Electrode, sensor, and method for producing sensor

Abstract

Provided is an electrode comprising a surface part that makes it possible to accurately form, on a prescribed region, a reagent layer having designed dimensions. An electrode (10) comprises: a surface part (11); a first region (110) that is formed on the surface part (11), that includes a first outer periphery part (111), and that has first surface free energy; a second region (120) that is formed on the surface part (11), that surrounds the first region (110), that includes a second inner periphery part (121) which is in contact with the first outer periphery part (111) of the first region (110) and a second outer periphery part (122) which is located more outward than the second inner periphery part (121), and that has second surface free energy higher than the first surface free energy; and a third region(130) that surrounds the second region (120), that includes a third inner periphery part (131) which is in contact with the second outer periphery part (122) of the second region (120), and that has third surface free energy lower than the second surface free energy.

Description

Electrode, sensor, and method for manufacturing the sensor

[0001] The present invention relates to, for example, an electrode, a sensor, and a method for manufacturing the sensor.

[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. The working electrode typically includes an electrode and a reagent layer disposed on the electrode and containing a reagent (e.g., an oxidoreductase and an electron carrier) involved in a redox reaction.

[0003] For example, Patent Document 1 describes a biosensor for detecting the presence of a target substance contained in a liquid sample, which comprises an insulating substrate having a recess formed in a portion thinner than the surrounding area, at least one of a working electrode and a counter electrode disposed in the recess, and a reaction reagent disposed in the recess and reacting with a specific substance in the liquid sample. An example of Patent Document 1 describes that the circular recess is formed on the surface of a polyethylene terephthalate substrate, and the working electrode is formed in the recess, and then the reagent layer is formed.

[0004] JP 2010-32501 A

[0005] However, the above-mentioned conventional sensor having a working electrode including an electrode and a reagent layer disposed in a recess in an insulating substrate has the following problems.

[0006] The detection sensitivity of a sensor including a working electrode for detecting a analyte depends on the amount and density of the reagent contained in the reagent layer on the working electrode, the contact area between the working electrode and the reagent layer, etc. Therefore, in order to improve the accuracy of detection of a analyte by a sensor including a working electrode, it is necessary to form a reagent layer with uniform dimensions (area, thickness, shape) on the electrode. In the biosensor described in Patent Document 1, the dimensions of the reagent layer on the working electrode and the contact area between the working electrode and the reagent layer can vary depending on the position and shape of the electrode within the recess of the insulating substrate, so it may be difficult to accurately form a reagent layer with the designed dimensions on a predetermined area on the surface of the electrode.

[0007] An object of the present invention is to provide an electrode having a surface portion that allows a reagent layer of designed dimensions to be formed accurately on a specified area, a sensor including the electrode, and a method for manufacturing the sensor.

[0008] The present invention relates to an electrode disposed on an insulating substrate, comprising: a surface portion located on the opposite side to the substrate when disposed on the substrate; a first region formed on the surface portion, including a first outer peripheral edge portion, at least a portion of which has a first surface free energy; a second region formed on the surface portion, including a second inner peripheral edge portion surrounding the first region and in contact with the first outer peripheral edge portion of the first region and a second outer peripheral edge portion located outside the second inner peripheral edge portion, the second region having a second surface free energy greater than the first surface free energy; and a third region formed on the surface portion, including a third inner peripheral edge portion surrounding the second region and in contact with the second outer peripheral edge portion of the second region, the third region having a third surface free energy smaller than the second surface free energy.

[0009] The present invention also relates to an electrode disposed on an insulating substrate, comprising: a surface portion located on the opposite side to the substrate when the electrode is disposed on the substrate; a first region formed on the surface portion, the first region including a first outer peripheral edge portion, at least a portion of which has a first surface free energy; a second region formed on the surface portion, the second region including a second inner peripheral edge portion surrounding the first region, in contact with the first outer peripheral edge portion of the first region, and a second outer peripheral edge portion located outside the second inner peripheral edge portion, the second region having a second surface free energy greater than the first surface free energy; and an insulating layer, at least a portion of which is disposed on the surface portion, the insulating layer having an inner peripheral edge portion formed on the first region and the second region, and defining the second outer peripheral edge portion of the second region, and including an opening penetrating through in the thickness direction.

[0010] The present invention also relates to a sensor comprising: the electrode; an outer periphery disposed on the first region and the second region of the electrode and positioned on the second outer periphery of the second region; and a reagent layer containing a reagent participating in an oxidation-reduction reaction.

[0011] The present invention also relates to a method for manufacturing a sensor comprising the electrode; and a reagent layer disposed on the first region and the second region of the electrode, the outer periphery being located on the second outer periphery of the second region, and containing a reagent participating in an oxidation-reduction reaction, the method comprising: applying a first liquid composition containing the reagent in a first solvent onto the first region and the second region of the electrode, and then drying the composition to form the reagent layer.

[0012] The present invention also relates to a method for manufacturing a sensor comprising the electrode; an outer periphery disposed on the first region and the second region of the electrode, the outer periphery being located on the second outer periphery of the second region, the reagent layer containing a reagent participating in an oxidation-reduction reaction; and a protective film covering the reagent layer, the method comprising: applying a first liquid composition containing the reagent in a first solvent onto the first region and the second region of the electrode, and then drying the composition to form the reagent layer; and applying a second liquid composition containing protective film components in a second solvent onto the surface region of the electrode so as to cover the reagent layer, and then drying the composition to form the protective film.

[0013] A preferred aspect of the present invention is an electrode arranged on an insulating substrate, comprising: a surface portion located on the opposite side to the substrate when the electrode is arranged on the substrate; a first region formed on the surface portion, including a first outer peripheral edge portion, at least a portion of which has a first surface free energy; a second region formed on the surface portion, including a second inner peripheral edge portion surrounding the first region and in contact with the first outer peripheral edge portion of the first region, and a second outer peripheral edge portion located outside the second inner peripheral edge portion, and having a second surface free energy greater than the first surface free energy; and a third region formed on the surface portion, including a third inner peripheral edge portion surrounding the second region and in contact with the second outer peripheral edge portion of the second region, and a third outer peripheral edge portion located outside the third inner peripheral edge portion, and having a third surface free energy smaller than the second surface free energy. an insulating layer, at least a portion of which is disposed on the surface portion, formed on the first region, the second region, the third region, and the fourth region, the insulating layer having an inner peripheral edge that contacts the fourth outer peripheral edge of the fourth region in a plan view perpendicular to the surface portion and including an opening that penetrates in the thickness direction;

[0014] Another preferred aspect of the present invention relates to a method for manufacturing the electrode comprising the surface portion, the first region, the second region, the third region, the fourth region, and the insulating layer, the method comprising: preparing an untreated electrode comprising an untreated surface portion having untreated regions for forming the first region, the second region, the third region, and the fourth region and having the first surface free energy; and an insulating layer having an opening formed on the untreated region and penetrating in a thickness direction, at least a portion of which is disposed on the untreated surface portion; and irradiating with laser light a ring-shaped fourth untreated region disposed on the outer periphery of the untreated region and adjacent to the inner periphery of the opening in the insulating layer, in a plan view from a direction perpendicular to the untreated surface portion, and a ring-shaped second untreated region disposed further inward than the fourth untreated region, among the untreated regions of the untreated surface portion of the untreated electrode, thereby converting the fourth untreated region into the fourth region and converting the second untreated region into the second region. In the electrode obtained by this method, preferably, the third surface free energy is the same as the first surface free energy, and the first untreated region, which is the portion of the untreated region that is more inward than the second untreated region, becomes the first region having the first surface free energy, and the third untreated region, which is the portion of the untreated region between the second untreated region and the fourth untreated region, becomes the third region having the third surface free energy that is the same as the first surface free energy.

[0015] The electrode according to the present invention allows a reagent layer of designed dimensions to be formed with high precision on the first and second regions formed on the surface portion of the electrode.

[0016] The sensor according to the present invention can detect a test substance.

[0017] According to the method for manufacturing a sensor of the present invention, a sensor having a reagent layer with designed dimensions on the first and second regions of the electrode can be efficiently manufactured.

[0018] In a preferred embodiment of the present invention, an electrode having a surface portion, a first region, a second region, a third region, a fourth region, and an insulating layer can accurately form a reagent layer of designed dimensions on the first region and the second region, and can accurately form a protective film that covers the reagent layer, the third region, and the fourth region.

[0019] According to another preferred aspect of the present invention, a method for manufacturing an electrode having a surface portion, a first region, a second region, a third region, a fourth region, and an insulating layer makes it easy to form each of the first region, the second region, the third region, and the fourth region accurately at the designed position.

[0020] 1 is a plan view showing a substrate on which two electrodes, a reference electrode conductive layer, a counter electrode, and wiring are arranged. A plan view showing a substrate on which a first region and a second region are formed on the surface of each of the two electrodes in FIG. 1. A plan view showing a substrate on which an insulating layer is further arranged in FIG. 2. A plan view showing a substrate on which a reagent layer is further arranged on the electrodes in FIG. 3. A plan view of a sensor according to an embodiment of the present invention. A schematic diagram explaining a method for measuring an analyte by immersing the sensor shown in FIG. 5 in a liquid sample containing cells. A plan view of a portion including the first region and the second region of an electrode according to an embodiment of the present invention, which is an enlarged view of the area surrounded by the dashed-dotted line in FIG. 2. A cross-sectional view taken along line D-D' of the electrode shown in FIG. 7. A cross-sectional view showing a first liquid composition containing a reagent participating in a redox reaction in a first solvent, which is applied to the first region and the second region of the electrode shown in FIG. 8. A cross-sectional view showing a reagent layer disposed on the first region and the second region of the electrode, which is formed by drying the first liquid composition shown in FIG. 9. A cross-sectional view taken along line A-A' of a portion including the working electrode (including the electrode, the reagent layer, and the protective film) of the sensor shown in FIG. 5. 19 is a cross-sectional view taken along line BB' of a portion of the sensor shown in FIG. 5 that includes a reference electrode (including a reference electrode conductive layer, a silver / silver chloride layer, and a protective film). 20 is a cross-sectional view taken along line CC' of a portion of the sensor shown in FIG. 5 that includes a counter electrode. 21 is a plan view of a portion of an electrode according to another embodiment of the present invention that includes a first region and a second region. 22 is a plan view of a portion of an electrode according to yet another embodiment of the present invention that includes a first region and a second region. 23 is a plan view of a portion of an electrode according to yet another embodiment of the present invention that includes a first region and a second region. 24 is a plan view of a portion of an electrode according to yet another embodiment of the present invention that includes a first region, a second region, a third region, and a fourth region. 25 is a cross-sectional view taken along line EE' of the electrode shown in FIG. 17. 26 is a cross-sectional view showing a first liquid composition that contains a reagent participating in a redox reaction in a first solvent, applied onto the first and second regions of the electrode shown in FIG. 18. 27 is a cross-sectional view showing a reagent layer disposed on the first and second regions of the electrode, formed by drying the first liquid composition shown in FIG. 19. 21 is a cross-sectional view showing a second liquid composition containing a protective film component in a second solvent, which is applied to cover the reagent layers on the first region, the second region, the third region, and the fourth region of the electrode shown in Fig. 20. 22 is a cross-sectional view showing protective films formed by drying the second liquid composition shown in Fig. 21 and arranged to cover the reagent layers on the first region, the second region, the third region, and the fourth region of the electrode.29A shows a plan view of a portion of an electrode according to yet another embodiment of the present invention, including an opening in the insulating layer, a first region, and a second region. A cross-sectional view of the electrode shown in FIG. 23 taken along line F-F'. A cross-sectional view of a sensor according to an embodiment of the present invention, in which a reagent layer and a protective film are disposed on the first and second regions within the opening in the insulating layer of the electrode shown in FIG. 23. A control block diagram of an analytical device including a sensor according to an embodiment of the present invention. Photographs showing the results of Experiment 1. Photographs showing the results of Experiment 3. FIG. 29A shows the results of continuous measurement of current values ​​over 12 days for a sensor in Experiment 5, in which a 0.40 mg application amount of lactate oxidase-containing liquid composition was used to create a reagent layer. FIG. 29B shows the results of continuous measurement of current values ​​over 12 days for a sensor in Experiment 5, in which a 0.45 mg application amount of lactate oxidase-containing liquid composition was used to create a reagent layer. FIG. 29C shows the results of continuous measurement of current values ​​over 12 days for a sensor in Experiment 5, in which a 0.50 mg application amount of lactate oxidase-containing liquid composition was used to create a reagent layer. 29D shows the results of continuous measurement of current values ​​for 12 days of a sensor coated with 0.55 mg of the lactate oxidase-containing liquid composition used to prepare the reagent layer in Experiment 5. FIG. 29E shows the results of continuous measurement of current values ​​for 12 days of a sensor coated with 0.60 mg of the lactate oxidase-containing liquid composition used to prepare the reagent layer in Experiment 5.

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[0155] 34 is a schematic diagram of a portion of the untreated electrode shown in Fig. 33, including a boundary between an untreated region of the untreated surface portion and an insulating layer around an opening, in a plan view from a direction perpendicular to the untreated surface portion. 35 is a schematic diagram of a portion of the untreated electrode shown in Fig. 34, obtained by converting a fourth untreated region of the untreated region of the untreated surface portion into a fourth region and a second untreated region of the untreated region of the untreated surface portion into a second region.Schematic diagram of a portion of the untreated electrode shown in Figure 34 obtained by converting the exposed untreated surface portion into a fourth untreated region by destroying the area surrounding the opening in the insulating layer with laser light irradiation, in addition to the fourth untreated region of the untreated surface portion, and converting the second untreated region of the untreated surface portion into a second region. The upper left of Figure 37 shows a photograph of the electrode prepared in Experiment 6-2 (including a circular second region and a surrounding third region within the opening in the insulating layer) after laser light irradiation. The lower left of Figure 37 shows a photograph of the working electrode obtained by forming a reagent layer and a protective film on the electrode prepared in Experiment 6-2. The upper right of Figure 37 shows a photograph of the electrode prepared in Experiment 6-1 (including a circular second region within the opening in the insulating layer, a surrounding third region, and a surrounding fourth region within the opening in the insulating layer) after laser light irradiation. The lower right of Figure 37 shows a photograph of the working electrode obtained by forming a reagent layer and a protective film on the electrode prepared in Experiment 6-1. The left side of Figure 38 shows the measurement results of the thickness of the protective film on the working electrode obtained by forming a reagent layer and a protective film on the electrode prepared in Experiment 6-2. The right side of Figure 38 shows the measurement results of the thickness of the protective film on the working electrode obtained by forming a reagent layer and a protective film on the electrode prepared in Experiment 6-1.

[0021] Hereinafter, embodiments of an electrode, a sensor, and a method for manufacturing a sensor according to the present invention will be described. In the present embodiments, unnecessary detailed description may be omitted. For example, detailed description of well-known matters or redundant description of substantially identical configurations may be omitted. This is to avoid unnecessary redundancy in the following description and to facilitate understanding by those skilled in the art.

[0022] Furthermore, the applicant provides the accompanying drawings and the following description to enable those skilled in the art to fully understand the present invention, and they are not intended to limit the subject matter described in the claims. This specification incorporates the disclosure of Japanese Patent Application No. 2022-124198, which is the basis of the priority of this application. Furthermore, all publications, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety.

