Sensor and manufacturing method thereof

Abstract

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

Description

Technical Field

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

[0002] Electrochemical sensors have been used to measure target substances in test samples such as cell culture fluids and blood samples. Such sensors, for example, have an insulating substrate, a working electrode disposed on the surface of the substrate, and further, a counter electrode and / or a reference electrode. The working electrode typically has a conductive layer, and a reagent layer disposed on the conductive layer and containing reagents participating in the redox reaction (e.g., oxidoreductase and electron transfer body). In order to prevent the reagent from flowing out of the reagent layer, the sensor may further have a protective film covering only the working electrode, or a protective film covering the working electrode and the counter electrode and / or the reference electrode.

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

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: U.S. Patent No. 9014774 Summary of the invention

[0007] However, the conventional sensor including the working electrode including the reagent layer and the protective film on the conductive layer has the following problems.

[0008] In Patent Document 1, an insulating substrate including a working electrode including a conductive layer and a reagent layer is immersed in a solution including a polymer compound and dried to form a protective film covering the entire insulating substrate.

[0009] However, in this method, it is difficult to control the properties of the protective film on the reagent layer of the working electrode (thickness of the protective film, amount of the polymer compound constituting the protective film, etc.) within a designed range. Deviations in the properties of the protective film of the working electrode may cause a decrease in the measurement accuracy of the sensor.

[0010] In Patent Document 1, a reagent layer including a reagent such as an enzyme is disposed on the surface of a flat electrode.

[0011] However, in the method of forming a reagent layer on the surface of a flat electrode, it is difficult to control the characteristics of the reagent layer (the thickness of the reagent layer, the amount of the reagent constituting the reagent layer, etc.) within a designed range. Deviations in the characteristics of the reagent layer in the working electrode may also cause a decrease in the measurement accuracy of the sensor.

[0012] The first disclosure aims to provide a sensor including a working electrode including a reagent layer and a protective film, the characteristics of which can be easily controlled within a desired range, and a method for manufacturing the sensor.

[0013] The sensor according to the first disclosure includes an insulating substrate and a working electrode disposed on the substrate.

[0014] The working electrode has:

[0015] A conductive layer disposed on the substrate;

[0016] a first insulating layer at least partially disposed on the conductive layer, the first insulating layer having a first opening extending through the thickness direction and formed at a position overlapping with a portion of the conductive layer when viewed from above in the thickness direction of the substrate, and a water-repellent surface;

[0017] a second insulating layer disposed on the first insulating layer, the second insulating layer having a second opening extending through the thickness direction and formed at a position overlapping with a portion of the first insulating layer that entirely encloses the first opening when viewed from above in the thickness direction, and a surface that is liquid-repellent to alcohol,

[0018] a reagent layer disposed in the first opening of the first insulating layer, the reagent layer including an outer periphery defined according to an inner periphery of the first opening of the first insulating layer and a reagent participating in a redox reaction,

[0019] The protective film is disposed in the second opening of the second insulating layer, and the protective film includes an outer peripheral edge defined according to an inner peripheral edge of the second opening of the second insulating layer.

[0020] The manufacturing method of the sensor according to the first disclosure includes:

[0021] forming droplets of a liquid composition A containing the reagent in water in the first opening of the first insulating layer of the substrate provided with the conductive layer, the first insulating layer, and the second insulating layer, and then drying the droplets to form the reagent layer; and

[0022] After the formation of the reagent layer, droplets of the liquid composition B containing protective film components in alcohol are formed in the second opening of the second insulating layer and then dried to form the protective film.

[0023] Effects of the Invention

[0024] According to the sensor and the manufacturing method according to the first disclosure, it is easy to control the characteristics of the reagent layer and the protective film of the working electrode of the sensor within a desired range. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a plan view showing a substrate on which a first working electrode, a second working electrode, a reference electrode, and a counter electrode and conductive layers and wirings are arranged.

[0026] Figure 2 It is a plan view showing a substrate on which a first working electrode, a second working electrode, a reference electrode, and a conductive layer and wiring of a counter electrode are arranged, and a first insulating layer is further arranged on these.

[0027] Figure 3 It is a plan view showing a substrate on which a first working electrode, a second working electrode, a reference electrode, and a counter electrode conductive layer and wiring are arranged, and a first insulating layer and a second insulating layer are further arranged thereon.

[0028] Figure 4 It is shown in Figure 3 A top view of a substrate further configured with a reagent layer of a working electrode and a silver / silver chloride layer of a reference electrode.

[0029] Figure 5 This is a top view of a sensor according to one embodiment of the first disclosure.

[0030] Figure 6 yes Figure 3 A cross-sectional view along line AA' of a portion of the substrate corresponding to the first working electrode.

[0031] Figure 7 is Figure 3 A cross-sectional view of a droplet of a liquid composition A formed in a first opening of a first insulating layer of a first working electrode of a substrate, wherein the liquid composition A contains a reagent participating in a redox reaction in water.

[0032] Figure 8 Yes Figure 3 The liquid composition A is dried by drying the liquid droplets, Figure 4 A cross-sectional view along line BB' of a portion of the substrate corresponding to the first working electrode.

[0033] Fig. 9 is Figure 4 A cross-sectional view of a droplet of a first liquid composition B formed in a second opening of a second insulating layer of a first working electrode of a substrate, wherein the first liquid composition B contains a protective film component in alcohol.

[0034] Fig.10 Yes Fig. 9 The first liquid composition B droplets are dried to obtain Figure 4 A cross-sectional view of a portion of a protective film within a second opening of a second insulating layer of a first working electrode of a substrate.

[0035] Fig.11 is included Fig.10 4 is a cross-sectional view of a droplet of the first liquid composition B further formed in the second opening of the second insulating layer of the first working electrode.

[0036] Fig.12 It includes Fig.11 The first protective film obtained by drying the droplets of the first liquid composition B Figure 5 A cross-sectional view taken along line CC' of a portion of the sensor including the first working electrode.

[0037] Fig.13 is Figure 4 A cross-sectional view of a droplet of a second liquid composition B formed in a second opening of a second insulating layer of a second working electrode of a substrate, wherein the second liquid composition B contains a protective film component in alcohol.

[0038] Fig.14 Yes Fig. 9 The second liquid composition B is dried by drying the droplets thereof, Figure 4 A cross-sectional view of the second protective film within the second opening of the second insulating layer of the second working electrode of the substrate.

[0039] Fig.15 is included Fig.14 A cross-sectional view of a droplet of a third liquid composition B formed in a second opening of a second insulating layer of a second working electrode, the third liquid composition B containing other protective film components in alcohol.

[0040] Fig.16 It includes Fig.15 The third protective film obtained by drying droplets of the third liquid composition B, Figure 5 A cross-sectional view taken along line DD' of a portion of the sensor including the second working electrode.

[0041] Fig.17It is a cross-sectional view of a droplet of a first liquid composition B formed in a second opening of a second insulating layer of a first working electrode of a sensor of a comparative example in a wet spread state, the first liquid composition B containing a protective film component in alcohol.

[0042] Fig.18 Yes Fig.17 4 is a cross-sectional view of a portion of a first protective film formed on the upper surface of the second insulating layer of the first working electrode and in the second opening, obtained by drying the first liquid composition B that has been wetted and spread.

[0043] Fig.19 is included Fig.18 4 is a cross-sectional view of a portion of the first protective film of the sensor of the comparative example, in a wet spread state, wherein a droplet of the first liquid composition B is further formed in the second opening of the second insulating layer of the first working electrode of the sensor.

[0044] Fig. 20 Yes Fig.19 4 is a cross-sectional view of a first protective film formed on the upper surface of the second insulating layer of the first working electrode and in the second opening, obtained by drying the first liquid composition B that has been wetted and spread.

[0045] Fig.21 is a schematic diagram for explaining a method for determining a substance to be tested. Figure 5 The sensor is immersed in the liquid sample.

[0046] Fig. 22 yes Figure 5 A cross-sectional view along line EE' of the portion of the sensor containing the reference electrode.

[0047] Fig.23 yes Figure 5 A cross-sectional view along line FF' of the portion of the sensor including the counter electrode.

[0048] Fig.24 This is a control block diagram of an analysis device including the sensor according to one embodiment of the first disclosure.

[0049] Fig.25 : Fig.25 A shows the measurement result of the current value of the first working electrode (for glucose measurement) of the sensor of the example. Fig.25 B shows the measurement result of the current value of the first working electrode (for glucose measurement) of the sensor of the comparative example.

[0050] Fig.26 : Fig.26 A shows the measurement result of the current value of the second working electrode (for measuring lactic acid) of the sensor of the example. Fig.26B shows the measurement result of the current value of the second working electrode (for measuring lactic acid) of the sensor of the comparative example.

[0051] Fig. 27 1 is a plan view showing a substrate on which a working electrode conductive layer, a reference electrode conductive layer, a counter electrode, and wiring are arranged.

[0052] Fig.28 It is a plan view showing a substrate on which a working electrode conductive layer, a reference electrode conductive layer, a counter electrode, and wiring are arranged, and a first insulating layer is further arranged thereon.

[0053] Fig.29 It is a plan view showing a substrate on which a working electrode conductive layer, a reference electrode conductive layer, a counter electrode, and wiring are arranged, and a first insulating layer and a second insulating layer are further arranged thereon.

[0054] Fig.30 It is shown in Fig.29 A top view of a substrate further configured with a reagent layer of a working electrode and a silver / silver chloride layer of a reference electrode.

[0055] Fig.31 It is a plan view of a sensor according to an embodiment of the second disclosure in which a front end opening and a flow path are formed on a second insulating layer.

[0056] Fig.32 It is a plan view of a sensor according to another embodiment of the second disclosure in which a front end opening and a flow path are formed in a first insulating layer and a second insulating layer.

[0057] Fig.33 yes Fig.31 A cross-sectional view along line J-J' of the portion of the sensor containing the working electrode.

[0058] Fig.34 yes Fig.31 A cross-sectional view along line K-K' of the portion of the sensor containing the reference electrode.

[0059] Fig.35 yes Fig.31 A cross-sectional view of a portion of the sensor including a counter electrode taken along line LL'.

[0060] Fig.36 yes Fig.31 A cross-sectional view taken along line MM' of a portion of the sensor including a counter electrode, a front end opening formed on a second insulating layer, and a flow path.

[0061] Fig.37This is a cross-sectional view of a portion including a counter electrode, a front end opening, and a flow path of a sensor according to another embodiment having a front end opening and a flow path with a structure surrounded by the front end formed on a portion of the upper surface in the thickness direction excluding the insulating layer.

[0062] Fig.38 yes Fig.32 A cross-sectional view of a portion of the sensor including a counter electrode, a front end opening formed on a first insulating layer and a second insulating layer, and a flow path taken along line NN′.

[0063] Fig.39 The substrate is immersed in the liquid sample with the front end of the substrate at the bottom. Fig.31 and Fig.36 The schematic diagram of the cross section of the sensor shown includes the counter electrode, the front end opening, and the flow path.

[0064] Fig.40 This is a schematic diagram of a cross section of a portion including a counter electrode of a sensor of a comparative example not having a tip opening and a flow path, which is immersed in a liquid sample with the tip of the substrate at the lower end.

[0065] Fig.41 This is an overhead view of a sensor involved in an embodiment of the second disclosure, which comprises: a substrate including a main body, a connecting portion and a base end portion; a first insulating layer arranged on the entire first surface of the substrate; and a second insulating layer including an insulating sheet arranged on the first surface of the substrate closer to the front end side than the bending portion.

[0066] Fig.42 There are multiple Fig.41 A top view of a multi-connected sensor in which the sensors are connected to the base end of the substrate. Fig.42 The insulation layer is omitted.

[0067] Fig.43 The main body of the substrate on which the detection electrodes are arranged is immersed in the liquid sample. Fig.41 Schematic diagram of the sensor. Fig.43 The insulation layer is omitted.

[0068] Fig.44 The main body of the substrate on which the detection electrodes are arranged is immersed in the liquid sample. Fig.41 Schematic side view of the sensor.

[0069] Fig.45 is Fig.41A schematic side view of a comparative example sensor in which a second insulating layer including an insulating sheet is arranged on the entire first surface of a substrate in a sensor, and a main body of a substrate having detection electrodes arranged thereon and bent at a bent portion of a connecting portion of the substrate is immersed in a liquid sample.

[0070] Fig.46 It is a top view of a sensor involved in another embodiment of the second disclosure, the sensor comprising: a substrate including a main body, a connecting portion and a base end portion; and a first insulating layer arranged on the entire first surface of the substrate; a second insulating layer including a first insulating sheet arranged on the first surface of the substrate closer to the front end side than the bending portion, and a second insulating layer including a second insulating sheet arranged on the first surface of the base end portion of the substrate, and no insulating sheet is included on the bending portion of the substrate.

[0071] Fig.47 is a top view of a sensor according to another embodiment of the second disclosure, wherein Fig.46 In the sensor shown, a second insulating layer including a plurality of third insulating sheets separated from the first insulating sheet and the second insulating sheet via the cutouts is further arranged in a region on the bent portion of the first surface of the substrate.

[0072] Fig.48 This is a control block diagram of an analysis device including a sensor according to one embodiment of the second disclosure.

[0073] Fig.49A It is a top view of the sensor S1 of the comparative example.

[0074] Fig.49B It is a top view of the sensor S2 of the comparative example.

[0075] Fig.49C FIG. 1 is a top view of the sensor S3 according to the embodiment.

[0076] Fig.49D FIG. 1 is a top view of the sensor S4 according to the embodiment.

[0077] Fig.49E 1 is a top view of the sensor S5 according to the embodiment.

[0078] Fig.50A The measurement results of the current value of the working electrode of the sensor S1 are shown.

[0079] Fig.50B The measurement results of the current value of the working electrode of the sensor S2 are shown.

[0080] Fig.50C The measurement results of the current value of the working electrode of the sensor S3 are shown.

[0081] Fig.50DThe measurement results of the current value of the working electrode of the sensor S4 are shown.

[0082] Fig.50E The measurement results of the current value of the working electrode of the sensor S5 are shown.

