1005 are as described in the <Material> column.

[0180] The insulating layer 1050 of the sensor 1101 is disposed on the first surface 1202 of the substrate 1200 and is formed so as to cover at least a portion of the detection electrodes 1300 and the wiring 1005 .

[0181] In the illustrated example, 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. However, in another embodiment not shown, the insulating layer 1050 may consist of only one layer, or may include three or more layers.

[0182] The insulating layer 1050 may be any insulating layer, and the material is not particularly limited, but it may be, for example, an insulating layer containing an insulating cured resin, an insulating layer containing an insulating sheet, or a combination of multiple insulating layers.

[0183] An insulating layer containing an insulating cured resin can be formed by curing a curable resin composition. The curable resin composition can be a composition that polymerizes and / or crosslinks upon irradiation with active energy rays to produce an insulating cured resin. Examples of active energy rays include ultraviolet rays, electron beams, X-rays, infrared rays, and visible light, with ultraviolet rays or electron beams being preferred. A negative resist composition can be used as the curable resin composition. A preferred insulating cured resin that can be formed by curing a curable resin composition is a fluororesin. The fluororesin may be any resin containing a polymer containing fluorine atoms on the carbon backbone and / or side chain, such as a poly(meth)acrylate resin or polyester resin containing fluorine atoms on the carbon backbone and / or side chain. The thickness of the insulating layer containing an insulating cured resin is not particularly limited, but can be, for example, 1 μm or more and 50 μm or less.

[0184] The insulating layer including the insulating sheet may include an insulating sheet and a bonding layer including a pressure-sensitive adhesive or adhesive disposed on one side of the insulating sheet. The insulating layer including the insulating sheet can be formed by attaching the insulating sheet including the bonding layer to the first surface 1202 of the substrate 1200 directly or via another insulating layer. The insulating sheet may be an insulating resin sheet, such as a polyethylene terephthalate sheet. The bonding layer may be, for example, a double-sided tape or a layer of pressure-sensitive adhesive or adhesive. The outer surface of the insulating sheet is preferably a water-repellent surface, and may be a water-repellent surface imparted with water-repellent properties, for example, by a water-repellent coating or a surface roughening treatment. The thickness of the insulating layer including the insulating sheet is not particularly limited, but the thickness of the insulating sheet may be, for example, 10 μm or more and 200 μm or less, and preferably 20 μm or more and 100 μm or less. The thickness of the bonding layer disposed on one side of the insulating sheet may be, for example, 1 μm or more and 50 μm or less, and preferably 5 μm or more and 20 μm or less.

[0185] A preferred embodiment of the insulating layer 1050 includes a first insulating layer 1051 containing an insulating cured resin and disposed on the first surface 1202 of the substrate 1200, and a second insulating layer 1052 containing an insulating sheet and disposed on the first insulating layer 1051. In this embodiment, the second insulating layer 1052 can include an insulating sheet and a bonding layer disposed on the surface of the insulating sheet facing the first insulating layer 1051, and the insulating sheet is preferably attached to the first insulating layer 1051 via the bonding layer. In this embodiment, the thickness of the first insulating layer 1051 containing an insulating cured resin is not particularly limited, but the thickness of the portion covering the wiring 1005 of the detection electrode 1300 and each conductive layer (working electrode conductive layer 1311, reference electrode conductive layer 1321, counter electrode 1330) can be, for example, 0.5 μm or more and 50 μm or less, preferably 2 μm or more and 20 μm or less. Examples of the thickness of the second insulating layer 1052 containing the insulating sheet are as described in the previous paragraph.

[0186] The working electrode 1310 of the sensor 1101 will be described with reference to the drawings, particularly Fig. 33. 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. 31.

[0187] 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. The working electrode 1310 preferably further includes a working electrode protective film 1316 disposed on the reagent layer 1315 as shown. Preferred embodiments of the materials for the working electrode conductive layer 1311, the reagent layer 1315, and the working electrode protective film 1316 are as described in the <Materials> section. Wiring 1005 is connected to the working electrode conductive layer 1311 of the working electrode 1310 (see FIGS. 27 and 31 ).

[0188] The working electrode 1310 includes the working electrode protective film 1316, which provides the following effects: The working electrode protective film 1316 prevents leakage of the reagent from the reagent layer 1315. In addition, the working electrode protective film 1316 is porous, allowing the liquid sample X1 to permeate. Therefore, when the sensor 1101 is immersed in the liquid sample X1, the liquid sample X1 can easily reach the working electrode 1310, and the immersion of the liquid sample X1 into the working electrode 1310 is less likely to be hindered.

[0189] The insulating layer 1050 includes a working electrode opening 1501 that penetrates in the thickness direction T1 and is formed at a position that overlaps at least a portion of the working electrode 1310 in a plan view from the thickness direction T1 of the substrate 1200. In the illustrated example, 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 disposed within the working electrode opening 1501. Disposing the working electrode protective film 1316 within the working electrode opening 1501 more effectively suppresses leakage of the reagent from the reagent layer 1315. The outer peripheral edges of the reagent layer 1315 and the working electrode protective film 1316 are defined by the inner peripheral edge of the working electrode opening 1501. In the illustrated example, the shape of the inner peripheral edge of the working electrode opening 1501 of the insulating layer 1050 in a plan view from the thickness direction T1 is circular, but is not limited to this and may be a polygon (e.g., square, triangle) or any other shape. The opening width (widest width) of the working electrode opening 1501 of the insulating layer 1050 is not particularly limited, but can be, for example, 0.5 mm or more and 10 mm or less, and preferably 1 mm or more and 5 mm or less.

[0190] In the illustrated example, the working electrode opening 1501 of the insulating layer 1050 is composed of 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 overlapping a portion of the working electrode conductive layer 1311 in a plan view from the thickness direction T1 and penetrates the first insulating layer 1051 in the thickness direction T1. The working electrode second opening 1521 is formed in the second insulating layer 1052 at a position overlapping a portion of the first insulating layer 1051 that entirely encompasses the working electrode first opening 1511 in a plan view from the thickness direction T1 and penetrates the second insulating layer 1052 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 periphery defined by the inner periphery of the working electrode first opening 1511. The working electrode protective film 1316 is disposed within the working electrode second opening 1521 of the second insulating layer 1052 and includes an outer periphery defined by the inner periphery of the working electrode second opening 1521. In the illustrated example, the inner peripheries 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 each have a circular shape when viewed from above in the thickness direction T1. However, this is not limited thereto and each may be a polygon (e.g., square, triangle, etc.) or any other shape. The opening width (widest portion) of the working electrode first opening 1511 of the first insulating layer 1051 is not particularly limited and may be, for example, 0.5 mm to 5 mm, preferably 1 mm to 2 mm. The opening width (widest portion) of the working electrode second opening 1521 of the second insulating layer 1052 is not particularly limited and may be, for example, 0.5 mm to 10 mm, preferably 1 mm to 5 mm.

[0191] Next, the reference electrode 1320 of the sensor 1101 will be described with reference to the drawings, particularly Fig. 34. Fig. 34 is a cross-sectional view of a portion including the reference electrode 1320 of the sensor 1101 of Fig. 31, taken along the line KK' of Fig. 31.

[0192] 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. The reference electrode 1320 preferably further includes a reference electrode protective film 1326 disposed on the silver / silver chloride layer 1322 as shown in the figure. Preferred embodiments of the materials for the reference electrode conductive layer 1321 and the reference electrode protective film 1326 are as described in the <Materials> section. A wire 1005 is connected to the reference electrode conductive layer 1321 of the reference electrode 1320 (see FIGS. 27 and 31 ).

[0193] The reference electrode 1320 includes the reference electrode protective film 1326, which provides the following effects. The reference electrode protective film 1326 suppresses leakage of silver from the silver / silver chloride layer 1322. In addition, the reference electrode protective film 1326 is porous, allowing the liquid sample X1 to permeate. Therefore, when the sensor 1101 is immersed in the liquid sample X1, the liquid sample X1 can easily reach the reference electrode 1320, and the immersion of the liquid sample X1 into the reference electrode 1320 is less likely to be hindered.

[0194] 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 in a plan view from the thickness direction T1 of the substrate 1200. In the illustrated example, a portion of the reference electrode conductive layer 1321, the silver / silver chloride layer 1322, and the reference electrode protective film 1326 are disposed 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 peripheries of the silver / silver chloride layer 1322 and the reference electrode protective film 1326 are defined by the inner periphery of the reference electrode opening 1502. In the illustrated example, the shape of the inner periphery of the reference electrode opening 1502 in the insulating layer 1050 in a plan view from the thickness direction T1 is circular, but is not limited thereto and may be a polygon (e.g., square, triangle, etc.) or any other shape. The opening width (widest portion width) of the reference electrode opening 1502 in the insulating layer 1050 is not particularly limited, but can be, for example, 0.5 mm or more and 10 mm or less, and preferably 1 mm or more and 5 mm or less.

[0195] In the illustrated example, the reference electrode opening 1502 of the insulating layer 1050 is composed of a reference electrode first opening 1512 of the first insulating layer 1051 and a reference electrode second opening 1522 of the second insulating layer 1052. The reference electrode first opening 1512 is formed in the first insulating layer 1051 at a position overlapping with a part of the reference electrode conductive layer 1321 in a plan view from the thickness direction T1, and penetrates the first insulating layer 1051 in the thickness direction T1. The reference electrode second opening 1522 is formed in the second insulating layer 1052 at a position overlapping with a part of the first insulating layer 1051 that entirely contains the reference electrode first opening 1512 in a plan view from the thickness direction T1, and penetrates the second insulating layer 1052 in the thickness direction T1. A portion of the reference electrode conductive layer 1321 and the silver / silver chloride layer 1322 are disposed in the reference electrode first opening 1512 of the first insulating layer 1051, and include an outer periphery defined by the inner periphery of the reference electrode first opening 1512. 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 periphery defined by the inner periphery of the reference electrode second opening 1522. The shapes of the inner peripheries 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 when viewed in the thickness direction T1 are each circular in the illustrated example, but are not limited thereto and may each be a polygon (such as a square or a triangle) or any other arbitrary shape. The opening width (width of the widest portion) of the reference electrode first opening 1512 in the first insulating layer 1051 is not particularly limited, but can be, for example, 0.5 mm to 5 mm, and preferably 1 mm to 2 mm. The opening width (width of the widest portion) of the reference electrode second opening 1522 in the second insulating layer 1052 is not particularly limited, but can be, for example, 0.5 mm to 10 mm, and preferably 1 mm to 5 mm.

[0196] Next, the counter electrode 1330 of the sensor 1101 will be described with reference to the drawings, particularly Fig. 35. Fig. 35 is a cross-sectional view of a portion including the counter electrode 1330 of the sensor 1101 of Fig. 31, taken along line LL' in Fig. 31.

[0197] The counter electrode 1330 is disposed on the first surface 1202 of the substrate 1200. In the illustrated embodiment, the counter electrode 1330 is entirely made of a conductive layer. To specifically refer to the conductive layer portion of the counter electrode, it may be referred to as the "counter electrode conductive layer" or the "conductive layer of the counter electrode." Preferred aspects of the material of the counter electrode 1330 are as described in the <Material> column as the material of the conductive layer of the counter electrode. Wiring 1005 is connected to the counter electrode 1330 (see FIGS. 27 and 31).

[0198] In the sensor 1101, the insulating layer 1050 has a counter electrode opening 1503 formed in a position overlapping at least a portion of the counter electrode 1330 in a plan view from the thickness direction T1 of the substrate 1200, the counter electrode opening 1503 penetrating the insulating layer 1050 in the thickness direction T1, and the upper surface 1331 of the counter electrode 1330 is exposed through the counter electrode opening 1503. When the sensor 1101 having this configuration is immersed in a liquid sample X1, the liquid sample X1 can contact the upper surface 1331 of the counter electrode 1330 through the counter electrode opening 1503 of the insulating layer 1050. In the illustrated example, the shape of the inner peripheral edge of the counter electrode opening 1503 of the insulating layer 1050 in a plan view from the thickness direction T1 is rectangular, but is not limited thereto and may be a circle, a polygon other than a rectangle (such as a triangle), or any other shape. The opening width (width of the widest portion) of the counter electrode opening 1503 in the insulating layer 1050 is not particularly limited, but can be, for example, 0.5 mm or more and 10 mm or less, and preferably 1 mm or more and 5 mm or less. In the example shown in the figure, a portion of the upper surface 1331 of the counter electrode 1330 is exposed through the counter electrode opening 1503, but this is not limiting. For example, the counter electrode opening 1503 may have a shape that encompasses 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.

[0199] In the illustrated example, the counter electrode opening 1503 of the insulating layer 1050 is composed of 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 portion of the counter electrode 1330 in a plan view from the thickness direction T1, and penetrates the first insulating layer 1051 in the thickness direction T1. The counter electrode second opening 1523 is formed in the second insulating layer 1052 at a position coinciding with the counter electrode first opening 1513 of the first insulating layer 1051 in a plan view from the thickness direction T1, and penetrates the second insulating layer 1052 in the thickness direction T1. It is not necessary for 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 to coincide with each other; they may at least partially overlap. The shape and opening width of the inner peripheral edge of the counter electrode first opening 1513 in the first insulating layer 1051 and the counter electrode second opening 1523 in the second insulating layer 1052 are not particularly limited, and can be within the ranges described for the counter electrode opening 1503 in the insulating layer 1050, for example.

[0200] Next, the tip opening 1061 and the flow path 1062 of the sensor 1101 will be described with reference to the drawings, particularly Fig. 31 and Fig. 36. Fig. 36 is a cross-sectional view taken along line MM' in Fig. 31 of a portion including the counter electrode 1330, the tip opening 1061, and the flow path 1062 of the sensor 1101 in Fig. 31.

[0201] The tip opening 1061 is formed in the insulating layer 1050, communicates with the upper surface 1331 of the counter electrode 1330, and opens to the side of the tip portion 1201 of the substrate 1200. The channel 1062 is formed in the insulating layer 1050, and connects the tip opening 1061 to the upper surface 1331 of the counter electrode 1330. The channel 1062 guides the liquid sample X1 from the tip opening 1061 to the upper surface 1331 of the counter electrode 1330. For example, 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.

[0202] The functions of the tip opening 1061 and the flow path 1062 of the sensor 1101 of the embodiment shown in Figures 31 and 36 will be described with reference to Figures 39 and 40. Figure 39 is a schematic cross-sectional view, corresponding to Figure 36, of the sensor 1101 of this embodiment, which is immersed in the liquid sample X1 from the tip portion 1201 of the substrate 1200 and includes the tip opening 1061 and the flow path 1062. Figure 40 is a schematic cross-sectional view of a comparative sensor 1150, which 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, immersed in the liquid sample X1.

[0203] 39 , when the sensor 1101 of this embodiment is immersed in the liquid sample X1 so that the tip 1201 of the substrate 1200 is at the bottom, the liquid sample X1 is guided from the tip opening 1061 through the flow path 1062 to the upper surface 1331 of the counter electrode 1330. As a result, the upper surface 1331 of the counter electrode 1330 of the sensor 1101 can be in sufficient contact with the liquid sample X1. This is particularly preferable because the upper surface 1331 of the counter electrode 1330 of the sensor 1101 can be in sufficient contact with the liquid sample X1 even when the liquid sample X1 is not deep enough. Therefore, use of the sensor 1101 of this embodiment improves the measurement accuracy of the analyte in the liquid sample X1.

[0204] 40 , in a comparative sensor 1150 in which the tip opening 1061 and the flow path 1062 are not formed in the insulating layer 1050, the region of the first surface 1202 of the substrate 1200 between the counter electrode 1330 and the tip 1201 is covered with the insulating layer 1050. When the comparative sensor 1150 having this structure is immersed in a liquid sample X1 so that the tip 1201 of the substrate 1200 is the tip, the liquid sample X1 is likely to be repelled away from the counter electrode 1330 due to the liquid repellency of the insulating layer 1050 between the counter electrode 1330 and the tip 1201 of the substrate 1200. Therefore, in the comparative sensor 1150, contact between the counter electrode 1330 and the liquid sample X1 is likely to be hindered, and the analyte in the liquid sample X1 may not be measured normally. In particular, when the counter electrode 1330 is a carbon conductive layer, the upper surface 1331 of the counter electrode 1330 is highly water-repellent, and therefore, the liquid sample X1 containing water as a solvent is particularly likely to be prevented from contacting the upper surface 1331 of the counter electrode 1330. This problem of the sensor 1150 of the comparative example can be solved by the sensor 1101 of the present embodiment, in which the tip opening 1061 and the flow path 1062 are formed in the insulating layer 1050.

[0205] 31 and 36 includes a tip opening 1061 and a flow path 1062 formed in a portion of the insulating layer 1050 between the counter electrode 1330 and the tip portion 1201 of the substrate 1200, when viewed in a plan view from the thickness direction T1 of the substrate 1200. In the sensor 1101 of the present embodiment, 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. This particularly enhances the effect of bringing the upper surface 1331 of the counter electrode 1330 into contact with the liquid sample X1 when the sensor 1101 is immersed in the liquid sample X1.

[0206] 31 and 36 are recesses formed in the insulating layer 1050 that are closed on the side where the substrate 1200 is present and open on the side of the insulating layer 1053, but are not limited to this form. For example, as in a sensor 1101' according to another embodiment shown in Fig. 37, the tip opening 1061 and the channel 1062 may be formed in a part of the thickness direction of the insulating layer 1050 that does not include the top surface 1053, and the tip opening 1061 and the channel 1062 may be surrounded when viewed from the direction from the counter electrode 1330 to the tip 1201 of the substrate 1200 (the M-M' direction in Fig. 31).

[0207] The flow channel 1062 of the sensor 1101 shown in Figures 31 and 36 has an expansion portion 1062A that expands the flow channel width W (see Figure 31) toward the tip opening 1061. The flow channel 1062 has the expansion portion 1062A, which promotes 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. Here, the flow channel width W refers to the width of the flow channel 1062 in a direction (LL' direction in Figure 31) perpendicular to the direction from the counter electrode 1330 to the tip 1201 of the substrate 1200 (MM' direction in Figure 31) when viewed from above in the thickness direction T1 of the substrate 1200.