[0023] <Materials> Examples of materials that can be used for the electrodes and sensors disclosed herein are described below. The material of the insulating substrate on which the electrodes disclosed herein are arranged is not particularly limited, but examples include resin materials such as polyethylene terephthalate, polycarbonate, polyimide, polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyoxymethylene, monomer cast nylon, cycloolefin polymer, polyethylene naphthalate, polybutylene terephthalate, methacrylic resin, and ABS resin, or 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, but a substrate with a thickness of, for example, 0.05 mm or more and 2 mm or less, preferably 0.1 mm or more and 1 mm or less, can be used.

[0024] The electrodes disclosed herein can be formed from conductive materials such as carbon, gold, platinum, and palladium. Examples of carbon that can be used include conductive carbon materials such as glassy carbon, carbon black, graphite, diamond-like carbon, graphene, carbon nanotubes, and fullerenes. The electrodes disclosed herein are preferably layers of conductive materials (conductive layers). The conductive layer can be formed from the above-described conductive materials using a sputtering method, a vapor deposition method, a screen printing method, or the like. The conductive layer can be processed into a predetermined pattern using a laser trimming method, if necessary.

[0025] The reference electrode conductive layer, counter electrode, and wiring provided in the sensor disclosed in this specification can also be made of the same conductive materials as those described above.

[0026] The electrodes and sensors disclosed herein may include an insulating layer. The insulating layer preferably includes a water-repellent surface. Here, a water-repellent surface refers to a surface having a water contact angle 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 insulating layer is not particularly limited, but may be, for example, 160° or less. That is, the water contact angle of the water-repellent surface of the insulating layer may be, for example, 90° or more and 160° or less, and preferably within a narrower range defined by the upper and / or lower limits. The water contact angle of the insulating layer surface may be measured at 20°C. The water contact angle of the insulating layer surface may be measured using a commercially available analytical device, for example, a handheld contact angle and surface free energy analyzer MSA manufactured by KRUSS. The water contact angle of the surface is preferably measured by discharging a 1 μL droplet of water onto the surface to be measured and measuring the contact angle between the droplet and the surface after 2 seconds.

[0027] The insulating layer provided in the electrodes and sensors disclosed herein 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 insulating layer can be entirely made of a fluororesin.

[0028] Another preferred example of the insulating layer provided in the electrodes and sensors disclosed herein is an insulating layer containing a compound containing a perfluoroalkyl group. Examples of compounds containing a perfluoroalkyl group include fluorine-based surface-modifying additives. An insulating layer containing a compound containing a perfluoroalkyl group can be formed by applying a composition containing an insulating matrix resin and a compound containing a perfluoroalkyl group (fluorine-based surface-modifying additive) to a solvent and drying the composition. The 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, in the range of 2 to 20 carbon atoms. The perfluoroalkyl group may contain a trifluoromethyl group at its terminal.

[0029] The insulating layer may have a single layer structure made of one type of insulating layer, or may have a laminated structure in which two or more insulating layers are laminated.

[0030] The sensor disclosed herein 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 include 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.

[0031] The sensor disclosed herein comprises a working electrode including the electrode disclosed herein and a reagent layer containing a reagent involved in a redox reaction. The reagent involved in the redox reaction may be any reagent that participates in a redox reaction with a 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.

[0032] 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.

[0033] 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.

[0034] 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, and the upper limit of the weight-average molecular weight is, for example, less than 10,000,000, preferably less than 1,000,000. That is, the weight-average molecular weight of the polymer compound can be 10,000 or more and less than 10,000,000, preferably within a narrower range defined by the upper and / or lower limits. 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.

[0035] The reagent layer of the sensor disclosed herein may contain, in addition to the reagent, components such as a buffer, a hydrophilic polymer, a conductive carbon filler, and a crosslinking agent. Examples of the hydrophilic polymer include cellulose derivatives, such as methyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose, and hydroxypropyl methyl cellulose. Examples of the conductive carbon filler include carbon black, graphite powder, porous carbon, and nanocarbon.

[0036] The reagent layer provided in the sensor disclosed herein can be formed by applying a first liquid composition containing a reagent involved in a redox reaction to a first solvent and drying the composition. The first liquid composition can further contain the components described above as components contained in the reagent layer. The first solvent contained in the first liquid composition can be a solvent capable of dissolving the reagent, and examples of the first solvent include a solvent containing one or more selected from water and alcohol. One or more selected from water and alcohol are preferred, water or a mixed solvent of water and alcohol is more preferred, and water is most preferred. Examples of alcohols include monohydric alcohols having 1 to 5 carbon atoms, with methanol, ethanol, or isopropyl alcohol being particularly preferred, and ethanol being most preferred.

[0037] The sensor disclosed herein may further include a protective film that covers the reagent layer. The protective film may be a film that prevents or inhibits leakage of the reagent contained in the reagent layer outside the protective film and allows permeation of 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 such as a proton conductive group.

[0038] 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 such as polyethylene glycol diglycidyl ether (PEGDGE). Examples of the crosslinking agent include crosslinking agents containing two or more epoxy groups, such as PEGDGE. 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 a pyridyl group (tertiary amine) derived from 4-vinylpyridine with an epoxy group.

[0039] Examples of polymer compounds containing cation-exchange functional groups such as proton-conducting groups include polymer compounds containing structural units having sulfonic acid groups in their side chains, preferably polymer compounds containing, as structural units, perfluorocompounds having sulfonic acid groups in their side chains, more preferably copolymer compounds containing, as structural units, perfluorocompounds having sulfonic acid groups in their side chains and perfluorocompounds having no ionic functional groups in their side chains, particularly preferably copolymers of tetrafluoroethylene and perfluoro[2-(fluorosulfonylethoxy)propylvinylether], and particularly preferably Nafion®. A protective membrane containing a polymer compound having 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.

[0040] The protective film may be a laminate of two or more protective films, for example, a laminate of a protective film containing a polymer compound containing a cation-exchange functional group, which is provided on the side in contact with a reagent layer containing a reagent that generates protons by an oxidation-reduction reaction of a test substance, and a protective film containing a polymer compound containing 4-vinylpyridine as a constituent unit, which is provided thereon.

[0041] Another suitable example of the protective film is a protective film containing a polymer compound containing 4-vinylpyridine as a constituent unit.

[0042] The reference electrode of the sensor disclosed herein may include a protective film, which may be made of the materials described above for the protective film of the working electrode.

[0043] The protective film provided in the sensor disclosed herein can be formed by applying a second liquid composition containing protective film components in a second solvent and then drying. Examples of the second solvent include alcohol or a mixed solvent of alcohol and water. Examples of the alcohol include monohydric alcohols having 1 to 5 carbon atoms, with methanol, ethanol, or isopropyl alcohol being particularly preferred, and ethanol being most preferred. The protective film components are components that can form a protective film after drying. The protective film components are, for example, one or more selected from polymer compounds that form the protective film, their monomers, and their prepolymers, and may further include a crosslinking agent.

[0044] <Surface Free Energy> In this specification, the "surface free energy" of a predetermined portion of the surface of an electrode refers to the surface free energy determined by the Owens-Wendt method. This surface free energy γs (mN / m) is the sum of a polar component γsp (mN / m) and a dispersive component γsd (mN / m).

[0045] Specifically, the polar component γsp (mN / m), the dispersion component γsd (mN / m), and the surface free energy γs (mN / m) can be calculated by measuring the contact angles (1 μL drop, measured 2 seconds after drop) of water and diiodomethane at a predetermined portion of the surface of the electrode using a contact angle meter at 20° C. and a relative humidity of 40%, and then calculating the contact angles using the simultaneous equations (1), (2), and (3) below. Equation (1): γs = γsd + γsp Equation (2): 72.8(1 + cos θH) = 2(21.8γsd) １／２ +2 (51.0γsp) １／２ Formula (3): 50.8(1+cosθI)=2(48.5γsd) １／２ +2 (2.3 γsp) １／２ γs: surface free energy γsd: dispersive component of surface free energy γsp: polar component of surface free energy θH: contact angle with water θI: contact angle with diiodomethane

[0046] The contact angles of water and diiodomethane in a predetermined region of the electrode surface can be measured using a commercially available analyzer, for example, a handy contact angle and surface free energy analyzer MSA manufactured by KRUSS GmbH. The contact angles of water and diiodomethane in a predetermined region of the electrode surface are preferably measured by discharging 1 μL of water and diiodomethane droplets onto the surface of the region to be measured at 20° C. and a relative humidity of 40%, and then measuring the contact angle between the droplet and the surface after 2 seconds.

[0047] In this specification, the second surface free energy is greater than the first surface free energy, and the difference therebetween is not limited. For example, the second surface free energy is preferably greater than the first surface free energy by 3.0 mN / m or more, more preferably 5.0 mN / m or more, more preferably 7.0 mN / m or more, more preferably 10.0 mN / m or more, more preferably 12.0 mN / m or more, and most preferably 15.0 mN / m or more. There is no particular upper limit to the difference between the second surface free energy and the first surface free energy, but the difference can be, for example, 50 mN / m or less, preferably 30 mN / m or less. That is, the difference between the second surface free energy and the first surface free energy can be, for example, 3.0 mN / m or more and 50 mN / m or less, and preferably within a narrower range defined by the upper and / or lower limits. In the case described in this paragraph, the region of the surface of the electrode having the first surface free energy has relatively low wettability to the first liquid composition containing a reagent involved in an oxidation-reduction reaction in a first solvent, and the region having the second surface free energy has relatively high wettability, thereby achieving the effect described below of being able to form a dimensionally stable reagent layer on the first and second regions of the electrode.

[0048] In particular, it is preferable that the polar component of the second surface free energy is greater than the polar component of the first surface free energy. For example, the polar component of the second surface free energy is preferably 2.0 mN / m or more, more preferably 3.0 mN / m or more, more preferably 5.0 mN / m or more, more preferably 8.0 mN / m or more, and most preferably 11.0 mN / m or more than the polar component of the first surface free energy. There is no particular upper limit to the difference between the polar components of the second surface free energy and the first surface free energy, but the difference can be, for example, 40 mN / m or less, preferably 25 mN / m or less. That is, the difference between the polar components of the second surface free energy and the polar components of the first surface free energy can be, for example, 2.0 mN / m or more and 40 mN / m or less, and preferably within a narrower range defined by the upper and / or lower limits. In the case described in this paragraph, the region of the surface of the electrode having the first surface free energy has relatively low wettability to the first liquid composition containing a reagent involved in an oxidation-reduction reaction in a first solvent, and the region having the second surface free energy has relatively high wettability, so the effect described below of being able to form a dimensionally stable reagent layer on the first and second regions of the electrode is particularly pronounced.

[0049] The value of the first surface free energy is not particularly limited, but is, for example, 20.0 mN / m or more, preferably 30.0 mN / m or more, more preferably 40.0 mN / m or more, particularly preferably 45.0 mN / m or more, and can be, for example, 70.0 mN / m or less, preferably 60.0 mN / m or less, more preferably 50.0 mN / m or less. That is, the value of the first surface free energy is, for example, 20.0 mN / m or more and 70.0 mN / m or less, preferably, in a narrower range defined by the upper and / or lower limits. The value of the polar component of the first surface free energy is not particularly limited, but is, for example, 5.0 mN / m or less, preferably 3.0 mN / m or less, and, for example, 0.0 mN / m or more, preferably 2.0 mN / m or more. That is, the value of the polar component of the first surface free energy is 0.0 mN / m or more and 5.0 mN / m or less, preferably within a narrower range defined by the upper and / or lower limits. The ratio of the dispersive component to the polar component of the first surface free energy (γsd / γsp) is, for example, 8.0 or more, preferably 10.0 or more, more preferably 12.0 or more, particularly preferably 15.0 or more, and can be, for example, 50.0 or less, preferably 30.0 or less. That is, the ratio of the dispersive component to the polar component of the first surface free energy is, for example, 8.0 or more and 50.0 or less, preferably within a narrower range defined by the upper and / or lower limits. The surface region of the electrode having the first surface free energy described in this paragraph has high repellency to water and alcohol, particularly water, and low wettability. Before performing the treatment to increase the surface free energy described below, an example of an electrode having a surface region having the first surface free energy described in this paragraph is a carbon electrode.

[0050] The value of the second surface free energy is not particularly limited, but may be, for example, 45.0 mN / m or more, preferably 50.0 mN / m or more, more preferably 55.0 mN / m or more, more preferably 59.0 mN / m or more, and particularly preferably 60.0 mN / m or more, and may be, for example, 100.0 mN / m or less, preferably 80.0 mN / m or less, and more preferably 70.0 mN / m or less. That is, the value of the second surface free energy may be, for example, 45.0 mN / m or more and 100.0 mN / m or less, and preferably, may be within a narrower range defined by the upper and / or lower limits. The value of the polar component of the second surface free energy is not particularly limited, but is, for example, 5.0 mN / m or more, preferably 8.0 mN / m or more, more preferably 10.0 mN / m or more, particularly preferably 14.0 mN / m or more, and for example, 50.0 mN / m or less, preferably 30.0 mN / m or less, more preferably 20.0 mN / m or less. That is, the value of the polar component of the second surface free energy is, for example, 5.0 mN / m or more and 50.0 mN / m or less, preferably, it can be in a narrower range defined by the upper and / or lower limits. The ratio of the dispersive component to the polar component of the second surface free energy (γsd / γsp) is, for example, 10.0 or less, preferably 8.0 or less, more preferably 6.0 or less, particularly preferably 5.0 or less, and can be, for example, 1.0 or more. That is, the ratio of the dispersive component to the polar component of the second surface free energy is, for example, 1.0 or more and 10.0 or less, and preferably can be within a narrower range defined by the upper and / or lower limits. The region of the surface portion of the electrode having the second surface free energy described in this paragraph has low liquid repellency and high wettability with respect to water and alcohol, particularly water.

[0051] In this specification, the third surface free energy is smaller than the second surface free energy. In other words, the second surface free energy is larger than the third surface free energy, and the specific difference therebetween can be selected from the same range as the difference between the second surface free energy and the first surface free energy. In particular, it is preferable that the polar component of the second surface free energy is larger than the polar component of the third surface free energy, and the specific value of this difference can be selected from the same range as the difference between the polar components of the second surface free energy and the first surface free energy. The value of the third surface free energy, the value of the polar component of the third surface free energy, and the ratio of the dispersive component of the third surface free energy to the polar component (γsd / γsp) can be selected from the ranges described for the value of the first surface free energy, the value of the polar component of the first surface free energy, and the ratio of the dispersive component of the first surface free energy to the polar component, respectively, and are particularly preferably the same as the value of the first surface free energy, the value of the polar component of the first surface free energy, and the ratio of the dispersive component of the first surface free energy to the polar component.