[0083] Fig.51 This is an exploded perspective view of a sensor assembly according to an embodiment of the second disclosure.

[0084] Fig.52 is a cross-sectional view showing a sensor held between an upper support plate and a lower support plate.

[0085] Fig.53 is an exploded perspective view of the adapter assembly including the sensor assembly.

[0086] Fig.54 It is a top view of a sensor according to one embodiment of the third disclosure.

[0087] Fig.55 yes Fig.54 The shown section is a cross-sectional view of a portion of the sensor including a counter electrode taken along line PP'.

[0088] Fig.56 yes Fig.54 The illustrated sensor is a cross-sectional view of a portion including the first working electrode taken along line QQ'.

[0089] Fig.57 yes Fig.54 The sensor shown is a cross-sectional view of a portion including the second working electrode taken along line RR'.

[0090] Fig.58 The results of cyclic voltammetry tests using a carbon electrode without platinum particles (Experiment 3-1), a carbon electrode containing 1% platinum particles (Experiment 3-2), and a carbon electrode containing 5% platinum particles (Experiment 3-3) as a working electrode in Experiment 4 of the third disclosure are shown.

[0091] Fig.59 The results of Experiment 5 of Publication 3 show that a carbon electrode without platinum particles (A) or a carbon electrode containing 1% platinum particles (B) was used as a working electrode, a potential of 0.3 V or -0.2 V was applied, and the current value was measured over time.

[0092] Fig.60The results are shown of using the sensor of Experiment 2-1 (the working electrode conductive layer and the counter electrode are carbon conductive layers that do not contain platinum particles) and the sensor of Experiment 2-2 (the counter electrode is a carbon conductive layer whose surface is modified with platinum nanoparticles, and the working electrode conductive layer is a carbon conductive layer that does not contain platinum particles) in Experiment 6 of the third disclosure, and measuring the current value (A) corresponding to the glucose concentration in the liquid sample and the current value (B) corresponding to the lactic acid concentration over time (N=2).

[0093] Fig.61 The results are shown of using the sensor of Experiment 3-1 (the working electrode conductive layer and the counter electrode are carbon conductive layers that do not contain platinum particles), the sensor of Experiment 3-2 (the working electrode conductive layer and the counter electrode are carbon conductive layers that contain 1 wt % of platinum particles relative to carbon), and the sensor of Experiment 3-3 (the working electrode conductive layer and the counter electrode are carbon conductive layers that contain 5 wt % of platinum particles relative to carbon) in Experiment 7 of the third disclosure, and measuring the current value (A) corresponding to the glucose concentration in the liquid sample and the current value (B) corresponding to the lactic acid concentration over time (N=2).

[0094] Fig.62 It is a top view of a sensor according to one embodiment of the fourth disclosure.

[0095] Fig.63 yes Fig.62 The shown section is a cross-sectional view of a portion of the sensor including a counter electrode taken along line SS'.

[0096] Fig.64 yes Fig.62 The portion of the sensor shown includes a working electrode and is a cross-sectional view taken along line TT'.

[0097] Fig.65 The results of Experiment 8 of Publication No. 4 are shown. Fig.65 A shows the measurement results using the sensor of Experiment 8-1, Fig.65 B shows the measurement results using the sensor of Experiment 8-2.

[0098] Fig.66 The results of Experiment 9 of Publication No. 4 are shown. Fig.66 A shows the measurement results using the sensor of Experiment 9-1, Fig.66 B shows the measurement results using the sensor of Experiment 9-2. Fig.66 C shows the measurement results using the sensor of Experiment 9-3.

[0099] Fig.67 The results of Experiment 10 of Publication No. 4 are shown. DETAILED DESCRIPTION

[0100] One or more embodiments of the first disclosure to the fourth disclosure in this specification will be described. In this specification, when the temperature is not explicitly stated for a method or a physical property, the method and the physical property refer to those at room temperature (25° C.±3° C.).

[0101] This specification incorporates the disclosure of Japanese Patent Application Nos. 2022-107320 and 2022-116287 on which the priority of this application is based.

[0102] In addition, all publications, patents and patent applications cited in this specification are incorporated into this specification by direct reference.

[0103] <First disclosed embodiment>

[0104] The following is a description of an embodiment of the sensor and the method for manufacturing the sensor involved in the first disclosure. In this embodiment, detailed descriptions beyond the necessary scope are sometimes omitted. For example, detailed descriptions of well-known matters and repeated descriptions of substantially the same structures are sometimes omitted. This is to avoid the following description from becoming unnecessarily lengthy and to facilitate the understanding of those skilled in the art.

[0105] In addition, the applicant provides the drawings and the following description in order to allow those skilled in the art to fully understand the first disclosure, and does not intend to limit the subject matter described in the claims by these.

[0106] <Materials>

[0107] Examples of materials that can be used for the sensor and the method for producing the sensor according to the first disclosure of this specification will be described.

[0108] The material of the insulating substrate of the sensor disclosed in the first aspect of this specification is not particularly limited, and for example, resin materials such as polyethylene terephthalate, polycarbonate, polyimide, polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyoxymethylene, monomer cast nylon, polybutylene terephthalate, methacrylic resin, ABS resin, etc., or glass materials can be used. Polyethylene terephthalate, polycarbonate and polyimide are preferred, and polyethylene terephthalate is more preferred. The thickness and other dimensions of the substrate are not particularly limited, and 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.

[0109] The conductive layer of the working electrode of the sensor disclosed in the first aspect of this specification is a layer containing conductive materials such as carbon, gold, platinum, palladium, etc. The conductive layers of the working electrode, reference electrode, and counter electrode can be manufactured by forming a layer of the above-mentioned conductive material on the surface of the substrate using sputtering, evaporation, screen printing, etc. If necessary, the conductive layer can be processed into a specified pattern using a laser trimming method. The wiring of the sensor involved in the embodiments described later can also be composed of the same conductive material.

[0110] The sensor disclosed in the first aspect of the present specification can be used to detect a predetermined test substance in a liquid sample by being immersed in the liquid sample. Examples of the test substance include: glucose, lactic acid, cholesterol, bilirubin, amino acids such as glutamine and glutamic acid, glycated amino acids (Japanese: glycosylated peptides), glycated peptides, ketone bodies (3-hydroxybutyric acid), alcohols, and the like.

[0111] The working electrode of the sensor disclosed in the first aspect of the present specification has a reagent layer containing a reagent participating in a redox reaction. The reagent participating in the redox reaction can be any reagent participating in the redox reaction of the substance to be tested, and can be appropriately selected according to the substance to be tested. As a reagent participating in the redox reaction, a combination of an oxidoreductase and a mediator (electron transfer substance) may be included, or an oxidoreductase may be included. The oxidoreductase may include a coenzyme.

[0112] As oxidoreductase, oxidase or dehydrogenase can be mentioned. As specific examples of oxidoreductase, for example, glucose oxidase, lactate oxidase, cholesterol oxidase, bilirubin oxidase, glucose dehydrogenase, lactate dehydrogenase, amino acid oxidase, amino acid dehydrogenase, glutamate oxidase, glutamate dehydrogenase, fructosyl amino acid oxidase, fructosyl peptide oxidase, 3-hydroxybutyrate dehydrogenase, alcohol oxidase, alcohol dehydrogenase, etc. These oxidoreductases can be used for the detection of the test substances shown in the above examples.

[0113] In addition, the medium is not limited and can be one or more selected from: metal complexes (for example, osmium complexes, ruthenium complexes, iron complexes, etc.), quinone compounds (for example, benzoquinone, naphthoquinone, phenanthroquinone, phenanthroline quinone, anthraquinone, and derivatives thereof, etc.), phenazine compounds, viologen compounds, phenothiazine compounds, and phenolic compounds. Specific examples of the mediator include potassium ferrocyanide, hexaamineruthenium, ferrocene, poly(1-vinylimidazole)-bis(bipyridyl)chloroosmium, hydroquinone, 2-methyl-1,4-benzoquinone, 1,2-naphthoquinone-4-sulfonate, 9,10-phenanthrolquinone-2-sulfonate, 9,10-phenanthrolquinone-2,7-disulfonate, 1,10-phenanthroline-5,6-dione, anthraquinone-2-sulfonate, phenazine derivatives (1-methoxy-5-methylphenazinium methylsulfate, 1-methoxy-5-ethylphenazinium ethylsulfate, etc.), methyl viologen, benzyl viologen, methylene blue, methylene green, 2-aminophenol, 2-amino-4-methylphenol, and 2,4-diaminophenol. The salt is not limited, and includes sodium salt, potassium salt, calcium salt, magnesium salt, lithium salt, etc.

[0114] For the mediator, from the viewpoint of durability of the sensor and suppression of the outflow of the mediator to the outside of the sensor, it is preferably a polymerized mediator bonded to a polymer compound. For the polymer compound bonded to the mediator, it can be a homopolymer, a random copolymer, a block copolymer, or a polymer compound bonded or mixed therewith. 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. In addition, the polymer compound is not particularly limited, and a polymer compound that is formed by a plurality of atoms selected from at least one of carbon atoms, nitrogen atoms, oxygen atoms, and sulfur atoms bonded into a chain to form a main chain can be cited. As specific examples thereof, 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 can be cited. 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 should be noted that the polymerized medium is preferably hydrophilic as a whole, and it is further preferred that the polymer compound of the bonding medium is hydrophilic.

[0115] As a polymerized medium, a polymerized medium obtained by bonding a medium to a polymer compound via a covalent bond can be used. As the covalent bond, for example, a covalent bond described in Japanese Patent Publication No. 2003-514924 can be used, specifically, ether bonds, thioether bonds, ester bonds, carbamate bonds, amide bonds, etc. can be cited. The covalent bond can be formed by a reactive group originally possessed by a monomer forming a polymer compound, or can be formed by a reactive group possessed by a connecting group separately introduced into a medium or a polymer compound. As a specific example of a polymerized medium, for example, a polymerized medium obtained by bonding a phenazine compound to poly(L-lysine) via an amide bond can be cited.

[0116] The reagent layer provided by the working electrode of the first disclosed sensor of this specification can be formed by drying a liquid composition A containing a reagent participating in a redox reaction in water. The liquid composition A contains the above-mentioned reagent in water, and further, as required, may also contain components such as a buffer, a hydrophilic polymer compound, a conductive carbon filler, and a cross-linking agent. As the hydrophilic polymer compound, a cellulose derivative can be cited, and as the cellulose derivative, one or more selected from: methylcellulose, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxyethylmethylcellulose and hydroxypropylmethylcellulose can be cited. As the conductive carbon filler, one or more selected from: carbon black, graphite powder, porous carbon and nanocarbon can be cited.

[0117] The protective film provided by the working electrode of the sensor disclosed in the first aspect of this specification may be a film that prevents or inhibits the leakage of the reagent contained in the reagent layer to the outside of the protective film and that can permeate the test substance present outside the protective film. The protective film having such a property preferably contains a polymer compound. Examples of the polymer compound contained in the protective film include: a polymer compound containing 4-vinylpyridine in the structural unit, a polymer compound containing a cation exchange functional group, and the like.

[0118] Examples of polymer compounds containing 4-vinylpyridine in the structural unit include poly(4-vinylpyridine), copolymers of 4-vinylpyridine and alkyl methacrylate (preferably block copolymers), and copolymers of styrene, 4-vinylpyridine, and oligopropylene glycol methyl ether methacrylate (preferably random copolymers). Examples of the above-mentioned alkyl methacrylate include tert-butyl methacrylate. Examples of the above-mentioned oligopropylene glycol methyl ether methacrylate include tripropylene glycol methyl ether methacrylate. The polymer compound containing 4-vinylpyridine in the structural unit is preferably cross-linked by a cross-linking agent containing two or more epoxy groups, such as polyethylene glycol diglycidyl ether (PEGDGE). The polymer compound containing 4-vinylpyridine in the structural unit, which is cross-linked by a cross-linking agent containing two or more epoxy groups, contains a functional group containing a quaternary ammonium cation generated by the reaction of a pyridyl group (tertiary amine) derived from 4-vinylpyridine and an epoxy group. As a preferred embodiment of the polymer compound containing 4-vinylpyridine in a structural unit and crosslinked by a crosslinking agent containing two or more epoxy groups, the specific embodiment of the "second polymer compound containing a cationic functional group" described in the fourth publication is preferred.

[0119] As the polymer compound containing cation exchange functional groups, there can be cited polymer compounds containing structural units having sulfonic acid groups on the side chains, preferably polymer compounds containing perfluoro compounds having sulfonic acid groups on the side chains in the structural units, more preferably copolymer compounds containing perfluoro compounds having sulfonic acid groups on the side chains and perfluoro compounds without ionic functional groups on the side chains in the structural units, particularly preferably copolymer compounds of tetrafluoroethylene and perfluoro-2-(2-fluorosulfonylethoxy)propyl vinyl ether, particularly preferably Nafion (registered trademark). As a preferred embodiment of the polymer compound containing cation exchange functional groups, the specific embodiment of the "first polymer compound containing cation exchange functional groups" described in the fourth disclosure is preferred. In order to transport cations such as protons between the reagent layer and the liquid sample, a protective film containing a polymer compound containing cation exchange functional groups is preferably provided on the reagent layer.

[0120] The protective film possessed by the working electrode of the sensor disclosed in the first aspect of this specification may be a stack of two or more protective films, for example, it may be a stack of a protective film comprising a polymer compound containing a cation exchange functional group disposed on the side in contact with the reagent layer, and a protective film comprising a polymer compound containing 4-vinylpyridine in the structural unit disposed thereon.

[0121] The protective film of the working electrode of the sensor disclosed in the first aspect of the present specification can be formed by drying a liquid composition B containing a protective film component in an alcohol. Examples of the protective film component include a polymer compound contained in the protective film, a cross-linking agent, etc. Examples of the alcohol in the liquid composition B include, for example, a monohydric alcohol having 1 to 5 carbon atoms, particularly preferably methanol, ethanol, or isopropanol, and most preferably ethanol.