[0208] In the sensor 1101 shown in FIGS. 31 and 36 , 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, with the tip opening 1061 and the flow path 1062 formed in the second insulating layer 1052. In a particularly preferred aspect of the sensor 1101 of this embodiment, the first insulating layer 1051 includes an insulating cured resin, and the second insulating layer 1052 includes an insulating sheet. The first insulating layer 1051 including the 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 it with active energy rays. The second insulating layer 1052 including the insulating sheet can be formed by attaching the insulating sheet to the first insulating layer 1051 including the insulating cured resin via a bonding layer. After forming second insulating layer 1052 including an insulating sheet, a portion of the insulating sheet between counter electrode 1330 and tip portion 1201 of substrate 1200 is peeled off, thereby forming tip opening 1061 and flow channel 1062 in second insulating layer 1052. Alternatively, second insulating layer 1052 including an insulating sheet in which tip opening 1061 and flow channel 1062 are formed can be formed by attaching an insulating sheet having a notch pre-cut in the portion between counter electrode 1330 and tip portion 1201 of substrate 1200 via a bonding layer onto first insulating layer 1051 containing an insulating cured resin.

[0209] In a sensor 1101'' of yet another embodiment shown in Figures 32 and 38, a tip opening 1061 and a flow path 1062 are further formed in the first insulating layer 1051 in the sensor 1101 shown in Figures 31 and 36. The sensor 1101'' shown in Figures 32 and 38 is similar to the sensor 1101 shown in Figures 31 and 36, but a tip opening 1061 and a flow path 1062 are further formed in a portion 1515 (see Figure 36) of the first insulating layer 1051 between the counter electrode 1330 and the tip portion 1201 of the substrate 1200. The sensor 1101″ shown in FIGS. 32 and 38 does not include an insulating layer between the counter electrode 1330 on the first surface 1202 of the substrate 1200 and the tip portion 1201. This further promotes contact between the liquid sample X1 and the counter electrode 1330 when the sensor 1101″ is immersed in the liquid sample X1, enabling more accurate detection of the analyte in the liquid sample X1. In a particularly preferred embodiment of the sensor 1101″ shown in FIGS. 32 and 38, the first insulating layer 1051 is a layer containing an insulating cured resin, and the second insulating layer 1052 is a layer containing an insulating sheet. The sensor 1101″ according to this particularly preferred embodiment can be manufactured by manufacturing the sensor 1101 including the insulating layer 1050, which includes the first insulating layer 1051 containing an insulating cured resin and the second insulating layer 1052 containing an insulating sheet, according to the procedure described in the previous paragraph, and then peeling off the portion 1515 (see FIG. 36 ) of the first insulating layer 1051 containing the insulating cured resin. Alternatively, when curing a curable resin composition on the first surface 1202 of the substrate 1200 to form a first insulating layer 1051 containing an insulating cured resin, the first insulating layer 1051 may be formed without the portion 1515, and the second insulating layer 1052 may be formed thereon to manufacture the sensor 1101''.

[0210] <Method for Manufacturing Sensor 1101> An example of a method for manufacturing the sensor 1101 according to the embodiment shown in FIG. 31 of the second disclosure will be described with reference to the drawings, particularly FIGS. 27 to 31.

[0211] First, as shown in FIG. 27 , working electrode conductive layers 1311, 1311, a reference electrode conductive layer 1321, a counter electrode 1330 (counter electrode conductive layer), and wiring 1005 electrically connected to each of them are arranged on a first surface 1202 of an insulating substrate 1200.

[0212] 28 , 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 positions overlapping with parts of the working electrode conductive layers 1311, 1311, a reference electrode first opening 1512 at a position overlapping with part of the reference electrode conductive layer 1321, and a counter electrode first opening 1513 at a position overlapping with part of the counter electrode 1330.

[0213] As described above, in a particularly preferred aspect of sensor 1101 of this embodiment, first insulating layer 1051 contains an insulating curable resin. First insulating layer 1051 containing an insulating curable resin can be formed by applying a curable resin composition to first surface 1202 of substrate 1200 and curing the coating by irradiating the coating with active energy rays.

[0214] 29 , a second insulating layer 1052 is further laminated on the first insulating layer 1051 of the substrate 1200 of Fig. 28 . The second insulating layer 1052 has working electrode second openings 1521, 1521 shaped to entirely enclose the working electrode first openings 1511, 1511 at positions overlapping with the working electrode first openings 1511, 1511 of the first insulating layer 1051, a reference electrode second opening 1522 shaped to entirely enclose the reference electrode first opening 1512 at a position overlapping with the reference electrode first opening 1512, and a counter electrode second opening 1523 shaped to coincide with the counter electrode first opening 1513 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 channel 1062 in a portion between the counter electrode 1330 and the tip portion 1201 of the substrate 1200 when viewed in a plan view in the thickness direction T1 of the substrate 1200.

[0215] As described above, in a particularly preferred aspect of the sensor 1101 of this embodiment, the second insulating layer 1052 includes an insulating sheet. The second insulating layer 1052 including the insulating sheet may further include a bonding layer including a pressure-sensitive adhesive or adhesive disposed on one surface of the insulating sheet. The second insulating layer 1052 including the insulating sheet may be formed by attaching the insulating sheet onto the first insulating layer 1051 via the bonding layer.

[0216] Next, as shown in FIG. 30 , a reagent layer 1315 is placed in the working electrode first openings 1511, 1511 of the first insulating layer 1051, and a silver / silver chloride layer 1322 is placed in the reference electrode first opening 1512 of the first insulating layer 1051.

[0217] 31 , working electrode protective films 1316 are disposed in the working electrode second openings 1521 of the second insulating layer 1052 to form working electrodes 1310, and a reference electrode protective film 1326 is disposed in the reference electrode second opening 1522 of the second insulating layer 1052 to form a reference electrode 1320. Note that the order of forming the layers in FIGS. 30 and 31 is not particularly limited, and they may be formed in a different order.

[0218] Further Embodiments of the Sensor 1101 Further preferred embodiments of the sensor 1101 of the second disclosure will be described with reference to FIGS. In the sensor 1101 according to the embodiment shown in Figure 41, the substrate 1200 includes: a main body 1210 including a tip portion 1201 and a first end 1211 opposite the tip portion 1201, and in which the detection electrode 1300 is arranged; a connection portion 1220 including a second end 1222 connected to the first end 1211 of the main body 1210 and a third end 1223 opposite the second end 1222, and extending from the second end 1222 to the third end 1223; and a base end 1230 including a fourth end 1234 connected to the third end 1223 of the connection portion 1220, and the connection 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.

[0219] 41, 42, 46, and 47 show plan views of the sensor 1101 before the substrate 1200 is bent at the bending portion 1225.

[0220] 41 , the wiring 1005 is preferably arranged on the first surface 1202 of the substrate 1200 from the main body portion 1210 via the connection portion 1220 to the base end portion 1230. As shown in FIG. 41 , the terminals 1006 are arranged on the first surface 1202 of the base end portion 1230, and each of the wirings 1005 preferably electrically connects one of the detection electrodes 1300 on the first surface 1202 of the main body portion 1210 to one of the terminals 1006 on the first surface 1202 of the base end portion 1230.

[0221] The substrate 1200 of the sensor 1101 shown in Fig. 41 is bent at a bending portion 1225 so that the side of the first surface 1202 on which the detection electrodes 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 1222 than the bending portion 1225 protrude from the base end 1230. Preferably, as shown in Figs. 43 and 44 , the main body portion 1210 of the substrate 1200 and a portion of the connecting portion 1220 closer to the second end 1222 than the bending portion 2125 protrude substantially perpendicularly from the base end 1230 of the substrate 1200.

[0222] The sensor 1101 shown in Figure 41, which has a substrate 1200 bent at the bending portion 1225 of the connection portion 1220, can be immersed in a liquid sample X1 for measuring the test substance by orienting the main body portion 1210 of the substrate 1200, on which the detection electrode 1300 is arranged, so that the tip portion 1201 is at the bottom, without tilting the base end portion 1230 of the substrate 1200 (for example, with the base end portion 1230 of the substrate 1200 held horizontally as shown in Figures 43 and 44).

[0223] As shown in Fig. 42, a plurality of sensors 1101 shown in Fig. 41 can be connected and integrated to form a multiple sensor 1105. The multiple sensor 1105 shown in Fig. 42 is formed by integrating the base ends 1230 of the substrates 1200 of four sensors 1101.

[0224] 41 , the insulating layer 1050 includes a second insulating layer 1052 including a first insulating sheet, which is disposed in a region of the first surface 1202 of the substrate 1200, in the main body portion 1210, and in a region closer to the second end 1222 than the bent portion 1225 of the connecting portion 1220. Here, the region closer to the second end 1222 than the bent portion 1225 of the connecting portion 1220 refers to the portion of the connecting portion 1220 from the end of the bent portion 1225 on the second end 1222 side to the second end 1222. In the sensor 1101 shown in FIG. 41 , the insulating layer 1050 further includes a first insulating layer 1051 including an insulating cured resin formed on 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 adhesive, and is attached to the above-mentioned region of the first surface 1202 of the substrate 1200 from the bonding layer side via the first insulating layer 1051. The sensor 1101 shown in Figure 41 is preferable because the portion to be immersed in the liquid sample X1 is integrally covered by the second insulating layer 1052 including the first insulating sheet, thereby achieving the necessary insulation between the detection electrode 1300 and the wiring 1005 and making it easy to form the tip opening 1061 and the flow path 1062 in the first insulating sheet of the second insulating layer 1052.

[0225] 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 connection portion 1220. The effects achieved by this configuration will be described with reference to FIGS.

[0226] 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. As shown in Fig. 44 , in the sensor 1101, the substrate 1200 is bent at the bent portion 1225 of the connecting portion 1220 so that the first surface 1202 side protrudes and the portion of the main body 1210 and the connecting portion 1220 closer to the second end 1222 than the bent portion 1225 is substantially perpendicular to the base end 1230. 41 and 44 , 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 containing a first insulating sheet that covers, via the first insulating layer 1051, a region of the first surface 1202 of the substrate 1200 that is closer to the second end 1222 than the main body 1210 and the bent portion 1225 of the connecting portion 1220. The insulating layer 1050 includes a non-insulating region 1055 that does not contain an insulating sheet and that includes the first insulating layer 1051 containing an insulating cured resin, on the region of the first surface 1202 of the substrate 1200 that is closer to the bent portion 1225 of the connecting portion 1220. In this embodiment, the second insulating layer 1052 containing the first insulating sheet is flat and not deformed. Therefore, substrate 1200 is not affected by the contraction or expansion that occurs after the first insulating sheet is deformed, and the portion of main body 1210 and connecting portion 1220 closer to second end 1222 than bent portion 1225 is maintained in a flat shape. As a result, as shown in Fig. 44, main body 1210 of substrate 1200 of sensor 1101 is maintained in a vertical position with tip 1201 at the bottom in liquid sample X1, allowing detection electrode 1300 to normally measure the test substance.

[0227] On the other hand, Figure 45 shows a comparative sensor 1151 having the same structure as the sensor 1101 shown in Figures 41 and 44 except that a second insulating layer 1052 including a first insulating sheet is formed over the entire first surface 1202 of the substrate 1200, including the region above the folded portion 1225. In the comparative sensor 1151, 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 folded portion 1225. The stretched second insulating layer 1052 including the insulating sheet attempts to contract, causing warping as shown in Figure 45 in the main body portion 1210 and the connection portion 1220 of the substrate 1200. The main body portion 1210 of the warped substrate 1200 is inclined with the side of the first surface 1202 on which the detection electrode 1300 is arranged facing upward. 45, contact between the liquid sample X1 and the detection electrode 1300 on the first surface 1202 of the main body 1210 is hindered, reducing the accuracy of measurement of the analyte by the detection electrode 1300. The sensor 1101 shown in FIGS. 41 and 44 solves this problem of the comparative sensor 1151 by including a non-insulating region 1055 in the insulating layer 1050 in the region above the bent portion 1225 of the first surface 1202 of the substrate 1200.

[0228] Another embodiment of the sensor 1101 shown in Figures 41 and 44 will be described with reference to Figure 46. In the sensor 1101 shown in Figure 46, the insulating layer 1050 includes a second insulating layer 1052a including a first insulating sheet, which is arranged on a region of the first surface 1202 of the substrate 1200 that is closer to the second end 1222 than the main body portion 1210 and the bent portion 1225 of the connecting portion 1220, and a second insulating layer 1052b including a second insulating sheet, which is arranged on a region of the first surface 1202 of the substrate 1200 that is closer to the base end 1230. Here, the first insulating sheet of the second insulating layer 1052a and the second insulating sheet of the second insulating layer 1052b are separated from each other in the region of the first surface 1202 of the substrate 1200 that is closer to the bent portion 1225 of the connecting portion 1220. 46 , 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 and includes the first insulating layer 1051 on a region of the first surface 1202 of the substrate 1200 at the bent portion 1225 of the connection portion 1220. A second insulating layer 1052a including the first insulating sheet and a second insulating layer 1052b including the second insulating sheet are each disposed on the first insulating layer 1051 in the above-mentioned region of the first surface 1202 of the substrate 1200. According to this embodiment, it is possible to suppress a decrease in detection accuracy due to warping of the substrate 1200 of the sensor 1101 and to improve insulation between the wiring 1005 and the terminal 1006 on the base end portion 1230 of the substrate 1200.

[0229] Another embodiment of the sensor 1101 shown in Fig. 46 will be described with reference to Fig. 47. In the sensor 1101 shown in Fig. 47, the first insulating sheet of the second insulating layer 1052a and the second insulating sheet of the second insulating layer 1052b are separated by a notch 1056 formed in a direction intersecting the direction in which the connecting portion 1220 extends, on the region of the bent portion 1225 of the connecting portion 1220 on the first surface 1202 of the substrate 1200. In this embodiment, the insulating layer 1050 includes a second insulating layer 1052c including a plurality of third insulating sheets separated from the second insulating layers 1052a and 1052b by the notch 1056, on the region of the bent portion 1225 of the connecting portion 1220 on the first surface 1202 of the substrate 1200. According to this embodiment, it is possible to suppress a decrease in detection accuracy due to warping of the substrate 1200 of the sensor 1101, and also to improve the insulation between the wirings 1005 on the bent portion 1225 of the connection portion 1220 of the substrate 1200.

[0230] <Sensor Unit 900> The sensor unit 900 will be described with reference to Figures 51, 52, and 53. The illustrated sensor unit 900 is used to position the main body 1210 of the substrate 1200, which includes the detection electrodes 1300 of the sensor 1101, relative to each well of a 24-well plate 925.

[0231] As shown in FIG. 51, the sensor unit 900 includes, arranged in this order from the bottom, a lower support plate 957, six sets of multiple sensors 1105 (see FIG. 42), an upper support plate 959 equipped with a port 961 for supplying additives, and a gasket sheet 960.

[0232] In this embodiment, the multiple sensor 1105 is formed by connecting and integrating four sensors 1101 shown in Fig. 42 at a base end 1230 of a substrate 1200. Each of the four sensors 1101 is bent at a bent portion 1225 of a connecting portion 1220 of the substrate 1200 so that the side of the first surface 1202 of the substrate 1200 protrudes, and the portion of the main body 1210 and the connecting portion 1220 closer to the second end 1222 than the bent portion 1225 is perpendicular to the base end 1230. As shown in Fig. 51 , six sets of multiple sensors 1105 each including four sensors 1101 are attached to the lower support plate 957.

[0233] The lower support plate 957 has through holes 957b at positions corresponding to the main body portions 1210 and connecting portions 1220 of the substrates 1200 of the 24 sensors 1101 included in the six sets of multiple sensors 1105. The main body portions 1210 and connecting portions 1220 of the substrates 1200 of the 24 sensors 1101 are inserted into the through holes 957b of the lower support plate 957. When the sensors 1101 are inserted into the through holes 957b of the lower support plate 957, the upper surface of the lower support plate 957 and the lower surface of the base end portions 1230 of the substrates 1200 of the sensors 1101 are engaged with each other, as shown in FIG. In addition, a first engagement portion 957d is provided on the inner wall of the through hole 957b of the lower support plate 957, at a portion that engages with the inside of the bent portion 1225 of the connection portion 1220 of the substrate 1200 of the sensor 1101, supporting the bent portion 1225 of the connection portion 1220 of the substrate 1200 from below.

[0234] On the other hand, the lower surface of the upper support plate 959 is provided with pin-shaped second engagement portions 959 a that are inserted into the 24 through holes 957 b of the lower support plate 957 .

[0235] When the six multiple sensors 1105 are sandwiched and assembled between the lower support plate 957 and the upper support plate 959, as shown in FIG. 52 , the upper surface of the first engagement portion 957d of the lower support plate 957 and the lower surface of the second engagement portion 959a of the upper support plate 959 engage and support the bent portion 1225 of the connection portion 1220 of the substrate 1200 of the sensor 1101. As shown in FIG. 52 , the first engagement portion 957d of the lower support plate 957 has a curved upper surface that includes a curved surface on the upper surface. The second engagement portion 959a of the upper support plate 959 has a curved lower surface that includes a curved lower surface. With this configuration, the sensor 1101 is supported so that the tip portion 1201 of the substrate 1200 is the lower end, and the portion of the substrate 1200 distal to the bent portion 1225 of the main body portion 1210 and the connection portion 1220 is vertical. As a result, the attitude of the main body 1210 of the substrate 1200 of the sensor 1101 relative to the liquid sample contained in each well of the well plate can be stabilized, and the detection accuracy of the sensor 1101 can be improved.

[0236] Finally, as shown in FIG. 51, a gasket sheet 960 having through holes is placed on the top surface of the upper support plate 959 to complete the sensor unit 900.

[0237] The sensor unit 900 can be combined with other components to form an adapter unit 920. As shown in Fig. 53, the adapter unit 920 has, from the bottom up, an adapter bottom (culture vessel mounting portion) 924, a well plate (culture vessel) 925, an adapter top 926, and the sensor unit 900 mounted in this order. In this embodiment, the well plate 925 has 24 wells in a 4 x 6 arrangement.

[0238] The adapter top 926 is provided to adjust the height of the well plate 925, and a different adapter top 926 is 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.

[0239] There are several types of well plates 925, including general-purpose ones, and different adaptor tops 926 are used depending on the type.

[0240] The sensor unit 900 placed on the adapter top 926 has four legs (supports) 940 on its underside that pass through through holes 941 in the lower adapter top 926 and are inserted into positioning holes 942 in the adapter bottom 924, which serves as a culture vessel installation section.

[0241] In the adapter unit 920 thus formed, which includes the sensor unit 900 and the well plate 925, the main body 1210 of the substrate 1200, which includes the detection electrodes 1300 of the sensor 1101, is held in a stable position within each well of the well plate 925.

[0242] Although not shown, in the adapter unit 920, the terminal 1006 on the base end 1230 of the substrate 1200 of the sensor 1101 included in the sensor unit 900 can be electrically connected to the electrochemical measurement section 1111 (see FIG. 48 ) of the external analysis unit 1102 through the through-hole in the gasket sheet 960 of the sensor unit 900. In other words, the adapter unit 920 equipped with the sensor unit 900 can be combined with the external analysis unit 1102 and control unit 1104 to form the analysis device 1100 shown in FIG. 48 .