[0052] In this specification, the fourth surface free energy is greater than the third surface free energy. The fourth surface free energy may be the same as or different from the second surface free energy. The specific difference between the fourth surface free energy and the third surface free energy can be selected from the same range as the difference between the second surface free energy and the first surface free energy. In particular, it is preferable that the polar component of the fourth surface free energy is greater than the polar component of the third surface free energy, and the specific value of this difference can be selected from the same range as the value described for the difference between the polar component of the second surface free energy and the polar component of the first surface free energy. The value of the fourth surface free energy, the value of the polar component of the fourth surface free energy, and the ratio of the dispersive component of the fourth surface free energy to the polar component (γsd / γsp) can be selected from the ranges described for the value of the second surface free energy, the value of the polar component of the second surface free energy, and the ratio of the dispersive component of the second surface free energy to the polar component, respectively, and are particularly preferably the same as the value of the second surface free energy, the value of the polar component of the second surface free energy, and the ratio of the dispersive component of the second surface free energy to the polar component.

[0053] In this specification, the fifth surface free energy is smaller than the fourth surface free energy. In other words, the fourth surface free energy is larger than the fifth surface free energy, and the specific difference therebetween can be selected from the same range as the difference between the second surface free energy and the first surface free energy. In particular, it is preferable that the polar component of the fourth surface free energy is larger than the polar component of the fifth surface free energy, and the specific value of this difference can be selected from the same range as the difference between the polar component of the second surface free energy and the polar component of the first surface free energy. The value of the fifth surface free energy, the value of the polar component of the fifth surface free energy, and the ratio of the dispersive component of the fifth surface free energy to the polar component (γsd / γsp) can be selected from the ranges described for the value of the first surface free energy, the value of the polar component of the first surface free energy, and the ratio of the dispersive component of the first surface free energy to the polar component, respectively, and are particularly preferably the same as the value of the first surface free energy, the value of the polar component of the first surface free energy, and the ratio of the dispersive component of the first surface free energy to the polar component.

[0054] In this specification, examples of treatments for increasing the surface free energy of a specific region on the surface of an electrode include laser processing, plasma treatment, corona discharge treatment, and mask exposure, with laser processing being particularly preferred. These treatments are particularly suitable because, compared to printing methods, they enable the formation of a region with the designed dimensions (outer shape and area) with high precision. Laser processing is a process in which a specific region on the surface of an electrode is scanned and irradiated with laser light to increase the surface free energy of the region. Ultraviolet (UV) lasers and SHG (Second Harmonic Generation) lasers can be used as laser light. UV lasers typically have a wavelength of 355 nm. Preferred examples of processing conditions using a UV laser are described in the examples. SHG lasers have a wavelength of, for example, 532 nm and a green color in the visible light region. Examples of processing conditions using an SHG laser include a wavelength of 532 nm, a laser output of 450 mW, a scanning speed of 2000 mm / s, and a frequency of 15 kHz.

[0055] <Electrode 10> The electrode 10 according to one embodiment of the present invention will be described with reference to Fig. 2, Fig. 7, and Fig. 8. Fig. 7 is an enlarged view of region X in Fig. 2, and Fig. 8 is a cross-sectional view taken along line DD' in Fig. 7.

[0056] The electrode 10 is disposed on an insulating substrate 20. In Figures 2, 7 and 8, the electrode 10 is shown disposed on a first surface 21 of the substrate 20.

[0057] When placed on the substrate 20 , the electrode 10 comprises a surface portion 11 located on the opposite side to the substrate 20 , and a first region 110 and a second region 120 formed on the surface portion 11 .

[0058] The first region 110 includes a first outer peripheral edge 111. At least a portion of the first region 110 has a first surface free energy. In the electrode 10 according to the embodiment shown in FIGS. 2, 7, and 8, the entire first region 110 surrounded by the second region 120 has the first surface free energy. The portion of the first region 110 having the first surface free energy is not limited to the examples shown in FIGS. 2, 7, and 8. For example, in the first region 110, all or a portion of the first outer peripheral edge 111 in the circumferential direction may have the first surface free energy. Preferably, all or a portion of the first outer peripheral edge 111 in the circumferential direction and a portion adjacent thereto on the inside may have the first surface free energy. Other preferred embodiments of the first region 110 will be described later with reference to FIGS. 14, 15, 16, etc.

[0059] The second region 120 is formed on the surface portion 11 and is a region surrounding the first region 110. The second region 120 includes a second inner peripheral edge portion 121 that contacts the first outer peripheral edge portion 111 of the first region 110, and a second outer peripheral edge portion 122 that is located outside the second inner peripheral edge portion 121. The second region 120 has a second surface free energy that is greater than the first surface free energy.

[0060] The electrode 10 according to the embodiment shown in Figures 2, 7 and 8 further comprises a third region 130 formed on the surface portion 11, which includes a third inner peripheral portion 131 that surrounds the second region 120 and is in contact with the second outer peripheral portion 122 of the second region 120, and which has a third surface free energy that is smaller than the second surface free energy.

[0061] The electrode 10 according to the embodiment shown in Figures 2, 7, and 8 can be manufactured by selectively applying a treatment for increasing the surface free energy described in the <Surface Free Energy> column to a portion of the surface portion 11 (see Figures 7 and 8) of the electrode 10 having a first surface free energy, surrounding the first region 110, to form a second region 120 having a second surface free energy greater than the first surface free energy. In this case, the outside of the second region 120 becomes a third region 130 having a third surface free energy equal to the first surface free energy. In this embodiment, the entire first region 110 surrounded by the second region 120 has the first surface free energy.

[0062] In this embodiment, the first surface free energy, the second surface free energy, and the third surface free energy are as described in the <Surface free energy> column.

[0063] 2, 7, and 8, the electrode 10 having the surface portion 11, the first region 110, the second region 120, and the third region 130 has the effect of enabling the reagent layer 30 to be formed with high precision in the designed dimensions on the first region 110 and the second region 120. The contact area between the reagent layer 30 and the electrode 10 is defined by the first region 110 and the second region 120 of the electrode 10. This effect will be described with reference to FIGS. 9 and 10.

[0064] As shown in FIG. 9 , a first liquid composition P containing a reagent participating in a redox reaction in a first solvent is applied to the first region 110 and the second region 120 of the electrode 10. At this time, the electrode 10 may be disposed on the first surface 21 of the substrate 20 in advance. The first liquid composition P has a relatively low wettability to the first region 110 having a first surface free energy, a relatively high wettability to the second region 120 having a second surface free energy, and a relatively low wettability to the third region 130 having a third surface free energy. Therefore, the first liquid composition P applied to the first region 110 and the second region 120 rises while attempting to remain on the central first region 110, forming a droplet whose periphery is defined by the second outer peripheral edge 122 of the second region 120. The first liquid composition P applied to the first region 110 and the second region 120 of the electrode 10 is prevented from spreading outward by the third inner peripheral edge 131 of the third region 130. The first region 110 and the second region 120 of the electrode 10 can carry a larger amount of the first liquid composition P as droplets than solid circular regions having a second surface free energy formed in island shapes in a region having a first surface free energy on the surface of the electrode, as in the comparative example described below, and can therefore form a reagent layer 30 containing a larger amount of reagent. The greater the amount of reagent carried in the reagent layer 30, the longer the continuous measurement of the test substance can be performed (see Experiment 5).

[0065] Next, the first liquid composition P applied to the first region 110 and the second region 120 of the electrode 10 is dried to form a reagent layer 30 including the reagent and an outer peripheral edge 31 located on the second outer peripheral edge 122 of the second region 120, as shown in FIG. 10 . As shown in FIG. 9 , the outer periphery of the first liquid composition P applied to the first region 110 and the second region 120 of the electrode 10 is defined by the second outer peripheral edge 122 of the second region 120. Therefore, as shown in FIG. 10 , the outer peripheral edge 31 of the reagent layer 30 obtained by drying is also located on the second outer peripheral edge 122 of the second region 120. Therefore, the outer peripheral shape and area of ​​the reagent layer 30 can be adjusted by adjusting the outer shape and area of ​​the first region 110 and the second region 120. Meanwhile, the thickness of the reagent layer 30 can be adjusted by adjusting the concentration of the reagent in the first liquid composition P and the amount of the first liquid composition P applied. That is, according to the electrode 10 of this embodiment, it is possible to form the reagent layer 30 with the designed dimensions with high precision on the first region 110 and the second region 120 formed on the surface portion 11 of the electrode 10. The contact area between the reagent layer 30 and the electrode 10 is determined by the first region 110 and the second region 120 of the electrode 10.

[0066] The first solvent contained in the first liquid composition P can be a solvent capable of dissolving the reagent, and examples of the first solvent include a solvent containing one or more selected from water and alcohol. Preferred examples of the first solvent are as described in the <Materials> section.

[0067] As described above, the outward spread of the first liquid composition P applied to the first region 110 and the second region 120 of the electrode 10 is prevented by the third inner peripheral edge portion 131 of the third region 130. The electrode 10 according to the embodiment shown in Figures 2, 7, and 8 is advantageous in that it is low cost, unlike the electrode 10 according to the embodiment shown in Figures 23 and 24 described below, in which an insulating layer 50 is disposed to prevent the applied first liquid composition P from spreading outward.

[0068] In the electrode 10 according to the embodiments shown in Figures 2, 7, and 8, the maximum width M (diameter in the illustrated example) of the outer periphery of the second region 120 can be adjusted as appropriate depending on the desired dimensions of the reagent layer 30, and can be, for example, 300 µm to 5,000 µm, and preferably 900 µm to 2,000 µm. The radial width N of the second region 120 can also be adjusted as appropriate depending on the desired dimensions of the reagent layer 30, and can be, for example, 0.5% to 40% of the maximum width M of the outer periphery, and preferably 5% to 30%. The radial width N of the second region 120 can be, for example, 50 µm to 500 µm, and preferably 90 µm to 400 µm.

[0069] <Sensor 1> A sensor 1 according to one embodiment of the present invention will be described with reference to the drawings. As shown in Fig. 5 and its cross-sectional views in Figs. 11, 12, and 13, the sensor 1 of this embodiment includes an electrode 10, an outer peripheral edge portion 31 disposed on the first region 110 and the second region 120 of the electrode 10 and located on the second outer peripheral edge portion 122 of the second region 120, and a reagent layer 30 containing a reagent involved in an oxidation-reduction reaction.

[0070] In the sensor 1 of this embodiment, the electrode 10 and the reagent layer 30 constitute a working electrode 2. The electrode 10 is preferably disposed on a first surface 21 of an insulating substrate 20.

[0071] 11 is a cross-sectional view taken along line AA' of a portion of the sensor 1 shown in FIG. 5 including the working electrode 2. A first region 110 and a second region 120 are formed on a surface portion 11 of the electrode 10 located on the side opposite the substrate 20, and the reagent layer 30 is disposed on the first region 110 and the second region 120. As shown in FIG. 11, the sensor 1 further includes a protective film 40 that covers the reagent layer 30. The protective film 40 prevents or suppresses leakage of the reagent contained in the reagent layer 30. The electrode 10 is connected to wiring 5 disposed on the first surface 21 of the substrate 20.

[0072] Specific examples of materials for the electrode 10, substrate 20, reagent layer 30 and protective film 40 are as described in the <Materials> section.

[0073] Although the sensor 1 of this embodiment includes two working electrodes 2, in another embodiment not shown, the number of working electrodes may be one or three or more. In a sensor 1 including two or more working electrodes 2, the reagent layers 30 of the two or more working electrodes 2 may contain reagents involved in the oxidation-reduction reaction of different test substances. For example, some of the two or more working electrodes 2 may include reagent layers 30 including reagents involved in the oxidation-reduction reaction of lactate, and other parts may include reagent layers 30 including reagents involved in the oxidation-reduction reaction of glucose.

[0074] In the sensor 1 of this embodiment, the electrode 10 further includes an insulating layer 50, at least a portion of which is disposed on the surface portion 11. The insulating layer 50 has openings 52 formed in the first region 110, the second region 120, and the third region 130 of the surface portion 11 of the electrode 10. In this embodiment, as shown in Fig. 11 , the reagent layer 30 and the protective film 40 are disposed in the openings 52. The insulating layer 50 covers not only the surface portion 11 of the electrode 10 but also other portions of the first surface 21 of the substrate 20.

[0075] The sensor 1 of this embodiment may further comprise a reference electrode 3 and / or a counter electrode 4 arranged on the substrate 20, and preferably further comprises a reference electrode 3 and a counter electrode 4 as in the example shown in the figure.

[0076] FIG. 12 is a cross-sectional view taken along line B-B' of a portion of the sensor 1 shown in FIG. 5 , including the reference electrode 3. The reference electrode 3 includes a reference electrode conductive layer 301 disposed on the first surface 21 of the substrate 20 and a silver / silver chloride layer 302 disposed on the reference electrode conductive layer 301. The reference electrode 3 preferably further includes a reference electrode protective film 303 disposed on the silver / silver chloride layer 302, as shown in the figure. Preferred embodiments of the materials for the reference electrode conductive layer 301 and the reference electrode protective film 303 are as described in the <Materials> section. A wiring 5 is connected to the reference electrode conductive layer 301 of the reference electrode 3. The insulating layer 50 has a reference electrode opening 53 formed at a position overlapping the reference electrode 3 in a plan view. The reference electrode conductive layer 301, silver / silver chloride layer 302, and reference electrode protective film 303 of the reference electrode 3 are disposed within the reference electrode opening 53, as shown in FIG. 12 .

[0077] 13 is a cross-sectional view taken along line CC' of a portion of the sensor 1 shown in FIG. 5 including the counter electrode 4. The counter electrode 4 is a conductive layer disposed on the first surface 21 of the substrate 20. Preferred embodiments of the material of the conductive layer constituting the counter electrode 4 are as described in the <Material> section. A wire 5 is connected to the counter electrode 4. The insulating layer 50 has a counter electrode opening 54 formed in a position overlapping a portion of the upper surface 4a of the counter electrode 4 in a plan view. As shown in FIG. 13, the counter electrode 4 is disposed within the counter electrode opening 54, and a portion of the upper surface 4a of the counter electrode 4 is exposed through the counter electrode opening 54.

[0078] As shown in Fig. 6, the sensor 1 is immersed in a liquid sample L and used to detect a predetermined test substance in the liquid sample L. Specific examples of the liquid sample and the test substance are as described in the <Materials> section. Fig. 6 shows a cell culture medium containing cells C as an example of the liquid sample L.

[0079] When the reagent layer 30 of the working electrode 2 of the sensor 1 contains a reagent that oxidizes the analyte in the liquid sample L, electrons are transferred from the analyte to the electrode 10 of the working electrode 2 under conditions in which a predetermined voltage is applied to the working electrode 2, reference electrode 3, and counter electrode 4 of the sensor 1. Similarly, when the reagent layer 30 contains a reagent that reduces the analyte in the liquid sample L, electrons are transferred from the electrode 10 of the working electrode 2 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 L can be measured based on the value of the current or a change in the current flowing through the electrode 10 of the working electrode 2 of the sensor 1.

[0080] An example of an analytical device 200 equipped with the sensor 1 for analyzing a test substance in a liquid sample will be described with reference to FIG.

[0081] 26 , the analysis device 200 includes a sensor 1, an analysis unit 202, and a control unit 204. The working electrode 2, reference electrode 3, and counter electrode 4 of the sensor 1 are each connected to the analysis unit 202 via wiring 5. The analysis unit 202 is capable of communicating with the control unit 204.

[0082] The analysis unit 202 includes an electrochemical measurement section 211 , a control section 212 , a storage section 213 , and a communication section 214 .