[0122] The first insulating layer of the working electrode of the sensor disclosed in the first aspect of the present specification includes a water-repellent surface. The water-repellent surface here refers to a surface having a contact angle with respect to water of, for example, 90° or more, more preferably 100° or more, and most preferably 110° or more. The upper limit of the contact angle of the water-repellent surface of the first insulating layer with respect to water is not particularly limited, and may be, for example, 160° or less (the range of the contact angle with respect to water is, for example, 90° or more and 160° or less).

[0123] The second insulating layer of the working electrode of the sensor disclosed in the first aspect of the present specification includes a surface that is liquid-repellent (alcohol-repellent) to alcohol. The liquid-repellent surface to alcohol herein refers to a surface having a contact angle to alcohol of, for example, 45° or more, more preferably 50° or more, and most preferably 55° or more. The upper limit of the contact angle to alcohol of the liquid-repellent surface of the second insulating layer to alcohol is not particularly limited, and may be, for example, 100° or less (the above-mentioned contact angle to alcohol ranges from, for example, 45° or more to 100° or less). The alcohol here may be, for example, a monohydric alcohol having 1 to 5 carbon atoms, particularly preferably methanol, ethanol or isopropanol, and most preferably ethanol.

[0124] The contact angle of the surface of the first insulating layer with respect to water and the contact angle of the surface of the second insulating layer with respect to alcohol can be measured at 20°C. The contact angle of the surface of the first insulating layer with respect to water and the contact angle of the surface of the second insulating layer with respect to alcohol can be measured using a commercially available analyzer, for example, a handheld contact angle / surface free energy analyzer MSA manufactured by KRUSS. The contact angle of the surface with respect to water or alcohol is preferably measured by spraying a 2 μL droplet of water or alcohol onto the surface of the object to be measured and measuring the contact angle between the droplet and the surface after 2 seconds.

[0125] The water-repellent surface of the first insulating layer of the working electrode of the sensor disclosed in the first aspect of the present specification preferably contains a fluororesin. As the fluororesin, a polymer compound of a fluorinated hydrocarbon can be used, for example, a polymer compound of one or more selected from vinylidene fluoride, tetrafluoroethylene, hexafluoropropylene and perfluoro(alkyl vinyl ether) can be cited, and a copolymer containing two or more selected from vinylidene fluoride, tetrafluoroethylene, hexafluoropropylene and perfluoro(alkyl vinyl ether) is particularly preferred, and a copolymer containing vinylidene fluoride and hexafluoropropylene is more preferred. The first insulating layer can be composed of a fluororesin as a whole.

[0126] The surface of the second insulating layer of the working electrode of the first disclosed sensor of this specification that is liquid-repellent to alcohol preferably contains a compound containing a perfluoroalkyl group. As a compound containing a perfluoroalkyl group, a fluorine-based surface modification additive can be cited. Such a second insulating layer can be formed by applying a composition containing an insulating base resin and a compound containing a perfluoroalkyl group (fluorine-based surface modification additive) in a solvent to the first insulating layer and drying it. At this time, the compound containing a perfluoroalkyl group segregates on the surface to form a surface that is liquid-repellent to alcohol. The second insulating layer formed in this way is preferably a layer of an insulating base resin containing a compound containing a perfluoroalkyl group on the surface. The type of insulating base resin is not particularly limited, for example, it can be a polyester resin. The number of carbons in the perfluoroalkyl group is not particularly limited, for example, it can be in the range of 2 to 20 carbons. The perfluoroalkyl group may contain a trifluoromethyl group at the end.

[0127] <Overview of Sensor 1>

[0128] A sensor 1 according to an embodiment of the first disclosure will be described with reference to the drawings.

[0129] The sensor 1 of this embodiment is as follows Figure 5As shown, it has: an insulating substrate 2, a first working electrode 10a, a second working electrode 10b, a reference electrode 20 and a counter electrode 30 arranged on the substrate 2, and a wiring 50 electrically connected to the first working electrode 10a, the second working electrode 10b, the reference electrode 20 and the counter electrode 30, respectively. The sensor 1 of this embodiment includes two working electrodes, but in other embodiments not shown in the figure, the working electrode may be only one, or may be three or more. In the following description, when the first working electrode 10a and the second working electrode 10b are not distinguished, they are sometimes described as working electrodes 10a, 10b. In addition, the first working electrode 10a, the second working electrode 10b, the reference electrode 20 and the counter electrode 30 are sometimes collectively described as electrodes. In addition, the sensor 1 of this embodiment is a three-pole sensor including a working electrode, a reference electrode and a counter electrode as electrodes, but it may also be a two-pole sensor including only a working electrode and a counter electrode without a reference electrode. Although not shown in the drawings, the reference electrode and / or the counter electrode may be disposed on a substrate different from the substrate on which the working electrode is disposed.

[0130] Sensor 1 Fig.21 As shown, it can be used to immerse in a liquid sample X to detect a predetermined test substance in the liquid sample X. As the liquid sample X, for example, a culture solution containing cells C as shown in the figure, or a liquid sample prepared from blood obtained from a living body can be cited. Specific examples of the test substance are described in the column of <Materials>.

[0131] The working electrodes 10a, 10b of the sensor 1 are as follows Fig.12 and Fig.16 As shown, the conductive layers 11a and 11b include reagent layers 15a and 15b containing a reagent participating in the oxidation-reduction reaction of the test substance in the liquid sample X. Preferred embodiments of the reagent participating in the oxidation-reduction reaction are described in the column of <Materials>.

[0132] When the reagent layers 15a, 15b of the working electrodes 10a, 10b of the sensor 1 contain a reagent that oxidizes the test substance in the liquid sample, electrons move from the test substance to the conductive layers 11a, 11b under the condition that a predetermined voltage is applied to the electrodes of the sensor 1. Similarly, when the reagent layers 15a, 15b contain a reagent that reduces the test substance in the liquid sample X, electrons move from the conductive layers 11a, 11b to the test substance. Since the amount of electrons that move depends on the concentration of the test substance, the concentration of the test substance in the liquid sample X or the concentration change can be measured based on the current value or the change in the current value flowing through the working electrodes 10a, 10b of the sensor 1.

[0133] Reference Fig.24An example of an analysis device 100 for analyzing a test substance in a liquid sample and including the sensor 1 will be described.

[0134] The analysis device 100 is as follows Fig.24 As shown, the sensor 1, the analysis unit 102, and the control unit 104 are provided. The working electrodes 10a, 10b, the reference electrode 20, and the counter electrode 30 of the sensor 1 are connected to the analysis unit 102 via wiring 50. The analysis unit 102 can communicate with the control unit 104.

[0135] The analysis unit 102 includes an electrochemical measurement unit 111 , a control unit 112 , a storage unit 113 , and a communication unit 114 .

[0136] The electrochemical measuring unit 111 is a potentiostat that applies a predetermined voltage to each electrode of the sensor 1 to measure the concentration of the test substance, and includes a voltage applying unit 111a and a current measuring unit 111b, and preferably further includes a voltage measuring unit (counter electrode terminal voltage measuring unit) 111c.

[0137] The voltage application unit 111 a applies a predetermined voltage for measuring the concentration of the test substance contained in the liquid sample X to the electrodes of the sensor 1 .

[0138] The current measuring unit 111b detects the current value or the change thereof flowing between the working electrodes 10a, 10b and the counter electrode 30 of the sensor 1, measured in a state where a voltage is applied from the voltage applying unit 111a to the electrodes of the sensor 1. As described above, the current value or the change in the current value detected by the current measuring unit 111b is used as an indicator of the concentration of the test substance in the liquid sample X or the concentration change.

[0139] The voltage measuring unit 111 c measures the terminal voltage of each electrode of the sensor 1 .

[0140] The control unit 112 is connected to the voltage applying unit 111a, the current measuring unit 111b, the voltage measuring unit 111c, the storage unit 113, and the communication unit 114. The control unit 112 controls the voltage applying unit 111a so as to apply a predetermined voltage to each electrode of the sensor 1, and controls the communication unit 114 so as to transmit the measurement results of the current measuring unit 111b and the voltage measuring unit 111c to the control unit 104.

[0141] The memory unit 113 is connected to the control unit 112 and stores data such as a value of applied voltage preset for each measurement object, measurement values ​​of the current measurement unit 111 b and the voltage measurement unit 111 c , and a calibration curve measured in advance.

[0142] The communication unit 114 is controlled by the control unit 112 , and transmits data such as measurement results of the current measurement unit 111 b and the voltage measurement unit 111 c to the analysis unit 142 of the control unit 104 .

[0143] The control unit 104 can communicate with the analysis unit 102 via the communication unit 114 , and includes a display unit 141 and an analysis unit 142 .

[0144] The display unit 141 displays, for example, the concentration of the test substance in the liquid sample X as the result of the analysis in the analysis unit 142 based on the current value detected in the current measurement unit 111 b .

[0145] The analyzing unit 142 is, for example, a PC (Personal Computer), and calculates the concentration of the test substance based on the current value flowing between the working electrodes 10 a and 10 b and the counter electrode 30 measured by the current measuring unit 111 b .

[0146] <Working electrode>

[0147] For the features of the working electrodes 10a, 10b of the sensor 1, refer to the accompanying drawings, in particular to Fig.12 and Fig.16 to explain. Fig.12 The portion of the sensor 1 including the first working electrode 10a is Figure 5 Cross-sectional view of the C-C' line. Fig.16 The portion of the sensor 1 including the second working electrode 10b is Figure 5 Cross-sectional view of the D-D' line.

[0148] The first working electrode 10a includes a conductive layer 11a, a first insulating layer 3a, a second insulating layer 4a, a reagent layer 15a, and a protective film 16a ( Fig.12 The second working electrode 10b includes a conductive layer 11b, a first insulating layer 3b, a second insulating layer 4b, a reagent layer 15b, and a protective film 16b ( Fig.16 In the following description, when the elements of the first working electrode 10a and the second working electrode 10b are mentioned without distinction, they are expressed as the conductive layers 11a, 11b, the first insulating layers 3a, 3b, the second insulating layers 4a, 4b, the reagent layers 15a, 15b, and the protective films 16a, 16b.

[0149] The conductive layers 11a and 11b of the working electrodes 10a and 10b are disposed on an insulating substrate 2. The main surface on which the conductive layers 11a and 11b are formed is referred to as a first surface 2a.

[0150] Preferred embodiments of the materials of the substrate 2 and the conductive layers 11 a and 11 b are as described in the column of <Material>.

[0151] At least a portion of the first insulating layers 3a and 3b of the working electrodes 10a and 10b is disposed on the conductive layers 11a and 11b. Fig.12 and Fig.16 As shown, a part of the first insulating layers 3a and 3b may be disposed on the conductive layers 11a and 11b. Although not shown, the first insulating layers 3a and 3b may be entirely disposed on the conductive layers 11a and 11b.

[0152] In the illustrated embodiment, the first insulating layer 3a, 3b of the working electrodes 10a, 10b refers to a portion near the conductive layer 11a, 11b of the working electrodes 10a, 10b in the first insulating layer 3 that covers almost the entire first surface 2a of the substrate 2. However, the present invention is not limited to this embodiment, and the first insulating layer 3a, 3b of the working electrodes 10a, 10b may be disposed only in the vicinity of the conductive layer 11a, 11b of the working electrodes 10a, 10b.

[0153] The first insulating layer 3a, 3b of the working electrodes 10a, 10b is characterized by having a first opening 3a1, 3b1 extending through the substrate 2 in the thickness direction T and formed at a position overlapping a portion of the conductive layer 11a, 11b when viewed from the thickness direction T of the substrate 2, and a water-repellent surface 3a2, 3b2. In the example shown in the figure, the shape of the inner periphery 3a10, 3b10 of the first opening 3a1, 3b1 in the top view from the thickness direction T is circular, but the shape is not limited thereto and may be a polygon (quadrilateral, triangle, etc.) or any other shape.

[0154] The thickness of the portion of the first insulating layers 3a and 3b of the working electrodes 10a and 10b covering the conductive layers 11a and 11b is not particularly limited, and can be, for example, 0.5 μm to 50 μm, preferably 2 μm to 20 μm.

[0155] The opening width of the first openings 3a1 and 3b1 of the first insulating layers 3a and 3b of the working electrodes 10a and 10b is not particularly limited, and can be, for example, 0.5 mm to 5 mm, preferably 1 mm to 2 mm.

[0156] The first insulating layer 3a, 3b of the working electrodes 10a, 10b has a water-repellent surface 3a2, 3b2. As described later, the reagent layer 15a, 15b of the working electrodes 10a, 10b is formed by forming droplets of a liquid composition A containing a reagent participating in a redox reaction in water in the first opening 3a1, 3b1 of the first insulating layer 3a, 3b of the working electrodes 10a, 10b, and then drying. When the droplets are formed, since the surfaces 3a2, 3b2 of the first insulating layer 3a, 3b of the working electrodes 10a, 10b are water-repellent, the droplets of the liquid composition A are easily retained and do not disintegrate. Therefore, it is easy to set the characteristics of the reagent layers 15a, 15b after drying (for example, the thickness of the reagent layers 15a, 15b, the amount of the reagent constituting the reagent layers 15a, 15b) within the designed range.

[0157] Preferred embodiments of the material constituting the first insulating layers 3a and 3b having the water-repellent surfaces 3a2 and 3b2 are as described in the column of <Material>.

[0158] The second insulating layer 4a, 4b of the working electrodes 10a, 10b is arranged on the first insulating layer 3a, 3b. In the illustrated embodiment, the second insulating layer 4a, 4b of the working electrodes 10a, 10b refers to a portion near the conductive layer 11a, 11b of the working electrodes 10a, 10b in the second insulating layer 4 that covers almost the entire first surface 2a of the substrate 2. However, the present invention is not limited to this embodiment, and the second insulating layer 4a, 4b of the working electrodes 10a, 10b may be arranged only in the vicinity of the conductive layer 11a, 11b of the working electrodes 10a, 10b.

[0159] The second insulating layer 4a, 4b of the working electrodes 10a, 10b has a position overlapping with the portion 3a3, 3b3 of the first insulating layer 3a, 3b that encloses the entire first opening 3a1, 3b1 when viewed from above in the thickness direction T of the substrate 2, a second opening 4a1, 4b1 extending along the thickness direction T, and surfaces 4a2, 4b2 that are liquid-repellent to alcohol.