[0243] <Examples of the Second Disclosure> The following five sensors were produced as examples of the second disclosure or comparative examples. Note that the sensors S1 to S5 used in the following experiments were not provided with the reagent layers 1315, 1315 and working electrode protective films 1316, 1316 of the working electrodes 1310, 1310, or the reference electrode protective film 1326 of the reference electrode 1320.

[0244] Sensor S1 Sensor S1 is a comparative example of the structure shown in FIG. 49A . In sensor S1, a first insulating layer 1051 and a second insulating layer 1052 including an insulating sheet are laminated over the entire first surface 1202 of substrate 1200. In sensor S1, a tip opening 1061 and a flow path 1062 are not formed, and the portion of first surface 1202 of substrate 1200 between counter electrode 1330 and tip 1201 is also covered with insulating layer 1050 including first insulating layer 1051 and second insulating layer 1052. That is, sensor S1 does not have non-insulating region 1055 of insulating layer 1050 for preventing warping of substrate 1200, nor does it have tip opening 1061 or flow path 1062 for guiding a liquid sample to upper surface 1331 of counter electrode 1330.

[0245] Sensor S2 Sensor S2 is a comparative example of the structure shown in FIG. 49B . Instead of the second insulating layer 1052 covering the entire sensor S1, sensor S2 has a second insulating layer 1052a including a first insulating sheet disposed in a portion distal to the bent portion 1225 of the connection portion 1220 of the substrate 1200, and a second insulating layer 1052b including a second insulating sheet disposed in a base end portion 1230 of the substrate 1200. Furthermore, a non-insulating region 1055 including the first insulating layer 1051 but not an insulating sheet is disposed on the bent portion 1225 of the connection portion 1220 of the substrate 1200. That is, sensor S2 has a non-insulating region 1055 of the insulating layer 1050 for preventing warping of the substrate 1200, but does not have a tip opening 1061 or a flow channel 1062 for guiding a liquid sample to the upper surface 1331 of the counter electrode 1330.

[0246] Sensor S3 is an embodiment of sensor 1101 having the structure shown in Fig. 49C. Similar to sensor 1101 shown in Fig. 46, sensor S3 has a non-insulating region 1055 of insulating layer 1050 to prevent warping of substrate 1200, and a tip opening 1061 and a flow path 1062 (see Fig. 36) formed only in second insulating layer 1052.

[0247] Sensor S4 Sensor S4 is an example of sensor 1101 having the structure shown in FIG. 49D . Sensor S4 differs from sensor S3 in that second insulating layer 1052a including the first insulating sheet and second insulating layer 1052b including the second insulating sheet of insulating layer 1050 are replaced with second insulating layer 1052 including an integrated insulating sheet that covers the entire first surface 1202 of substrate 1200, and a portion 1515 (see FIGS. 36 and 49C ) of first insulating layer 1051 between counter electrode 1330 and tip portion 1201 of substrate 1200 is peeled off to form tip opening 1061 and flow channel 1062. That is, sensor S4 does not have non-insulating region 1055 of insulating layer 1050 to prevent warping of substrate 1200, but has tip opening 1061 and flow channel 1062 (see FIG. 38 ) formed in first insulating layer 1051 and second insulating layer 1052.

[0248] Sensor S5 Sensor S5 is an example of sensor 1101 having the structure shown in Fig. 49E. Sensor S5 is obtained by removing portion 1515 (see Figs. 36 and 49C ) of first insulating layer 1051 between counter electrode 1330 and tip portion 1201 of substrate 1200 in sensor S3 to form tip opening 1061 and flow channel 1062. That is, sensor S5 has non-insulating region 1055 of insulating layer 1050 for preventing warping of substrate 1200, and tip opening 1061 and flow channel 1062 (see Fig. 38 ) formed in first insulating layer 1051 and second insulating layer 1052.

[0249] The materials used in the manufacture of the five sensors are listed below.

[0250] (Substrate) As an insulating substrate, a substrate 1200 made of polyethylene terephthalate and having a thickness of 188 μm, including a main body portion 1210, a connecting portion 1220, and a base end portion 1230, was used, having the shape shown in FIGS. 49A to 49E.

[0251] The main body 1210 of the substrate 1200 was rectangular with a width of 11 mm and a length of 4.2 mm.

[0252] After the insulating layer 1050 is laminated to complete the sensor, the bending portion 1225 of the connection portion 1220 of the substrate 1200 is bent so that it protrudes toward the first surface 1202, and the portion of the main body portion 1210 and the connection portion 1220 closer to the tip than the bending portion 1225 is bent perpendicular to the base end portion 1230.

[0253] (Conductive Layer) On the first surface 1202 of the substrate 1200, working electrode conductive layers 1311, 1311, a reference electrode conductive layer 1321, a counter electrode (counter electrode conductive layer) 1330, a wiring 1005, and a terminal 1006 having the shapes illustrated in FIGS. 27 and 46 were formed using a carbon conductive layer having a thickness of 5 μm obtained by applying and heating a carbon paste.

[0254] (First Insulating Layer) As shown in FIG. 28 , a first insulating layer 1051 made of fluororesin was laminated on the first surface 1202 of the substrate 1200 on which the carbon conductive layer was disposed. The first insulating layer 1051 covered the first surface 1202 of the substrate 1200 and the carbon conductive layer, and had a thickness of 5 μm above the carbon conductive layer. The first insulating layer 1051 was formed by applying a curable resin composition (negative resist composition) that hardens upon exposure to active energy rays to produce an insulating cured resin, and then irradiating the coating with active energy rays. After appropriate masking, working electrode first openings 1511, 1511, reference electrode first opening 1512, and counter electrode first opening 1513 were formed in the first insulating layer 1051.

[0255] The silver / silver chloride layer 1322 was disposed in the reference electrode first opening 1512 of the first insulating layer 1051 .

[0256] (Second insulating layer) Second insulating layer 1052 was disposed in a predetermined region on first surface 1202 of substrate 1200 on which the carbon conductive layer and first insulating layer 1051 were disposed. Second insulating layer 1052 was a laminate of a 50 μm thick polyethylene terephthalate sheet (insulating sheet) with a water-repellent surface and a 10 μm thick bonding layer containing an adhesive, and was attached to a predetermined region on first insulating layer 1051 disposed on first surface 1202 of substrate 1200 via the bonding layer.

[0257] 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 counter electrode second opening 1523 was rectangular 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.

[0258] In the following experiments, sensors S1 to S5 were used which were not provided with the reagent layers 1315, 1315 and working electrode protective films 1316, 1316 of the working electrodes 1310, 1310, and the reference electrode protective film 1326 of the reference electrode 1320.

[0259] 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 μM ascorbic acid (hereinafter referred to as "liquid sample X1") was added to each well of the 24-well plate.

[0260] As shown in Figures 43 to 45, sensors S1 to S5 were held horizontally with the base end 1230 of the substrate 1200 facing upward, and the main body 1210 of the substrate 1200 was immersed in each amount of liquid sample X1 in the well with the tip end 1201 at the bottom.

[0261] The voltage difference between the working electrodes 1310, 1310 (excluding the reagent layer and protective film) of each of the sensors S1 to S5 and the reference electrode 1320 (excluding the protective film) was controlled to 500 mV by a potentiostat, and the current values ​​between each of the two working electrodes 1310, 1310 and the counter electrode 1330 were measured over time up to 0.7 hours. In addition, the voltage of the counter electrode 1330 was measured over time to confirm whether the liquid sample X1 was in contact with the counter electrode 1330.

[0262] In order to promote contact between each of the detection electrodes 1300 and the liquid sample X1, the measurement was performed while vibrating the 24-well plate after a certain time had elapsed since the start of the measurement.

[0263] The measurement results of the current values ​​of the two working electrodes 1310, 1310 when each of the four volumes of liquid sample was used for each of sensors S1 to S5 are shown in Fig. 50. 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, and Fig. 50E shows the results for sensor S5.

[0264] In the comparative example sensor S1 (see FIG. 49A ), when no vibration was applied, the current value at the working electrode bottomed out in all volumes of liquid sample X1, and normal current values ​​were not measured. When vibration was applied, normal current values ​​were measured at the working electrode in 1.2 mL, 1.0 mL, and 0.94 mL of liquid sample X1, but the current value at the working electrode bottomed out in 0.85 mL of liquid sample X1, and normal measurement was not possible.

[0265] In the comparative example, sensor S2 (see FIG. 49B ), when no vibration was applied, the current value at the working electrode bottomed out and normal current values ​​were not measured in 1.2 mL, 1.0 mL, and 0.85 mL of liquid sample X1, but normal current values ​​were measured at the working electrode in 0.94 mL of liquid sample X1. When vibration was applied, normal current values ​​were measured at the working electrode in all volumes of liquid sample X1.

[0266] In the sensor S3 of the example (see FIG. 49C), normal current values ​​were measured at the working electrode in all volumes of the liquid sample X1 both when vibration was applied and when vibration was not applied.

[0267] In the case of the sensor S4 of the example (see FIG. 49D ), when no vibration was applied, normal current values ​​were measured at the working electrode in 1.2 mL, 1.0 mL, and 0.94 mL of liquid sample X1, but in the case of 0.85 mL of liquid sample, the current value at the working electrode bottomed out and normal measurement was not possible. When vibration was applied, normal current values ​​were measured at the working electrode in all volumes of liquid sample X1.

[0268] In the sensor S5 of the example (see FIG. 49E), normal current values ​​were measured at the working electrode in all volumes of the liquid sample X1 both when vibration was applied and when vibration was not applied.

[0269] The above results are summarized in the table below.

[0270]

[0271] Regardless of the sensor used, when the current value at the working electrode bottomed out and normal measurement was not possible, the voltage value at the counter electrode also bottomed out, and when the current value at the working electrode was normal, the voltage value at the counter electrode was also normal (data not shown). This indicates that sufficient contact between the counter electrode and the liquid sample is necessary for normal measurement by the sensor. Furthermore, the current measurement results confirmed that forming a tip opening and a flow path in the insulating layer allows the liquid sample to be guided to the upper surface of the counter electrode, thereby suppressing contact inhibition between the counter electrode and the liquid sample, and that suppressing warping of the substrate further suppresses contact inhibition.

[0272] <Notes on the second disclosure> In the configuration of a conventional cell culture analyzer equipped with a sensor for analyzing a cell culture medium, the sensor is fixed to a through-hole portion provided in a base, and a lead wire for extracting a signal is connected to this sensor.

[0273] For example, Patent Document 2 (US Pat. No. 9,170,255) discloses a cell culture analyzer having a cartridge that fits with a plate on which a plurality of cell culture vessels are provided.

[0274] The cell culture analyzer of Patent Document 2 has sensors that measure the inside of each culture vessel, and a cartridge is provided with multiple openings into which these sensors are inserted, and the sensors are connected to fiber cables within each opening. These fiber cables are then connected to an external control unit.

[0275] The sensor of the cell culture analyzer described in Patent Document 2 has the following problems.

[0276] That is, in the cell culture analyzer disclosed in Patent Document 2, it is difficult to accurately position each of the sensors inserted into the plurality of culture vessels with respect to the culture vessels.

[0277] This can lead to variations in the immersion depth of the sensor into the culture medium placed in the culture vessel, which can reduce measurement accuracy. In particular, if the sensor is not immersed deep enough into the culture medium, the liquid sample will not come into sufficient contact with the sensor's detection electrode, which can reduce measurement accuracy.

[0278] The second object of the disclosure is to provide a sensor and a sensor unit including the same, in which the inhibition of contact of the liquid sample with the detection electrodes of the sensor is suppressed when the sensor is immersed in the liquid sample.

[0279] Therefore, this specification discloses, as a second disclosure, a sensor and a sensor unit described in any one of Supplementary Notes 1 to 11 below.

[0280] (Supplementary Note 1) A sensor used while immersed in a liquid sample, comprising: an insulating substrate including a tip portion immersed in the liquid sample, a detection electrode disposed on a first surface of the substrate near the tip portion and including a working electrode, a counter electrode, and a reference electrode, wiring disposed 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 so as to cover at least a portion of the detection electrode and the wiring, a tip opening formed in the insulating layer and communicating with an upper surface of the counter electrode and opening to the side of the substrate toward the tip portion, and a flow path formed in the insulating layer connecting the tip opening and the upper surface of the counter electrode, the flow path leading from the tip opening to the upper surface of the counter electrode. (Supplementary Note 2) The sensor according to Supplementary Note 1, wherein the insulating layer has a counter electrode opening formed at a position overlapping the counter electrode in a plan view from the thickness direction of the substrate, the counter electrode opening penetrating the thickness direction, and the upper surface of the counter electrode being exposed through the counter electrode opening. (Supplementary Note 3) The sensor according to Supplementary Note 1 or 2, wherein the insulating layer comprises: a working electrode opening formed at a position overlapping the working electrode in a plan view from the thickness direction of the substrate, the working electrode opening penetrating the thickness direction; and a reference electrode opening formed at a position overlapping the reference electrode, the working electrode including a working electrode protective film disposed in the working electrode opening; and the reference electrode including a reference electrode protective film disposed in the reference electrode opening. (Supplementary Note 4) The sensor according to any one of Supplements 1 to 3, wherein the insulating layer includes: a first insulating layer disposed on the first surface of the substrate and including an insulating cured resin; and a second insulating layer disposed on the first insulating layer and including an insulating sheet, the tip opening and the flow path being formed in the second insulating layer. (Supplementary Note 5) The sensor according to Supplementary Note 4, wherein the tip opening and the flow path are further formed in the first insulating layer. (Supplementary Note 6) The sensor according to any one of Supplements 1 to 5, wherein the flow path has an expansion portion that expands a flow path width toward the tip opening.(Supplementary Note 7) The sensor according to any one of Supplementary Notes 1 to 6, wherein the substrate includes: a main body portion including the tip portion and a first end opposite the tip portion, and in which the detection electrode is arranged; a connection portion including a second end connected to the first end of the main body portion and a third end opposite the second end, and extending from the second end to the third end; and a base end portion including a fourth end connected to the third end of the connection portion, and the connection portion of the substrate includes, near the third end, a bending portion that bends the substrate so that the first surface side protrudes. (Supplementary Note 8) The sensor according to Supplementary Note 7, wherein the insulating layer includes a first insulating sheet arranged on a region of the first surface of the substrate that is on the main body portion and a portion of the connection portion that is closer to the second end than the bending portion. (Supplementary Note 9) The sensor according to Supplementary Note 8, wherein the insulating layer has a non-insulating region that does not include an insulating sheet, on the first surface of the substrate, in a region of the bending portion of the connection portion. (Supplementary Note 10) The sensor according to Supplementary Note 8 or 9, wherein the insulating layer further includes a second insulating sheet arranged on a region of the base end of the first surface of the substrate, and the first insulating sheet and the second insulating sheet are separated on a region of the first surface of the substrate where the connecting portion is bent. (Supplementary Note 11) A sensor unit comprising: the sensor according to any one of Supplementary Notes 1 to 10; and a support member having an engaging portion that engages with the substrate of the sensor and supports the sensor so that the tip end of the substrate is at a lower end.

[0281] <Embodiment of the Third Disclosure> An embodiment of a sensor according to the third disclosure will be described below. In this embodiment, more detailed explanation than necessary may be omitted. For example, detailed explanation of already well-known matters or redundant explanation of substantially identical configurations may be omitted. This is to avoid unnecessary redundancy in the following explanation and to facilitate understanding by those skilled in the art. Note that, for components in the sensor according to the third disclosure that are assigned the same reference numerals as those in the sensor according to the first disclosure, reference may be made to the explanation of the corresponding components in the sensor according to the first disclosure.

[0282] Furthermore, the applicant provides the accompanying drawings and the following description so that those skilled in the art can fully understand the third disclosure, and does not intend for them to limit the matters described in the "Additional Notes Regarding the Third Disclosure" below.

[0283] <Materials> Examples of materials that can be used in the sensor of the third disclosure of this specification will be described.

[0284] When the sensor of the third disclosure of this specification has an insulating substrate, the material thereof is not particularly limited, but for example, the same material as that of the insulating substrate of the sensor of the first disclosure of this specification can be used.

[0285] The conductive layers of the working electrode, reference electrode, and counter electrode of the sensor of the third disclosure of this specification are layers containing a conductive material such as carbon or gold. The conductive layer can be manufactured by forming a layer of such a conductive material on the surface of a substrate using a sputtering method, a vapor deposition method, a screen printing method, or the like. If necessary, the conductive layer can be processed into a predetermined pattern using a laser trimming method. In the third disclosure, the conductive layer of the working electrode may be referred to as the working electrode conductive layer, and the conductive layer of the reference electrode may be referred to as the reference electrode conductive layer. In the third disclosure, the conductive layer of the counter electrode is simply referred to as the counter electrode, since 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 described below.

[0286] The wiring of the sensor disclosed in the third specification can also be made of the same conductive material as the conductive layer.

[0287] The sensor of the third disclosure of this specification is used to detect a predetermined test substance (analyte) in a liquid sample by immersion in the liquid sample. The liquid sample is preferably a liquid sample containing water as a solvent. Examples of liquid samples include cell culture medium and liquid samples prepared using blood obtained from a living organism. Examples of the test substance are as described with respect to the test substance using the sensor of the first disclosure of this specification, and are particularly preferably one or more selected from glucose and lactic acid. The liquid sample is preferably a liquid sample containing cells, particularly living cells, and is particularly preferably a cell culture medium.

[0288] The working electrode of the sensor disclosed in the third aspect of the present specification includes a reagent layer disposed on the working electrode conductive layer and containing a reagent involved in the oxidation-reduction reaction of the test substance. The reagent can be appropriately selected depending on the test substance. The reagent can include a combination of an oxidoreductase and a mediator (electron carrier), or an oxidoreductase itself. The oxidoreductase can include a coenzyme.

[0289] The oxidoreductase may be an oxidase or a dehydrogenase. Specific examples of the oxidoreductase are as described in relation to the sensor of the first disclosure of this specification, and particularly preferably, the oxidoreductase may be glucose oxidase, lactate oxidase, glucose dehydrogenase, or lactate dehydrogenase.

[0290] Furthermore, the mediator is not particularly limited, and examples thereof are as described in relation to the sensor of the first disclosure of this specification.

[0291] The reagent layer provided on the working electrode of the sensor disclosed in the third disclosure of this specification may further contain, in addition to the reagent, components such as a buffer, a hydrophilic polymer compound, a conductive carbon filler, a crosslinking agent, etc. Examples of the hydrophilic polymer compound and the conductive carbon filler are as described in relation to the sensor disclosed in the first disclosure of this specification.