[0083] The electrochemical measurement unit 211 is a potentiostat that applies a predetermined voltage to the working electrode 2, reference electrode 3, and counter electrode 4 of the sensor 1 to measure the concentration of the test substance, and includes a voltage application unit 211a and a current measurement unit 211b, and preferably further includes a voltage measurement unit (counter electrode terminal voltage measurement unit) 211c.

[0084] The voltage application unit 211a applies a predetermined voltage to the working electrode 2, the reference electrode 3, and the counter electrode 4 of the sensor 1 in order to measure the concentration of the test substance contained in the liquid sample L.

[0085] The current measuring unit 211b detects the value of a current flowing between the working electrode 2 and the counter electrode 4 of the sensor 1 or a change therein, which is measured while a voltage is applied from the voltage applying unit 211a to the working electrode 2, the reference electrode 3, and the counter electrode 4 of the sensor 1. As described above, the current value or a change in the current value detected by the current measuring unit 211b is an index of the concentration or a change in concentration of the test substance in the liquid sample L.

[0086] The voltage measurement unit 211 c measures the terminal voltage of the counter electrode 4 of the sensor 1 .

[0087] The control unit 212 is connected to the voltage application unit 211a, the current measurement unit 211b, the voltage measurement unit 211c, the storage unit 213, and the communication unit 214. The control unit 212 controls the voltage application unit 211a to apply a predetermined voltage to the working electrode 2, the reference electrode 3, and the counter electrode 4 of the sensor 1, and controls the communication unit 214 to transmit the measurement results of the current measurement unit 211b and the voltage measurement unit 211c to the control unit 204.

[0088] The memory unit 213 is connected to the control unit 212 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 211b and the voltage measurement unit 211c, and a calibration curve that has been measured in advance.

[0089] The communication section 214 is controlled by the control section 212 and transmits data such as the measurement results of the current measurement section 211 b and the voltage measurement section 211 c to the analysis section 242 of the control unit 204 .

[0090] The control unit 204 is capable of communicating with the analysis unit 202 via a communication unit 214 , and includes a display unit 241 and an analysis unit 242 .

[0091] The display unit 241 displays the results of the analysis in the analysis unit 242, such as the concentration of the test substance in the liquid sample L from the current value detected in the current measurement unit 211b.

[0092] The analysis unit 242 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 2 and the counter electrode 4 measured by the current measurement unit 211b.

[0093] <Method for Manufacturing Sensor 1> An example of a method for manufacturing the sensor 1 according to the embodiment shown in Fig. 5 will be described with reference to the drawings, particularly Fig. 1 to Fig. 5. First, as shown in Fig. 1, an electrode (working electrode conductive layer) 10, a reference electrode conductive layer 301, a counter electrode (counter electrode conductive layer) 4, and wiring 5 electrically connected to each of them are arranged on a first surface 21 of an insulating substrate 20.

[0094] 2, a first region 110, a second region 120, and a third region 130 are formed on the surface portion 11 of the electrode 10 located on the opposite side from the substrate 20. Here, when the entire surface portion 11 of the electrode 10 before processing has a first surface free energy, a part of the surface portion 11 is made into the first region 110, and a treatment for increasing the surface free energy as described above is selectively performed on an annular region surrounding the first region 110, thereby forming the first region 110 having the first surface free energy, the second region 120 having a second surface free energy greater than the first surface free energy, and the third region 130 having a third surface free energy equal to the first surface free energy.

[0095] 3 , an insulating layer 50 is laminated on the first surface 21 of the substrate 20 on which the electrode 10, the reference electrode conductive layer 301, the counter electrode 4, and the wiring 5 are arranged. The insulating layer 50 has openings 52 formed in the first region 110, the second region 120, and the third region 130 of the surface portion 11 of the electrode 10, a reference electrode opening 53 formed on the reference electrode conductive layer 301, and a counter electrode opening 54 formed on the counter electrode 4.

[0096] Next, as shown in FIGS. 4 and 10 , a reagent layer 30 containing a reagent participating in a redox reaction and an outer peripheral edge 31 located on the second outer peripheral edge 122 of the second region 120 is formed on the first region 110 and the second region 120 of the electrode 10 within the opening 52 of the insulating layer 50. As described above with reference to FIGS. 9 and 10 , the reagent layer 30 can be formed by applying a first liquid composition P containing the reagent in a first solvent to the first region 110 and the second region 120 of the electrode 10 and then drying the applied composition. The method for forming the reagent layer 30, the first solvent, the first liquid composition P, and preferred aspects of the electrode 10 on which the reagent layer 30 is formed are as described above. The reagent layer 30 formed on the electrode 10 by this method contains a large amount of reagent. Therefore, the sensor 1 manufactured by the method according to this embodiment is suitable for a method of continuously detecting a test substance by immersing the sensor 1 in a liquid sample L for a long period of time.

[0097] Next, as shown in Figures 5 and 11, a protective film 40 is formed to cover the reagent layer 30. The protective film 40 can be formed by applying a second liquid composition containing protective film components in a second solvent to the surface portion 11 of the electrode 10 so as to cover the reagent layer 30, and then drying the composition. In the example shown in Figures 5 and 11, the protective film 40 can be formed by applying the second liquid composition to the opening 52 of the insulating layer 50, including the reagent layer 30 formed on the surface portion 11 of the electrode 10, and then drying the composition. An example of the second solvent is alcohol. Specific examples of the second solvent, protective film components, polymer compound, and crosslinking agent that constitute the protective film are as described in the <Materials> section.

[0098] 5 and 12 , a silver / silver chloride layer 302 and a reference electrode protective film 303 are laminated on the reference electrode conductive layer 301 in the reference electrode opening 53 of the insulating layer 50 to complete the reference electrode 3. The reference electrode protective film 303 can be formed by the same method as the protective film 40 of the working electrode 2.

[0099] <Embodiment of Electrode 10 Shown in FIG. 14> Another embodiment of the electrode 10 will be described with reference to FIG. 14. FIG. 14 is an enlarged view of a region of the electrode 10 according to this embodiment, which corresponds to region X in the electrode 10 shown in FIG. 2. The electrode 10 according to this embodiment shown in FIG. 14 includes a surface portion 11, a first region 110 formed on the surface portion 11 and having the following characteristics, and a second region 120 formed on the surface portion 11 and having a second surface free energy greater than the first surface free energy. The electrode 10 according to this embodiment further includes a third region 130 formed on the surface portion 11 and having a third surface free energy smaller than the second surface free energy.

[0100] In the electrode 10 according to this embodiment, the first region 110 includes: a first outer peripheral edge portion 111; and a central region 112 that includes an outer peripheral edge portion 113 that is disposed inside the first region 110 and is located more inward than the first outer peripheral edge portion 111 of the first region 110, and has a surface free energy greater than the first surface free energy; and a peripheral region 115 that surrounds the central region 112 and includes an inner peripheral edge portion 114 that is in contact with the outer peripheral edge portion 113 of the central region 112 and the first outer peripheral edge portion 111, and has the first surface free energy.

[0101] 14 , the peripheral region 115 is a region of the first region 110 other than the central region 112, and includes an inner peripheral edge 114 that surrounds the central region 112 and contacts the entire outer peripheral edge 113 of the central region 112, and the entire first outer peripheral edge 111. The central region 112 is preferably disposed at a position that includes the center of gravity of the first region 110, and more preferably, the center of gravity of the central region 112 and the center of gravity of the first region 110 coincide with each other.

[0102] In the electrode 10 according to this embodiment shown in FIG. 14 , the surface free energy of the central region 112 is greater than the first surface free energy. The specific difference between the surface free energy of the central region 112 and the first surface free energy can be selected from the same range as the difference between the second surface free energy and the first surface free energy. In particular, it is preferable that the polar component of the surface free energy of the central region 112 is greater than the polar component of the first surface free energy, and the specific value of this difference can be selected from the same range as the difference between the polar component of the second surface free energy and the polar component of the first surface free energy. The value of the surface free energy of the central region 112, the value of the polar component of the surface free energy of the central region 112, and the ratio of the dispersive component to the polar component (γsd / γsp) of the surface free energy of the central region 112 can be selected from the ranges described for the second surface free energy, and are particularly preferably the same as the second surface free energy.

[0103] The electrode 10 according to this embodiment shown in FIG. 14 has the following effects.

[0104] 9 on the first region 110 and the second region 120 of the electrode 10 according to the embodiment shown in Fig. 7 , in which the entire first region 110 has a first surface free energy and the surrounding second region 120 has a second surface free energy, unlike Fig. 14 , typically a nozzle (not shown) is brought close to the first region 110, the first liquid composition P is dispensed from the nozzle and dispensed, and then the nozzle is moved away from the electrode 10. In this case, because the entire first region 110 of the electrode 10 according to the embodiment shown in Fig. 7 has low wettability with the first liquid composition P, when the first liquid composition P is dispensed, the droplets of the first liquid composition P formed on the first region 110 become high and do not separate from the tip of the nozzle, and when the nozzle is moved away from the electrode 10, the first liquid composition P attached to the nozzle may also separate from the electrode 10.

[0105] In the electrode 10 according to this embodiment shown in FIG. 14 , the first region 110 includes a central region 112 having a surface free energy greater than the first surface free energy and a peripheral region 115 surrounding the central region 112 and having the first surface free energy. The central region 112 has relatively higher wettability with respect to the first liquid composition P than the peripheral region 115 of the first region 110. Therefore, when a nozzle is brought close to the central region 112 of the first region 110 and the first liquid composition P is dropped from the nozzle, the droplets of the first liquid composition P on the central region 112 are low in height and easily separate from the tip of the nozzle. Therefore, when the nozzle is moved away from the electrode 10, the first liquid composition P is unlikely to separate from the electrode 10. Therefore, with the electrode 10 according to this embodiment shown in FIG. 14 , it is easy to apply the intended amount of the first liquid composition P onto the first region 110 and the second region 120.

[0106] In the electrode 10 according to this embodiment shown in Fig. 14, the maximum width M (diameter in the illustrated example) of the outer periphery of the second region 120 and the radial width N of the second region 120 may have the same values ​​as M and N in the electrode 10 according to the embodiments shown in Figs. 2, 7, and 8. The maximum width W (diameter in the illustrated example) of the central region 112 may be, for example, 0.5% to 30%, preferably 5% to 25%, of the maximum width M of the outer periphery of the second region 120. The maximum width W of the central region 112 may be, for example, 50 µm to 500 µm, and preferably 90 µm to 300 µm.

[0107] <Embodiment of electrode 10 shown in Fig. 15> Another embodiment of the electrode 10 will be described with reference to Fig. 15. This embodiment shown in Fig. 15 is a modification of the embodiment shown in Fig. 14, and a description of features common to the embodiment shown in Fig. 14 will be omitted.

[0108] In the electrode 10 according to this embodiment, the first region 110 includes: a central region 112 including a first outer peripheral edge 111, and further including an outer peripheral edge 113 disposed inside the first region 110 and located more inward than the first outer peripheral edge 111 of the first region 110, and having a surface free energy greater than the first surface free energy; a peripheral region 115 surrounding the central region 112 and including an inner peripheral edge 114 in contact with the outer peripheral edge 113 of the central region 112 and the first outer peripheral edge 111, and having a first surface free energy; and a connection region 116 extending through the peripheral region 115 from a portion of the outer peripheral edge 113 of the central region 112 to a portion of the second inner peripheral edge 121 of the second region 120 opposite the central region 112 via the peripheral region 115, and having a surface free energy greater than the first surface free energy.

[0109] The connection regions 116 extend in the radial direction of the first region 110. As shown in the figure, it is preferable that a plurality of connection regions 116, for example, 3 to 10 connection regions 116, are included. The plurality of connection regions 116 are preferably arranged at equal intervals in the circumferential direction of the first outer peripheral edge portion 111.

[0110] 15 , the peripheral region 115 is divided into a plurality of parts by a plurality of connection regions 116, each of which has a first surface free energy. In the electrode 10 according to the present embodiment shown in FIG. 15 , the first outer peripheral edge portion 111 and the inner peripheral edge portion 114 are also divided into a plurality of parts by a plurality of connection regions 116.

[0111] The surface free energy of the central region 112 and the surface free energy of the connection region 116 are each greater than the first surface free energy. The surface free energy of the central region 112 and the surface free energy of the connection region 116 may be the same or different, but are preferably the same. The surface free energy of the central region 112 and the surface free energy of the connection region 116 may be the same or different from the second surface free energy of the second region 120, but are preferably both the same as the second surface free energy of the second region 120. A preferred range for the surface free energy of the central region 112 is as described with respect to the embodiment shown in FIG. 14 . The specific difference between the surface free energy of the connection region 116 and the first surface free energy can be selected from the same range as the difference between the second surface free energy and the first surface free energy. In particular, the polar component of the surface free energy of the connection region 116 is preferably greater than the polar component of the first surface free energy, and the specific value of this difference can be selected from the same range as the value described with respect to the difference between the polar component of the second surface free energy and the polar component of the first surface free energy. The value of the surface free energy of the connection region 116, the value of the polar component of the surface free energy of the connection region 116, and the ratio of the dispersive component to the polar component of the surface free energy of the connection region 116 (γsd / γsp) can be selected from the ranges described for the second surface free energy, and are particularly preferably the same as the second surface free energy.

[0112] The electrode 10 according to this embodiment shown in FIG. 15 has the following effect. Compared with the peripheral region 115 of the first region 110, which has a first surface free energy, the central region 112, the connection region 116, and the second region 120, which have higher surface free energies, have relatively high wettability with the first liquid composition P. Therefore, the first liquid composition P dispensed from a nozzle onto the central region 112 of the first region 110 can easily spread from the central region 112, along the connection region 116, to the second region 120. Therefore, with the electrode 10 according to this embodiment, when a nozzle is brought close to the central region 112 of the first region 110, the first liquid composition P is dispensed from the nozzle, and then the nozzle is moved away from the electrode 10, the possibility of the first liquid composition P adhering to the nozzle and being separated from the electrode 10 is even lower than with the electrode 10 according to the embodiment shown in FIG. 14 .

[0113] 7 , 14 , and 15 , the first outer peripheral edge 111 of the first region 110, the second inner peripheral edge 121 of the second region 120, the second outer peripheral edge 122 of the second region 120, and the third inner peripheral edge 131 of the third region 130 each have a circular shape in plan view. Furthermore, in the electrode 10 according to the embodiment shown in FIGS. 14 and 15 , the outer peripheral edge 113 of the central region 112 of the first region 110 and the inner peripheral edge 114 of the peripheral region 115 each have a circular shape in plan view. However, these shapes are not limited to circles and may be other shapes such as polygons (triangles, rectangles, hexagons, etc.).

[0114] For example, the electrode 10 according to the embodiment shown in FIG. 16 is an example in which the first outer peripheral edge portion 111 of the first region 110, the second inner peripheral edge portion 121 of the second region 120, the second outer peripheral edge portion 122 of the second region 120, and the third inner peripheral edge portion 131 of the third region 130 each have a rectangular shape in plan view. In the electrode 10 according to the embodiment shown in FIG. 16, the central region 112 of the first region 110 and the outer peripheral edge portion 113 of the inner peripheral edge portion 114 of the peripheral region 115 each have a circular shape in plan view, but this is not limited thereto and may be another shape such as a polygon. Other features of the electrode 10 according to the embodiment shown in FIG. 16 are the same as those of the electrode 10 according to the embodiment shown in FIG. 14 , and therefore description thereof will be omitted.