[0160] In the example shown in the figure, the inner edges 4a10 and 4b10 of the second openings 4a1 and 4b1 of the second insulating layers 4a and 4b of the working electrodes 10a and 10b are circular in shape when viewed from the thickness direction T, but the shape is not limited thereto and may be a polygon (a quadrilateral, a triangle, etc.) or any other shape. Figure 3As shown, it is preferred that the inner periphery 3a10, 3b10 of the first opening 3a1, 3b1 of the first insulating layer 3a, 3b and the inner periphery 4a10, 4b10 of the second opening 4a1, 4b1 of the second insulating layer 4a, 4b are parallel to each other when viewed from the thickness direction T, but the present invention is not limited thereto. The second opening 4a1, 4b1 of the second insulating layer 4a, 4b is a concave portion including the first opening 3a1, 3b1 of the first insulating layer 3a, 3b and the reagent layer 15a, 15b therein, and the region surrounding the first opening 3a1, 3b1 in the water-repellent surface 3a2, 3b2 of the first insulating layer 3a, 3b at its bottom.

[0161] The thickness of the second insulating layers 4a and 4b of the working electrodes 10a and 10b is not particularly limited, and can be, for example, 5 μm or more and 100 μm or less, and preferably 10 μm or more and 50 μm or less.

[0162] The opening width of the second openings 4a1 and 4b1 of the second insulating layers 4a and 4b of the working electrodes 10a and 10b is not particularly limited, and can be, for example, 0.5 mm to 5 mm, preferably 1 mm to 3 mm.

[0163] The second insulating layer 4a, 4b of the working electrodes 10a, 10b has a surface 4a2, 4b2 that is liquid-repellent (alcohol-repellent) to alcohol. As described later, the protective film 16a, 16b of the working electrodes 10a, 10b is formed by forming droplets of a liquid composition B containing protective film components in alcohol in the second openings 4a1, 4b1 of the second insulating layer 4a, 4b of the working electrodes 10a, 10b, and then drying. When the droplets of the liquid composition B are formed, since the surfaces 4a2, 4b2 of the second insulating layer 4a, 4b of the working electrodes 10a, 10b are liquid-repellent to alcohol, the droplets of the liquid composition B are easily retained without disintegration. Therefore, it is easy to set the characteristics of the protective films 16a, 16b after drying (for example, the thickness of the protective films 16a, 16b, the amount of the components constituting the protective films 16a, 16b) within the designed range.

[0164] Preferred embodiments of the material constituting the second insulating layers 4 a , 4 b having the surfaces 4 a 2 , 4 b 2 that are liquid-repellent (alcohol-repellent) to alcohol are as described in the column of <Material>.

[0165] The reagent layers 15a and 15b of the working electrodes 10a and 10b are arranged in the first openings 3a1 and 3b1 of the first insulating layers 3a and 3b, and include outer peripheries 15a10 and 15b10 defined by the inner peripheries 3a10 and 3b10 of the first openings 3a1 and 3b1 of the first insulating layers 3a and 3b, and reagents participating in the redox reaction. The thickness of the reagent layers 15a and 15b is not particularly limited, but is preferably substantially the same as the thickness of the portion of the first insulating layers 3a and 3b covering the conductive layers 11a and 11b, or smaller than the thickness of the portion as shown in the figure.

[0166] Preferred embodiments of the reagents participating in the redox reaction in the reagent layers 15 a and 15 b are as described in the column of <Materials>.

[0167] The protective films 16a and 16b of the working electrodes 10a and 10b are arranged in the second openings 4a1 and 4b1 of the second insulating layers 4a and 4b, and include outer peripheral edges 16a10 and 16b10 defined by the inner peripheral edges 4a10 and 4b10 of the second openings 4a1 and 4b1 of the second insulating layers 4a and 4b. The thickness of the protective films 16a and 16b is not particularly limited, but is preferably substantially the same as the thickness of the second insulating layers 4a and 4b, or smaller than the thickness of the second insulating layers 4a and 4b, as shown in the figure.

[0168] Preferred embodiments of the protective films 16a and 16b are as described in the column of <Material>.

[0169] In the following description, in order to distinguish between the "reagent layer 15a" of the first working electrode 10a and the "reagent layer 15b" of the second working electrode 10b, the former is sometimes expressed as the "first reagent layer 15a" of the first working electrode 10a, and the latter is sometimes expressed as the "second reagent layer 15b" of the second working electrode 10b. In addition, the reagent contained in the reagent layer 15a of the first working electrode 10a is sometimes expressed as the "first reagent", and the reagent contained in the second reagent layer 15b of the second working electrode 10b is sometimes expressed as the "second reagent".

[0170] In a preferred embodiment, the sensor 1 includes a plurality of working electrodes 10a and 10b, wherein the first reagent contained in the first reagent layer 15a of the first working electrode 10a and the second reagent contained in the second reagent layer 15b of the second working electrode 10b different from the first working electrode 10a are different from each other. The sensor 1 according to this preferred embodiment can be used to detect a plurality of test substances including the first test substance and the second test substance in the liquid sample X because the first reagent can participate in the oxidation-reduction reaction of the first test substance in the liquid sample X and the second reagent can participate in the oxidation-reduction reaction of the second test substance different from the first test substance in the liquid sample X.

[0171] In the sensor 1 involved in the above-mentioned preferred embodiment having a plurality of working electrodes 10a, 10b, the first reagent preferably includes an enzyme participating in the redox reaction of glucose, more preferably includes glucose dehydrogenase or glucose oxidase, and the second reagent preferably includes an enzyme participating in the redox reaction of lactic acid, more preferably includes lactate oxidase or lactate dehydrogenase. The sensor 1 involved in this embodiment can detect glucose and lactic acid.

[0172] <Reference electrode>

[0173] With reference to the accompanying drawings, in particular with reference to Fig. 22 Next, the structure of reference electrode 20 of sensor 1 will be described. Fig. 22 is the portion of the sensor 1 that includes the reference electrode 20 Figure 5 Cross-sectional view of line E-E'.

[0174] The reference electrode 20 includes a reference electrode conductive layer 21 disposed on the first surface 2 a of the insulating substrate 2 , a silver / silver chloride layer 22 disposed on the reference electrode conductive layer 21 , and a reference electrode protective film 23 disposed on the silver / silver chloride layer 22 .

[0175] The reference electrode conductive layer 21 can be formed of the materials described with respect to the conductive layers 11 a and 11 b of the working electrodes 10 a and 10 b .

[0176] The reference electrode protective film 23 can be formed of the materials described in the column of <Material> for the protective films 16a and 16b of the working electrodes 10a and 10b.

[0177] The first insulating layer 3 disposed on the first surface 2a of the substrate 2 includes a reference electrode first opening 301 which is partially disposed on the reference electrode conductive layer 21 and is formed at a position overlapping with a portion of the reference electrode conductive layer 21 when viewed from above in the thickness direction T of the substrate 2 and passes through in the thickness direction T. The silver / silver chloride layer 22 of the reference electrode 20 is disposed in the reference electrode first opening 301 of the first insulating layer 3.

[0178] The second insulating layer 4 disposed on the first insulating layer 3 includes a reference electrode second opening 401 formed at a position overlapping with a portion of the first insulating layer 3 that entirely encloses the reference electrode first opening 301 when viewed from above in the thickness direction T and extending through the reference electrode in the thickness direction T. The reference electrode protective film 23 of the reference electrode 20 is disposed in the reference electrode second opening 401 of the second insulating layer 4.

[0179] <Counter Electrode>

[0180] With reference to the accompanying drawings, in particular with reference to Fig.23Next, the structure of the counter electrode 30 of the sensor 1 will be described. Fig.23 The portion of the sensor 1 including the counter electrode 30 is Figure 5 Cross-sectional view of the F-F' line.

[0181] The counter electrode 30 includes a counter electrode conductive layer 31 disposed on the first surface 2 a of the insulating substrate 2 .

[0182] The counter electrode conductive layer 31 can be formed of the materials described with respect to the conductive layers 11 a and 11 b of the working electrodes 10 a and 10 b .

[0183] The first insulating layer 3 arranged on the first surface 2a of the substrate 2 includes a first opening portion 302 for the counter electrode, which is partially arranged on the counter electrode conductive layer 31 and extends through the thickness direction T and is formed at a position overlapping with a portion of the counter electrode conductive layer 31 when viewed from above in the thickness direction T of the substrate 2.

[0184] The second insulating layer 4 disposed on the first insulating layer 3 includes a second counter electrode opening 402 penetrating in the thickness direction T and formed at a position overlapping with the first counter electrode opening 302 in the first insulating layer 3 in a plan view in the thickness direction T.

[0185] The counter electrode conductive layer 31 of the counter electrode 30 is exposed to the outside through the counter electrode first opening 302 of the first insulating layer 3 and the counter electrode second opening 402 of the second insulating layer 4. Fig.21 As shown, when the sensor 1 is immersed in the liquid sample X, the counter electrode conductive layer 31 of the counter electrode 30 is in direct contact with the liquid sample X.

[0186] <Method of manufacturing sensor 1>

[0187] In a preferred embodiment of the method for manufacturing the sensor 1, referring to the drawings, in particular to Figures 1 to 16 To explain.

[0188] First, Figure 1 As shown, on the first surface 2a of the insulating substrate 2 are arranged: conductive layers 11a, 11b of working electrodes 10a, 10b, reference electrode conductive layer 21 of reference electrode 20, counter electrode conductive layer 31 of counter electrode 30, and wiring 50 electrically connected to them respectively.

[0189] Then, if Figure 2As shown in the figure, on the first surface 2a of the substrate 2 on which the conductive layers 11a and 11b of the working electrodes 10a and 10b, the reference electrode conductive layer 21, the counter electrode conductive layer 31, and the wiring 50 are arranged, the first insulating layer 3 is further stacked. In the first insulating layer 3, the portion near the conductive layers 11a and 11b of the working electrodes 10a and 10b is the first insulating layer 3a and 3b of the working electrodes 10a and 10b. A portion of the first insulating layer 3a and 3b of the working electrodes 10a and 10b is arranged on the conductive layers 11a and 11b, covering the upper surface of the conductive layers 11a and 11b. The first insulating layer 3a and 3b of the working electrodes 10a and 10b has the first openings 3a1 and 3b1 of the working electrodes 10a and 10b at a position overlapping with a portion of the conductive layers 11a and 11b of the working electrodes 10a and 10b. The first insulating layer 3 further includes a reference electrode first opening 301 at a position overlapping with a portion of the reference electrode conductive layer 21 , and includes a counter electrode first opening 302 at a position overlapping with a portion of the counter electrode conductive layer 31 .

[0190] Then, if Figure 3 As shown in FIG. 1 , a second insulating layer 4 is further stacked on the first insulating layer 3. In the second insulating layer 4, the portion near the conductive layer 11a, 11b of the working electrodes 10a, 10b is the second insulating layer 4a, 4b of the working electrodes 10a, 10b. The second insulating layer 4a, 4b of the working electrodes 10a, 10b has second openings 4a1, 4b1 formed at positions overlapping with portions 3a3, 3b3 that entirely enclose the first openings 3a1, 3b1 in the first insulating layer 3a, 3b of the working electrodes 10a, 10b. The second insulating layer 4 further has a reference electrode second opening 401 at a position overlapping with the reference electrode first opening 301 of the first insulating layer 3, and has a counter electrode second opening 402 at a position overlapping with the counter electrode first opening 302 of the first insulating layer 3. The second insulating layer 4 is preferably formed by applying ink containing an insulating matrix resin and a compound containing a perfluoroalkyl group (fluorine-based surface modification additive) in a solvent on the first insulating layer and drying the ink.

[0191] Will be in Figure 3 The cross section of the portion of the substrate 2 corresponding to the first working electrode 10a along the line AA', which is provided with the conductive layers 11a and 11b of the working electrodes 10a and 10b, the reference electrode conductive layer 21, the counter electrode conductive layer 31 and the wiring 50, the first insulating layer 3 and the second insulating layer 4, is shown in FIG. Figure 6 .like Figure 6 As shown, the first opening 3a1 of the first insulating layer 3a of the working electrode 10a is a concave portion including the conductive layer 11a of the working electrode 10a at its bottom. The first insulating layer 3a of the working electrode 10a has a water-repellent surface 3a2.

[0192] Then, if Figure 7 As shown in FIG. 1 , a droplet of a liquid composition A containing a reagent participating in a redox reaction in water is formed in the first opening 3a1 of the first insulating layer 3a of the working electrode 10a. At this time, since the surface 3a2 of the first insulating layer 3a of the working electrode 10a is water-repellent, the internal angle θa (shown in FIG. 1 ) at the contact point between the droplet of the liquid composition A and the surface 3a2 of the first insulating layer 3a is θa. Figure 7 ) until the water contact angle of the surface 3a2 of the first insulating layer 3a is reached, the droplet is maintained without disintegrating.

[0193] In this way, a droplet of the liquid composition A is formed in the first opening 3a1 of the first insulating layer 3a of the working electrode 10a and then dried. Figure 8 As shown, the reagent layer 15a is formed. As described above, since the droplets of the liquid composition A containing the reagent participating in the redox reaction in water are easily retained without disintegrating in the first opening 3a1 of the first insulating layer 3a having the water-repellent surface 3a2, the characteristics of the reagent layer 15a after drying (for example, the thickness of the reagent layer 15a, the amount of the reagent constituting the reagent layer 15a) are easily set within the designed range. If the surface of the first insulating layer 3a is not water-repellent, when the droplets of the liquid composition A are formed in the first opening 3a1 of the first insulating layer 3a, there is a high possibility that the droplets disintegrate and the liquid composition A wets the outside of the first opening 3a1 of the first insulating layer 3a, and it is not easy to adjust the characteristics of the reagent layer obtained by drying to within the designed range.

[0194] Preferred embodiments of the composition of the liquid composition A containing a reagent participating in the redox reaction in water are as described in the column of <Materials>.