[0292] The working electrode of the sensor of the third disclosure of this specification may further include a protective film. The protective film on the working electrode may be a film that prevents or inhibits leakage of the reagent contained in the reagent layer outside the protective film and is permeable to the analyte present outside the protective film. A 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 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 the cation exchange functional group include an anionic functional group.

[0293] Examples of the polymer compound containing 4-vinylpyridine as a constituent unit and the polymer compound having proton conductivity are as described in relation to the sensor disclosed in the first aspect of this specification.

[0294] The protective film provided on the working electrode of the sensor of the third disclosure of this specification can be a laminate of two or more protective films, examples of which are as described for the sensor of the first disclosure of this specification.

[0295] Another suitable example of the protective film provided on the working electrode of the sensor of the third disclosure of this specification is a protective film containing a polymer compound containing 4-vinylpyridine as a constituent unit.

[0296] The reference electrode of the sensor disclosed in the third specification may be provided with a protective film. The protective film of the reference electrode may be made of the materials described above for the protective film of the working electrode.

[0297] The counter electrode of the sensor of the third disclosure of this specification may be covered with a protective film. Preferred embodiments of the protective film that covers the counter electrode will be described later.

[0298] In the sensor of the third disclosure of this specification, the electrodes, including the working electrode and counter electrode, arranged on an insulating substrate, are preferably covered with an insulating layer except for the portion that comes into contact with the liquid sample. The insulating layer may include an insulating resin. The insulating layer may have a multilayer structure of two or more layers. The multilayer insulating layer may include, for example, a first insulating layer arranged on the insulating substrate and the conductive layer, and a second insulating layer arranged on the first insulating layer. Preferred aspects of the materials constituting the first insulating layer and the second insulating layer are as described for the sensor according to the first disclosure.

[0299] <Outline of Sensor 300> A sensor 300 according to an embodiment of the third disclosure will be described with reference to the drawings.

[0300] As shown in FIGS. 54 to 57 , the sensor 300 of this embodiment includes an insulating substrate 2, a first working electrode 10a, a second working electrode 10b, a reference electrode 20, and a counter electrode 60 arranged on a first surface 2a of the substrate 2, and 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. While the sensor 300 of this embodiment includes two working electrodes, in another embodiment not shown, the number of working electrodes may be only one or may be three or more. In the following description, when the first working electrode 10a and the second working electrode 10b are not to be distinguished, they may be referred to as the working electrodes 10a and 10b. Furthermore, 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 electrodes. Although the sensor 300 of this embodiment is a three-electrode sensor including a working electrode, a reference electrode, and a counter electrode as electrodes, it may also be a two-electrode sensor including only a working electrode and a counter electrode without including a reference electrode. Although not shown, the reference electrode and / or the counter electrode may be provided on a substrate separate from the substrate on which the working electrode is disposed.

[0301] 21 for sensor 1 of the first disclosure, sensor 300 is immersed in a liquid sample X and used to detect a predetermined analyte in the liquid sample X. Specific examples of the liquid sample and the analyte are as described in the <Materials> section.

[0302] 56 and 57, 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 a reagent involved in the oxidation-reduction reaction of the analyte in the liquid sample. Preferred embodiments of the reagent are as described in the <Materials> section.

[0303] When the reagent layers 15a, 15b of the working electrodes 10a, 10b of the sensor 300 contain a reagent that oxidizes an analyte in a liquid sample, electrons are transferred from the analyte to the working electrode conductive layers 11a, 11b under conditions in which a predetermined voltage is applied to the electrodes of the sensor 300. Similarly, when the reagent layers 15a, 15b contain a reagent that reduces an analyte in a liquid sample, electrons are transferred from the working electrode conductive layers 11a, 11b to the analyte. Because the amount of transferred electrons depends on the concentration of the analyte, the concentration or change in concentration of the analyte in the liquid sample can be measured based on the value of the current or a change in the current value flowing through the working electrodes 10a, 10b of the sensor 300.

[0304] The sensor 300 can constitute an analytical device for analyzing a test substance in a liquid sample. An example of the analytical device is an analytical device including a sensor 300, an analytical unit 102, and a control unit 104, in which the sensor 1 according to the first disclosure in the analytical device 100 shown in Fig. 24 is replaced with the sensor 300 according to the third disclosure. The functions of this analytical device are as described for the analytical device 100 shown in Fig. 24.

[0305] <Structure of Sensor 300> The structure of the sensor 300 according to the third disclosure will be described with reference to Fig. 54 and its cross-sectional views, Figs. 55, 56, and 57. The structure of the reference electrode 20 of the sensor 300 according to the third disclosure is the same as the structure of the reference electrode 20 of the sensor 1 according to the first disclosure. Therefore, the description of the reference electrode 20 of the sensor 1 according to the first disclosure, with reference to Fig. 22, will be cited as the description of the reference electrode 20 of the sensor 300 according to the third disclosure.

[0306] The sensor 300 includes an insulating substrate 2 and working electrode conductive layers 11a and 11b, a counter electrode 60, a reference electrode conductive layer 21, and a wiring 50 disposed on a first surface 2a of the substrate 2. In the illustrated embodiment of the sensor 300 according to the third disclosure, the counter electrode 60 is entirely made of a conductive layer. The conductive layer portion of the counter electrode may also be referred to as a "counter electrode conductive layer" or a "counter electrode conductive layer" to specifically refer to the counter electrode. A first insulating layer 3 is further disposed on the first surface 2a of the substrate 2, and a second insulating layer 4 is further disposed 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 greater than the heights of the working electrode conductive layers 11a and 11b, the counter electrode 60, the reference electrode conductive layer 21, and the wiring 50 from the first surface 2a of the substrate 2. Therefore, a portion of the first insulating layer 3 is disposed on the working electrode conductive layers 11a and 11b, the counter electrode 60, the reference electrode conductive layer 21, and the wiring 50.

[0307] 55 , the first insulating layer 3 has a counter electrode first opening 302 formed at a position overlapping a portion of the counter electrode 60 and penetrating in the thickness direction T of the substrate 2. Furthermore, the second insulating layer 4 has a counter electrode second opening 402 formed at a position overlapping the entire counter electrode first opening 302 of the first insulating layer 3 and penetrating in the thickness direction T of the substrate 2. The counter electrode 60 is exposed to the outside through the counter electrode first opening 302 and the counter electrode second 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 on the bottom surface of a recess formed by the counter electrode first opening 302.

[0308] 56 and 57, the working electrodes 10a and 10b include working electrode conductive layers 11a and 11b, and reagent layers 15a and 15b disposed on the working electrode conductive layers 11a and 11b and containing a reagent participating in the oxidation-reduction reaction of the test substance. Preferred embodiments of the reagent in the reagent layers 15a and 15b are as described in the <Materials> section.

[0309] As in the first disclosure, 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 may be referred to as the "first reagent layer 15a" of the first working electrode 10a and the latter as the "second reagent layer 15b" of the second working electrode 10b. Furthermore, the reagent contained in the reagent layer 15a of the first working electrode 10a may be referred to as the "first reagent," and the reagent contained in the second reagent layer 15b of the second working electrode 10b may be referred to as the "second reagent." In a preferred embodiment, the sensor 300 includes a plurality of working electrodes 10a, 10b, and the first reagent contained in the first reagent layer 15a of the first working electrode 10a 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 sensor 300 according to this preferred embodiment can be used to detect multiple test substances, including a first test substance and a second test substance, in a liquid sample, because the first reagent is involved in an oxidation-reduction reaction of a first test substance in the liquid sample, and the second reagent is involved in an oxidation-reduction reaction of a second test substance different from the first test substance in the liquid sample.

[0310] As shown in Figures 56 and 57, 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 described in the <Materials> section. In the illustrated example, a first protective film 16a consisting of one layer is disposed on the first reagent layer 15a, and 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. However, the present invention is not limited to this example, and the protective films 16a and 16b may have the same structure or another structure not shown.

[0311] 56 and 57 , the first insulating layer 3 has working electrode first openings 303a and 303b formed in positions overlapping with portions of the working electrode conductive layers 11a and 11b, respectively, and penetrating in the thickness direction T of the substrate 2. Furthermore, the second insulating layer 4 has working electrode second openings 403a and 403b formed in positions overlapping with portions of the first insulating layer 3 that entirely encompass the working electrode first openings 303a and 303b, penetrating in the thickness direction T of the substrate 2. The working electrodes 10a and 10b are exposed to the outside through the working electrode first openings 303a and 303b and the working electrode second openings 403a and 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 on the bottom surfaces of the recesses formed by the working electrode first openings 303a and 303b. Furthermore, the reagent layers 15a and 15b are enclosed in the working electrode first openings 303a and 303b, and the outer peripheries of the reagent layers 15a and 15b are defined by the inner peripheries of the working electrode first openings 303a and 303b. The protective films 16a and 16b are enclosed in the working electrode second openings 403a and 403b, and the outer peripheries of the protective films 16a and 16b are defined by the inner peripheries of the working electrode second openings 403a and 403b.

[0312] <Suppression of Hydrogen Peroxide Generation by Sensor 300> The sensor 300 according to the third disclosure is characterized in that, when immersed in a liquid sample and electrochemically measuring a test substance in the liquid sample, the generation of hydrogen peroxide is suppressed and / or the concentration of the generated hydrogen peroxide is reduced during the measurement, thereby maintaining the hydrogen peroxide concentration in the liquid sample at less than 15 μM during the measurement. Hydrogen peroxide is known to be toxic to cells. Therefore, when using an electrochemical sensor to continuously measure a test substance in a liquid sample containing cells, such as a cell culture medium, over an extended period of several days or more, it is desirable to suppress an increase in the hydrogen peroxide concentration during the measurement.

[0313] The sensor 300 allows the hydrogen peroxide concentration in the liquid sample to be maintained at a low concentration of less than 15 μM during measurement without causing a significant decrease in the detection sensitivity of the analyte.

[0314] Here, "electrochemically measuring a test substance in a liquid sample" refers to, for example, connecting the sensor 300 immersed in the liquid sample to a potentiostat, applying a predetermined potential to the working electrodes 10a, 10b relative to the reference electrode 20, detecting the current between the working electrodes 10a, 10b and the counter electrode 60, and measuring the test substance based on the detected current. Electrochemical measurements using the sensor 300 are typically performed continuously for a period of several hours or several days or more, for example, for a period of one hour or more, preferably 72 hours (3 days) or more, more preferably 96 hours (4 days) or more, even more preferably 168 hours (7 days) or more, and most preferably 240 hours (10 days) or more. The upper limit of the period is not particularly limited, but is, for example, within 336 hours (14 days). The sensor 300 maintains the hydrogen peroxide concentration in the liquid sample at less than 15 μM during this period. The hydrogen peroxide concentration in the liquid sample during this period is preferably maintained at 10 μM or less.

[0315] In a preferred embodiment of the sensor 300 having the above-described features, the counter electrode 60 includes a catalyst that decomposes hydrogen peroxide at the portion 60a that comes into contact with the liquid sample. The generation of hydrogen peroxide occurs mainly due to the reaction of oxygen (O ２ ) is hydrogen peroxide (H ２ O ２ ) in the liquid sample. In particular, the generation of hydrogen peroxide on the counter electrode 60 can be a problem when measuring a test substance that undergoes an oxidation reaction on the working electrodes 10a and 10b. Measurement of a test substance that undergoes an oxidation reaction on the working electrodes 10a and 10b can be performed using the working electrodes 10a and 10b that are provided with reagent layers 15a and 15b containing oxidases, dehydrogenases, etc. that are involved in the oxidation of the test substance. With the sensor 300 that is provided with the counter electrode 60 that contains a catalyst that decomposes hydrogen peroxide in the portion 60a that comes into contact with the liquid sample, oxygen (O ２ ) is water (H ２O), the generation of hydrogen peroxide is suppressed. Furthermore, some of the reagents contained in the reagent layers 15a and 15b of the working electrodes 10a and 10b undergo a reaction that generates hydrogen peroxide during measurement (e.g., oxidase and dehydrogenase). According to this embodiment, the hydrogen peroxide generated on the working electrodes 10a and 10b is also decomposed into water by the catalyst contained in the counter electrode 60, and therefore an increase in the hydrogen peroxide concentration in the liquid sample during measurement can be suppressed.

[0316] In the third disclosure, the catalyst can be, for example, a catalyst containing a metal. Metal-containing catalysts are preferred because they have higher stability than organic catalysts such as catalase. As the metal-containing catalyst, a catalyst containing one or more selected from platinum, nickel, palladium, iron, manganese, and tungsten is preferred, and a platinum-containing catalyst is particularly preferred. These metal-containing catalysts are preferred because they have high activity for decomposing hydrogen peroxide, and among them, platinum-containing catalysts are particularly preferred from the viewpoints of catalytic activity and stability.

[0317] In the third disclosure, the catalyst may be contained in the portion of the counter electrode that comes into contact with the liquid sample, and may be contained only in the portion that comes into contact with the liquid sample, or may be contained in the entire counter electrode. Furthermore, the catalyst may be contained in the counter electrode as a film that covers at least the surface of the portion that comes into contact with the liquid sample, or may be contained in a dispersed state in a conductive material that constitutes the entire counter electrode or at least the portion that comes into contact with the liquid sample.

[0318] In the third disclosure, examples of counter electrodes containing the catalyst as a film covering the surface of the portion in contact with the liquid sample include counter electrodes obtained by forming a film of a metal usable as the catalyst on the surface of the portion in contact with the liquid sample by means of sputtering, plating, or the like, and counter electrodes obtained by applying a dispersion containing particles of the catalyst in a liquid medium to the surface of the portion in contact with the liquid sample and drying it. Of these, counter electrodes obtained using a dispersion containing particles of the catalyst in a liquid medium are preferred because they can be produced by simple methods such as coating or printing. Examples of catalyst particles include particles of the metals mentioned above, with platinum particles being particularly preferred. Platinum nanoparticles with an average particle diameter of less than 1 μm, for example, 800 nm or less, preferably 600 nm or less, are preferred.

[0319] In the third disclosure, an example of a counter electrode in which the catalyst is dispersed in a conductive material that constitutes the entire counter electrode or at least the portion that contacts the liquid sample is a counter electrode obtained by forming the entire counter electrode or at least the portion that contacts the liquid sample using a paste or liquid composition containing particles of the catalyst and particles of the conductive material. This type of counter electrode can be produced with a small number of steps because the catalyst can be incorporated simultaneously with the formation of the counter electrode on the substrate. Examples of the catalyst particles include particles of the metals mentioned above, with platinum particles being particularly preferred. Preferred embodiments of platinum particles are as described above.

[0320] In the third disclosure, the counter electrode may contain carbon, gold, or the like as a conductive material, and preferably contains carbon as a conductive material. Examples of the carbon that can be used include conductive carbon materials such as glassy carbon, carbon black, graphite, diamond-like carbon, graphene, carbon nanotubes, and fullerene.

[0321] In the third disclosure, the content of the catalyst in the counter electrode can be appropriately set depending on the type and activity of the catalyst. For example, if the entire counter electrode or at least the portion that contacts the liquid sample contains carbon as a conductive material and platinum particles as the catalyst, the platinum particles can be contained in an amount of, for example, 0.5 wt.% or more, preferably 1 wt.% or more, more preferably 3 wt.% or more, particularly preferably 5 wt.% or more, relative to the carbon. Although the upper limit is not particularly limited, from a cost perspective, it is preferably 50 wt.% or less (the ratio of the platinum particles to the carbon is, for example, 0.5 wt.% or more and 50 wt.% or less), even more preferably 30 wt.% or less, and most preferably 10 wt.% or less. By setting the ratio of carbon to platinum particles within this range, an increase in the hydrogen peroxide concentration in the liquid sample during the measurement period can be suppressed.

[0322] Although not shown, in the third disclosure, the surface of the counter electrode may be further coated with a protective film. The protective film coating 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 its side chain. Examples of polymer compounds having a cation-exchange functional group include polymer compounds containing structural units having sulfonic acid groups, preferably polymer compounds containing perfluorocompounds having sulfonic acid groups in their side chains, particularly preferably copolymers of tetrafluoroethylene and perfluoro-2-(2-fluorosulfonylethoxy)propyl vinyl ether, and most preferably Nafion®. Anions such as chloride ions and organic acid ions that may be contained in a liquid sample may adversely affect the activity of catalysts that decompose hydrogen peroxide, such as platinum catalysts. By coating the counter electrode with a protective film containing a polymer compound having a cation-exchange functional group, it is possible to prevent anions from contacting the counter electrode and suppress a decrease in the activity of the catalyst.

[0323] On the other hand, in the sensor 300 according to the present disclosure, it is preferable that at least the portions 11a1, 11b1 of the working electrode conductive layers 11a, 11b of the working electrodes 10a, 10b that come into contact with the liquid sample, preferably the entirety, do not contain a catalyst that decomposes hydrogen peroxide. If the working electrode conductive layers 11a, 11b of the working electrodes 10a, 10b of the sensor 300 contain such a catalyst, particularly a catalyst containing platinum, there is a possibility that background noise will increase during measurement and the current response value will decrease. Examples of the working electrode conductive layers 11a, 11b or the portions 11a1, 11b1 that do not contain a catalyst that decomposes hydrogen peroxide include layers or portions of conductive materials such as carbon or gold that do not contain a catalyst that decomposes hydrogen peroxide.

[0324] More preferably, the working electrode conductive layers 11a and 11b of the working electrodes 10a and 10b have a current value of ±5.5 nA / mm when a three-electrode electrolytic cell is used, the three-electrode electrolytic cell 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, and a potential in the range of −0.2 V to 0.3 V (vs. the silver-silver chloride (saturated KCl) electrode) is applied to the working electrode conductive layers in phosphate buffered saline. ２ The sensor 300 having the working electrode conductive layers 11a, 11b that satisfy this condition is preferable because the background current when measuring the analyte is sufficiently small and the sensor has high responsiveness. Examples of such working electrode conductive layers 11a, 11b include carbon conductive layers containing carbon, and more preferably, carbon conductive layers that do not contain a catalyst that decomposes hydrogen peroxide, such as a catalyst containing platinum. Here, the carbon that can be used is the same as that exemplified for the conductive material of the counter electrode 60. Note that the current value is ±5.5 nA / mm ２ The term "less than or equal to" means that the vertical projection area of ​​the portion of the working electrode conductive layer 11a, 11b used as the working electrode that comes into contact with phosphate buffered saline is 1 mm ２ This means that the absolute value of the current value per electrode is 5.5 nA or less. Specifically, the measurement of the current value using the above three-electrode electrolytic cell can be carried out by the procedure described in <Third Disclosure / Experiment 5> above.