[0115] 17 and 18 , the electrode 10 includes a surface portion 11 located on the side opposite to the insulating substrate 20 when the electrode 10 is placed on the insulating substrate 20; a first region 110 formed on the surface portion 11 and including a first outer peripheral edge portion 111, at least a portion of which has a first surface free energy; a second region 120 formed on the surface portion 11 and including a second inner peripheral edge portion 121 surrounding the first region 110 and in contact with the first outer peripheral edge portion 111 of the first region 110, and a second outer peripheral edge portion 122 located outside the second inner peripheral edge portion 121, the second region 120 having a second surface free energy greater than the first surface free energy; The insulating film is provided with: a third region 130 formed on the surface portion 11, which includes a third inner peripheral edge portion 131 surrounding the second region 120 and in contact with the second outer peripheral edge portion 122 of the second region 120, and a third outer peripheral edge portion 132 located outside the third inner peripheral edge portion 131, and which has a third surface free energy smaller than the second surface free energy; and a fourth region 140 formed on the surface portion 11, which includes a fourth inner peripheral edge portion 141 surrounding the third region 130 and in contact with the third outer peripheral edge portion 132 of the third region 130, and a fourth outer peripheral edge portion 142 located outside the fourth inner peripheral edge portion 141, and which has a fourth surface free energy larger than the third surface free energy.

[0116] The features of the first region 110 and the second region 120 in this embodiment shown in Figures 17 and 18 are the same as those described for the electrode 10 according to other embodiments, and therefore will not be described again. Note that the first region 110 in this embodiment shown in Figures 17 and 18 includes a central region 112 having a surface free energy greater than the first surface free energy and a peripheral region 115 having the first surface free energy, similar to the embodiment shown in Figure 14.

[0117] The third surface free energy of the third region 130 may be relatively smaller than the second surface free energy of the second region 120, and may be the same as or different from the first surface free energy of the peripheral region 115 of the first region 110. Preferably, the third surface free energy of the third region 130 is the same as the first surface free energy of the peripheral region 115 of the first region 110. The difference between the third surface free energy of the third region 130 and the second surface free energy and the preferred range of the third surface free energy are as explained in the <Surface Free Energy> section.

[0118] The fourth surface free energy of the fourth region 140 only needs to be relatively larger than the third surface free energy of the third region 130, and may be the same as or different from the second surface free energy of the second region 120. The difference between the fourth surface free energy and the third surface free energy, and specific examples of the value of the fourth surface free energy are as described in the <Surface free energy> section.

[0119] By using the electrode 10 having such a surface portion 11, first region 110, second region 120, third region 130, and fourth region 140, it is possible to form the reagent layer 30 and the protective film 40 with high precision. This mechanism will be described with reference to Figures 19 to 22.

[0120] As shown in FIG. 19 , a first liquid composition P containing a reagent participating in a redox reaction in a first solvent is applied to the first region 110 and the second region 120 of the electrode 10, followed by drying. As shown in FIG. 20 , a reagent layer 30 including an outer peripheral edge 31 located on the second outer peripheral edge 122 of the second region 120 can be formed. This process is the same as the process for forming the reagent layer 30 on the electrode 10 according to the embodiment shown in FIGS. 7 and 8 , which was described with reference to FIGS. 9 and 10 , and therefore further description will be omitted. The electrode 10 of this embodiment shown in FIGS. 17 and 18 allows the reagent layer 30 to be accurately formed with the designed dimensions on the first region 110 and the second region 120 of the surface portion 11. The contact area between the reagent layer 30 and the electrode 10 is determined by the first region 110 and the second region 120 of the electrode 10.

[0121] 17 and 18 includes a third region 130 having a third surface free energy that is less wettable by the first liquid composition P than the second region 120 having the second surface free energy. Therefore, as shown in FIG. 19 , the outward spread of the first liquid composition P applied to the first region 110 and the second region 120 is prevented by a third inner peripheral edge 131 of the third region 130, and the outer periphery is defined by a second outer peripheral edge 122 of the second region 120.

[0122] 21 , a second liquid composition Q containing a protective film component in a second solvent is applied to the first region 110, the second region 120, the third region 130, and the fourth region 140 of the electrode 10 so as to cover the reagent layer 30. Examples of the second solvent and the protective film component of the second liquid composition Q are as described above. The applied second liquid composition Q forms a droplet whose outer periphery is defined by the fourth outer periphery 142 of the fourth region 140.

[0123] The applied second liquid composition Q is then dried to form a protective film 40, as shown in FIG. 22 . The formed protective film 40 is disposed on the first region 110, the second region 120, the third region 130, and the fourth region 140 of the electrode 10 so as to cover the reagent layer 30, and includes an outer peripheral edge 41 located on the fourth outer peripheral edge 142 of the fourth region 140. The area of ​​the protective film 40 is defined by the fourth outer peripheral edge 142 of the fourth region 140. In this manner, the outer shape and area of ​​the protective film 40 can be adjusted by adjusting the outer shapes and areas of the first region 110, the second region 120, the third region 130, and the fourth region 140. Meanwhile, the thickness of the protective film 40 can be adjusted by adjusting the concentrations of the protective film components in the second liquid composition Q and the amount of the second liquid composition Q applied. According to the electrode 10 of this embodiment, it is possible to form a protective film of designed dimensions on the first region 110, the second region 120, the third region 130, and the fourth region 140 with high precision.

[0124] 17 and 18 , the electrode 10 according to this embodiment further includes a fifth region 150 formed on the surface portion 11, the fifth region 150 including a fifth inner peripheral edge 151 surrounding the fourth region 140 and in contact with a fourth outer peripheral edge 142 of the fourth region 140, the fifth region 150 having a fifth surface free energy smaller than the fourth surface free energy. The second liquid composition Q has lower wettability with the fifth region 150 having the fifth surface free energy than the fourth region 140 having the fourth surface free energy. Therefore, the second liquid composition Q applied to the first region 110, the second region 120, the third region 130, and the fourth region 140 of the electrode 10 so as to cover the reagent layer 30 is prevented from spreading outward by the fifth inner peripheral edge 151 of the fifth region 150, thereby more easily achieving the effect of defining the periphery of the droplet at the fourth outer peripheral edge 142 of the fourth region 140. The difference between the fourth surface free energy and the fifth surface free energy, and specific examples of the value of the fifth surface free energy are as described in the <Surface Free Energy> section. In an embodiment in which the surface portion 11 of the electrode 10, which has a uniform surface free energy, is partially subjected to a treatment to increase the surface free energy, thereby forming the second region 120 having the second surface free energy and the fourth region 140 having the fourth surface free energy, the first region 110, the third region 130, and the fifth region 150 have the same surface free energy.

[0125] 23 and 24 of the electrode 10 In the electrode 10 according to the embodiments shown in FIGS. 7, 8, 14, 15, 16, 17, and 18, the second outer peripheral edge 122 of the second region 120 is defined by the third inner peripheral edge 131 of the third region 130. However, this is not limited to this embodiment, and the second outer peripheral edge 122 of the second region 120 may be defined by the insulating layer 50, as in the electrode 10 according to the embodiments shown in FIGS. 23 and 24. FIG. 23 is an enlarged view of a region of the electrode 10 according to this embodiment, which corresponds to region X in the electrode 10 shown in FIG. 2. FIG. 24 is a cross-sectional view taken along line F-F' in FIG. 23.

[0126] The electrode 10 according to this embodiment shown in FIGS. 23 and 24 includes a surface portion 11, a first region 110, a second region 120, and an insulating layer 50, at least a portion of which is disposed on the surface portion 11. The insulating layer 50 has openings 52 formed on the first region 110 and the second region 120, penetrating the insulating layer 50 in the thickness direction. The openings 52 in the insulating layer 50 include an inner peripheral edge 55 that defines a second outer peripheral edge 122 of the second region 120 in a plan view. The electrode 10 according to this embodiment shown in FIGS. 23 and 24 having this configuration achieves the following effect. That is, when a first liquid composition containing a reagent participating in a redox reaction in a first solvent is applied to a recess having the first region 110 and the second region 120 as its bottom surface and surrounded by the openings 52 in the insulating layer 50 of the electrode 10, the first liquid composition is prevented from spreading outward by the inner peripheral edge 55 of the insulating layer 50, and the first liquid composition is applied only onto the first region 110 and the second region 120. The reagent layer 30 formed by drying the applied first liquid composition includes an outer peripheral edge 31 located on the second outer peripheral edge 122 of the second region 120 defined by the inner peripheral edge 55 of the insulating layer 50, and is disposed only on the first region 110 and the second region 120 (see FIG. 25 ). Therefore, with the electrode 10 according to this embodiment shown in FIGS. 23 and 24 , it is possible to form the reagent layer 30 with the designed dimensions on the first region 110 and the second region 120 with even greater precision. Specific examples of materials constituting the insulating layer 50 are as described above in the <Materials> section. The insulating layer 50 preferably has a water-repellent surface, more preferably a water-repellent surface containing a fluororesin.

[0127] The electrode 10 according to this embodiment shown in Figures 23 and 24 can be manufactured by stacking an insulating layer 50 including an opening 52 whose inner peripheral edge 55 is located outside a second inner peripheral edge 121 of the second region 120 and above the second region 120 on the surface portion 11 of the electrode 10, the surface portion 11 including the first region 110 and the second region 120. In this case, the inner peripheral edge 55 of the opening 52 in the insulating layer 50 defines the second outer peripheral edge 122 of the second region 120 within the opening 52. Features of other components (e.g., the first region 110 and the second region 120) of the electrode 10 according to this embodiment shown in Figures 23 and 24 are similar to the features of the respective components of the electrode 10 according to the embodiments shown in Figures 7, 8, 14, 15, 16, 17, and 18.

[0128] 23 and 24 , a reagent layer 30 can be disposed on the first region 110 and the second region 120 of the electrode 10 according to this embodiment, and a protective film 40 can be further disposed thereon (see FIG. 25 ). The protective film 40 can be formed by applying a second liquid composition containing protective film components in a second solvent to a recess having the first region 110 and the second region 120 of the electrode 10 as its bottom surface and surrounded by an opening 52 in the insulating layer 50 in which the reagent layer 30 is disposed, and then drying the second liquid composition. In this case, the applied second liquid composition is also prevented from spreading outward by the inner peripheral edge 55 of the insulating layer 50, and therefore, after drying, the protective film 40 is disposed so as to cover the reagent layer 30 only on the first region 110 and the second region 120.

[0129] <Embodiment of electrode 10 shown in Figs. 30 and 31> Yet another embodiment of the electrode 10 will be described with reference to Figs. 30 and 31. Fig. 30 is an enlarged view of a region of the electrode 10 according to this embodiment, which corresponds to region X in the electrode 10 shown in Fig. 2. Fig. 31 is a cross-sectional view taken along line GG' in the figure. This embodiment is a modification of the embodiment of the electrode 10 shown in Figs. 17 and 18.

[0130] The electrode 10 according to this embodiment shown in FIGS. 30 and 31 includes: a surface portion 11 located on the opposite side of an insulating substrate 20 when placed on the substrate 20; a first region 110 formed on the surface portion 11 and including a first outer peripheral edge portion 111, and having a first surface free energy; a second region 120 formed on the surface portion 11 and including a second inner peripheral edge portion 121 surrounding the first region 110 and in contact with the first outer peripheral edge portion 111 of the first region 110, and a second outer peripheral edge portion 122 located outside the second inner peripheral edge portion 121, and having a second surface free energy greater than the first surface free energy; a third region 130 formed on the surface portion 11, surrounding the second region 120 and including a third inner peripheral edge portion 131 in contact with the second outer peripheral edge portion 122 of the second region 120, and a third outer peripheral edge portion 132 located outside the third inner peripheral edge portion 131, and having a third surface free energy smaller than the second surface free energy; a fourth region 140 formed on the surface portion 11, surrounding the third region 130 and including a fourth inner peripheral edge portion 141 in contact with the third outer peripheral edge portion 132 of the third region 130, and a fourth outer peripheral edge portion 142 located outside the fourth inner peripheral edge portion 141, and having a fourth surface free energy larger than the third surface free energy; and an insulating layer (50) formed on a first region (110), a second region (120), a third region (130) and a fourth region (140), at least a portion of which is disposed on the surface portion (11), the insulating layer (50) having an inner peripheral edge (55) that contacts a fourth outer peripheral edge (142) of the fourth region (140) in a plan view perpendicular to the surface portion (11), and including an opening (52) that penetrates in the thickness direction.

[0131] 30 and 31 , the features of the surface portion 11, the first region 110, the second region 120, the third region 130, the fourth region 140, and the insulating layer 50 other than those described below are the same as those described for the electrode 10 according to other embodiments, and therefore further description thereof will be omitted. Note that the first region 110 in the present embodiment shown in FIG. 30 and 31 has a first surface free energy as a whole, similar to the embodiment shown in FIG. 7 .

[0132] Fig. 32 shows a cross-sectional view of a sensor 1 including a working electrode 2 including the electrode 10 according to the present embodiment shown in Fig. 30 and Fig. 31. The working electrode 2 (sensor 1) shown in Fig. 32 includes a substrate 20, the electrode 10 according to the present embodiment shown in Fig. 30 and Fig. 31 arranged on the substrate 20, a reagent layer 30 arranged on the first region 110 and the second region 120 in the opening 52 of the insulating layer 50, and a protective film 40 arranged on the first region 110, the second region 120, the third region 130, and the fourth region 140 in the opening 52 of the insulating layer 50 so as to cover the reagent layer 30.

[0133] 32, the reagent layer 30 includes an outer peripheral portion 31 located on the second outer peripheral portion 122 of the second region 120 and a reagent participating in an oxidation-reduction reaction. The reagent layer 30 can be formed by the method described with reference to FIGS. 9 and 19.

[0134] In the working electrode 2 (sensor 1) shown in Figure 32, the protective film 40 is located on the fourth outer peripheral edge 142 of the fourth region 140 and includes an outer peripheral edge 41 defined by the inner peripheral edge 55 of the insulating layer 50. As described with reference to Figures 11 and 21, the protective film 40 is formed by applying a second liquid composition containing protective film components in a second solvent to the first region 110, the second region 120, the third region 130, and the fourth region 140 within the opening 52 of the insulating layer 50 so as to cover the reagent layer 30, and then drying the composition. In this case, using the electrode 10 shown in Figures 30 and 31 has the following advantages.

[0135] Since the applied second liquid composition is prevented from spreading outward by the inner peripheral edge 55 of the insulating layer 50, the outer peripheral edge 41 of the protective film 40 after drying can be positioned on the fourth outer peripheral edge 142 of the fourth region 140.