[0195] After the formation of the reagent layer 15a, as Fig. 9 As shown in FIG. 1 , a droplet of a liquid composition B containing a protective film component in alcohol is formed in the second opening 4a1 of the second insulating layer 4a of the working electrode 10a. At this time, since the surface 4a2 of the second insulating layer 4a is liquid-repellent to alcohol, the internal angle θb (shown in FIG. 1 ) at the contact point between the droplet of the liquid composition B and the surface 4a2 of the second insulating layer 4a is Fig. 9 ) until the contact angle of the surface 4a2 of the second insulating layer 4a with the alcohol reaches , the droplet is maintained without disintegrating.

[0196] In this way, the droplets of the liquid composition B in the second opening 4a1 of the second insulating layer 4a of the working electrode 10a are formed and then dried. Fig.10As shown, a protective film 16a is formed. As described above, since droplets of the liquid composition B containing protective film components in alcohol are easily retained without disintegrating in the second opening 4a1 of the second insulating layer 4a having a surface 4a2 that is liquid-repellent to alcohol, it is easy to set the properties of the protective film 16a after drying (for example, the thickness of the protective film 16a, the amount of the components constituting the protective film 16a) within a designed range. If the surface of the second insulating layer 4a does not have liquid-repellent properties to alcohol, when droplets of the liquid composition B are formed in the second opening 4a1 of the second insulating layer 4a, the droplets disintegrate, and there is a high possibility that the liquid composition B wets the outside of the second opening 4a1 of the second insulating layer 4a, making it difficult to adjust the properties of the protective film obtained by drying to within a designed range (see reference 2). Figures 17-20 The comparative examples described below will be described below).

[0197] Preferred embodiments of the composition of the liquid composition B containing the protective film component in alcohol are as described in the column of <Materials>.

[0198] Figures 9 to 12 FIG. 4 shows an example in which the protective film 16a in the second opening 4a1 of the second insulating layer 4a of the working electrode 10a is formed in two stages. In this embodiment of the manufacturing method of the sensor 1, Fig. 9 As shown, after the reagent layer 15a is formed, droplets of the liquid composition B containing the protective film component in alcohol are formed in the second opening 4a1 of the second insulating layer 4a and then dried. Fig.10 As shown in FIG. 1 , a portion of the protective film 16a in the thickness direction is formed. Fig.11 As shown, droplets of the liquid composition B are further formed in the second opening 4a1 of the second insulating layer 4a and then dried. Fig.12 The entire protective film 16a is formed as shown. However, the present invention is not limited to this example, and the protective film may be formed in one step or in three or more steps.

[0199] As described above, the sensor 1 in a preferred embodiment includes a plurality of working electrodes 10a, 10b, and the first reagent contained in the first reagent layer 15a of the first working electrode 10a of the plurality of working electrodes 10a, 10b is different from the second reagent contained in the second reagent layer 15b of the second working electrode 10b different from the first working electrode 10a. The manufacturing method of the sensor 1 involved in this preferred embodiment preferably includes, as a step of forming the reagent layer:

[0200] In the first opening 3a1 of the first insulating layer 3a of the first working electrode 10a, a droplet of the first liquid composition A containing the first reagent in water is formed and then dried to form the first reagent layer 15a of the first working electrode 10a included in the plurality of working electrodes 10a and 10b (see Figure 6 to Figure 8 );as well as

[0201] In the first opening 3b1 of the first insulating layer 3b of the second working electrode 10b, a droplet of a second liquid composition A containing a second reagent different from the first reagent in water is formed and then dried to form a second reagent layer 15b (not shown, which is different from the first working electrode 10a) of the second working electrode 10b contained in the plurality of working electrodes 10a and 10b. Figure 6 to Figure 8 Same process).

[0202] Since the reagent layers 15a, 15b of the plurality of working electrodes 10a, 10b can be formed independently in the manufacturing method of the sensor 1 involved in this embodiment, it is easy to form reagent layers 15a, 15b containing different reagents on the plurality of working electrodes 10a, 10b by adjusting the composition of the liquid composition A containing the reagent in water.

[0203] In the sensor 1 including different reagents in the plurality of working electrodes 10a and 10b manufactured by the method according to the preferred embodiment described above, the first reagent layer 15a of the first working electrode 10a includes, for example, a first reagent containing glucose dehydrogenase or glucose oxidase. Fig.12 As shown, the first protective film 16a includes a polymer compound containing 4-vinylpyridine in a structural unit. The formation of such a first protective film 16a may include:

[0204] After the first reagent layer 15a of the first working electrode 10a is formed, a droplet of the first liquid composition B containing a polymer compound containing 4-vinylpyridine in a structural unit in alcohol is formed in the second opening 4a1 of the second insulating layer 4a of the first working electrode 10a, and then dried to form the first protective film 16a. The first protective film 16a can be formed as follows: Figures 9 to 12 Although the process is performed in two stages as shown, it may be performed in one stage or three or more stages not shown.

[0205] In the sensor 1 in which the plurality of working electrodes 10a and 10b contain different reagents and are manufactured by the method according to the preferred embodiment, the second reagent layer 15b of the second working electrode 10b contains, for example, a second reagent containing lactate oxidase or lactate dehydrogenase. Fig.16 As shown, the protective film 16b including the second protective film 16ba and the third protective film 16bb can include:

[0206] After the second reagent layer 15b of the second working electrode 10b is formed, droplets of the second liquid composition B containing the polymer compound having a cation exchange functional group in alcohol are formed in the second opening 4b1 of the second insulating layer 4b of the second working electrode 10b, and then dried to form a second protective film 16ba (see Fig.13 and Fig.14 );as well as

[0207] After the second protective film 16ba is formed, droplets of the third liquid composition B containing the polymer compound containing 4-vinylpyridine in a structural unit in alcohol are formed in the second opening 4b1 of the second insulating layer 4b of the second working electrode 10b, and then dried to form the third protective film 16bb (see Fig.15 and Fig.16 ).

[0208] In the method involved in this embodiment, since the protective films 16a, 16b of multiple working electrodes 10a, 10b can be formed independently, by adjusting the composition of the liquid composition B containing the protective film component in alcohol, it is easy to form protective films 16a, 16b with different compositions optimized corresponding to the reagent layers 15a, 15b on the reagent layers 15a, 15b containing different reagents of the multiple working electrodes 10a, 10b.

[0209] The method according to the present embodiment may include a step of forming a reference electrode 20. The step of forming the reference electrode 20 may include:

[0210] exist Figure 3 In the substrate 2 shown in FIG. 2 , which is provided with a reference electrode conductive layer 21, a first insulating layer 3, and a second insulating layer 4, a silver / silver chloride layer 22 ( Figure 4 );as well as

[0211] After the silver / silver chloride layer 22 is disposed, a reference electrode protective film 23 ( Figure 5 , Fig. 22 ).

[0212] <First disclosed embodiment>

[0213] As a first disclosed embodiment, manufacturing Figure 5 The structure of the sensor 1 is shown. Figure 5 The materials used in the sensor 1 having the structure shown and the outline of the manufacturing method are as follows.

[0214] (Substrate)

[0215] As the insulating substrate 2, a Figure 1 A substrate having a thickness of 188 μm and made of polyethylene terephthalate having the shape shown in the drawings, etc.

[0216] (Conductive layer)

[0217] On the first surface 2a of the insulating substrate 2, a carbon paste was applied and heated at 140°C for 1 hour to form a carbon conductive layer with a thickness of 5 μm. Figure 1 The conductive layers 11a and 11b of the working electrodes 10a and 10b, the reference electrode conductive layer 21 and the counter electrode conductive layer 31 of the shape shown in the figure, and the wirings 50 electrically connected to them respectively.

[0218] (1st insulation layer)

[0219] Then, if Figure 2 As shown in the figure, the first insulating layer 3 having a thickness of 5 μm on the portion on the carbon conductive layer is laminated on the first surface 2a of the substrate 2 provided with the carbon conductive layer, and the first insulating layer 3 is formed of a fluororesin containing a copolymer of vinylidene fluoride and hexafluoropropylene, and covers the first surface 2a of the substrate 2 and the carbon conductive layer. The composition formed by curing the fluororesin is applied to the first surface 2a of the substrate 2 provided with the carbon conductive layer, and heated at 140° C. for 1 hour, thereby forming the first insulating layer 3.

[0220] The first opening 3a1 of the first insulating layer 3a of the first working electrode 10a is formed into a circle with a diameter of 1.2 mm. The first opening 3b1 of the first insulating layer 3b of the second working electrode 10b is formed into a circle with a diameter of 1.4 mm. The first opening 301 of the reference electrode is formed into a circle with a diameter of 1.1 mm, and the first opening 302 of the counter electrode is formed into a rectangle with a size of 1.8 mm×2.1 mm.

[0221] The water contact angle of the surface of the first insulating layer 3 (including the surfaces 3a2 and 3b2 of the first insulating layers 3a and 3b of the working electrodes 10a and 10b) was 134.2°. The water contact angle was measured by spraying a 2 μL droplet of water onto the surface of the measurement object at 20°C using a handheld contact angle / surface free energy analyzer MSA manufactured by KRUSS, and measuring the contact angle between the droplet and the surface after 2 seconds.

[0222] (Second insulation layer)

[0223] Then, if Figure 3 As shown, a composition containing a polyester resin and a fluorine-based surface modification additive containing a perfluoroalkyl group in a solvent is applied on the first insulating layer 3 and heated at 140° C. for 1 hour to laminate the second insulating layer 4 having a thickness of 40 μm.

[0224] The second opening 4a1 of the second insulating layer 4a of the first working electrode 10a and the second opening 4b1 of the second insulating layer 4b of the second working electrode 10b are each formed into a circle with a diameter of 2 mm. The second opening 401 of the reference electrode is formed into a circle with a diameter of 2 mm, and the second opening 402 of the counter electrode is formed into a rectangle with a size of 1.8 mm×2.1 mm.

[0225] The contact angle of the surface of the second insulating layer 4 (including the surfaces 4a2 and 4b2 of the second insulating layers 4a and 4b of the working electrodes 10a and 10b) with respect to ethanol was 60.7°. The contact angle with respect to ethanol was measured by spraying a 2 μL droplet of ethanol onto the surface of the measurement object at 20° C. using a handheld contact angle / surface free energy analyzer MSA manufactured by KRUSS, and measuring the contact angle between the droplet and the surface after 2 seconds.

[0226] (First working electrode (for glucose measurement) reagent layer)

[0227] In the first opening 3a1 of the first insulating layer 3a of the first working electrode 10a, a 0.4 μL droplet of the first liquid composition A containing sodium phosphate buffer (pH 7.4), carbon black dispersion, polymer-bonded mediator, glucose oxidase and a cross-linking agent in water is formed and dried to form a first reagent layer 15a containing glucose oxidase and the mediator.

[0228] At this time, the droplets of the first liquid composition A do not overflow from the first opening 3a1, and the dried first reagent layer 15a is formed only in the first opening 3a1 (see Figure 7 and Figure 8 ).

[0229] (Second working electrode (for lactic acid measurement) reagent layer)

[0230] In the first opening 3b1 of the first insulating layer 3b of the second working electrode 10b, a 0.6 μL droplet of the second liquid composition A containing a carbon black dispersion, hydroxypropyl cellulose, a polymer-bonded medium, lactate oxidase, polyimidazole, poly-L-lysine and a cross-linking agent in water is formed and dried to form a second reagent layer 15b containing lactate oxidase and the medium.

[0231] At this time, the droplets of the second liquid composition A do not overflow from the first opening 3b1, and the dried second reagent layer 15b is formed only in the first opening 3b1 (see Figure 7 and Figure 8 ).

[0232] (Protective film for the first working electrode (for glucose measurement))

[0233] The following reagents were mixed in ethanol and reacted for about 1 hour to achieve the following final concentrations, thereby preparing a first liquid composition B (P4VP-tBuMA polymer dispersion).

[0234] P4VP-tBuMA (Mn of poly-4-vinylpyridine: 74,000, Mn of poly-tert-butyl methacrylate: 87,000, Mw / Mn: 1.16, manufactured by NARD), final concentration: 3.55 wt%

[0235] · Random copolymer of tripropylene glycol methyl ether methacrylate and styrene-4-vinylpyridine (manufactured by NARD), final concentration 4.45 wt%

[0236] PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ~1000, manufactured by Sigma-Aldrich), final concentration 7 mM

[0237] In the second opening 4a1 of the second insulating layer 4a of the first working electrode 10a, a 0.65 μL droplet of the first liquid composition B is formed and dried, and then a 0.65 μL droplet of the first liquid composition B is further formed and dried to form a first protective film 16a (see Fig.12 ).

[0238] At this time, the droplets of the first liquid composition B do not overflow from the second opening 4a1, and the first protective film 16a after drying is formed only in the second opening 4a1 (see Fig. 9 , Fig.10 , Fig.11 as well as Fig.12 ).

[0239] (Second working electrode (for lactic acid measurement) protective film)

[0240] To 23093.67 mg of a 21.5 wt% Nafion (registered trademark) dispersion (manufactured by Sigma-Aldrich), 6472.79 mg of ethanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 1226.46 mg of a 5 mol / L sodium hydroxide aqueous solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added to adjust the pH of the dispersion (neutralization treatment of the cation exchange group), and the precipitate was dissolved with a vortex mixer to prepare 30792.92 mg of a 16.12 wt% Nafion (registered trademark) dispersion. The obtained 16.12 wt% Nafion (registered trademark) dispersion was used as the second liquid composition B. The second liquid composition B contains Nafion (registered trademark) in a solvent containing a lower alcohol.

[0241] A 0.60 μL droplet of the second liquid composition B was formed in the second opening 4 b 1 of the second insulating layer 4 b of the second working electrode 10 b and dried to form a second protective film 16 ba (see Fig.14 ).

[0242] At this time, the droplets of the second liquid composition B do not overflow from the second opening 4b1, and the dried second protective film 16ba is formed only in the second opening 4b1 (see Fig.13 and Fig.14 ).

[0243] Next, in the following process, the same composition as the first liquid composition B (P4VP-tBuMA polymer dispersion) was used as the third liquid composition B. That is, a 0.65 μL droplet of the third liquid composition B was formed in the second opening 4b1 of the second insulating layer 4b of the second working electrode 10b on which the second protective film 16ba was formed and dried to form the third protective film 16bb (see Fig.16 ).