[0325] The inventors further discovered an unexpected effect in the sensor 300 according to the present disclosure: when the portion 60 a of the counter electrode 60 that contacts the liquid sample contains a platinum-containing catalyst, a large absolute value of negative current can flow at a low potential. Therefore, the counter electrode 60 that contains a platinum-containing catalyst in the portion 60 a that contacts the liquid sample can function as a counter electrode with a smaller area than a counter electrode that does not contain a platinum-containing catalyst, thereby contributing to a reduction in sensor size. Therefore, in the sensor 300 according to the present disclosure, the projected area of ​​the counter electrode 60 can be preferably 250% or less, more preferably 100% or less, and preferably 3% or more of the projected area of ​​the working electrode conductive layers 11 a, 11 b (the projected area of ​​the counter electrode is, for example, 3% or more and 250% or less of the projected area of ​​the working electrode conductive layers), and more preferably 10% or more. Furthermore, the sensor 300, which includes a counter electrode 60 containing a platinum-containing catalyst in a portion 60a that contacts the liquid sample, is also suitable for a case in which multiple working electrodes 10a, 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 counter electrode first opening 302). The projected area of ​​the working electrode conductive layers 11a, 11b refers to the vertical projected area of ​​the portions 11a1, 11b1 of the working electrode conductive layers 11a, 11b that contact the liquid sample (corresponding to the bottom surface of the recess formed by the working electrode first openings 303a, 303b). When multiple working electrodes 10a, 10b are present, the projected area refers to the total area of ​​the working electrodes.

[0326] <Example of the 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 of the sensor according to the third disclosure and a comparative example.

[0327] The characteristics of the sensors used in each experiment will be explained individually in Experiments 1 to 7. First, an outline of the materials and manufacturing methods common to the sensors used in each experiment will be explained.

[0328] (Substrate) As in the first disclosed example, a substrate made of polyethylene terephthalate and having a thickness of 188 μm, having the shape shown in FIG. 54, was used as the insulating substrate 2 .

[0329] (Conductive Layer) A predetermined carbon paste described in Experiments 1 to 7 was applied to the first surface 2 a of the insulating substrate 2, and heated at 140° C. for 1 hour, thereby forming working electrode conductive layers 11 a and 11 b, a reference electrode conductive layer 21, and a counter electrode (counter electrode conductive layer) 60, all of which had the same shapes as those shown in FIG. 1 relating to the first disclosure, as well as wiring 50 electrically connected to each of them, using a 5 μm-thick carbon conductive layer.

[0330] (First Insulating Layer) Next, a first insulating layer 3 was laminated on the first surface 2a of the substrate 2 on which the carbon conductive layer was disposed. The first insulating layer 3 was made of a fluororesin containing a copolymer containing vinylidene fluoride and hexafluoropropylene, and had a thickness of 5 μm on the carbon conductive layer, covering the first surface 2a of the substrate 2 and the carbon conductive layer. The method for manufacturing the first insulating layer 3 was as described in the first disclosed example.

[0331] The working electrode first opening 303a on the first working electrode conductive layer 11a of the first insulating layer 3 was circular and 1.2 mm in diameter, and the working electrode first opening 303b on the second working electrode conductive layer 11b was circular and 1.4 mm in diameter. The reference electrode first opening 301 was circular and 1.1 mm in diameter, and the counter electrode first opening 302 was rectangular and 1.8 mm × 2.1 mm.

[0332] (Second insulating layer) Next, a composition containing a polyester resin and a fluorine-based surface-modifying additive containing a perfluoroalkyl group in a solvent was applied onto the first insulating layer 3, and heated at 140°C for 1 hour to laminate a second insulating layer 4 having a thickness of 40 µm.

[0333] 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 a circle with a diameter of 2 mm. The reference electrode second opening 401 was a circle with a diameter of 2 mm, and the counter electrode second opening 402 was a rectangle measuring 1.8 mm x 2.1 mm.

[0334] (First Working Electrode (for Glucose Measurement) Reagent Layer) A 0.4 μL droplet of a first liquid composition A containing a sodium phosphate buffer solution (pH 7.4), a carbon black dispersion, a polymer-bound mediator, glucose oxidase, and a crosslinker in water was formed in the working electrode first opening 303 a of the first insulating layer 3 on the first working electrode conductive layer 11 a, and then dried to form a first reagent layer 15 a containing glucose oxidase and the mediator.

[0335] (Second Working Electrode (for Lactic Acid Measurement) Reagent Layer) A 0.6 μL droplet of a second liquid composition A containing a carbon black dispersion, hydroxypropyl cellulose, a polymer-bound mediator, lactate oxidase, polyimidazole, poly-L-lysine, and a crosslinking agent in water was formed in the first working electrode opening 303 b of the first insulating layer 3 of the second working electrode conductive layer 11 b, and then dried to form a second reagent layer 15 b containing lactate oxidase and the mediator.

[0336] (Protective film for first working electrode (for measuring glucose)) The following reagents were mixed with ethanol to the following final concentrations and allowed to react for about 1 hour to prepare a first liquid composition B (P4VP-tBuMA polymer dispersion): P4VP-tBuMA (poly-4-vinylpyridine Mn: 74,000, poly-tert-butyl methacrylate Mn: 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%

[0337] A 0.65 μL droplet of the first liquid composition B was formed in the working electrode second opening 403 a of the second insulating layer 4 on the first working electrode 10 a and dried, and then another 0.65 μL droplet of the first liquid composition B was formed and dried to form a first protective film 16 a.

[0338] (Second Working Electrode (for Lactic Acid Measurement) Protective Film) 23,093.67 mg of a 21.5 wt% Nafion® dispersion (Sigma-Aldrich Corporation) was mixed with 6,472.79 mg of ethanol (Fujifilm Wako Pure Chemical Industries, Ltd.) and 1,226.46 mg of a 5 mol / L aqueous sodium hydroxide solution (Fujifilm Wako Pure Chemical Industries, Ltd.) to adjust the pH of the dispersion (neutralization of the cation exchange groups). The precipitate was dissolved using a vortex mixer to prepare 30,792.92 mg of a 16.12 wt% Nafion® dispersion. The resulting 16.12 wt% Nafion® dispersion was designated as second liquid composition B. This second liquid composition B contains Nafion® in a solvent containing a lower alcohol.

[0339] A 0.60 μL droplet of the second liquid composition B was formed in the second working electrode opening 403 b of the second insulating layer 4 on the second working electrode 10 b and dried to form a second protective film 16 ba.

[0340] Subsequently, the same composition as the first liquid composition B (P4VP-tBuMA polymer dispersion) was used in the following process as a third liquid composition B. That is, a 0.65 μL droplet of the third liquid composition B was formed in the second working electrode opening 403b of the second insulating layer 4 on which the second protective film 16ba had been formed, and then dried to form a third protective film 16bb. In this way, a protective film 16b was formed on the second working electrode 10b, in which the third protective film 16bb was laminated on the second protective film 16ba.

[0341] (Reference Electrode) The cross-sectional structure of the reference electrode 20 is the same as that shown in Fig. 22 for the sensor of the first disclosure, and therefore, the manufacturing method of the reference electrode 20 will be outlined below with reference to Fig. 22. A silver-silver chloride paste was applied to 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. Subsequently, 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, thereby forming the reference electrode 20.

[0342] <Third Disclosure / Experiment 1> Experiment 1-1: In the above-described method, a carbon conductive layer was formed using a normal carbon paste that did not contain a catalyst such as platinum, to fabricate a sensor 300 in Experiment 1-1. In the sensor in 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 decomposes hydrogen peroxide.

[0343] Experiment 1-2: Sensor 300 of Experiment 1-2 was fabricated using the above procedure, except that a carbon conductive layer was formed using a normal carbon paste that did not contain a catalyst such as platinum, and then platinum was vapor-deposited only on counter electrode 60 before forming first insulating layer 3. In the sensor of Experiment 1-2, counter electrode 60 had a portion 60a that was exposed through first counter electrode opening 302 of first insulating layer 3 and second counter electrode opening 402 of second insulating layer 4 and that came into contact with the liquid sample, and included a carbon conductive layer and a platinum vapor-deposited film laminated thereon, and working electrode conductive layers 11a, 11b and reference electrode conductive layer 21 were made of carbon conductive layers that did not contain a catalyst such as platinum.

[0344] As a liquid sample containing glucose and lactic acid as test substances, phosphate-buffered saline (PBS) containing 30 mM glucose and 15.6 mM lactic acid and adjusted to a pH of 7.0 was prepared using PBS (Takara Bio PBS Tablets T9181), glucose (Fujifilm Wako Pure Chemical Industries, Ltd.), and sodium lactate (Sigma-Aldrich).

[0345] The sensor of Experiment 1-1 or Experiment 1-2 was immersed in the liquid sample, a voltage of 100 mV was applied to the first working electrode 10a and the second working electrode 10b relative to the reference electrode 20 (Ag / AgCl), and the current value between the first working electrode 10a and the counter electrode 60 and the current value between the second working electrode 10b and the counter electrode 60 were measured for seven consecutive days. N=4 measurements were performed.

[0346] Using the liquid sample after 7 days of measurement, a sample for measuring hydrogen peroxide concentration was prepared according to the formulation shown in the table below. The absorbance of the sample for measuring hydrogen peroxide concentration at 666 nm was measured for 5 minutes using a plate reader (at 37°C).

[0347]

[0348] In addition, a sample for measuring hydrogen peroxide concentration was prepared by compounding a solution containing hydrogen peroxide (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) of a known concentration in place of the liquid sample in the above table, and similarly measuring the absorbance at 666 nm to create a calibration curve showing the relationship between hydrogen peroxide concentration and absorbance.

[0349] The hydrogen peroxide concentration in the liquid sample was determined from the calibration curve. The determined hydrogen peroxide concentration was corrected for the amount of water evaporated from the liquid sample during the 7-day continuous measurement. The results are shown in the table below.

[0350]

[0351] From the above results, it was confirmed that the sensor in Experiment 1-2, which had a counter electrode consisting of a carbon conductive layer with a platinum vapor-deposited film laminated on the part that came into contact with the liquid sample, was able to suppress or reduce the generation of hydrogen peroxide in the liquid sample during measurement of the test substance in the liquid sample, compared to the sensor in Experiment 1-1, which had a counter electrode consisting of only a carbon conductive layer.

[0352] <Third Disclosure / Experiment 2> Experiment 2-1: The sensor 300 in Experiment 2-1 was the same as the sensor in Experiment 1-1, except that all conductive layers including the counter electrode 60 were carbon conductive layers.

[0353] Experiment 2-2: In the sensor of Experiment 2-1, a platinum nanoparticle dispersion (manufactured by Sigma-Aldrich, platinum particle diameter: 70±6 nm) was dropped onto the surface of the counter electrode 60 exposed through the counter electrode first opening 302 and the counter electrode second opening 402 and dried, and then a 21.5 wt % Nafion (registered trademark) dispersion (manufactured by Sigma-Aldrich) was dropped onto the surface and dried to form a protective film (not shown), thereby producing the sensor 300 of Experiment 2-2. In the sensor 300 of Experiment 2-2 thus produced, 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 covered with a protective film (not shown) containing Nafion (registered trademark), a polymer compound having cation-exchange functional groups.

[0354] The sensor 300 of Experiment 2-1 or Experiment 2-2 was immersed in a liquid sample having the composition described in Experiment 1, a voltage of 100 mV was applied to the first working electrode 10a and the second working electrode 10b relative to the reference electrode 20 (Ag / AgCl), and the current value between the first working electrode 10a and the counter electrode 60 and the current value between the second working electrode 10b and the counter electrode 60 were measured for seven consecutive days. The measurements were performed with N=2.

[0355] Using the liquid sample after 7 days of measurement, a sample for measuring hydrogen peroxide concentration was prepared according to the formulation shown in the table below. The absorbance of the sample for measuring hydrogen peroxide concentration at 666 nm was measured for 5 minutes using a plate reader (at 37°C).

[0356]

[0357] In addition, a sample for measuring hydrogen peroxide concentration was prepared by blending a solution containing a known concentration of hydrogen peroxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) instead of the liquid sample in the above table, and similarly measuring the absorbance at 666 nm to create a calibration curve of hydrogen peroxide concentration versus absorbance.

[0358] The hydrogen peroxide concentration in the liquid sample was determined from the calibration curve. The determined hydrogen peroxide concentration was corrected for the amount of water evaporated from the liquid sample during the 7-day continuous measurement. The results are shown in the table below.

[0359]

[0360] From the above results, it was confirmed that the sensor 300 of Experiment 2-2, which has a counter electrode 60 consisting of a carbon conductive layer modified with platinum nanoparticles on the portion 60a that comes into contact with the liquid sample, was able to suppress or reduce the generation of hydrogen peroxide in the liquid sample during measurement of the test substance in the liquid sample, compared to the sensor 300 of Experiment 2-1, which has a counter electrode 60 consisting of only a carbon conductive layer.

[0361] <Third Disclosure / Experiment 3> Experiment 2-1: The sensor 300 in Experiment 3-1 was the same as the sensors in Experiments 1-1 and 2-1, except that all conductive layers including the counter electrode 60 were carbon conductive layers.

[0362] Experiment 3-2: The sensor 300 of Experiment 3-2 was fabricated according to the above procedure, except that the working electrode conductive layers 11a, 11b, the reference electrode conductive layer 21, the counter electrode (counter electrode conductive layer) 30, and the wiring 50 were formed using a carbon paste containing carbon (containing graphite and carbon black) and 1% by weight of platinum particles (average particle diameter 390 nm) relative to the carbon.

[0363] Experiment 3-3: Sensor 300 of Experiment 3-3 was fabricated using the sensor and procedure of Experiment 3-2, except that a carbon paste containing the carbon and 5% by weight of platinum particles relative to the carbon was used.

[0364] The sensor of Experiment 3-1, Experiment 3-2, or Experiment 3-3 was immersed in a liquid sample having the composition described in Experiment 1, and a voltage of 100 mV was applied to the first working electrode 10a and the second working electrode 10b relative to the reference electrode 20 (Ag / AgCl), and the current value between the first working electrode 10a and the counter electrode 60 and the current value between the second working electrode 10b and the counter electrode 60 were measured for seven consecutive days. N=4 measurements were performed.

[0365] Using the liquid sample after 7 days of measurement, a sample for measuring hydrogen peroxide concentration was prepared according to the formulation shown in the table below. The absorbance of the sample for measuring hydrogen peroxide concentration at 666 nm was measured for 5 minutes using a plate reader (at 37°C).

[0366]

[0367] In addition, a sample for measuring hydrogen peroxide concentration was prepared by blending a solution containing a known concentration of hydrogen peroxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) instead of the liquid sample in the above table, and similarly measuring the absorbance at 666 nm to create a calibration curve of hydrogen peroxide concentration versus absorbance.

[0368] The hydrogen peroxide concentration in the liquid sample was determined from the calibration curve. The determined hydrogen peroxide concentration was corrected for the amount of water evaporated from the liquid sample during the 7-day continuous measurement. The results are shown in the table below.

[0369]

[0370] From the above results, it was confirmed that the sensors 300 of Experiments 3-2 and 3-3, which had a counter electrode 60 consisting of a carbon conductive layer containing platinum particles, were able to suppress or reduce the generation of hydrogen peroxide in the liquid sample during measurement of the test substance in the liquid sample, compared to the sensor 300 of Experiment 3-1, which had a counter electrode 60 consisting of only a carbon conductive layer.

[0371] <Third Disclosure / Experiment 4> The following experiment was conducted to investigate the electrochemical properties of a carbon conductive layer containing no platinum (hereinafter referred to as a "carbon electrode without platinum particles"), a carbon conductive layer containing 1% by weight of platinum particles relative to the carbon (hereinafter referred to as a "carbon electrode containing 1% platinum particles"), and a carbon conductive layer containing 5% by weight of platinum particles relative to the carbon (hereinafter referred to as a "carbon electrode containing 5% platinum particles"), which were used as the counter electrode 60 in Experiments 3-1, 3-2, and 3-3.

[0372] A three-electrode electrolytic cell was constructed using a carbon electrode without platinum particles, a carbon electrode containing 1% platinum particles, or a carbon electrode containing 5% platinum particles as the working electrode, a silver-silver chloride (saturated KCl) electrode (manufactured by BAS) as the reference electrode, and a platinum electrode as the counter electrode, and each electrode was connected to a potentiostat. The working electrode, counter electrode, and reference electrode were placed in phosphate-buffered saline (PBS) (Takara Bio PBS Tablets T9181; 0.14 M NaCl, 0.0027 M KCl, 0.010 M PO4) adjusted to pH 7.4 ± 0.05 (25°C). ３＋ ) and a potential in the range of ±0.5 V relative to the reference electrode was applied to the working electrode at a sweep rate of 10 mV / s to perform cyclic voltammetry. The resulting cyclic voltammogram is shown in FIG.

[0373] The higher the platinum particle content of the electrode, the larger the absolute value of the positive current value at the high potential side and the larger the absolute value of the negative current value at the low potential side. The negative current value at the low potential side is thought to be due to the reduction of oxygen, etc. in PBS. This suggests that when an electrode containing platinum particles is used as a working electrode, there is a possibility that the sensitivity will decrease due to increased background noise, decreased current response, and increased influence of environmental disturbances such as oxygen concentration.

[0374] On the other hand, when an electrode containing platinum particles is used as a counter electrode, the increase in current due to the reduction of oxygen and the like allows a large absolute negative current to flow at a low potential (a potential with a small absolute value) in the negative (negative) potential region. 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, thereby contributing to the reduction of sensor size. Furthermore, by operating the sensor of the present disclosure using an electrode containing platinum particles as a counter electrode at a low potential in the negative potential region, it is also possible to reduce the load on the potentiostat, which is the measuring device.

[0375] <Third Disclosure / Experiment 5> In order to further investigate the electrochemical properties of the carbon electrode without platinum particles and the carbon electrode containing 1% platinum particles used in Experiment 4, the following experiment was carried out.

[0376] A three-electrode electrolysis cell was constructed using either a carbon electrode without platinum particles or a carbon electrode containing 1% platinum particles as the working electrode, a silver-silver chloride (saturated KCl) electrode (manufactured by BAS) as the reference electrode, and a platinum electrode as the counter electrode, and each electrode was connected to a potentiostat. The working electrode, counter electrode, and reference electrode were placed in phosphate-buffered saline (PBS) (Takara Bio PBS Tablets T9181; 0.14 M NaCl, 0.0027 M KCl, 0.010 M PO) adjusted to pH 7.4 ± 0.05 (25°C). ４ ３＋ ) and a potential of 0.3 V or −0.2 V relative to the reference electrode was applied to the working electrode, and amperometry measurement was performed to measure the current value over time. The measurement results of the current value per area of ​​the working electrode are shown in FIG.