[0136] On the other hand, when the insulating layer 50 having the openings 52 is formed on the surface portion 11 of the electrode 10 by a printing method such as screen printing, the inner periphery 55 of the openings 52 may have fine irregularities in a plan view from a direction perpendicular to the surface portion 11 due to constraints on processing accuracy. When the electrode 10 including such an insulating layer 50 is an electrode 10 in which the outer periphery of the bottom surface of the recess formed by the openings 52 of the insulating layer 50 and the surface portion 11 of the electrode 10 is a third region 130 having a third surface free energy that has low wettability with the second liquid composition, as shown in FIG. 11 , the following problem may occur. That is, the applied second liquid composition cannot adhere to the inner periphery 55 of the openings 52 of the insulating layer 50 and does not wet and spread over the entire openings 52 of the insulating layer 50. This may result in a problem that the area of ​​the protective film 40 formed after drying is smaller than designed, and the protective film 40 is prone to peeling. Furthermore, within the opening 52 of the insulating layer 50, the outer peripheral edge 41 of the protective film 40 after drying may not adhere sufficiently to the third region 130 of the surface portion 11 of the electrode 10, which may result in a problem that the protective film 40 is easily peeled off. In the electrode 10 shown in Figures 30 and 31, the outer peripheral edge of the bottom surface of the recess formed by the opening 52 of the insulating layer 50 and the surface portion 11 of the electrode 10 is a fourth region 140 having a fourth surface free energy that is highly wettable with the second liquid composition. Therefore, even if the inner peripheral edge 55 of the opening 52 of the insulating layer 50 has fine irregularities, the applied second liquid composition can wet and spread to the fourth outer peripheral edge 142 of the fourth region 140 and can adhere to the inner peripheral edge 55 of the opening 52 of the insulating layer 50 that is in contact with the fourth outer peripheral edge 142 in a planar view. As a result, after drying, the protective film 40 is formed with the designed area over the entire opening 52 of the insulating layer 50, and the outer peripheral edge 41 of the protective film 40 can adhere to the fourth outer peripheral edge 142 of the fourth region 140, which is preferable because it is not easily peeled off.

[0137] Next, a method for manufacturing the electrode 10 according to this embodiment shown in FIGS. 30 and 31 will be described with reference to FIGS.

[0138] A method for manufacturing an electrode 10 according to this embodiment includes preparing an untreated electrode 10A shown in Fig. 33. The untreated electrode 10A includes an untreated surface portion 11A having a first surface free energy and including an untreated region 500 for forming the first region 110, the second region 120, the third region 130, and the fourth region 140, and an insulating layer 50 having an opening 52 formed on the untreated region 500 and penetrating in the thickness direction, at least a portion of which is disposed on the untreated surface portion 11A. The materials constituting the untreated electrode 10A and the insulating layer 50 can be selected from the same range as the materials constituting the electrode 10 and the insulating layer 50 in the electrode 10 and the sensor 1 of each embodiment disclosed herein.

[0139] The manufacturing method of the electrode 10 according to this embodiment further includes irradiating with laser light a fourth annular untreated region 540 arranged on the outer periphery of the untreated region 500 of the untreated surface portion 11A of the untreated electrode 10A, which is adjacent to the inner peripheral edge portion 55 of the opening 52 of the insulating layer 50 in a planar view perpendicular to the untreated surface portion 11A, and a second annular untreated region 520 arranged further inward than the fourth untreated region 540, thereby converting the fourth untreated region 540 into a fourth region 140 and converting the second untreated region 520 into a second region 120.

[0140] 34 is an enlarged schematic view of a portion of the untreated electrode 10A shown in FIG. 33 , including the boundary portion between the untreated region 500 of the untreated surface portion 11A and the insulating layer 50 around the opening 52, in the plan view. In the illustrated example, the fine irregularities of the inner peripheral edge 55 of the opening 52 of the insulating layer 50 are emphasized. Of the untreated region 500, the portion contained within the second untreated region 520 is the first untreated region 510, and the portion between the second untreated region 520 and the fourth untreated region 540 is the third untreated region 530. That is, the untreated area 500 consists of a first untreated area 510, a ring-shaped second untreated area 520 surrounding the first untreated area 510, a ring-shaped third untreated area 530 surrounding the second untreated area 520, and a ring-shaped fourth untreated area 540 surrounding the third untreated area 530, and the entire untreated area 500 has a first surface free energy.

[0141] FIG. 35 schematically illustrates a portion of the electrode 10 obtained by irradiating the electrode 10A shown in FIG. 34 with laser light to convert the fourth untreated region 540 into the fourth region 140 and the second untreated region 520 into the second region 120. Here, the laser light irradiation is, for example, a process of scanning and irradiating the laser light to increase the surface free energy of the second untreated region 520 and the fourth untreated region 540, thereby converting them into the second region 120 having the second surface free energy and the fourth region 140 having the fourth surface free energy, respectively. The first untreated region 510 and the third untreated region 530 of the untreated region 500 in the untreated electrode 10A are preferably not irradiated with laser light and become the first region 110 and the third region 130 of the electrode 10, each having a first surface free energy. In this case, the third region 130 has a third surface free energy equal to the first surface free energy. The preferred mode of the irradiated laser light is as described above.

[0142] 34 , when the fourth untreated region 540 of the untreated region 500 of the untreated surface portion 11A is irradiated with laser light, it is preferable to irradiate the laser light not only to the fourth untreated region 540 but also to an opening peripheral region 58 of the insulating layer 50 that surrounds the opening 52 and is adjacent to the outside of the fourth untreated region 540 in a plan view from a direction perpendicular to the untreated surface portion 11A. According to this aspect, even if the inner peripheral edge 55 of the opening 52 of the insulating layer 50 has minute irregularities that are intricately formed on the inside, as shown in the figure, the fourth untreated region 540 of the untreated region 500 can be converted into the fourth region 140 by irradiating the laser light up to the inner peripheral edge 55. According to the electrode 10 of the embodiment shown in Figures 30 and 31 manufactured in this manner, the second liquid composition applied within the opening 52 of the insulating layer 50 can wet and spread to the fourth outer peripheral edge 142 of the fourth region 140, and the protective film 40 obtained upon drying can have the designed area and is not easily peeled off, which is particularly likely to achieve the above-mentioned effects.

[0143] In a more preferred embodiment of the embodiment in which laser light is irradiated to the opening peripheral region 58 of the insulating layer 50 in addition to the fourth untreated region 540 in the untreated region 500 of the untreated surface portion 11A shown in Figure 34, the method further includes irradiating the opening peripheral region 58 of the insulating layer 50 with laser light to destroy the insulating layer 50 in the opening peripheral region 58, thereby converting the portion of the untreated surface portion 11A exposed by the destruction into a fourth region 140. Figure 36 schematically shows a portion of the electrode 10 manufactured by this preferred embodiment. The fourth region 140 of the electrode 10 manufactured by this preferred embodiment is a region in the untreated surface portion 11A in which the surface free energy of the fourth untreated region 540 of the untreated region 500 and the portion of the insulating layer 50 exposed by the destruction of the opening peripheral region 58 have been increased to a fourth surface free energy by laser light irradiation. According to this preferred aspect, fourth outer peripheral edge 142 of fourth region 140 that defines the outer periphery of protective film 40 and inner peripheral edge 55 of opening 52 in insulating layer 50 are formed by laser light irradiation, so it is possible to define the outer periphery of protective film 40 with higher accuracy compared to when insulating layer 50 having opening 52 is formed by printing. Furthermore, according to this preferred aspect, second outer peripheral edge 122 of second region 120 that defines the outer periphery of reagent layer 30, and fourth outer peripheral edge 142 of fourth region 140 that defines the outer periphery of protective film 40 and inner peripheral edge 55 of opening 52 in insulating layer 50 are all formed by laser light irradiation, so it is possible to manufacture electrode 10 in which reagent layer 30 and protective film 40 can be formed coaxially with each other more accurately.

[0144] In the following experiments, operations for which no particular temperature is specified were performed at room temperature (21°C ± 3°C). <Experiment 1> As shown in Figure 7, a ring-shaped portion of the surface portion 11 of the electrode 10 was laser processed to increase the surface free energy and form a second region 120, with the inside of the second region 120 designated as the first region 110 and the outside as the third region 130. A reagent layer 30 was formed on the first region 110 and the second region 120 of the obtained electrode 10, thereby producing a sensor 1 equipped with a working electrode 2.

[0145] (Substrate) As the insulating substrate 20, a substrate made of polyethylene terephthalate and having a thickness of 188 μm, having the shape shown in FIG. 1 etc., was used.

[0146] (Electrodes) A ​​carbon paste was applied to the first surface 21 of the insulating substrate 20 and heated at 140° C. for 1 hour to form two electrodes 10, 10, a reference electrode conductive layer 301, a counter electrode 4, and wiring 5 electrically connected to each of them, each made of a 5 μm-thick carbon conductive layer, in the shape shown in FIG. 1 .

[0147] (Laser Processing) Laser processing was performed on a circular portion of the surface portion 11 of the electrode 10 placed on the substrate 20 to increase the surface free energy and form the second region 120. Laser processing was performed under the following conditions on a circular portion having a diameter M of 1.2 mm and a width N of 0.1 mm, 0.2 mm, or 0.3 mm as shown in Figure 7, to form the second region 120 of the electrode 10 of the example. As a comparative example, laser processing was performed under the same conditions on a circular region having a diameter of 1.2 mm on the surface portion 11 of the electrode 10 placed on the substrate 20.

[0148]

[0149] The surface free energy (dispersive component, polar component) of the surface portion 11 of the electrode 10 was determined before laser processing and after a predetermined time had elapsed since laser processing. The surface free energy was calculated using simultaneous equations consisting of the above-mentioned formulas (1), (2), and (3) by measuring the contact angles (1 μL drop amount, measured 2 seconds after dropping) of water and diiodomethane using a contact angle meter at 20° C. and a relative humidity of 40%.

[0150] The surface free energy (dispersive component, polar component) of the second region 120 of the electrode 10 of the example formed by laser processing and the circular region of the electrode of the comparative example at each elapsed time after laser processing is shown in the table below. The surface free energy at 0 minutes elapsed time is the surface free energy of the surface portion 11 of the electrode 10 before laser processing, and was considered to be the surface free energy of the first region 110 and the third region 130.

[0151]

[0152] (First Liquid Composition) As the first liquid composition for forming a reagent layer, an aqueous solution containing a carbon black dispersion, hydroxypropyl cellulose, a polymer-bound mediator, lactate oxidase, polyimidazole, poly-L-lysine, and a crosslinking agent in water was prepared.

[0153] (Formation of Reagent Layer) 24 hours after the laser processing, 0.65 mg of the first liquid composition was dropped onto the center of the first region 110 of the electrode 10 of the example, which had a first region 110 and a laser-processed annular second region 120 of each width surrounding it, to form a droplet, which was then dried to form a reagent layer 30 containing lactate oxidase and a mediator, thereby producing a sensor 1 equipped with a working electrode 2.

[0154] On the other hand, 24 hours after the laser processing, 0.65 mg of the first liquid composition was dropped onto the center of the circular region of the comparative electrode having the laser-processed circular region to form a droplet, which was then dried to form a reagent layer containing lactate oxidase and a mediator.

[0155] A photograph of the reagent layer formed on each electrode is shown in Figure 27. In all of the electrodes 10 of the example, the reagent layer 30 was formed only on the first region 110 and the second region 120, and the outer peripheral edge 31 of the reagent layer 30 was located on the second outer peripheral edge 122 of the second region 120.

[0156] On the other hand, in 8 out of 12 samples of the comparative electrode, the first liquid composition applied to the circular region spilled over the circular region, resulting in an irregularly shaped reagent layer after drying.

[0157] <Experiment 2> Using the same material and following the same procedure as in Experiment 1, a circular second region 120 having a diameter M of 1.2 mm and a width N of 0.3 mm as shown in FIG. 7 was formed on the surface portion 11 of the electrode 10, with the first region 110 inside and the third region 130 outside.

[0158] A nozzle was brought close to the center of the first region 110 of this electrode 10 to a distance of 0.5 mm, and 0.65 mg of the same first liquid composition as in Experiment 1 was dropped from the nozzle, and then the nozzle was moved away and dried to form a reagent layer 30 containing lactate oxidase and a mediator.

[0159] Formation of the reagent layer 30 was carried out ten times, and in five of these, when the nozzle was moved away, the first liquid composition that had adhered to the nozzle separated from the electrode 10 and could not be applied.

[0160] <Experiment 3> Using the same material and performing laser processing using the same procedure as in Experiment 1, an electrode 10 was created that had the shape shown in Fig. 14 and included a first region 110 including a central region 112 (laser-processed portion) and a peripheral region 115 (unprocessed portion), an annular second region 120 (laser-processed portion), and a third region 130 outside the second region 120. As shown in Fig. 14, the diameter W of the central region 112 was 0.25 mm, the diameter M of the second region 120 was 1.2 mm, and the width N of the second region 120 was 0.25 mm.

[0161] As in Experiment 2, the nozzle was brought close to the central region 112 of the first region 110 of the electrode 10 to a distance of 0.5 mm, and 0.65 mg of the same first liquid composition as in Experiments 1 and 2 was dropped from the nozzle. The nozzle was then moved away and dried to form a reagent layer 30 containing lactate oxidase and a mediator.

[0162] The first liquid composition was applied and dried approximately 200 times, and each time the first liquid composition was applied normally. The reagent layer 30 obtained by drying the applied first liquid composition was formed only on the first region 110 and the second region 120, and the outer periphery 31 of the reagent layer 30 was accurately positioned on the second outer periphery 122 of the second region 120. A photograph of a portion of the formed reagent layer 30 is shown in Figure 28.

[0163] <Experiment 4> Using the same material and under the same conditions as in Experiment 1, laser processing was performed under the same conditions. As in Experiment 1, a circular second region 120 having a diameter M of 1.2 mm and a width N of 0.1 mm or 0.3 mm as shown in Fig. 7 was formed on the surface portion 11 of the electrode 10 disposed on the first surface 21 of the insulating substrate 20, with the inside of the second region 120 designated as the first region 110 and the outside of the first region 110 designated as the third region 130. As in Experiment 1, as a comparative example, laser processing was performed under the same conditions on a circular region having a diameter of 1.2 mm on the surface portion 11 of the electrode 10 disposed on the substrate 20.

[0164] As a first liquid composition for forming a reagent layer, a liquid containing a carbon black dispersion, a polymer-bound mediator, lactate oxidase, polyimidazole, and ε-poly-L-lysine in ultrapure water (Milli-Q (registered trademark) water) was prepared.

[0165] Twenty-four hours after the laser processing, an amount of the first liquid composition described below was dropped onto the center of the first region 110 of the electrode 10 of the example, which had a first region 110 and a laser-processed circular second region 120 of each width surrounding it, to form a droplet, which was then dried to form a reagent layer 30 for detecting lactic acid. Meanwhile, 24 hours after the laser processing, an amount of the first liquid composition described below was dropped onto the center of the circular region of the electrode of the comparative example, which had a laser-processed circular region, to form a droplet, which was then dried to form a reagent layer for detecting lactic acid.

[0166] In this test, the first liquid composition was dropped to form the reagent layer in amounts gradually increased by 0.05 g starting from 0.40 g. The maximum amount of the first liquid composition capable of forming a circular reagent layer without overflowing on the first region 110 and the second region 120 of each example electrode 10 or on the circular region of the comparative electrode (the maximum amount capable of forming a circular reagent layer without overflowing after 10 tests) was confirmed. As a result, it was confirmed that the maximum amount of the first liquid composition was 0.75 mg for the example electrode 10 having a circular second region 120 with a diameter M of 1.2 mm and a width N of 0.1 mm, as shown in FIG. 7 , 0.70 mg for the example electrode 10 having a circular second region 120 with a diameter M of 1.2 mm and a width N of 0.3 mm, and 0.60 mg for the comparative electrode having a circular region with a diameter of 1.2 mm. That is, it was confirmed that by forming a first region 110 in the center having a first surface free energy with low wettability to the first liquid composition, and forming a second region 120 around it having a second surface free energy with high wettability to the first liquid composition, a larger amount of the first liquid composition can be supported, and that the smaller the width N of the second region 120, the larger the amount of the first liquid composition that can be supported.