[0244] At this time, the liquid droplets of the third liquid composition B do not overflow from the second opening 4b1, and the dried third protective film 16bb is formed only in the second opening 4b1 (see Fig.15 and Fig.16 ).

[0245] (reference electrode)

[0246] A silver / silver chloride paste was applied to the reference electrode conductive layer 21 in the reference electrode first opening 301 of the first insulating layer 3 and heated at 140° C. for 1 hour to form a silver / silver chloride layer 22 .

[0247] Next, the reference electrode protective film 23 is disposed on the silver / silver chloride layer 22 in the reference electrode second opening 401 of the second insulating layer 4 to form the reference electrode 20 ( Fig. 22 ).

[0248] <Comparative Example>

[0249] A sensor of the comparative example was produced in the same manner as the sensor 1 of the above-described embodiment except that the second insulating layer was produced in the following steps.

[0250] A composition containing the same polyester resin as in Example and no fluorine-based surface modification additive in a solvent was applied on the first insulating layer 3 and heated at 140° C. for 1 hour to form a second insulating layer having a thickness of 38 μm.

[0251] The contact angle of the surface of the second insulating layer of the sensor of the comparative example obtained in this way with respect to ethanol was 11.2°.

[0252] When forming the protective films of the first working electrode and the second working electrode of the sensor of the comparative example, the following problems occurred.

[0253] In the second opening 4a1 of the second insulating layer 4a of the first working electrode 10a of the sensor of the comparative example, a droplet of 0.65 μL of the first liquid composition B (P4VP-tBuMA polymer dispersion) used in the example was formed. Fig.17 As shown in FIG. 1 , the droplet does not remain in the second opening 4a1, and the first liquid composition B wets and diffuses to the upper surface of the second insulating layer 4a. The droplet is dried, and the result is as shown in FIG. Fig.18 As shown in FIG. 1 , a portion of the first protective film 16a is formed not only in the second opening 4a1 but also on the upper surface of the second insulating layer 4a. Drying is performed, and then a 0.65 μL droplet of the first liquid composition B is further formed. Fig.19 As shown in FIG. 1 , the first liquid composition B wets and diffuses to the upper surface of the second insulating layer 4a. The liquid composition B is dried, and the result is as shown in FIG. Fig. 20 As shown in FIG. 1 , a portion of the first protective film 16 a is formed not only in the second opening 4 a 1 but also on the upper surface of the second insulating layer 4 a .

[0254] Although not shown in the figure, when droplets of the second liquid composition B (Nafion (registered trademark) dispersion) used in the example were formed in the second opening 4b1 of the second insulating layer 4b of the second working electrode 10b of the sensor of the comparative example and dried, the second protective film 16ba was formed irregularly not only in the second opening 4b1 but also on the upper surface of the second insulating layer 4b. Furthermore, when droplets of the third liquid composition B (P4VP-tBuMA polymer dispersion) used in the example were formed and dried, the third protective film 16bb was formed irregularly not only in the second opening 4b1 but also on the upper surface of the second insulating layer 4b.

[0255] <Glucose and lactic acid measurement experiment>

[0256] (culture medium)

[0257] MES (2-Morpholinoethanesulfonic acid monohydrate) (manufactured by Dojindo Chemical Co., Ltd.) and MOPS (3-Morpholinopropanesulfonic acid) (manufactured by Dojindo Chemical Co., Ltd.) as buffer components were added to a solution of RPMI-1640 Medium (R1383, manufactured by Sigma-Aldrich) to a final concentration of 25 mM, respectively. The concentration of glucose, lactic acid, and pH were further adjusted to 30 mM, 15 mM, and 7.4, respectively, to prepare a culture solution.

[0258] (Current value measurement)

[0259] The sensor 1 of the above-mentioned embodiment and the sensor of the comparative example were immersed in the above-mentioned culture solution, a voltage of 100 mV relative to the reference electrode was applied to the first working electrode and the second working electrode, and the current value between the first working electrode and the counter electrode and the current value between the second working electrode and the counter electrode were measured for about 11 days. The measurement was performed with N=2.

[0260] The measurement results of the current value of the first working electrode (for glucose measurement) of the sensor 1 of the embodiment are as follows: Fig.25 A. The measurement results of the current value of the first working electrode (for glucose measurement) of the sensor of the comparative example are shown in Fig.25 B. The current value of the sensor 1 of the embodiment is stable, while the current value of the sensor of the comparative example decreases over time. It is presumed that in the sensor of the comparative example, since it is difficult to control the thickness of the first protective film, the permeability of glucose is large from the beginning, and the reagent containing glucose oxidase leaks.

[0261] The measurement results of the current value of the second working electrode (for lactic acid measurement) of the sensor 1 of the embodiment are as follows: Fig.26 A. The measurement results of the current value of the second working electrode (for lactic acid measurement) of the sensor of the comparative example are shown in Fig.26 B. The current value of the sensor 1 of the embodiment is stable, while the current value of the sensor of the comparative example decreases over time. It is presumed that in the sensor of the comparative example, since it is difficult to control the thickness of the second protective film and the third protective film, the permeability of lactic acid is large from the beginning, and the reagent containing lactate oxidase leaks.

[0262] <Embodiment of the second disclosure>

[0263] The following describes an embodiment of the sensor and sensor assembly involved in the second disclosure. In this embodiment, a detailed description that is more than necessary is sometimes omitted. For example, a detailed description of known matters and a repeated description of substantially the same structure are sometimes omitted. This is to avoid the following description from becoming unnecessarily lengthy and to facilitate understanding by those skilled in the art.

[0264] In addition, the applicant provides the attached drawings and the following description in order to enable those skilled in the art to fully understand the second disclosure, but does not intend to limit the matters described in the following "Supplementary Notes Related to the Second Disclosure" by these.

[0265] <Materials>

[0266] Examples of materials that can be used for the sensor disclosed in the second aspect of this specification will be described.

[0267] The material of the insulating substrate of the sensor disclosed in the second aspect of the present specification is not particularly limited, and for example, the same material as that of the insulating substrate of the sensor disclosed in the first aspect of the present specification can be used.

[0268] The conductive layer of the working electrode, reference electrode and counter electrode of the sensor disclosed in the second aspect of this specification is a layer containing a conductive material such as carbon, gold, platinum, palladium, etc. The conductive layer can be manufactured by forming a layer of the above conductive material on the surface of the substrate by sputtering, evaporation, screen printing, etc. The conductive layer can be processed into a predetermined pattern by laser trimming as needed.

[0269] The wiring of the sensor disclosed in the second aspect of the present specification may be made of the same conductive material as the conductive layer.

[0270] The sensor disclosed in the second aspect of this specification is used to detect a specified test substance in the liquid sample by being immersed in a liquid sample. The liquid sample is preferably a liquid sample containing water as a solvent. As the liquid sample, a cell culture fluid or a liquid sample prepared by blood obtained from a living body can be cited. Examples of the test substance are the same as those described for the test substance of the sensor disclosed in the first aspect of this specification.

[0271] The working electrode of the sensor disclosed in the second aspect of the present specification has a reagent layer containing a reagent participating in a redox reaction. The reagent participating in the redox reaction can be any reagent participating in the redox reaction of the substance to be tested, and can be appropriately selected according to the substance to be tested. As a reagent participating in the redox reaction, a combination of an oxidoreductase and a mediator (electron transfer substance) may be included, or an oxidoreductase may be included. The oxidoreductase may include a coenzyme.

[0272] Examples of the oxidoreductase include oxidase and dehydrogenase. Specific examples of the oxidoreductase are as described in connection with the sensor disclosed in Section 1 of the present specification.

[0273] In addition, the medium is not particularly limited, and examples thereof are as described in connection with the sensor disclosed in the first aspect of this specification.

[0274] The reagent layer of the working electrode of the sensor disclosed in the second aspect of this specification may further include components such as a buffer, a hydrophilic polymer compound, a conductive carbon filler, and a crosslinking agent in addition to the above reagents. Examples of the hydrophilic polymer compound and the conductive carbon filler are as described for the sensor disclosed in the first aspect of this specification.

[0275] The protective film provided by the working electrode of the sensor disclosed in the second aspect of this specification may be a film that prevents or inhibits the leakage of the reagent contained in the reagent layer to the outside of the protective film and that can permeate the test substance present outside the protective film. The protective film having such a property preferably contains a polymer compound. Examples of the polymer compound contained in the protective film include: a polymer compound containing 4-vinylpyridine in the structural unit, a polymer compound containing a cation exchange functional group, and the like.

[0276] Examples of the polymer compound containing 4-vinylpyridine in a structural unit and the polymer compound containing a cation-exchange functional group are as described in connection with the sensor disclosed in the first aspect of the present specification.

[0277] The protective film provided in the working electrode of the sensor disclosed in the second aspect of the present specification may be a laminate of two or more protective films, and examples thereof are as described in connection with the sensor disclosed in the first aspect of the present specification.

[0278] Another suitable example of the protective film provided in the working electrode of the sensor disclosed in the second aspect of the present specification is a protective film comprising a polymer compound containing 4-vinylpyridine as a structural unit.

[0279] The reference electrode of the sensor disclosed in the second aspect of the present specification may include a protective film. The protective film of the reference electrode may be made of the material described above with respect to the protective film of the working electrode.

[0280] <Overview of sensor 1101>

[0281] A sensor 1101 according to an embodiment of the second disclosure will be described with reference to the drawings.

[0282] The sensor 1101 of this embodiment is as follows Fig.31 and as its cross-sectional view Fig.33 , Fig.34 , Fig.35 as well as Fig.36 As shown, it has:

[0283] An insulating substrate 1200 including a front end portion 1201 immersed in a liquid sample;

[0284] A detection electrode 1300 disposed near the front end portion 1201 on the first surface 1202 of the substrate 1200 and including a working electrode 1310 , a reference electrode 1320 , and a counter electrode 1330 ;

[0285] A wiring 1005 disposed on the first surface 1202 of the substrate 1200 and connected to the detection electrode 1300;

[0286] an insulating layer 1050 disposed on the first surface 1202 of the substrate 1200 and formed so as to cover at least a portion of the detection electrode 1300 and the wiring 1005;

[0287] a front end opening 1061 formed in the insulating layer 1050 , communicating with the upper surface 1331 of the counter electrode 1330 , and opening on the front end portion 1201 side of the substrate 1200 ; and

[0288] A flow path 1062 is formed in the insulating layer 1050 , is connected to the front end opening 1061 and the upper surface 1331 of the counter electrode 1330 , and conducts the liquid sample from the front end opening 1061 to the upper surface 1331 of the counter electrode 1330 .

[0289] The sensor 1101 of this embodiment includes two working electrodes 1310 , but in other embodiments not shown in the figure, there may be only one working electrode, or there may be three or more working electrodes.

[0290] Sensor 1101 such as Fig.39and Fig.43 As shown, it can be used to detect a predetermined test substance in the liquid sample X1 by being immersed in the liquid sample X1. Specific examples of the liquid sample and the test substance are described in the column of <Material>.

[0291] The working electrode 1310 of the sensor 1101 is as follows Fig.33 As shown, it includes a working electrode conductive layer 1311 and a reagent layer 1315 including a reagent participating in the oxidation-reduction reaction of the test substance in the liquid sample X1 disposed on the working electrode conductive layer 1311. Preferred embodiments of the reagent participating in the oxidation-reduction reaction are described in the column of <Materials>.

[0292] When the reagent layer 1315 of the working electrode 1310 contains a reagent that oxidizes the test substance in the liquid sample X1, electrons move from the test substance to the working electrode 1310 under the condition that a predetermined voltage is applied to the detection electrode 1300 of the sensor 1101. Similarly, when the reagent layer 1315 contains a reagent that reduces the test substance in the liquid sample X1, electrons move from the working electrode 1310 to the test substance. Since the amount of electrons that move depends on the concentration of the test substance, the concentration of the test substance in the liquid sample X1 or the concentration change can be measured based on the current value or the change in the current value flowing through the working electrode 1310 of the sensor 1101.

[0293] Reference Fig.48 , an example of an analysis device 1100 including a sensor 1101 for analyzing a test substance in a liquid sample will be described.

[0294] The analysis device 1100 is as follows Fig.48 As shown, a sensor 1101, an analysis unit 1102 and a control unit 1104 are provided. The detection electrode 1300 including the working electrode 1310, the reference electrode 1320 and the counter electrode 1330 of the sensor 1101 is connected to the analysis unit 1102 via wiring 1005. The analysis unit 1102 can communicate with the control unit 1104.

[0295] The analysis unit 1102 includes an electrochemical measurement unit 1111 , a control unit 1112 , a storage unit 1113 , and a communication unit 1114 .

[0296] The electrochemical measuring unit 1111 is a potentiostat that applies a predetermined voltage to the detection electrode 1300 of the sensor 1101 to measure the concentration of the test substance, and includes a voltage applying unit 1111a and a current measuring unit 1111b, and preferably further includes a voltage measuring unit (counter electrode terminal voltage measuring unit) 1111c.

[0297] The voltage application unit 1111 a applies a predetermined voltage for measuring the concentration of the test substance contained in the liquid sample X1 to the detection electrode 1300 of the sensor 1101 .

[0298] The current measuring unit 1111b detects the current value or the change thereof flowing between the working electrode 1310 and the counter electrode 1330 of the sensor 1101, which is measured when a voltage is applied from the voltage applying unit 1111a to the detection electrode 1300 of the sensor 1101. As described above, the current value or the change in the current value detected by the current measuring unit 1111b is used as an indicator of the concentration of the test substance in the liquid sample X1 or the concentration change.

[0299] The voltage measuring unit 1111 c measures the terminal voltage of the counter electrode 1330 of the sensor 1101 .

[0300] The control unit 1112 is connected to the voltage applying unit 1111a, the current measuring unit 1111b, the voltage measuring unit 1111c, the storage unit 1113, and the communication unit 1114. The control unit 1112 controls the voltage applying unit 1111a so as to apply a predetermined voltage to the detection electrode 1300 of the sensor 1101, and controls the communication unit 1114 so as to transmit the measurement results of the current measuring unit 1111b and the voltage measuring unit 1111c to the control unit 1104.