[0377] Figure 59A shows the measurement results when a carbon electrode without platinum particles was used as the working electrode. Figure 59B shows the measurement results when a carbon electrode containing 1% platinum particles was used as the working electrode. It was confirmed that the current value (absolute value) when a carbon electrode without platinum particles was used as the working electrode was significantly smaller than the current value (absolute value) when a carbon electrode containing 1% platinum particles was used as the working electrode (the vertical scales of Figures A and B are different). This result indicates that a conductive layer without platinum particles is suitable for the working electrode conductive layers 11a and 11b because it generates a small background current.

[0378] <Third Disclosure / Experiment 6> In the sensor of Experiment 2-1 described in Experiment 2 above, all conductive layers including the working electrode conductive layers 11a and 11b and the counter electrode 60 are carbon conductive layers that do not contain platinum particles.

[0379] On the other hand, in the sensor of Experiment 2-2, the portion 60a of the counter electrode 60 in the sensor of Experiment 2-1 that comes into contact with the liquid sample to be measured is a carbon conductive layer whose surface is modified with platinum nanoparticles, and the working electrode conductive layers 11a and 11b are carbon conductive layers that do not contain platinum particles.

[0380] In this experiment 6, the sensor of experiment 2-1 or experiment 2-2 was immersed in a liquid sample having the composition described in experiment 1, a voltage of 100 mV was applied to the first working electrode 10a and the second working electrode 10b relative to the reference electrode 20 (Ag / AgCl), and the current value between the first working electrode 10a and the counter electrode 60 and the current value between the second working electrode 10b and the counter electrode 60 were continuously measured for a predetermined time. Measurements were performed with N=2.

[0381] The measurement results of the current values ​​of the first working electrode 10a and the second working electrode 10b are shown in A and B of FIG. 60, 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. No significant difference in responsiveness was observed between the sensor of Experiment 2-2, in which the working electrode conductive layers 11a and 11b did not contain platinum particles and only the counter electrode 60 contained platinum particles, and the sensor of Experiment 2-1, in which neither the working electrode conductive layers 11a and 11b nor the counter electrode 60 contained platinum particles.

[0382] <Third Disclosure / Experiment 7> In the sensor of Experiment 3-1 described in Experiment 3 above, all conductive layers including the working electrode conductive layers 11a and 11b and the counter electrode 60 are carbon conductive layers that do not contain platinum particles.

[0383] In the sensor of Experiment 3-2, all conductive layers including the working electrode conductive layers 11a and 11b and the counter electrode 60 are carbon conductive layers containing platinum particles in an amount of 1% by weight relative to the carbon.

[0384] In the sensor of Experiment 3-3, all conductive layers including the working electrode conductive layers 11a and 11b and the counter electrode 60 are carbon conductive layers containing platinum particles in an amount of 5% by weight relative to the carbon.

[0385] In this experiment 7, the sensor of experiment 3-1, experiment 3-2, or experiment 3-3 was immersed in a liquid sample having the composition described in experiment 1, a voltage of 100 mV was applied to the first working electrode 10a and the second working electrode 10b relative to the reference electrode 20 (Ag / AgCl), and the current value between the first working electrode 10a and the counter electrode 60 and the current value at the second working electrode 10b were continuously measured for a predetermined time. Measurements were performed with N=2.

[0386] The measurement results of the current values ​​of the first working electrode 10a and the second working electrode 10b are shown in Figure 61A and B, respectively. The current value of the first working electrode 10a shown in Figure 61A corresponds to the glucose concentration. The current value of the second working electrode 10b shown in Figure 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 platinum particles at 1 wt% relative to the carbon, did not exhibit a significant decrease in current response value compared to the sensor of Experiment 3-1, in which the working electrode conductive layers 11a, 11b and the counter electrode 60 did not contain platinum particles. On the other hand, the sensor of Experiment 3-3, in which the working electrode conductive layers 11a, 11b and the counter electrode 60 contained platinum particles at 5 wt% relative to the carbon, exhibited a clearly decreased current response value compared to the sensor of Experiment 3-1, in which the working electrode conductive layers 11a, 11b and the counter electrode 60 did not contain platinum particles. This suggests that the presence of platinum in the working electrode conductive layers 11a and 11b reduces the responsiveness of the sensor.

[0387] <Supplementary Note on the Third Disclosure> Sensors that include a working electrode and a counter electrode for electrochemically measuring a test substance (allanite) in a liquid sample such as a cell culture medium have been known.

[0388] Such sensors are sometimes used for continuous monitoring, where they are immersed in a liquid sample and continuously measure a test substance over a long period of time, such as several days. In this case, it is desirable for the sensor to have high detection sensitivity for the test substance and to have minimal negative effects (e.g., cytotoxicity) on cultured cells contained in the liquid sample.

[0389] It has been discovered that when a test substance in a liquid sample is electrochemically measured using a sensor comprising a working electrode including 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, hydrogen peroxide may be generated during long-term measurement. If the hydrogen peroxide concentration in the liquid sample exceeds a certain level, it may become toxic to cells.

[0390] Therefore, the third disclosure aims to provide a sensor equipped with a working electrode and a counter electrode that can suppress the generation of hydrogen peroxide during measurement or reduce the amount of hydrogen peroxide generated without causing a significant decrease in sensitivity.

[0391] When used to measure a test substance in a liquid sample such as a cell culture medium, the sensor according to the third disclosure can suppress the generation of hydrogen peroxide or reduce the amount of hydrogen peroxide generated during measurement without a significant decrease in sensitivity, thereby suppressing an increase in the hydrogen peroxide concentration in the liquid sample. Therefore, the sensor according to the third disclosure has little negative effect on cells in the liquid sample and can be suitably used for long-term continuous monitoring.

[0392] Therefore, this specification discloses, as a third disclosure, a sensor described in any one or more of Supplementary Notes 3-1 to 3-16 below.

[0393] (Supplementary Note 3-1) A sensor for measuring a 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 including a reagent participating in an oxidation-reduction reaction of the analyte; and a counter electrode, wherein 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 μM during measurement. (Supplementary Note 3-2) The sensor according to Supplementary Note 3-1, wherein the counter electrode includes a catalyst that decomposes hydrogen peroxide in a portion that comes into contact with the liquid sample. (Supplementary Note 3-3) The sensor according to Supplementary Note 3-2, wherein the working electrode conductive layer does not include a catalyst that decomposes hydrogen peroxide in a portion that comes into contact with the liquid sample. (Supplementary Note 3-4) The sensor according to Supplementary Note 3-2 or 3-3, wherein the catalyst includes a metal. (Appendix 3-5) The sensor according to Appendix 3-4, wherein the metal comprises one or more selected from platinum, nickel, palladium, iron, manganese, and tungsten. (Appendix 3-6) The sensor according to Appendix 3-4, wherein the metal comprises platinum. (Appendix 3-7) The sensor according to Appendix 3-6, wherein the platinum is platinum particles. (Appendix 3-8) The sensor according to Appendix 3-7, wherein the portion of the counter electrode that comes into contact with the liquid sample comprises carbon and platinum particles in an amount of 1% by weight or more relative to the carbon. (Appendix 3-9) The sensor according to Appendix 3-8, wherein the portion of the counter electrode that comes into contact with the liquid sample comprises carbon and platinum particles in an amount of 50% by weight or less relative to the carbon. (Appendix 3-10) When a three-electrode electrolytic cell including the working electrode conductive layer as a working electrode, a silver-silver chloride (saturated KCl) electrode as a reference electrode, and a platinum electrode as a counter electrode is used, a potential in the range of −0.2 V to 0.3 V (vs. the silver-silver chloride (saturated KCl) electrode) is applied to the working electrode conductive layer in phosphate buffered saline, and a current value measured is ±5.5 nA / mm ２The sensor according to any one of Appendices 3-1 to 3-9, satisfying the following condition: (Appendix 3-11) The sensor according to Appendices 3-10, wherein the working electrode conductive layer is a carbon conductive layer. (Appendix 3-12) The sensor according to any one of Appendices 3-1 to 3-11, wherein the projected area of ​​the counter electrode is 250% or less of the projected area of ​​the working electrode conductive layer. (Appendix 3-13) The sensor according to Appendices 3-12, wherein the projected area of ​​the counter electrode is 100% or less of the projected area of ​​the working electrode conductive layer. (Appendix 3-14) The sensor according to Appendices 3-12 or 3-13, comprising a plurality of the working electrodes. (Appendix 3-15) The sensor according to any one of Appendices 3-1 to 3-14, further comprising a protective film covering the counter electrode. (Appendix 3-16) The sensor according to Appendices 3-15, wherein the protective film contains a polymer compound having a cation exchange functional group.

[0394] <Embodiment of the Fourth Disclosure> An embodiment of a sensor according to the fourth disclosure will be described below. In this embodiment, more detailed explanation than necessary may be omitted. For example, detailed explanation of already well-known matters or redundant explanation of substantially identical configurations may be omitted. This is to avoid unnecessary redundancy in the following explanation and to facilitate understanding by those skilled in the art. Note that, for components in the sensor according to the fourth disclosure that are assigned the same reference numerals as those in the sensor according to the first disclosure, reference may be made to the description of the corresponding components in the sensor according to the first disclosure.

[0395] Furthermore, the applicant provides the accompanying drawings and the following description so that those skilled in the art can fully understand the fourth disclosure, and does not intend for them to limit the matters described in the "Additional Notes Regarding the Fourth Disclosure" below.

[0396] <Materials> Examples of materials that can be used in the sensor of the fourth disclosure of this specification will be described.

[0397] The material of the insulating substrate used in the sensor of the fourth disclosure of this specification is not particularly limited, but for example, the same material as the insulating substrate of the sensor of the first disclosure of this specification can be used.

[0398] The conductive layers of the working electrode, reference electrode, and counter electrode of the sensor of the fourth disclosure of this specification are layers containing a conductive material such as carbon, gold, platinum, or palladium. The conductive layer can be manufactured by forming a layer of such a conductive material on the surface of a substrate using a sputtering method, a vapor deposition method, a screen printing method, or the like. If necessary, the conductive layer can be processed into a predetermined pattern using a laser trimming method. In the fourth disclosure, the conductive layer of the working electrode may be referred to as the working electrode conductive layer, and the conductive layer of the reference electrode may be referred to as the reference electrode conductive layer. In the fourth disclosure, the conductive layer of the counter electrode is simply referred to as the counter electrode, since the conductive layer itself constitutes the counter electrode.

[0399] The wiring of the sensor disclosed in the fourth specification can also be made of the same conductive material as the conductive layer.

[0400] The sensor of the fourth disclosure of this specification is used to detect a predetermined test substance (analyte) in a liquid sample by immersion in the liquid sample. The liquid sample preferably contains water as a solvent. Examples of the liquid sample include a cell culture medium and a liquid sample prepared using blood obtained from a living body. Examples of the test substance are as described with respect to the test substance using the sensor of the first disclosure of this specification, and are particularly preferably one or more selected from glucose and lactic acid.

[0401] The working electrode of the sensor disclosed in the fourth specification includes a reagent layer disposed on the working electrode conductive layer and containing a reagent involved in the oxidation-reduction reaction of the test substance. The reagent can be appropriately selected depending on the test substance. The reagent can include a combination of an oxidoreductase and a mediator (electron carrier), or an oxidoreductase itself. The oxidoreductase can include a coenzyme.

[0402] The oxidoreductase may be an oxidase or a dehydrogenase. Specific examples of the oxidoreductase are as described in relation to the sensor of the first disclosure of this specification, and particularly preferably, the oxidoreductase may be glucose oxidase, lactate oxidase, glucose dehydrogenase, or lactate dehydrogenase.

[0403] Furthermore, the mediator is not particularly limited, and examples thereof are as described in relation to the sensor of the first disclosure of this specification.

[0404] The reagent layer provided on the working electrode of the sensor of the fourth disclosure of this specification may further contain, in addition to the reagent, components such as a buffer, a hydrophilic polymer compound, a conductive carbon filler, a crosslinking agent, etc. Examples of the hydrophilic polymer compound and the conductive carbon filler are as described in relation to the sensor of the first disclosure of this specification.

[0405] The working electrode of the sensor of 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 analyte present outside the protective film. Preferred embodiments of materials constituting the first protective film and the second protective film on the reagent layer that have such properties will be described later.

[0406] The reference electrode of the sensor disclosed in the fourth specification may include a protective film. The protective film of the reference electrode may contain a polymer compound. Examples of such a polymer compound 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 compound contained in the second protective film is particularly preferred.

[0407] In the sensor of the fourth disclosure of the present specification, the electrodes, including the working electrode and counter electrode, arranged on an insulating substrate, are preferably covered with an insulating layer except for the portion that comes into contact with the liquid sample. The insulating layer may include an insulating resin. The insulating layer may have a multilayer structure of two or more layers. The multilayer insulating layer may include, for example, a first insulating layer arranged on the insulating substrate and the conductive layer, and a second insulating layer arranged on the first insulating layer. Preferred aspects of the materials constituting the first insulating layer and the second insulating layer are as described for the sensor according to the first disclosure.

[0408] <Outline of Sensor 400> A sensor 400 according to an embodiment of the fourth disclosure will be described with reference to the drawings.

[0409] As shown in FIGS. 62 to 64 , the sensor 400 of this embodiment includes an insulating substrate 2, a first working electrode 10a, a second working electrode 10b, a reference electrode 20, and a counter electrode 30 arranged on a first surface 2a of the substrate 2, and 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. While the sensor 400 of this embodiment includes two working electrodes, in another embodiment not shown, the number of working electrodes may be only one or may be three or more. In the following description, when the first working electrode 10a and the second working electrode 10b are not to be distinguished, they may be referred to as the working electrodes 10a and 10b. Furthermore, 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. Although the sensor 400 of this embodiment is a three-electrode sensor including a working electrode, a reference electrode, and a counter electrode as electrodes, it may also be a two-electrode sensor including only a working electrode and a counter electrode without including a reference electrode. Although not shown, the reference electrode and / or the counter electrode may be provided on a substrate separate from the substrate on which the working electrode is disposed.

[0410] 21 for sensor 1 of the first disclosure, sensor 400 is immersed in a liquid sample X and used to detect a predetermined analyte in the liquid sample X. Specific examples of the liquid sample and the analyte are as described in the <Materials> section.

[0411] 64, the first working electrode 10a of the sensor 400 comprises a working electrode conductive layer 11a, a reagent layer 15a containing a reagent involved in the oxidation-reduction reaction of the analyte in the liquid sample, a first protective film 18aa disposed on the reagent layer 15a, and a second protective film 18ab disposed on the first protective film 18aa. Preferred aspects of the reagent are as described in the <Materials> section. Although not shown, 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. 64, and in FIG. 64, the first working electrode 10a is replaced by the second working electrode 10b, the working electrode conductive layer 11a is replaced by the working electrode conductive layer 11, the reagent layer 15a is replaced by the reagent layer 15b, the first protective film 18aa is replaced by the first protective film 18ba, the second protective film 18ab is replaced by the second protective film 18bb, and the protective film 18a is replaced by the protective film 18b.

[0412] When the reagent layers 15a, 15b of the working electrodes 10a, 10b of the sensor 400 contain a reagent that oxidizes an analyte in a liquid sample, electrons are transferred from the analyte to the working electrode conductive layers 11a, 11b under conditions in which a predetermined voltage is applied to the electrodes of the sensor 400. Similarly, when the reagent layers 15a, 15b contain a reagent that reduces an analyte in a liquid sample, electrons are transferred from the working electrode conductive layers 11a, 11b to the analyte. Because the amount of transferred electrons depends on the concentration of the analyte, the concentration or change in concentration of the analyte in the liquid sample can be measured based on the value of the current or a change in the current value flowing through the working electrodes 10a, 10b of the sensor 400.

[0413] The sensor 400 can constitute an analytical device for analyzing a test substance in a liquid sample. An example of the analytical device is an analytical device including a sensor 400, an analytical unit 102, and a control unit 104, in which the sensor 1 according to the first disclosure in the analytical device 100 shown in Fig. 24 is replaced with the sensor 400 according to the fourth disclosure. The functions of this analytical device are as described for the analytical device 100 shown in Fig. 24.

[0414] <Structure of Sensor 400> The structure of the sensor 400 according to the fourth disclosure will be described with reference to Fig. 62 and its cross-sectional views Figs. 63 and 64. The structure of the reference electrode 20 of the sensor 400 according to the fourth disclosure is the same as the structure of the reference electrode 20 of the sensor 1 according to the first disclosure. Therefore, the description of the reference electrode 20 of the sensor 1 according to the first disclosure with reference to Fig. 22 will be cited as the description of the reference electrode 20 of the sensor 400 according to the fourth disclosure.

[0415] The sensor 400 includes an insulating substrate 2 and working electrode conductive layers 11a and 11b, a counter electrode 30, a reference electrode conductive layer 21, and wiring 50 disposed on a first surface 2a of the substrate 2. In the illustrated embodiment of the sensor 400 according to the fourth disclosure, the counter electrode 30 is entirely made of a conductive layer. The conductive layer portion of the counter electrode may also be referred to as a "counter electrode conductive layer" or a "counter electrode conductive layer" to specifically refer to the counter electrode. A first insulating layer 3 is further disposed on the first surface 2a of the substrate 2, and a second insulating layer 4 is further disposed 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 greater than the height of the working electrode conductive layers 11a and 11b, the counter electrode 30, the reference electrode conductive layer 21, and the wiring 50 from the first surface 2a of the substrate 2. Therefore, a portion of the first insulating layer 3 is disposed on the working electrode conductive layers 11a and 11b, the counter electrode 30, the reference electrode conductive layer 21, and the wiring 50.

[0416] 63 , the first insulating layer 3 has a counter electrode first opening 302 formed in a position overlapping a portion of the counter electrode 30 and penetrating in the thickness direction T of the substrate 2. Furthermore, the second insulating layer 4 has a counter electrode second opening 402 formed in a position overlapping the entire counter electrode first opening 302 of the first insulating layer 3 and penetrating in the thickness direction T of the substrate 2. The counter electrode 30 is exposed to the outside through the counter electrode first opening 302 and the counter electrode second opening 402. When the sensor 400 is immersed in a liquid sample, a portion of the counter electrode 30 located on the bottom surface of the recess formed by the counter electrode first opening 302 comes into contact with the liquid sample.

[0417] 64, the working electrodes 10a, 10b include working electrode conductive layers 11a, 11b and reagent layers 15a, 15b disposed on the working electrode conductive layers 11a, 11b and containing a reagent participating in the oxidation-reduction reaction of the test substance. Preferred embodiments of the reagent in the reagent layers 15a, 15b are as described in the <Materials> section.