[0167] <Experiment 5> 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 an outline of the manufacturing method are described below. Of the two working electrodes 2, one was used as a working electrode for detecting lactate, and the other was used as a working electrode for detecting glucose. Since lactate was measured in the measurement test described below, only the manufacturing method of the working electrode for detecting lactate will be described below.

[0168] (Substrate) As the insulating substrate 20, a substrate made of polyethylene terephthalate and having a thickness of 188 μm, having the shape shown in FIG. 1 etc., was used.

[0169] (Conductive Layer) A carbon paste was applied to the first surface 21 of the insulating substrate 20 and heated at 140° C. for 1 hour to form the electrode 10, the reference electrode conductive layer 301, the counter electrode 4, and the wiring 5 electrically connected to each of them, each having the shape shown in FIG. 1 , using a 5 μm-thick carbon conductive layer.

[0170] (Laser Processing) Under the conditions described in Experiment 1, laser processing was performed on a circular portion of the surface portion 11 of the electrode 10 disposed on the substrate 20, the portion having a diameter M of 1.4 mm and a width N of 0.3 mm as shown in Fig. 7, to increase the surface free energy and form the second region 120. The portion of the surface portion 11 of the electrode 10 inside the second region 120 is the first region 110, and the portion outside the second region 120 is the third region 130.

[0171] (Insulating Layer) Subsequently, as shown in FIG. 3, an insulating layer 50 made of fluororesin was laminated on the first surface 21 of the substrate 20 on which the carbon conductive layer was disposed.

[0172] The openings 52 formed on the first region 110, the second region 120, and the third region 130 of the insulating layer 50 were circular and had a diameter of 2.0 mm. The reference electrode opening 53 was circular and had a diameter of 1.0 mm, and the counter electrode opening 54 was rectangular and had dimensions of 2.7 mm x 2.65 mm.

[0173] (Reagent layer) 24 hours after the laser processing, 0.40 mg, 0.45 mg, 0.50 mg, 0.55 mg, and 0.60 mg of the lactate oxidase-containing first liquid composition having the composition described in Experiment 4 were dropped onto the center of the first region 110 within the opening 52 of the insulating layer 50 to form droplets on the first region and the second region, which were then dried to form a reagent layer 30 for detecting lactate (see Figure 4).

[0174] (Protective film) Ethanol (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) and a sodium hydroxide aqueous solution (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) were added to a Nafion (registered trademark) dispersion (manufactured by Sigma-Aldrich Corporation) to adjust the pH of the dispersion (neutralization of the cation exchange group), and the precipitate was dissolved in a vortex mixer and adjusted to 9 wt % to prepare a second liquid composition A.

[0175] The following reagents were mixed with ethanol to the following final concentrations and allowed to react for about 1 hour to prepare a second liquid composition B: 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 or less, manufactured by Sigma-Aldrich), 0.9 wt%.

[0176] After the reagent layer 30 was formed, 0.6 mg of the second liquid composition A was dropped into the opening 52 of the insulating layer 50 to form droplets, which were then dried to form a first layer of the protective film 40, and then 0.6 mg of the second liquid composition B was dropped into the opening 52 to form droplets, which were then dried to form a second layer of the protective film 40, thereby creating a two-layer protective film 40. In this way, a working electrode 2 having the cross-sectional structure shown in FIG. 11 (although not shown, the protective film 40 has a two-layer structure) was completed.

[0177] (Reference Electrode) A silver / silver chloride paste was applied to the reference electrode conductive layer 301 in the reference electrode opening 53 of the insulating layer 50, and heated at 140° C. for 1 hour to form a silver / silver chloride layer 302. Subsequently, a reference electrode protective film 303 was placed on the silver / silver chloride layer 302 to form a reference electrode 3 having the cross-sectional structure shown in FIG.

[0178] (Storage under High Temperature Conditions) The sensor 1 manufactured as described above was stored at 60° C. for two weeks after manufacture, and then used to measure the lactic acid concentration.

[0179] (Continuous measurement of lactate concentration) Measurement medium: DMEM, no glucose (Gibco 11966025) Penicillin-streptomycin-amphotericin B suspension (x100) (antibiotic-antimycotic solution) (Fujifilm Wako Pure Chemical Industries, Ltd. 161-23181) Sodium pyruvate (100 mM) (Gibco 11360070) Using the above reagents, a DMEM medium containing a penicillin-streptomycin-amphotericin B suspension at a final concentration of 1% (v / v) and sodium pyruvate at a final concentration of 1 mM was prepared.

[0180] Measurement media were prepared using DMEM media as follows: Medium 1: DMEM medium adjusted to a final lactate concentration of 6 mM Medium 2: DMEM medium adjusted to a final lactate concentration of 12 mM Medium 3: DMEM medium adjusted to a final FBS (Fetal Bovine Serum Gibco 16140071) concentration of approximately 9% (v / v) and a final lactate concentration of 16 mM Blank medium: DMEM medium adjusted to a final lactate concentration of 10 mM

[0181] A voltage of 100 mV was applied to the working electrode 2 immersed in the measurement medium relative to the reference electrode 3 (Ag / AgCl), and the current value between the working electrode 2 and the counter electrode 4 was measured continuously for about 12 days (about 288 hours). ２ Incubator, CO ２ The test was carried out at 5% and a temperature of 37°C.

[0182] Measurement test (N=3): The above-mentioned media 1 to 3 were used as measurement media, and were replaced over time in the following order, and the above measurements were carried out: Medium 1 for about 24 hours → Medium 2 for about 24 hours → Medium 3 for about 72 hours → Medium 1 for about 9 hours → Medium 2 for about 15 hours → Medium 3 for about 72 hours → Medium 3 for about 72 hours

[0183] Blank test (N=1): The blank medium was used as the measurement medium, and was replaced with a new one at the same timing as in the above measurement test, and the above measurement was carried out for about 288 hours.

[0184] The results of measuring the current value between the working electrode 2 and counter electrode 4 for measuring lactate acid are shown in Figures 29A to 29E. The current value for sensor 1 when the amount of the lactate oxidase-containing first liquid composition having the composition described in Experiment 4 used to prepare the reagent layer 30 was applied in an amount of 0.40 mg is shown in Figure 29A, the current value for sensor 1 when the amount was 0.45 mg is shown in Figure 29B, the current value for sensor 1 when the amount was 0.50 mg is shown in Figure 29C, the current value for sensor 1 when the amount was 0.55 mg is shown in Figure 29D, and the current value for sensor 1 when the amount was 0.60 mg is shown in Figure 29E.

[0185] The decay rate of the current value of sensor 1 after 12 days was 37.8% when the applied amount of the lactate oxidase-containing first liquid composition described in Experiment 4 used to prepare the reagent layer 30 was 0.40 mg ( FIG. 29A ), 29.0% when it was 0.45 mg ( FIG. 29B ), 20.0% when it was 0.50 mg ( FIG. 29C ), 17.0% when it was 0.55 mg ( FIG. 29D ), and 11.0% when it was 0.60 mg ( FIG. 29E ). This result indicates that the greater the amount of reagent carried in the reagent layer 30, the longer the continuous measurement of the test substance can be performed. This suggests that sensor 1 equipped with electrode 10 disclosed herein is suitable for long-term continuous measurement of the test substance because it can carry a large amount of reagent in the reagent layer 30.

[0186] 30 and 31 was manufactured, and then a reagent layer 30 and a protective film 40 were formed to manufacture the working electrode 2 shown in Fig. 32. However, in this experiment 6, unlike the examples shown in Figs. 30 to 32, instead of the annular second region 120 containing the circular first region 110, the first region 110 was not provided, and instead a circular second region 120 having a second surface free energy as a whole was formed, and the reagent layer 30 was placed on the circular second region 120.

[0187] An untreated electrode 10A was formed by applying a carbon paste to the first surface 21 of an insulating substrate 20 made of polyethylene terephthalate and having a thickness of 188 μm, and heating the substrate at 140° C. for 1 hour, thereby forming an untreated electrode 10A with a carbon conductive layer having a thickness of 5 μm.

[0188] Subsequently, an insulating layer 50 made of fluororesin was laminated by screen printing on the first surface 21 of the substrate 20 on which the untreated electrode 10A was disposed. The thickness of the insulating layer 50 on the untreated electrode 10A was set to 5 μm. The opening 52 formed in the insulating layer 50 in the untreated region 500 of the untreated surface portion 11A of the untreated electrode 10A was a circle with a diameter (inner diameter) of 2.0 mm.

[0189] In Experiment 6-1, under the conditions described in Experiment 1, laser light was applied to a circular second untreated region 520 of the untreated surface portion 11A of the untreated electrode 10A within the opening 52 of the insulating layer 50, the second untreated region 520 being 1.4 mm in diameter from the center of the untreated region 500, and a ring-shaped fourth untreated region 540 located 1.75 mm from the center of the untreated region 500 on the outer periphery, adjacent to the inner periphery 55 of the opening 52 of the insulating layer 50. The second untreated region 520 was converted into a circular second region 120, and the fourth untreated region 540 was converted into a ring-shaped fourth region 140. At this time, the opening periphery region 58 of the insulating layer 50, adjacent to the outside of the fourth untreated region 540, was also irradiated with laser light. The portion between the second region 120 and the fourth region 140 after laser light irradiation was the third region 130. Since laser light irradiation was performed under the same conditions as in Experiment 1, the surface free energy γs of the untreated region 500 of the untreated surface portion 11A of the untreated electrode 10A and the third region 130 of the electrode 10 was 47.82 mN / m (the dispersion component γsd was 44.92 mN / m, and the polar component γsp was 2.90 mN / m). 24 hours after laser light irradiation, the surface free energy γs of the second region 120 and the fourth region 140 of the electrode 10 was 60.28 mN / m (the dispersion component γsd was 48.48 mN / m, and the polar component γsp was 11.80 mN / m). A photograph of the electrode 10 after laser light irradiation in Experiment 6-1 is shown in the upper right corner of Figure 37.

[0190] In Experiment 6-2, under the conditions described in Experiment 1, laser light was irradiated only to a circular second untreated region 520 having a diameter of 1.4 mm from the center of the untreated region 500 of the untreated surface portion 11A of the untreated electrode 10A in the opening 52 of the insulating layer 50, converting it into a circular second region 120. After laser light irradiation, the portion outside the second region 120 in the opening 52 of the insulating layer 50 is the third region 130. The surface free energies of the second region 120 and the third region 130 after laser light irradiation are the same as in Experiment 6-1. A photograph of the electrode 10 after laser light irradiation in Experiment 6-2 is shown in the upper left of Figure 37.

[0191] (Reagent layer) 24 hours after laser processing, 0.5 mg of the lactate oxidase-containing first liquid composition having the composition described in Experiment 4 was dripped onto the center of the second region 120 within the opening 52 of the insulating layer 50 of the electrode 10 of Experiment 6-1 or Experiment 6-2 to form a droplet on the second region 120, which was then dried to form a reagent layer 30 for detecting lactate (see Figure 32).

[0192] (Protective Film) After the reagent layer 30 was formed, 0.6 mg of the second liquid composition A described in Experiment 5 was dropped into the opening 52 of the insulating layer 50 of the electrode 10 of Experiment 6-1 or Experiment 6-2 to form a droplet, which was then dried to form a first layer of the protective film 40. Next, 0.6 mg of the second liquid composition B described in Experiment 5 was dropped into the opening 52 of the insulating layer 50 of the electrode 10 of Experiment 6-1 or Experiment 6-2 to form a droplet, which was then dried to form a second layer of the protective film 40, thereby creating a two-layer protective film 40. In this way, a working electrode 2 having the cross-sectional structure shown in FIG. 32 (although not shown, the protective film 40 had a two-layer structure) was completed. The thickness of the protective film 40 after drying on the electrode 10 of Experiment 6-1 or Experiment 6-2 was measured based on the difference in height measured before and after the formation of the protective film 40 using a white light interferometer displacement meter. The thickness measurement results are shown in FIG. 38.

[0193] (Shape of the Protective Film) A photograph of the working electrode 2 using the electrode 10 of Experiment 6-2 is shown in the lower left of Figure 37. In the working electrode 2 using the electrode 10 of Experiment 6-2, the outer diameter of the protective film 40 was 1.88 mm, which was smaller than the designed inner diameter of the opening 52 of the insulating layer 50. Furthermore, as shown in Figure 38, the thickness of the protective film 40 in the working electrode 2 using the electrode 10 of Experiment 6-2 was approximately 22 µm, which was thicker than that in the case using the electrode 10 of Experiment 6-1, and the standard deviation was approximately twice as large. These results suggest that the inner peripheral edge 55 of the opening 52 of the insulating layer 50 formed by screen printing had fine irregularities in a plan view, and that in the electrode 10 of Experiment 6-2, the second liquid compositions A and B used to form the protective film 40 applied to the opening 52 of the insulating layer 50 were unable to sufficiently adhere to the inner peripheral edge 55 of the opening 52 of the insulating layer 50 and the third outer peripheral edge 132 of the third region 130.

[0194] A photograph of the working electrode 2 using the electrode 10 of Experiment 6-1 is shown in the lower right of Figure 37. In the working electrode 2 using the electrode 10 of Experiment 6-1, the outer diameter of the protective film 40 matched 2.0 mm, which was the design value of the inner diameter of the opening 52 of the insulating layer 50. Furthermore, as shown in Figure 38, it was confirmed that the thickness of the protective film 40 in the working electrode 2 using the electrode 10 of Experiment 6-1 was approximately 18 µm, which was thinner than when the electrode 10 of Experiment 6-2 was used, and the standard deviation was smaller. In the electrode 10 of Experiment 6-1, the outer peripheral edge of the bottom within the opening 52 of the insulating layer 50 was the fourth region 140 having the fourth surface free energy, and therefore the second liquid compositions A and B for forming the protective film 40 were able to spread over the entire opening 52 of the insulating layer 50, and a part of the insulating layer 50 corresponding to the fine irregularities on the inner peripheral edge 55 of the opening 52 was destroyed by the laser light irradiation, and the exposed untreated surface portion 11A was converted into the fourth region 140, which suggests that the second liquid compositions A and B were able to adhere sufficiently to the inner peripheral edge 55 of the opening 52 of the insulating layer 50, and therefore the protective film 40 was able to be formed over the entire opening 52 of the insulating layer 50.

[0195] This specification includes disclosures related to the following Supplementary Notes 1 to 20. <Supplementary Note 1> An electrode to be placed on an insulating substrate, comprising: a surface portion located on an opposite side to the substrate when the electrode is placed on the substrate; a first region formed on the surface portion and including a first outer peripheral edge portion, at least a portion of which has a first surface free energy; a second region formed on the surface portion and including a second inner peripheral edge portion surrounding the first region and in contact with the first outer peripheral edge portion of the first region and a second outer peripheral edge portion located outside the second inner peripheral edge portion, the second region having a second surface free energy greater than the first surface free energy; and a third region formed on the surface portion and including a third inner peripheral edge portion surrounding the second region and in contact with the second outer peripheral edge portion of the second region, the third region having a third surface free energy smaller than the second surface free energy.