[0301] The memory unit 1113 is connected to the control unit 1112 and stores data such as a value of applied voltage preset for each measurement object, measurement values ​​in the current measurement unit 1111b and the voltage measurement unit 1111c, and a calibration curve measured in advance.

[0302] The communication unit 1114 is controlled by the control unit 1112 , and transmits data such as measurement results in the current measurement unit 1111 b and the voltage measurement unit 1111 c to the analysis unit 1142 of the control unit 1104 .

[0303] The control unit 1104 can communicate with the analysis unit 1102 via the communication unit 1114 , and includes a display unit 1141 and an analysis unit 1142 .

[0304] The display unit 1141 displays, for example, the concentration of the test substance in the liquid sample X1 as the result of the analysis performed by the analysis unit 1142 based on the current value detected by the current measurement unit 1111 b .

[0305] The analyzing unit 1142 is, for example, a PC (Personal Computer), and calculates the concentration of the test substance based on the current value flowing between the working electrode 1310 and the counter electrode 1330 measured by the current measuring unit 1111 b .

[0306] <Structure of sensor 1101>

[0307] Next, the main reference Fig.31 and as its cross-sectional view Fig.33 , Fig.34 , Fig.35 as well as Fig.36 , the structure of the sensor 1101 of this embodiment is described.

[0308] The substrate 1200 of the sensor 1101 of this embodiment includes a front end portion 1201 immersed in the liquid sample. The substrate 1200 may be a plate-shaped body. The front end portion 1201 is a portion of the peripheral portion of the substrate 1200 that becomes the front end when the sensor 1101 is immersed in the liquid sample X1 ( Fig.43 Reference). Preferred embodiments of the substrate 1200 are as described in the column of <Material>.

[0309] In the sensor 1101 , the detection electrode 1300 including the working electrode 1310 , the reference electrode 1320 , and the counter electrode 1330 is arranged near the tip portion 1201 on the first surface 1202 of the substrate 1200 .

[0310] The wiring 1005 of the sensor 1101 is disposed on the first surface 1202 of the substrate 1200 and is electrically connected to each electrode (working electrode 1310, reference electrode 1320, and counter electrode 1330) of the detection electrode 1300. Preferred embodiments of the material of the wiring 1005 are described in the column of <Material>.

[0311] The insulating layer 1050 of the sensor 1101 is disposed on the first surface 1202 of the substrate 1200 , and is formed so as to cover at least a portion of the detection electrode 1300 and the wiring 1005 .

[0312] In the example shown in the figure, the insulating layer 1050 includes a first insulating layer 1051 disposed on the first surface 1202 of the substrate 1200 and a second insulating layer 1052 disposed on the first insulating layer 1051. However, in other embodiments not shown, the insulating layer 1050 may include only one layer or three or more layers.

[0313] The insulating layer 1050 may be any insulating layer, and the material thereof is not particularly limited. For example, the insulating layer may be an insulating layer including an insulating cured resin, an insulating layer including an insulating sheet, or a combination of a plurality of insulating layers.

[0314] The insulating layer containing an insulating cured resin can be formed by curing a curable resin composition. As a curable resin composition, a composition that generates an insulating cured resin by polymerization and / or crosslinking by irradiation with active energy rays can be used. As active energy rays, ultraviolet rays, electron beams, X-rays, infrared rays, visible light, etc., preferably ultraviolet rays or electron beams. As a curable resin composition, for example, a negative resist composition can be used. As an insulating cured resin that can be formed by curing a curable resin composition, a fluororesin is preferably used. As a fluororesin, as long as it is a resin containing a polymer containing fluorine atoms on the main chain carbon and / or side chain, for example, poly (meth) acrylate resins, polyester resins, etc. containing fluorine atoms on the main chain carbon and / or side chain can be used. The thickness of the insulating layer containing an insulating cured resin is not particularly limited, for example, it can be set to be greater than 1 μm and less than 50 μm.

[0315] The insulating layer including the insulating sheet may include the insulating sheet and a bonding layer including an adhesive or a bonding agent disposed on one surface of the insulating sheet. The insulating layer including the insulating sheet may be formed by attaching the insulating sheet including the bonding layer directly or through other insulating layers to the first surface 1202 of the substrate 1200. The insulating sheet may be an insulating resin sheet, for example, a polyethylene terephthalate sheet may be exemplified. As the bonding layer, for example, a double-sided tape, an adhesive or a bonding agent layer may be used. The outer surface of the insulating sheet is preferably a water-repellent surface, for example, a water-repellent surface that is rendered water-repellent by water-repellent coating or surface concave-convex treatment. The thickness of the insulating layer including the insulating sheet is not particularly limited. The thickness of the insulating sheet may be, for example, set to be greater than 10 μm and less than 200 μm, preferably set to be greater than 20 μm and less than 100 μm, and the thickness of the bonding layer disposed on one surface of the insulating sheet may be, for example, set to be greater than 1 μm and less than 50 μm, preferably set to be greater than 5 μm and less than 20 μm.

[0316] A preferred embodiment of the insulating layer 1050 includes: a first insulating layer 1051 including an insulating curable resin disposed on the first surface 1202 of the substrate 1200; and a second insulating layer 1052 including an insulating sheet disposed on the first insulating layer 1051. In this embodiment, the second insulating layer 1052 may include an insulating sheet and a bonding layer disposed on the surface of the insulating sheet on the first insulating layer 1051 side, and the insulating sheet is preferably attached to the first insulating layer 1051 via the bonding layer. In this embodiment, the thickness of the first insulating layer 1051 including an insulating curable resin is not particularly limited, and the thickness of the portion covering the wiring 1005 of the detection electrode 1300 and each conductive layer (working electrode conductive layer 1311, reference electrode conductive layer 1321, counter electrode 1330) can be set to, for example, 0.5 μm or more and 50 μm or less, preferably 2 μm or more and 20 μm or less. Examples of the thickness of the second insulating layer 1052 including the insulating sheet are as described in the previous paragraph.

[0317] With reference to the accompanying drawings, in particular with reference to Fig.33 The working electrode 1310 of the sensor 1101 will be described. Fig.33 yes Fig.31 The portion of the sensor 1101 shown that includes the working electrode 1310 is Fig.31 Cross-sectional view of the J-J' line.

[0318] The working electrode 1310 includes a working electrode conductive layer 1311 disposed on the first surface 1202 of the substrate 1200, and a reagent layer 1315 disposed on the working electrode conductive layer 1311. The working electrode 1310 preferably further includes a working electrode protective film 1316 disposed on the reagent layer 1315 as shown in the figure. Preferred embodiments of the materials of the working electrode conductive layer 1311, the reagent layer 1315, and the working electrode protective film 1316 are described in the column of <Materials>. The wiring 1005 is connected to the working electrode conductive layer 1311 of the working electrode 1310 (see Fig. 27 and Fig.31 ).

[0319] The working electrode 1310 achieves the following effects by including the working electrode protective film 1316. The working electrode protective film 1316 suppresses leakage of the reagent from the reagent layer 1315. In addition, since the working electrode protective film 1316 is porous, the liquid sample X1 can penetrate. Therefore, when the sensor 1101 is immersed in the liquid sample X1, the liquid sample X1 easily reaches the working electrode 1310, and it is not easy to cause the liquid sample X1 to be impregnated into the working electrode 1310.

[0320] The insulating layer 1050 includes a working electrode opening 1501 formed at a position overlapping at least a portion of the working electrode 1310 when viewed from the thickness direction T1 of the substrate 1200, and penetrating in the thickness direction T1. In the example shown in the figure, the working electrode opening 1501 is located on the working electrode conductive layer 1311, and the reagent layer 1315 and the working electrode protective film 1316 are arranged in the working electrode opening 1501. The working electrode protective film 1316 is arranged in the working electrode opening 1501, thereby more effectively suppressing the leakage of the reagent from the reagent layer 1315. The outer periphery of each of the reagent layer 1315 and the working electrode protective film 1316 is defined according to the inner periphery of the working electrode opening 1501. The shape of the inner periphery of the working electrode opening 1501 of the insulating layer 1050 when viewed from the thickness direction T1 is circular in the example shown in the figure, but is not limited thereto, and may be a polygon (quadrilateral, triangle, etc.) or any other shape. The opening width (the width of the widest part) of the working electrode opening 1501 of the insulating layer 1050 is not particularly limited, and can be, for example, 0.5 mm to 10 mm, preferably 1 mm to 5 mm.

[0321] In the example shown in the figure, the working electrode opening 1501 of the insulating layer 1050 is composed of the working electrode first opening 1511 of the first insulating layer 1051 and the working electrode second opening 1521 of the second insulating layer 1052. The working electrode first opening 1511 is formed at a position of the first insulating layer 1051 that overlaps with a part of the working electrode conductive layer 1311 when viewed from the thickness direction T1, and penetrates in the thickness direction T1. The working electrode second opening 1521 is formed at a position of the second insulating layer 1052 that overlaps with a portion of the first insulating layer 1051 that includes the entire working electrode first opening 1511 when viewed from the thickness direction T1, and penetrates in the thickness direction T1. The reagent layer 1315 is arranged in the working electrode first opening 1511 of the first insulating layer 1051, and includes an outer peripheral edge defined by the inner peripheral edge of the working electrode first opening 1511. The working electrode protection film 1316 is arranged in the working electrode second opening 1521 of the second insulating layer 1052, and includes an outer periphery defined according to the inner periphery of the working electrode second opening 1521. The shapes of the working electrode first opening 1511 of the first insulating layer 1051 and the inner periphery of the working electrode second opening 1521 of the second insulating layer 1052 when viewed from the thickness direction T1 are each circular in the example shown in the figure, but are not limited thereto, and each may be a polygon (quadrilateral, triangle, etc.) or any other shape. The opening width (the width of the widest part) of the working electrode first opening 1511 of the first insulating layer 1051 is not particularly limited, and may be, for example, 0.5 mm or more and 5 mm or less, and preferably 1 mm or more and 2 mm or less. The opening width (the width of the widest part) of the working electrode second opening 1521 of the second insulating layer 1052 is not particularly limited, and can be, for example, 0.5 mm to 10 mm, and preferably 1 mm to 5 mm.

[0322] Next, refer to the drawings, especially to Fig.34 Reference electrode 1320 of sensor 1101 will be described. Fig.34 yes Fig.31 The portion of sensor 1101 that contains reference electrode 1320 is located along Fig.31 Cross-sectional view of the K-K' line.

[0323] The reference electrode 1320 includes a reference electrode conductive layer 1321 disposed on the first surface 1202 of the substrate 1200, and a silver / silver chloride layer 1322 disposed on the reference electrode conductive layer 1321. The reference electrode 1320 preferably further includes a reference electrode protective film 1326 disposed on the silver / silver chloride layer 1322 as shown in the figure. Preferred embodiments of the materials of the reference electrode conductive layer 1321 and the reference electrode protective film 1326 are described in the column of <Materials>. The wiring 1005 is connected to the reference electrode conductive layer 1321 (see Fig. 27 and Fig.31 ).

[0324] The reference electrode 1320 achieves the following effects by including the reference electrode protective film 1326. The reference electrode protective film 1326 suppresses the leakage of silver from the silver / silver chloride layer 1322. In addition, since the reference electrode protective film 1326 is porous, the liquid sample X1 can penetrate. Therefore, when the sensor 1101 is immersed in the liquid sample X1, the liquid sample X1 easily reaches the reference electrode 1320, and it is not easy to cause the liquid sample X1 to be impregnated into the reference electrode 1320.

[0325] The insulating layer 1050 includes a reference electrode opening 1502 formed at a position overlapping with at least a portion of the reference electrode 1320 when viewed from the thickness direction T1 of the substrate 1200. In the example shown in the figure, a portion of the reference electrode conductive layer 1321, the silver / silver chloride layer 1322, and the reference electrode protective film 1326 are arranged in the reference electrode opening 1502. The reference electrode protective film 1326 is arranged in the reference electrode opening 1502 to more effectively suppress the leakage of silver from the silver / silver chloride layer 1322. The outer peripheries of the silver / silver chloride layer 1322 and the reference electrode protective film 1326 are defined based on the inner periphery of the reference electrode opening 1502. The shape of the inner periphery of the reference electrode opening 1502 of the insulating layer 1050 when viewed from the thickness direction T1 is circular in the illustrated example, but is not limited thereto and may be a polygon (quadrilateral, triangle, etc.) or any other shape. The opening width (the width of the widest part) of the reference electrode opening 1502 of the insulating layer 1050 is not particularly limited, and may be, for example, 0.5 mm or more and 10 mm or less, preferably 1 mm or more and 5 mm or less.

[0326] In the example shown in the figure, the reference electrode opening 1502 of the insulating layer 1050 is composed of the reference electrode first opening 1512 of the first insulating layer 1051 and the reference electrode second opening 1522 of the second insulating layer 1052. The reference electrode first opening 1512 is formed at a position of the first insulating layer 1051 that overlaps with a portion of the reference electrode conductive layer 1321 when viewed from the thickness direction T1, and penetrates in the thickness direction T1. The reference electrode second opening 1522 is formed at a position of the second insulating layer 1052 that overlaps with a portion of the first insulating layer 1051 that includes the entire reference electrode first opening 1512 when viewed from the thickness direction T1, and penetrates in the thickness direction T1. A portion of the reference electrode conductive layer 1321 and the silver / silver chloride layer 1322 are disposed in the reference electrode first opening 1512 of the first insulating layer 1051, and include an outer periphery defined by the inner periphery of the reference electrode first opening 1512. The reference electrode protective film 1326 is disposed in the reference electrode second opening 1522 of the second insulating layer 1052, and includes an outer periphery defined by the inner periphery of the reference electrode second opening 1522. The shapes of the inner peripheries of the reference electrode first opening 1512 of the first insulating layer 1051 and the reference electrode second opening 1522 of the second insulating layer 1052 when viewed from the thickness direction T1 are each circular in the example shown in the figure, but are not limited thereto, and may each be a polygon (a quadrilateral, a triangle, etc.) or any other shape. The opening width (the width of the widest part) of the reference electrode first opening 1512 of the first insulating layer 1051 is not particularly limited, and can be set to, for example, 0.5 mm or more and 5 mm or less, preferably 1 mm or more and 2 mm or less. The opening width (the width of the widest part) of the reference electrode second opening 1522 of the second insulating layer 1052 is not particularly limited, and can be set to, for example, 0.5 mm or more and 10 mm or less, preferably 1 mm or more and 5 mm or less.