[0418] As in the first disclosure, 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 may be referred to as the "first reagent layer 15a" of the first working electrode 10a and the latter as the "second reagent layer 15b" of the second working electrode 10b. Furthermore, the reagent contained in the reagent layer 15a of the first working electrode 10a may be referred to as the "first reagent," and the reagent contained in the second reagent layer 15b of the second working electrode 10b may be referred to as the "second reagent." In a preferred embodiment, the sensor 400 includes a plurality of working electrodes 10a, 10b, and the first reagent contained in the first reagent layer 15a of the first working electrode 10a of the plurality of working electrodes 10a, 10b is different from 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. The sensor 400 according to this preferred embodiment can be used to detect multiple test substances, including a first test substance and a second test substance, in a liquid sample, because the first reagent is involved in an oxidation-reduction reaction of a first test substance in the liquid sample, and the second reagent is involved in an oxidation-reduction reaction of a second test substance different from the first test substance in the liquid sample.

[0419] As shown in FIG. 64, the working electrodes 10a, 10b are provided with protective films 18a, 18b consisting of first protective films 18aa, 18ba arranged on the reagent layers 15a, 15b and second protective films 18ab, 18bb ​​arranged on the first protective films 18aa, 18ba.

[0420] 64 , first insulating layer 3 has working electrode first openings 303a, 303b formed at positions overlapping with portions of working electrode conductive layers 11a, 11b, respectively, and penetrating in the thickness direction T of substrate 2. Furthermore, second insulating layer 4 has working electrode second openings 403a, 403b formed at positions overlapping with portions of first insulating layer 3 that entirely encompass working electrode first openings 303a, 303b, and penetrating in the thickness direction T of substrate 2. Reagent layers 15a, 15b are enclosed in working electrode first openings 303a, 303b, and the outer peripheries of reagent layers 15a, 15b are defined by the inner peripheries of working electrode first openings 303a, 303b. The first protective films 18aa, 18ba and the second protective films 18ab, 18bb ​​are enclosed within the working electrode second openings 403a, 403b, and the outer edges of the first protective films 18aa, 18ba and the second protective films 18ab, 18bb ​​are defined by the inner edges of the working electrode second openings 403a, 403b.

[0421] <Characteristics of the First and Second Protective Films of Sensor 400> The sensor 400 according to the fourth disclosure is characterized in that the first protective films 18aa, 18ba contain a first polymer compound having a cation exchange functional group, and the second protective films 18ab, 18bb ​​contain a second polymer compound having a cationic functional group. A sensor having this characteristic does not peel off from the first protective film and the second protective film, enabling stable measurement, even when used for continuous monitoring purposes in which a test substance is continuously measured over an extended period of time, such as several days, while immersed in a liquid sample.

[0422] Conventionally, sensors have been known that include a two-layer protective film structure, in which a first protective film containing a polymer compound (e.g., a copolymer compound of tetrafluoroethylene and perfluoro-2-(2-fluorosulfonylethoxy)propyl vinyl ether) containing a cation-exchange functional group for transporting cations such as protons between the reagent layer and the liquid sample is provided on the reagent layer, and a second protective film containing a polymer compound containing 4-vinylpyridine as a structural unit (e.g., P4VP-tBuMA (a block copolymer compound of poly-4-vinylpyridine and poly-tert-butyl methacrylate)) is further provided on the first protective film. The present inventors have found that when this sensor is immersed in a liquid sample and continuous electrochemical measurements of a test substance are performed, after the passage of a long period of time (more than three days), an abnormal value is detected in which the detected current value changes significantly, making accurate measurements impossible. They then confirmed that the sensor that showed the abnormal value had peeled off between the first and second protective films.

[0423] As a result of extensive research, the inventors have found that the above problems can be solved by a 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 protective film contains a cation-exchangeable functional group (i.e., anionic functional group), while the second polymer compound of the second protective film contains a cationic functional group. This is thought to be why the first protective film and the second protective film are closely attached to each other at their interface by electrostatic bonding, thereby preventing peeling.

[0424] In the fourth disclosure, the first polymer compound contained in the first protective film may be any compound as long as it has a cation exchange functional group, and preferably has a cation exchange functional group in its side chain. The cation exchange functional group is preferably a sulfonic acid group. The first polymer compound having a sulfonic acid group in its side chain is preferably a polymer compound containing, as a structural unit, a perfluoro compound having a sulfonic acid group in its side chain, more preferably a copolymer compound containing, as a structural unit, a perfluoro compound having a sulfonic acid group in its side chain and a perfluoro compound not having an ionic functional group in its side chain, particularly preferably a copolymer compound of tetrafluoroethylene and perfluoro-2-(2-fluorosulfonylethoxy)propyl vinyl ether, and most preferably Nafion (registered trademark).

[0425] In the fourth disclosure, the second polymer compound contained in the second protective film may contain a cationic functional group, and preferably has a cationic functional group in a side chain. Because the pH of the liquid sample to be analyzed may fluctuate during long-term measurement, the cationic functional group in the second polymer compound is preferably a pH-independent cationic functional group, and particularly preferably a functional group containing a quaternary ammonium cation. The quaternary ammonium cation is preferably derived from a reaction between a tertiary amine and an epoxy group. Examples of the tertiary amine include a pyridyl group, and particularly a 4-pyridyl group.

[0426] The second polymer compound preferably 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 constituent units of the second polymer compound is 50 mol % or more. Because such a second polymer compound is highly hydrophobic, even when the sensor according to the fourth disclosure is immersed in an aqueous liquid sample such as a cell culture medium for long-term measurements, the second protective film does not swell with water, 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, the ratio of the second unit to the total amount of constituent units of the second polymer compound can be 70 mol % or less. Examples of hydrophobic functional groups include saturated hydrocarbon groups having 1 to 10 carbon atoms. For example, second units derived from methacrylic acid esters or acrylic acid esters of monohydric alcohols containing saturated hydrocarbon groups having 1 to 10 carbon atoms, specifically tert-butyl methacrylate, are preferred.

[0427] The second polymer compound containing a first unit containing a cationic functional group and a second unit containing a hydrophobic functional group can be obtained, for example, by reacting an uncrosslinked polymer compound containing a third unit containing a tertiary amine and the second unit containing a hydrophobic functional group with a crosslinker compound containing two or more epoxy groups for a certain period of time, thereby reacting the tertiary amine of the third unit with the epoxy group of the crosslinker compound and converting the third unit to the first unit containing a quaternary ammonium cation. In this case, the epoxy group of the crosslinker compound is preferably 6.8 mol% or more, more preferably 23 mol% or less, relative to the tertiary amine in the third unit (for example, 6.8 mol% or more and 23 mol% or less, relative to the tertiary amine in the third unit), thereby resulting in a second polymer compound having a high density of quaternary ammonium cations, which is preferable because it strengthens the electrostatic bond between the first protective film and the second protective film. Here, when calculating the ratio (mol %) of the epoxy groups of the crosslinker compound to the tertiary amine of the third unit in the uncrosslinked polymer compound, the number of moles of the tertiary amine can be calculated based on the molecular weight of the monomer of the third unit, and the number of moles of the epoxy groups can be calculated based on the molecular weight of the crosslinker compound (when the crosslinker compound is a polymer compound, its number average molecular weight is regarded as a uniform molecular weight).

[0428] In the uncrosslinked polymer compound, the third unit containing a tertiary amine is preferably a structural unit derived from 4-vinylpyridine.

[0429] A preferred example of the uncrosslinked polymer compound is a copolymer compound of 4-vinylpyridine and tert-butyl methacrylate. More preferably, the copolymer compound contains second units derived from tert-butyl methacrylate in an amount of preferably 50 mol% or more, more preferably 70 mol% or less, based on the total amount of third units derived from 4-vinylpyridine and second units derived from tert-butyl methacrylate (the proportion of the second units to the total amount of the third units and the second units is, for example, 50 mol% or more and 70 mol% or less). It is particularly preferred that the copolymer compound of 4-vinylpyridine and tert-butyl methacrylate is a block copolymer compound. The molecular weight of the uncrosslinked polymer compound is not particularly limited, but the number average molecular weight (Mn) can be, for example, 150,000 to 500,000.

[0430] The crosslinker compound containing two or more epoxy groups is, for example, a linear compound having an epoxy group at each end, preferably a polyalkylene glycol compound having both ends modified with a glycidyl group, specifically poly(ethylene glycol) diglycidyl ether. The molecular weight of the poly(ethylene glycol) diglycidyl ether is not particularly limited, but the number average molecular weight (Mn) can be, for example, 200 to 2,000.

[0431] <Method for manufacturing sensor 400> A preferred method for manufacturing the sensor 400 according to the third disclosure, in which the second protective films 18ab, 18bb ​​include a second polymer compound including a first unit including a quaternary ammonium cation derived from a reaction between a tertiary amine and an epoxy group, and a second unit including a hydrophobic functional group, includes the following steps: a first step of holding a mixture containing, in alcohol, an uncrosslinked polymer compound including a third unit including a tertiary amine and a second unit including a hydrophobic functional group, and a crosslinker compound including two or more epoxy groups, at a temperature of 20°C or higher for 6 hours or more, thereby reacting the tertiary amine of the third unit with the epoxy group of the crosslinker compound and converting the third unit to the first unit including the quaternary ammonium cation, thereby obtaining a second polymer compound including the first unit and the second unit in alcohol; a second step of coating the first protective films 18aa, 18ba and drying the coating to form second protective films 18ab, 18bb ​​containing the second polymer compound on the first protective films 18aa, 18ba of a sensor component including the second polymer compound, the second polymer compound being disposed on the first protective films 18aa, 18ba and then dried.

[0432] According to this method, in the first step, the crosslinking reaction between the tertiary amine of the third unit in the uncrosslinked polymer compound and the epoxy group of the crosslinker compound proceeds sufficiently, resulting in a composition for forming a second protective film containing a second polymer compound with a high density of quaternary ammonium cations. The temperature conditions in the first step are preferably 30°C or higher, more preferably 40°C or higher, with the upper limit being preferably below the boiling point of the alcohol. For example, when ethanol is used as the alcohol, the upper limit is preferably below 78°C. The reaction time in the first step is preferably 12 hours or longer, with no particular upper limit, for example, 48 hours or shorter (for example, the reaction time in the first step is 12 hours or longer and 48 hours or shorter). Ethanol, methanol, and propanol are preferred as the alcohol, with ethanol being particularly preferred. The preferred embodiments of the copolymer compound and the crosslinker compound are as described above.

[0433] In the second step, the composition for forming the second protective film is applied or dropped onto the first protective films 18aa, 18ba to cover them, and then dried by evaporating the alcohol solvent to form the second protective films 18ab, 18bb.

[0434] In this method, crosslinking is performed in advance in the first step, so there is no need to perform a further crosslinking process after forming the second protective films 18ab and 18bb ​​in the second step, which reduces the risk that the reagent layers 15a and 15b containing reagents such as enzymes will be damaged by heat during the crosslinking process.

[0435] <Example of the Fourth Disclosure> In Experiments 8 to 10 of the sensor according to the fourth disclosure, a sensor 400 having a configuration in FIG. 62 that does not include a second working electrode 10b and only includes a first working electrode 10a (hereinafter referred to as "working electrode 10a") as a working electrode was used as an example and comparative example of the sensor according to the fourth disclosure.

[0436] The characteristics of the sensors used in each experiment will be explained individually in Experiments 8 to 10. First, an outline of the materials and manufacturing methods common to the sensors used in each experiment will be explained.

[0437] (Substrate) As in the first disclosed example, a substrate made of polyethylene terephthalate and having a thickness of 188 μm, having the shape shown in FIG. 62, was used as the insulating substrate 2 .

[0438] (Conductive Layer) A carbon paste was applied to the first surface 2 a of the insulating substrate 2 and heated at 140° C. for 1 hour, thereby forming a working electrode conductive layer 11 a, a reference electrode conductive layer 21, and a counter electrode (counter electrode conductive layer) 30, each having the same shape as that shown in FIG. 1 relating to the first disclosure, as well as wiring 50 electrically connected to each of them, using a 5 μm-thick carbon conductive layer.

[0439] (First Insulating Layer) Next, a first insulating layer 3 was laminated on the first surface 2a of the substrate 2 on which the carbon conductive layer was disposed. The first insulating layer 3 was made of a fluororesin containing a copolymer containing vinylidene fluoride and hexafluoropropylene, and had a thickness of 5 μm on the carbon conductive layer, covering the first surface 2a of the substrate 2 and the carbon conductive layer. The method for manufacturing the first insulating layer 3 was as described in the first disclosed example.

[0440] The working electrode first opening 303a on the working electrode conductive layer 11a of the first insulating layer 3 was circular and had a diameter of 1.2 mm. The reference electrode first opening 301 was circular and had a diameter of 1.1 mm, and the counter electrode first opening 302 was rectangular and had dimensions of 1.8 mm × 2.1 mm.

[0441] (Second insulating layer) Next, a composition containing a polyester resin and a fluorine-based surface-modifying additive containing a perfluoroalkyl group in a solvent was applied onto the first insulating layer 3, and heated at 140°C for 1 hour to laminate a second insulating layer 4 having a thickness of 40 µm.

[0442] The working electrode second opening 403a of the second insulating layer 4 on the working electrode conductive layer 11a was a circle with a diameter of 2 mm. The reference electrode second opening 401 was a circle with a diameter of 2 mm, and the counter electrode second opening 402 was a rectangle with dimensions of 1.8 mm × 2.1 mm.

[0443] (Working Electrode Reagent Layer) In the working electrode first opening 303a of the first insulating layer 3, 0.4 mg of a glucose measurement reagent solution containing a sodium phosphate buffer solution (pH 7.4) with a final concentration of 3 mM, a carbon black dispersion with a final concentration of 14 mg / mL (carbon black concentration), a polymer-bound mediator, FAD-dependent glucose dehydrogenase with a final concentration of 3200 U / mL, and a crosslinker with a final concentration of 0.1 mM (concentration of binding sites) was applied to the working electrode conductive layer 11a, and then dried to form a reagent layer 15a containing glucose dehydrogenase and the mediator.

[0444] (Working electrode protective film) A first protective film 18aa was formed on the reagent layer 15a in the second working electrode opening 403a of the second insulating layer 4, and a second protective film 18ab was formed on the first protective film 18aa to form a protective film 18a having a two-layer structure consisting of the first protective film 18aa and the second protective film 18ab. Specific methods for manufacturing the first protective film 18aa and the second protective film 18ab will be described in Experiments 8 to 10.

[0445] (Reference Electrode) The cross-sectional structure of the reference electrode 20 is the same as that shown in Fig. 22 for the sensor of the first disclosure, and therefore, the manufacturing method of the reference electrode 20 will be outlined below with reference to Fig. 22. A silver-silver chloride paste was applied to 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. Subsequently, 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, thereby forming the reference electrode 20.

[0446] <Fourth Disclosure / Experiment 8> (Working Electrode Protective Film) To 23,093.67 mg of a 21.5 wt % Nafion (registered trademark) dispersion (manufactured by Sigma-Aldrich Corporation), 6,472.79 mg of ethanol (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) and 1,226.46 mg of a 5 mol / L aqueous sodium hydroxide solution (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) were added to adjust the pH of the dispersion (neutralization of the cation exchange group), and the precipitate was dissolved using a vortex mixer to prepare 30,792.92 mg of a 16.12 wt % Nafion (registered trademark) dispersion. The obtained 16.12 wt % Nafion (registered trademark) dispersion was applied in an amount of 0.6 mg onto the reagent layer 15a in the working electrode second opening 403a of the second insulating layer 4 and dried to form a first protective film 18aa containing Nafion (registered trademark) as a polymer compound having a cation exchange functional group.

[0447] Next, the following two components were mixed with ethanol to the final concentrations shown below and reacted at room temperature (25°C ± 3°C) for about 1 hour to prepare two types of compositions for forming a second protective film. 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.

[0448] Composition for forming second protective film in Experiment 8-1: P4VP-tBuMA (poly-4-vinylpyridine Mn: 74,000, poly-tert-butyl methacrylate Mn: 87,000, Mw / Mn: 1.16, manufactured by Polymer Source), final concentration 5.72 wt% PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ∼1,000, manufactured by Sigma-Aldrich), final concentration 0.91 wt%

[0449] Composition for forming second protective film in Experiment 8-2: P4VP-tBuMA (poly-4-vinylpyridine Mn: 120,000, poly-tert-butyl methacrylate Mn: 270,000, Mw / Mn: 1.15, manufactured by Polymer Source), final concentration 5.72 wt% PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ∼1,000, manufactured by Sigma-Aldrich), final concentration 2.0 wt%

[0450] 0.65 mg of the composition for forming the second protective film of Experiment 8-1 or Experiment 8-2 was applied to the first protective film 18aa in the working electrode second opening 403a of the second insulating layer 4 and dried to form the second protective film 18ab of Experiment 8-1 or Experiment 8-2.

[0451] The sensor 400 provided with the second protective film 18ab of Experiment 8-1 was used as the sensor of Experiment 8-1.

[0452] The sensor 400 provided with the second protective film 18ab of Experiment 8-2 was used as the sensor of Experiment 8-2.

[0453] As a liquid sample containing a test substance, RPMI medium containing a final concentration of 30 mM glucose and 15 mM lactic acid and adjusted to a pH of 6.5 was prepared using RPMI-1640 Medium (Sigma-Aldrich R1383), glucose (Fujifilm Wako Pure Chemical Industries, Ltd.), and sodium lactate (Sigma-Aldrich).

[0454] The sensor of Experiment 8-1 or Experiment 8-2 was immersed in the liquid sample, a voltage of 100 mV was applied to the working electrode 10a relative to the reference electrode 20 (Ag / AgCl), and the current value between the working electrode 10a and the counter electrode 30 was measured continuously for about 14 days. N=12 measurements were performed.

[0455] The measurement results of the current values ​​are shown in Figure 65. Figure 65A shows the measurement results using the sensor of Experiment 8-1, and Figure 65B shows the measurement results using the sensor of Experiment 8-2.

[0456] In some samples of the sensor in Experiment 8-1, which had a second protective film 18ab formed from a composition for forming a second protective film containing P4VP-tBuMA, which contained P4VP with Mn: 74,000 and tBuMA with Mn: 87,000, and PEGDGE at a final concentration of 0.91 wt%, abnormally high current values ​​were detected after more than four days had passed since the start of measurement.

[0457] On the other hand, in the sensor of Experiment 8-2, which had a second protective film 18ab formed from a composition for forming a second protective film containing P4VP-tBuMA, which contained P4VP with Mn: 120,000 and tBuMA with Mn: 270,000, and PEGDGE at a final concentration of 2.0 wt%, no abnormally high current values ​​were detected even after more than four days had passed since the start of measurement.