[0196] <Supplementary Note 2> The electrode according to Supplementary Note 1, wherein the entire first region has the first surface free energy.

[0197] <Supplementary Note 3> The electrode according to Supplementary Note 1, wherein the first region includes: a central region disposed inside the first region, including an outer peripheral edge portion located more inward than the first outer peripheral edge portion of the first region, and having a surface free energy greater than the first surface free energy; and a peripheral region surrounding the central region, including an inner peripheral edge portion in contact with the outer peripheral edge portion of the central region and the first outer peripheral edge portion, and having the first surface free energy.

[0198] <Supplementary Note 4> The electrode according to Supplementary Note 3, wherein the first region further includes a connection region having a surface free energy greater than the first surface free energy, the connection region extending through the peripheral region from a part of the outer peripheral edge of the central region to a part of the second inner peripheral edge of the second region opposite the peripheral region across the peripheral region.

[0199] <Supplementary Note 5> The electrode according to any one of Supplementary Notes 1 to 4, wherein the third region further includes a third outer peripheral edge portion located outside the third inner peripheral edge portion, and further comprises a fourth region formed on the surface portion, the fourth region including a fourth inner peripheral edge portion surrounding the third region and in contact with the third outer peripheral edge portion of the third region, and a fourth outer peripheral edge portion located outside the fourth inner peripheral edge portion, and the fourth region having a fourth surface free energy greater than the third surface free energy.

[0200] <Supplementary Note 6> The electrode according to Supplementary Note 5, further comprising an insulating layer formed on the first region, the second region, the third region, and the fourth region, at least a portion of which is disposed on the surface portion, the insulating layer having an inner peripheral edge portion in contact with the fourth outer peripheral edge portion of the fourth region in a plan view from a direction perpendicular to the surface portion, and including an opening penetrating in a thickness direction.

[0201] <Supplementary Note 7> The electrode according to any one of Supplementary Notes 1 to 5, further comprising an insulating layer, at least a portion of which is disposed on the surface portion, the insulating layer including openings formed on the first region, the second region, and the third region and penetrating in a thickness direction.

[0202] <Supplementary Note 8> An electrode disposed on an insulating substrate, comprising: a surface portion located on an opposite side to the substrate when disposed on the substrate; a first region formed on the surface portion, including a first outer circumferential edge portion, at least a portion of which has a first surface free energy; a second region formed on the surface portion, surrounding the first region, including a second inner circumferential edge portion in contact with the first outer circumferential edge portion of the first region and a second outer circumferential edge portion located outward from the second inner circumferential edge portion, the second region having a second surface free energy greater than the first surface free energy; and an insulating layer, at least a portion of which is disposed on the surface portion, the insulating layer having an inner circumferential edge portion formed on the first region and the second region, and including an opening penetrating in a thickness direction.

[0203] <Supplementary Note 9> The electrode according to Supplementary Note 8, wherein the entire first region has the first surface free energy.

[0204] <Supplementary Note 10> The electrode according to Supplementary Note 8, wherein the first region includes: a central region disposed inside the first region, including an outer peripheral edge portion located more inward than the first outer peripheral edge portion of the first region, and having a surface free energy greater than the first surface free energy; and a peripheral region surrounding the central region, including an inner peripheral edge portion in contact with the outer peripheral edge portion of the central region and the first outer peripheral edge portion, and having the first surface free energy.

[0205] <Supplementary Note 11> The electrode according to Supplementary Note 10, wherein the first region further includes a connection region that extends through the peripheral region from a part of the outer peripheral edge of the central region to a part of the second inner peripheral edge of the second region that faces the peripheral region across the peripheral region, the connection region having a surface free energy greater than the first surface free energy.

[0206] <Supplementary Note 12> A sensor comprising: the electrode according to any one of Supplementary Notes 1 to 11; an outer periphery disposed on the first region and the second region of the electrode and positioned on the second outer periphery of the second region; and a reagent layer containing a reagent participating in an oxidation-reduction reaction.

[0207] <Supplementary Note 13> The sensor according to Supplementary Note 12, further comprising a protective film that covers the reagent layer.

[0208] <Supplementary Note 14> The sensor according to Supplementary Note 12 or 13, further comprising an insulating substrate on which the electrodes are arranged.

[0209] <Supplementary Note 15> A method for manufacturing a sensor comprising: the electrode according to any one of Supplementary Notes 1 to 11; and a reagent layer disposed on the first region and the second region of the electrode, the outer periphery being located on the second outer periphery of the second region, and the reagent layer containing a reagent participating in an oxidation-reduction reaction, the method comprising: applying a first liquid composition containing the reagent in a first solvent onto the first region and the second region of the electrode, and then drying the composition to form the reagent layer.

[0210] <Appendix 16> The method according to Appendix 15, wherein the first solvent comprises one or more selected from water and alcohol.

[0211] <Supplementary Note 17> A method for manufacturing a sensor comprising: the electrode according to any one of Supplementary Notes 1 to 11; a reagent layer disposed on the first region and the second region of the electrode, the reagent layer including an outer periphery located on the second outer periphery of the second region, the outer periphery being involved in a redox reaction; and a protective film covering the reagent layer, the method comprising: applying a first liquid composition including the reagent in a first solvent onto the first region and the second region of the electrode, and then drying the composition to form the reagent layer; and applying a second liquid composition including protective film components in a second solvent onto the surface portion of the electrode so as to cover the reagent layer, and then drying the composition to form the protective film.

[0212] <Supplementary Note 18> The method according to Supplementary Note 17, wherein the electrode is the electrode according to Supplementary Note 5 or 6, the protective film is disposed on the first region, the second region, the third region, and the fourth region of the electrode so as to cover the reagent layer, and includes an outer peripheral edge portion located on the fourth outer peripheral edge portion of the fourth region, and forming the protective film includes applying the second liquid composition to the first region, the second region, the third region, and the fourth region of the electrode so as to cover the reagent layer, and then drying the second liquid composition.

[0213] <Supplementary Note 19> A method for producing the electrode described in Supplementary Note 6, comprising: preparing an untreated electrode including an untreated surface portion having untreated regions in part thereof for forming the first region, the second region, the third region, and the fourth region and having the first surface free energy; and an insulating layer, at least a portion of which is disposed on the untreated surface portion, formed on the untreated region and including an opening penetrating in a thickness direction; and irradiating with laser light a ring-shaped fourth untreated region disposed on the outer periphery of the untreated region and adjacent to an inner periphery of the opening in the insulating layer in a plan view perpendicular to the untreated surface portion, and a ring-shaped second untreated region disposed more inward than the fourth untreated region, of the untreated regions of the untreated surface portion of the untreated electrode, thereby converting the fourth untreated region into the fourth region and converting the second untreated region into the second region.

[0214] <Supplementary Note 20> The method according to Supplementary Note 19, wherein irradiating the fourth untreated region of the untreated region with the laser light further includes irradiating with the laser light an opening peripheral region of the insulating layer that surrounds the opening and is adjacent to the outside of the fourth untreated region in a planar view from a direction perpendicular to the untreated surface portion.

[0215] <Supplementary Note 21> The method according to Supplementary Note 20, wherein irradiating the opening peripheral region of the insulating layer with the laser light further comprises irradiating the opening peripheral region of the insulating layer with the laser light to destroy the insulating layer in the opening peripheral region and converting a portion of the untreated surface portion exposed by the destruction into the fourth region.

[0216] <Supplementary Note 22> In the electrodes described in Supplements 1 to 11, preferably, the first outer peripheral edge of the first region and the second inner peripheral edge and second outer peripheral edge of the second region are circular. In the electrodes described in Supplements 1 to 10, more preferably, when one or more selected from the third inner peripheral edge and third outer peripheral edge of the third region and the fourth inner peripheral edge and fourth outer peripheral edge of the fourth region are present, they are circular.

[0217] 1: Sensor 2: Working electrode 3: Reference electrode 4: Counter electrode 5: Wiring 10: Electrode 10A: Untreated electrode 11: Surface portion 11A: Untreated surface portion 20: Substrate 21: First surface of substrate 30: Reagent layer 31: Outer peripheral edge portion of reagent layer 40: Protective film 41: Outer peripheral edge portion of protective film 50: Insulating layer 52: Opening 53: Reference electrode opening 54: Counter electrode opening 55: Inner peripheral edge portion of opening in insulating layer 58: Opening surrounding region 110: First region 111: First outer peripheral edge portion 112: Central region 113: Outer peripheral edge portion of central region 114: Inner peripheral edge portion of surrounding region 115: Surrounding region 116: Connection region 120: Second region 121 : Second inner peripheral edge portion 122 : Second outer peripheral edge portion 130 : Third region 131 : Third inner peripheral edge portion 132 : Third outer peripheral edge portion 140 : Fourth region 141 : Fourth inner peripheral edge portion 142 : Fourth outer peripheral edge portion 150 : Fifth region 151 : Fifth inner peripheral edge portion 500 : Untreated region 510 : First untreated region 520 : Second untreated region 530 : Third untreated region 540 : Fourth untreated region C : Cell L : Liquid sample P : First liquid composition Q : Second liquid composition

Claims

1. An electrode disposed on an insulating substrate, a surface portion located opposite the substrate when the surface portion is placed on the substrate; a first region formed on the surface portion, the first region including a first outer periphery and having a first surface free energy; a second region formed on the surface portion, the second region including a second inner peripheral edge portion surrounding the first region and contacting the first outer peripheral edge portion of the first region and a second outer peripheral edge portion positioned outside the second inner peripheral edge portion, the second region having a second surface free energy greater than the first surface free energy; a third region formed on the surface portion, the third region including a third inner peripheral edge portion surrounding the second region and contacting the second outer peripheral edge portion of the second region, the third region having a third surface free energy smaller than the second surface free energy; An electrode comprising:

2. The third region further includes a third outer circumferential edge portion located outside the third inner circumferential edge portion, a fourth region formed on the surface portion, the fourth region including a fourth inner peripheral edge portion surrounding the third region, the fourth inner peripheral edge portion being in contact with the third outer peripheral edge portion of the third region, and a fourth outer peripheral edge portion located outside the fourth inner peripheral edge portion, the fourth region having a fourth surface free energy greater than the third surface free energy; 2. The electrode of claim 1.

3. an insulating layer is formed on the first region, the second region, the third region, and the fourth region, at least a portion of which is disposed on the surface portion, and includes an opening having an inner peripheral edge portion that contacts the fourth outer peripheral edge portion of the fourth region in a plan view from a direction perpendicular to the surface portion and penetrating in a thickness direction; 3. The electrode of claim 2.

4. an insulating layer including an opening formed on the first region, the second region, and the third region and penetrating in a thickness direction, at least a portion of which is disposed on the surface portion; 2. The electrode of claim 1.

5. An electrode disposed on an insulating substrate, a surface portion located opposite the substrate when the surface portion is placed on the substrate; a first region formed on the surface portion, the first region including a first outer periphery and having a first surface free energy; a second region formed on the surface portion, the second region including a second inner peripheral edge portion surrounding the first region and contacting the first outer peripheral edge portion of the first region and a second outer peripheral edge portion positioned outside the second inner peripheral edge portion, the second region having a second surface free energy greater than the first surface free energy; an insulating layer, at least a portion of which is disposed on the surface portion, including an opening formed on the first region and the second region and having an inner peripheral edge portion that defines the second outer peripheral edge portion of the second region, the opening penetrating in a thickness direction; An electrode comprising:

6. An electrode according to any one of claims 1 to 5; a reagent layer including an outer periphery located on the second outer periphery of the second region and disposed on the first region and the second region of the electrode, and a reagent participating in an oxidation-reduction reaction; A sensor comprising:

7. Further comprising a protective film covering the reagent layer. The sensor of claim 6.

8. Further comprising an insulating substrate on which the electrodes are disposed. The sensor of claim 6.

9. An electrode according to any one of claims 1 to 5; a reagent layer including an outer periphery located on the second outer periphery of the second region and disposed on the first region and the second region of the electrode, and a reagent participating in an oxidation-reduction reaction; A method for manufacturing a sensor comprising: applying a first liquid composition containing the reagent in a first solvent onto the first region and the second region of the electrode and then drying the composition to form the reagent layer; The method includes:

10. The first solvent comprises one or more selected from water and alcohol; 10. The method of claim 9.

11. An electrode according to any one of claims 1 to 5; a reagent layer including an outer periphery located on the second outer periphery of the second region and disposed on the first region and the second region of the electrode, and a reagent participating in an oxidation-reduction reaction; a protective film covering the reagent layer; A method for manufacturing a sensor comprising: applying a first liquid composition including the reagent in a first solvent onto the first region and the second region of the electrode and then drying the first liquid composition to form the reagent layer; and applying a second liquid composition containing a protective film component in a second solvent onto the surface portion of the electrode so as to cover the reagent layer, and then drying the second liquid composition to form the protective film; The method includes:

12. An electrode according to claim 2 or 3, a reagent layer including an outer periphery located on the second outer periphery of the second region and disposed on the first region and the second region of the electrode, and a reagent participating in an oxidation-reduction reaction; a protective film covering the reagent layer, the protective film being disposed on the first region, the second region, the third region, and the fourth region of the electrode so as to cover the reagent layer, the protective film including an outer periphery portion located on the fourth outer periphery portion of the fourth region; A method for manufacturing a sensor comprising: applying a first liquid composition including the reagent in a first solvent onto the first region and the second region of the electrode and then drying the first liquid composition to form the reagent layer; and applying a second liquid composition containing a protective film component in a second solvent onto the first region, the second region, the third region, and the fourth region of the electrode so as to cover the reagent layer, and then drying the composition to form the protective film; The method includes:

13. A method for producing the electrode of claim 3, comprising the steps of: preparing an untreated electrode including an untreated surface portion having an untreated region for forming the first region, the second region, the third region, and the fourth region in a part thereof and having the first surface free energy, and an insulating layer including an opening formed on the untreated region and penetrating in a thickness direction, the insulating layer having at least a part disposed on the untreated surface portion; and irradiating a laser beam to an annular fourth untreated region disposed on the outer periphery of the untreated region adjacent to an inner peripheral edge of the opening of the insulating layer in a plan view perpendicular to the untreated surface portion of the untreated electrode, and an annular second untreated region disposed further inward than the fourth untreated region, thereby converting the fourth untreated region into the fourth region and converting the second untreated region into the second region; The method includes:

14. 14. The method of claim 13, wherein irradiating the fourth untreated area of ​​the untreated area with the laser light further comprises irradiating with the laser light an opening peripheral area of ​​the insulating layer surrounding the opening, the opening peripheral area being adjacent to the outside of the fourth untreated area in a planar view from a direction perpendicular to the untreated surface portion.

15. 15. The method of claim 14, wherein irradiating the insulating layer with the laser light at the opening perimeter region further comprises irradiating the insulating layer with the laser light at the opening perimeter region to destroy the insulating layer in the opening perimeter region and convert a portion of the untreated surface portion exposed by the destruction to the fourth region.