[0458] This result suggests that by increasing the ratio of tBuMA-derived units containing hydrophobic functional groups and the concentration of the crosslinker compound in the second protective film 18ab, it is possible to suppress the occurrence of abnormally high current values ​​during long-term measurements.

[0459] <Fourth Disclosure / Experiment 9> Two types of compositions were designed as compositions for forming a second protective film, each containing P4VP-tBuMA and PEGDGE in ethanol at the following final concentrations: In the experiments shown below regarding the fourth disclosure, the ratio (mol %) of epoxy groups derived from PEGDGE to pyridine derived from P4VP was calculated using the number of moles of pyridine derived from P4VP calculated based on the molecular weight of the P4VP monomer and the number of moles of epoxy groups derived from PEGDGE calculated based on the number average molecular weight of PEGDGE (regarded as a uniform molecular weight of PEGDGE).

[0460] Composition A (10.17 mol% of epoxy groups derived from PEGDGE relative to pyridine derived from 4-vinylpyridine): P4VP-tBuMA (poly-4-vinylpyridine Mn: 120,000, poly-tert-butyl methacrylate Mn: 270,000, Mw / Mn: 1.15, manufactured by Polymer Source), final concentration 5.72 wt% PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ∼1,000, manufactured by Sigma-Aldrich), final concentration 0.9 wt%

[0461] Composition B (6.77 mol% of epoxy groups derived from PEGDGE relative to pyridine derived from 4-vinylpyridine): P4VP-tBuMA (poly-4-vinylpyridine Mn: 120,000, poly-tert-butyl methacrylate Mn: 270,000, Mw / Mn: 1.15, manufactured by Polymer Source), final concentration 5.72 wt% PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ∼1,000, manufactured by Sigma-Aldrich), final concentration 0.6 wt%

[0462] A mixture containing the components of composition A in ethanol was held at 40° C. for 16 hours to perform pre-crosslinking, thereby preparing a composition for forming a second protective film in Experiment 9-1.

[0463] A mixture containing the components of composition B in ethanol was held at 40° C. for 16 hours to perform pre-crosslinking, thereby preparing a composition for forming a second protective film of Experiment 9-2.

[0464] A mixture containing the components of composition B in ethanol was held at 53° C. for 16 hours for pre-crosslinking to prepare a composition for forming a second protective film of Experiment 9-3.

[0465] The sensors of Experiments 9-1, 9-2, and 9-3 were prepared using the same procedure as Experiment 8, except that the second protective film 18ab of the working electrode 10a was formed using the compositions for forming the second protective film of Experiments 9-1, 9-2, and 9-3.

[0466] The sensor of Experiment 9-1, Experiment 9-2, or Experiment 9-3 was immersed in the same liquid sample as used in Experiment 8, a voltage of 100 mV was applied to the working electrode 10a relative to the reference electrode 20 (Ag / AgCl), and the current value between the working electrode 10a and the counter electrode 30 was measured continuously for about 14 days. The measurements were performed with N=8 in Experiment 9-1, N=12 in Experiment 9-2, and N=4 in Experiment 9-3.

[0467] The measurement results of the current values ​​are shown in Figure 66. Figure 66A shows the measurement results using the sensor of Experiment 9-1, Figure 66B shows the measurement results using the sensor of Experiment 9-2, and Figure 66C shows the measurement results using the sensor of Experiment 9-3.

[0468] It was suggested that a sensor having a second protective film 18ab formed using a composition for forming a second protective film, which was prepared by mixing P4VP-tBuMA and PEGDGE and then reacting them for a certain period of time to pre-crosslink the second protective film, on a first protective film 18aa containing Nafion (registered trademark) containing cation exchange functional groups, could suppress the occurrence of abnormally high current values ​​during long-term measurements. It was also suggested that increasing the temperature during pre-crosslinking could further suppress the occurrence of abnormally high current values ​​during long-term measurements.

[0469] <Fourth Disclosure / Experiment 10> Three types of compositions containing P4VP-tBuMA and PEGDGE in ethanol at the following final concentrations were designed as compositions for forming a second protective film.

[0470] Composition C (22.67 mol% of epoxy groups derived from PEGDGE relative to pyridine derived from 4-vinylpyridine): P4VP-tBuMA (poly-4-vinylpyridine Mn: 120,000, poly-tert-butyl methacrylate Mn: 270,000, Mw / Mn: 1.15, manufactured by Polymer Source), final concentration 5.72 wt% PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ∼1,000, manufactured by Sigma-Aldrich), final concentration 2.0 wt%

[0471] Composition D (10.17 mol% epoxy groups derived from PEGDGE relative to pyridine derived from 4-vinylpyridine): P4VP-tBuMA (poly-4-vinylpyridine Mn: 120,000, poly-tert-butyl methacrylate Mn: 270,000, Mw / Mn: 1.15, manufactured by Polymer Source), final concentration 5.72 wt% PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ∼1,000, manufactured by Sigma-Aldrich), final concentration 0.9 wt%

[0472] Composition E (6.77 mol% of epoxy groups derived from PEGDGE relative to pyridine derived from 4-vinylpyridine): P4VP-tBuMA (poly-4-vinylpyridine Mn: 120,000, poly-tert-butyl methacrylate Mn: 270,000, Mw / Mn: 1.15, manufactured by Polymer Source), final concentration 5.72 wt% PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ∼1,000, manufactured by Sigma-Aldrich), final concentration 0.6 wt%

[0473] A mixture containing the components of composition C in ethanol was held at room temperature (25°C ± 3°C) for about 1 hour to pre-crosslink the mixture, thereby preparing a composition for forming a second protective film for Experiment 10-1.

[0474] A mixture containing the components of composition D in ethanol was held at 40° C. for 16 hours to perform pre-crosslinking, thereby preparing a composition for forming a second protective film in Experiment 10-2.

[0475] A mixture containing the components of composition E in ethanol was held at 53° C. for 16 hours for pre-crosslinking to prepare a composition for forming a second protective film of Experiment 10-3.

[0476] The sensors of Experiments 10-1, 10-2, and 10-3 were prepared using the same procedure as Experiment 8, except that the second protective film 18ab of the working electrode 10a was formed using the compositions for forming the second protective film of Experiments 10-1, 10-2, and 10-3.

[0477] Subsequently, the fabricated sensors of Experiments 10-1, 10-2, and 10-3 were each stored in the presence of a desiccant at 60° C. for 14 days, with the aim of accelerating degradation.

[0478] After storage at 60°C for 14 days, each sensor was immersed in the same liquid sample as used in Experiment 8, a voltage of 100 mV was applied to the working electrode 10a relative to the reference electrode 20 (Ag / AgCl), and the current value between the working electrode 10a and the counter electrode 30 was measured continuously for 10 days. N=4 measurements were performed. The current value 24 hours after the start of measurement (average of N=4) was set to 100%, and current values ​​measured at later time points (average of N=4) were expressed as relative values.

[0479] The measurement results of the current values ​​are shown in FIG.

[0480] The sensors of Experiments 10-2 and 10-3, which had a second protective film 18ab formed using a composition for forming a second protective film, which was pre-crosslinked by mixing P4VP-tBuMA and PEGDGE and reacting for 6 hours or more, on a first protective film 18aa containing Nafion (registered trademark) containing cation exchange functional groups, were confirmed to have smaller fluctuations in current value after a temperature accelerated test compared to the sensor of Experiment 10-1. It was also confirmed that increasing the temperature during pre-crosslinking could further reduce fluctuations in current value after a temperature accelerated test. These results suggest that a sensor with excellent long-term stability can be achieved by forming a second protective film 18ab on a first protective film 18aa containing Nafion (registered trademark) using a composition for forming a second protective film, which was pre-crosslinked by mixing P4VP-tBuMA and PEGDGE and reacting for 6 hours or more at a temperature of 20°C or higher.

[0481] <Additional Note on the Fourth Disclosure> A sensor for electrochemically measuring a test substance (allanite) in a liquid sample such as a cell culture medium, which includes a working electrode including a reagent layer containing a reagent involved in a redox reaction, has been conventionally known.

[0482] Such sensors are sometimes used for continuous monitoring, where they are immersed in a liquid sample and measure a test substance continuously over a long period of time, such as several days, and in this case, the sensor is required to have a small change in response to enable stable measurement over the measurement period.

[0483] Therefore, an object of the fourth disclosure of this specification is to provide a sensor that can perform stable measurements with little change in responsiveness even when the sensor is immersed in a liquid sample for a long period of time, and a method for manufacturing the same. The sensor according to the fourth disclosure can be suitably used for long-term continuous monitoring.

[0484] Specifically, this specification discloses, as a fourth disclosure, a sensor and a manufacturing method thereof described in any one or more of Supplementary Notes 4-1 to 4-10 below.

[0485] (Appendix 4-1) A sensor comprising an insulating substrate and a working electrode disposed on the substrate, wherein the working electrode comprises: a working electrode conductive layer disposed on the substrate; a reagent layer disposed on the working electrode conductive layer and containing a reagent involved in a redox reaction; a first protective film disposed on the reagent layer and containing a first polymer compound containing a cation exchange functional group; and a second protective film disposed on the first protective film and containing a second polymer compound containing a cationic functional group. (Appendix 4-2) The sensor according to Appendix 4-1, wherein the second polymer compound contains a first unit containing the cationic functional group and a second unit containing a hydrophobic functional group, and the ratio of the second unit to the total amount of constituent units of the second polymer compound is 50 mol % or more. (Appendix 4-3) The sensor according to Appendix 4-1 or 4-2, wherein the cationic functional group is pH-independent. (Appendix 4-4) 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. (Appendix 4-5) The sensor according to Appendix 4-3, wherein the reaction is a reaction between the tertiary amine and 6.8 mol% or more of the epoxy groups relative to the tertiary amine. (Appendix 4-6) The sensor according to any one of Appendices 4-1 to 4-5, wherein the second polymer compound is a copolymer compound of 4-vinylpyridine and tert-butyl methacrylate crosslinked by a crosslinker compound containing two or more epoxy groups.(Appendix 4-7) A method for manufacturing a sensor comprising an insulating substrate and a working electrode disposed on the substrate, wherein the working electrode comprises: a working electrode conductive layer disposed on the substrate; a reagent layer disposed on the working electrode conductive layer and containing a reagent involved in a redox reaction; a first protective film disposed on the reagent layer and containing a first polymer compound containing a cation exchange functional group; and a second protective film disposed on the first protective film and containing a second polymer compound 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 step of holding a mixture containing, in alcohol, an uncrosslinked polymer compound including a third unit containing a tertiary amine and the second unit including the hydrophobic functional group, and a crosslinker compound including two or more epoxy groups at a temperature of 20°C or higher for 6 hours or more to react the tertiary amine of the third unit with the epoxy group of the crosslinker compound and convert the third unit to the first unit including the quaternary ammonium cation, thereby obtaining a composition for forming a second protective film, the second polymer compound including the first unit and the second unit, in alcohol; and a step of coating the composition for forming a second protective film on the first protective film of a sensor component including: the substrate; the working electrode conductive layer disposed on the substrate; the reagent layer disposed on the working electrode conductive layer; and the first protective film disposed on the reagent layer, and drying the composition to form the second protective film containing the second polymer compound. (Appendix 4-8) The method according to Appendices 4-7, wherein the uncrosslinked polymer compound contains the third units and the second units such that the ratio of the second units is 50 mol % or more relative to the total amount of the third units and the second units. (Appendix 4-9) The method according to Appendices 4-7 or 4-8, wherein the mixture contains the uncrosslinked polymer compound and the crosslinker compound such that the epoxy groups of the crosslinker compound are 6.8 mol % or more relative to the tertiary amines of the third units. (Appendix 4-10) The method according to any one of Appendices 4-7 to 4-9, wherein the uncrosslinked polymer compound is a copolymer compound of 4-vinylpyridine and tert-butyl methacrylate.

[0486] REFERENCE SIGNS LIST 1 Sensor 2 Substrate 3, 3a, 3b First insulating layer 3a1, 3b1 First opening 3a10, 3b10 Inner periphery of first opening 3a2, 3b2 Water-repellent surface 3a3, 3b3 Portion of first insulating layer enclosing the entire first opening 4, 4a, 4b Second insulating layer 4a1, 4b1 Second opening 4a10, 4b10 Inner periphery of second opening 4b2, 4b2 Surface repellent to alcohol 10a, 10b Working electrode 10a First working electrode 10b Second working electrode 11a, 11b Conductive layer of working electrode 11a Conductive layer of first working electrode 11b Conductive layer of second working electrode 15a, 15b Reagent layer 15a10, 15b10 Outer periphery of reagent layer 15a First reagent layer of first working electrode 15b Second reagent layer 16a, 16b of second working electrode Protective film 16a10,16b10 Outer periphery of protective film 16a First protective film 16ba Second protective film 16bb Third protective film 20 Reference electrode 30 Counter electrode 50 Wiring C Cell T Thickness direction of substrate X Liquid sample A Liquid composition containing a reagent involved in an oxidation-reduction reaction in water B Liquid composition containing a protective film component in alcohol 1101, 1101', 1101'' Sensor 1200 Insulating substrate 1201 Tip portion of substrate 1202 First surface of substrate 1210 Main body portion 1211 First end portion of main body portion 1220 Connection portion 1222 Second end portion of connection portion 1223 Third end portion of connection portion 1225 Bent portion 1230 Base end portion 1234 Fourth end portion of base end portion 1300 Detection electrode 1310 Working electrode 1311 Working electrode conductive layer 1315 Reagent layer 1316 Working electrode protective film 1320 Reference electrode 1321 Reference electrode conductive layer 1322 Silver / silver chloride layer 1326 Reference electrode protective film 1330 Counter electrode 1331 Upper surface of counter electrode 1501 Working electrode opening 1502 Reference electrode opening 1503 Counter electrode opening 1050 Insulating layer 1051 First insulating layer 1052, 1052a, 1052b, 1052c Second insulating layer 1055 Non-insulating region 1061 Tip opening 1062 Flow path 1062A Extension portion 1005 Wiring 1006 Terminal 900 Sensor unit 957 Lower support plate (support member) 957d First engaging portion 959 Upper support plate (support member) 959a Second engaging portion T1 Thickness direction of substrate X1 Liquid sample 300, 400 Sensor 60 Counter electrode 60a Portion of counter electrode in contact with liquid sample 11a, 11b Working electrode conductive layer 11a1, 11b1 Portion of working electrode conductive layer in contact with liquid sample 18a Protective film 18aa First protective film 18ab Second protective film

Claims

1. A sensor comprising an insulating substrate and a working electrode disposed on the substrate, The working electrode is a conductive layer disposed on the substrate; a first insulating layer having a water-repellent surface and a first opening formed at a position overlapping a portion of the conductive layer in a plan view from the thickness direction of the substrate, the first opening penetrating the thickness direction; and a second insulating layer disposed on the first insulating layer, the second opening being formed in a position overlapping a portion of the first insulating layer that entirely contains the first opening in a plan view in the thickness direction, the second insulating layer penetrating the first insulating layer in the thickness direction, and the second insulating layer having a surface that is liquid-repellent with respect to alcohol; a reagent layer disposed within the first opening of the first insulating layer, the reagent layer having an outer periphery defined by an inner periphery of the first opening of the first insulating layer and including a reagent participating in an oxidation-reduction reaction; a protective film disposed within the second opening of the second insulating layer, the protective film including an outer periphery defined by an inner periphery of the second opening of the second insulating layer; A sensor comprising:

2. The surface of the first insulating layer contains a fluororesin. The sensor of claim 1 .

3. the surface of the second insulating layer contains a compound containing a perfluoroalkyl group; The sensor of claim 1 .

4. A plurality of the working electrodes are provided, a first reagent contained in a first reagent layer of a first working electrode included in the plurality of working electrodes and a second reagent contained in a second reagent layer of a second working electrode different from the first working electrode are different from each other; The sensor of claim 1 .

5. the first reagent contains glucose dehydrogenase or glucose oxidase; The second reagent includes lactate oxidase or lactate dehydrogenase. The sensor of claim 4.

6. the protective film includes a first protective film containing a polymer compound containing 4-vinylpyridine as a structural unit; The sensor of claim 1 .

7. the protective film includes a second protective film disposed on the reagent layer and including a polymer compound having a cation exchange functional group, and a third protective film disposed on the second protective film and including a polymer compound having 4-vinylpyridine as a constituent unit; The sensor of claim 1 .

8. Further comprising a counter electrode and a reference electrode disposed on the substrate. The sensor of claim 1 .

9. A method for manufacturing the sensor according to claim 1, comprising the steps of: forming a droplet of liquid composition A containing the reagent 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, and then drying the droplet to form the reagent layer; and After the reagent layer is formed, droplets of a liquid composition B containing a protective film component in alcohol are formed in the second opening of the second insulating layer, and then dried to form the protective film. The method according to claim 1, further comprising:

10. The surface of the first insulating layer contains a fluororesin.

10. The method of claim 9.

11. the surface of the second insulating layer contains a compound containing a perfluoroalkyl group; 10. The method of claim 9.

12. The sensor includes a plurality of the working electrodes, The formation of the reagent layer includes forming a first reagent layer of a first working electrode included in the plurality of working electrodes by forming a droplet of a first liquid composition A containing a first reagent in water in the first opening of the first insulating layer of the first working electrode, and then drying the droplet; a second reagent layer of a second working electrode different from the first working electrode included in the plurality of working electrodes is formed in the first opening of the first insulating layer of the second working electrode by forming a droplet of a second liquid composition A containing a second reagent different from the first reagent in water and then drying the droplet; Including, 10. The method of claim 9.

13. the first reagent contains glucose dehydrogenase or glucose oxidase; The second reagent includes lactate oxidase or lactate dehydrogenase. The method of claim 12.

14. the protective film includes a first protective film disposed on the reagent layer and including a polymer compound including 4-vinylpyridine as a constituent unit; The formation of the protective film is After the reagent layer is formed, the first protective film is formed in the second opening of the second insulating layer by forming droplets of a first liquid composition B containing a polymer compound containing 4-vinylpyridine as a structural unit in alcohol, and then drying the droplets. Including, 10. The method of claim 9.

15. the protective film includes a second protective film disposed on the reagent layer and including a polymer compound having a cation exchange functional group, and a third protective film disposed on the second protective film and including a polymer compound having 4-vinylpyridine as a constituent unit; The formation of the protective film is After the formation of the reagent layer, the second protective film is formed in the second opening of the second insulating layer by forming droplets of a second liquid composition B containing a polymer compound having the cation exchange functional group in an alcohol, and then drying the droplets; and After the second protective film is formed, the third protective film is formed in the second opening of the second insulating layer by forming droplets of a third liquid composition B containing a polymer compound containing 4-vinylpyridine as a structural unit in alcohol, and then drying the droplets. Including, 10. The method of claim 9.