[0327] Next, refer to the drawings, especially to Fig.35 Next, the counter electrode 1330 of the sensor 1101 will be described. Fig.35 yes Fig.31 The portion of sensor 1101 that includes electrode 1330 is along Fig.31 Cross-sectional view of the line L-L'.

[0328] The counter electrode 1330 is disposed on the first surface 1202 of the substrate 1200. In the illustrated embodiment, the counter electrode 1330 is entirely formed of a conductive layer. In order to specify the portion of the conductive layer in the counter electrode, it is sometimes referred to as "counter electrode conductive layer" or "conductive layer of the counter electrode". Preferred forms of the material of the counter electrode 1330 are as described in the column of <Material> as the material of the conductive layer of the counter electrode. The wiring 1005 is connected to the counter electrode 1330 (see Fig. 27 and Fig.31 ).

[0329] In the sensor 1101, the insulating layer 1050 includes an electrode opening 1503, which is formed at a position overlapping with at least a portion of the counter electrode 1330 when viewed from the thickness direction T1 of the substrate 1200, and penetrates in the thickness direction T1, and the upper surface 1331 of the counter electrode 1330 is exposed through the electrode opening 1503. When the sensor 1101 having this structure is immersed in the liquid sample X1, the liquid sample X1 can contact the upper surface 1331 of the counter electrode 1330 through the electrode opening 1503 of the insulating layer 1050. The shape of the inner periphery of the electrode opening 1503 of the insulating layer 1050 when viewed from the thickness direction T1 is a quadrilateral in the example shown in the figure, but it is not limited to this, and can be a circle, a polygon other than a quadrilateral (such as a triangle), or any other shape. The opening width (the width of the widest part) of the electrode opening 1503 of the insulating layer 1050 is not particularly limited, and can be set to, for example, 0.5 mm or more and 10 mm or less, preferably 1 mm or more and 5 mm or less. In the example shown in the figure, a portion of the upper surface 1331 of the electrode 1330 is exposed through the electrode opening 1503, but the present invention is not limited thereto. For example, the electrode opening 1503 can also be a shape that encloses the entire electrode 1330, and the entire upper surface 1331 of the electrode 1330 can also be exposed through the electrode opening 1503.

[0330] In the example shown in the figure, the counter electrode opening 1503 of the insulating layer 1050 is composed of the counter electrode first opening 1513 of the first insulating layer 1051 and the counter electrode second opening 1523 of the second insulating layer 1052. The counter electrode first opening 1513 is formed at a position overlapping with a portion of the counter electrode 1330 when viewed from the thickness direction T1 of the first insulating layer 1051, and penetrates along the thickness direction T1. The counter electrode second opening 1523 is formed at a position consistent with the counter electrode first opening 1513 of the first insulating layer 1051 when viewed from the thickness direction T1 of the second insulating layer 1052, and penetrates along the thickness direction T1. The counter electrode first opening 1513 of the first insulating layer 1051 and the counter electrode second opening 1523 of the second insulating layer 1052 do not necessarily have to be consistent, as long as at least a portion overlaps. The shape and opening width of the inner periphery of the first opening 1513 for the electrode of the first insulating layer 1051 and the second opening 1523 for the electrode of the second insulating layer 1052 are not particularly limited, and for example, they may each be within the range described for the opening 1503 for the electrode of the insulating layer 1050 .

[0331] Next, refer to the drawings, especially to Fig.31 and Fig.36 , the front end opening 1061 and the flow path 1062 of the sensor 1101 are described. Fig.36 yes Fig.31 The sensor 1101 includes the electrode 1330, the front end opening 1061 and the flow path 1062. Fig.31 Cross-sectional view of the M-M' line.

[0332] The front end opening 1061 is formed in the insulating layer 1050, communicates with the upper surface 1331 of the counter electrode 1330, and opens on the front end portion 1201 side of the substrate 1200. The flow path 1062 is formed in the insulating layer 1050, and is connected to the front end opening 1061 and the upper surface 1331 of the counter electrode 1330. The flow path 1062 guides the liquid sample X1 from the front end opening 1061 to the upper surface 1331 of the counter electrode 1330. For example, the flow path 1062 can guide the liquid sample X1 from the front end opening 1061 to the upper surface 1331 of the counter electrode 1330 using a capillary phenomenon.

[0333] Reference Fig.39 and Fig.40 ,right Fig.31 and Fig.36 The functions of the front end opening 1061 and the flow path 1062 of the sensor 1101 of the illustrated embodiment will be described. Fig.39The sensor 1101 of this embodiment is immersed in the liquid sample X1 from the front end portion 1201 of the substrate 1200 and includes the front end opening 1061 and the flow path 1062. Fig.36 Schematic diagram of the cross section. Fig.40 1 is a schematic diagram of a cross section of a sensor 1150 of a comparative example which is immersed in the liquid sample X1 and has the same structure as the sensor 1101 of the present embodiment except that the sensor 1150 does not include the front end opening 1061 and the flow path 1062 .

[0334] like Fig.39 As shown, when the sensor 1101 of the present embodiment is immersed in the liquid sample X1 in a manner that the front end portion 1201 of the substrate 1200 becomes the lower end, the liquid sample X1 is guided from the front end opening 1061 through the flow path 1062 to the upper surface 1331 of the counter electrode 1330. As a result, the upper surface 1331 of the counter electrode 1330 of the sensor 1101 can fully contact the liquid sample X1. In particular, even if the depth of the liquid sample X1 is insufficient, the upper surface 1331 of the counter electrode 1330 of the sensor 1101 can fully contact the liquid sample X1, so it is preferred. Therefore, by using the sensor 1101 of the present embodiment, the measurement accuracy of the test substance in the liquid sample X1 is improved.

[0335] On the other hand, Fig.40 As shown, in the sensor 1150 of the comparative example in which the front end opening 1061 and the flow path 1062 are not formed in the insulating layer 1050, the region between the counter electrode 1330 and the front end portion 1201 of the first surface 1202 of the substrate 1200 is covered by the insulating layer 1050. When the sensor 1150 of the comparative example of such a structure is immersed in the liquid sample X1 in such a manner that the front end portion 1201 of the substrate 1200 becomes the front end, the liquid sample X1 is easily separated from the counter electrode 1330 by the liquid repellency caused by the insulating layer 1050 between the counter electrode 1330 and the front end portion 1201 of the substrate 1200. Therefore, in the sensor 1150 of the comparative example, the contact between the counter electrode 1330 and the liquid sample X1 is easily hindered, and the test substance in the liquid sample X1 may not be measured normally. When the counter electrode 1330 is a carbon conductive layer, the contact between the liquid sample X1 containing water as a solvent and the upper surface 1331 of the counter electrode 1330 is particularly likely to be hindered due to the high water repellency of the upper surface 1331 of the counter electrode 1330. This problem of the sensor 1150 of the comparative example can be solved by using the sensor 1101 of the present embodiment in which the front end opening 1061 and the flow path 1062 are formed in the insulating layer 1050.

[0336] Fig.31 and Fig.36The sensor 1101 of the present embodiment shown in the figure includes a front end opening 1061 and a flow path 1062 formed in a portion between the counter electrode 1330 and the front end portion 1201 of the substrate 1200 in the insulating layer 1050 when viewed from above in the thickness direction T1 of the substrate 1200. In the sensor 1101 of the present embodiment, the flow path 1062 is connected to the counter electrode opening 1503 of the insulating layer 1050, and since the upper surface 1331 of the counter electrode 1330 is exposed through the counter electrode opening 1503, when immersed in the liquid sample X1, the contact effect between the upper surface 1331 of the counter electrode 1330 and the liquid sample X1 is particularly high.

[0337] Fig.31 and Fig.36 The front end opening 1061 and the flow path 1062 of the sensor 1101 shown in the figure are recessed portions formed in the insulating layer 1050, which are closed on the side where the substrate 1200 exists and open on the upper surface 1053 side of the insulating layer 1050, but the present invention is not limited to this embodiment. Fig.37 As in the sensor 1101′ according to another embodiment shown in FIG. 1 , the front end opening 1061 and the flow path 1062 are formed in a portion of the thickness direction of the upper surface 1053 excluding the insulating layer 1050, and may also have: a direction from the counter electrode 1330 to the front end portion 1201 of the substrate 1200 ( Fig.31 When viewed in the M-M' direction), the front end opening 1061 and the flow path 1062 are surrounded.

[0338] Fig.31 and Fig.36 The flow path 1062 of the sensor 1101 shown in FIG. 1 has a flow path width W ( Fig.31 The expansion portion 1062A expands toward the front end opening 1061. The flow path 1062 has the expansion portion 1062A, and when the sensor 1101 is immersed in the liquid sample X1, the movement of the liquid sample X1 from the front end opening 1061 to the upper surface 1331 of the counter electrode 1330 is promoted. The flow path width W here refers to: when viewed from the thickness direction T1 of the substrate 1200 of the sensor 1101, the width W of the flow path is the width from the counter electrode 1330 to the substrate 1200 ( Fig.31 The direction orthogonal to the M-M' direction ( Fig.31 The width of the flow path 1062 in the L-L' direction).

[0339] Fig.31 and Fig.36In the sensor 1101 shown, the insulating layer 1050 includes: a first insulating layer 1051 disposed on the first surface 1202 of the substrate 1200, and a second insulating layer 1052 disposed on the first insulating layer 1051, and a front end opening 1061 and a flow path 1062 are formed in the second insulating layer 1052. In a particularly preferred mode of the sensor 1101 of this embodiment, the first insulating layer 1051 includes an insulating curable resin, and the second insulating layer 1052 includes an insulating sheet. The first insulating layer 1051 including the insulating curable resin can be formed by applying a curable resin composition to the first surface 1202 of the substrate 1200 and irradiating the coating with active energy rays to cure it. The second insulating layer 1052 including the insulating sheet can be formed by attaching the insulating sheet to the first insulating layer 1051 including the insulating curable resin via a bonding layer. After forming the second insulating layer 1052 including the insulating sheet, by peeling off the portion between the counter electrode 1330 and the front end portion 1201 of the substrate 1200, the front end opening 1061 and the flow path 1062 can be formed in the second insulating layer 1052. Alternatively, by attaching the insulating sheet having a pre-cut portion to the first insulating layer 1051 including the insulating cured resin via a bonding layer at the portion between the counter electrode 1330 and the front end portion 1201 of the substrate 1200, the second insulating layer 1052 including the insulating sheet with the front end opening 1061 and the flow path 1062 formed therein can be formed.

[0340] for Fig.32 and Fig.38 As for the sensor 1101'' of another embodiment shown in FIG. Fig.31 and Fig.36 In the illustrated sensor 1101 , a front end opening 1061 and a flow path 1062 are further formed in the first insulating layer 1051 . Fig.32 and Fig.38 The sensor 1101'' is shown in Fig.31 and Fig.36 In the sensor 1101 shown in FIG. 1 , a portion 1515 (see FIG. 1 ) between the counter electrode 1330 and the front end portion 1201 of the substrate 1200 is further formed in the first insulating layer 1051. Fig.36 ) is formed with a front end opening 1061 and a flow path 1062. Fig.32 and Fig.38 The sensor 1101'' shown does not include an insulating layer between the counter electrode 1330 and the front end portion 1201 on the first surface 1202 of the substrate 1200. Therefore, when the sensor 1101'' is immersed in the liquid sample X1, the contact between the liquid sample X1 and the counter electrode 1330 is further promoted, and the test substance in the liquid sample X1 can be detected with higher accuracy. Fig.32 and Fig.38In a particularly preferred embodiment of the sensor 1101'' shown in FIG. 1 , the first insulating layer 1051 is a layer containing an insulating cured resin, and the second insulating layer 1052 is a layer containing an insulating sheet. After manufacturing the sensor 1101 having the insulating layer 1050, the portion 1515 of the first insulating layer 1051 containing an insulating cured resin can be peeled off (see FIG. 1 ). Fig.36 ), thereby manufacturing the sensor 1101'' involved in this particularly preferred embodiment, wherein the insulating layer 1050 includes: a first insulating layer 1051 including an insulating cured resin and a second insulating layer 1052 including an insulating sheet. Alternatively, when the first insulating layer 1051 including an insulating cured resin is formed by curing a curable resin composition on the first surface 1202 of the substrate 1200, the first insulating layer 1051 lacking the portion 1515 is formed, and the second insulating layer 1052 is formed thereon, thereby manufacturing the sensor 1101''.

[0341] <Method of Manufacturing Sensor 1101>

[0342] With reference to the accompanying drawings, in particular Figure 27 to Figure 31 , for the second public Fig.31 An example of a method for manufacturing the sensor 1101 according to the illustrated embodiment will be described.

[0343] First, Fig. 27 As shown, on the first surface 1202 of the insulating substrate 1200, there are arranged: working electrode conductive layers 1311, 1311, a reference electrode conductive layer 1321 and a counter electrode 1330 (counter electrode conductive layer), and wirings 1005 electrically connected thereto.

[0344] Then, if Fig.28 As shown, a first insulating layer 1051 is stacked on the first surface 1202 of the substrate 1200 on which the working electrode conductive layers 1311, 1311, the reference electrode conductive layer 1321, the counter electrode 1330, and the wiring 1005 are arranged. The first insulating layer 1051 has working electrode first openings 1511, 1511 at positions overlapping with a portion of the working electrode conductive layers 1311, 1311, has a reference electrode first opening 1512 at a position overlapping with a portion of the reference electrode conductive layer 1321, and has a counter electrode first opening 1513 at a position overlapping with a portion of the counter electrode 1330.

[0345] As described above, in a particularly preferred embodiment of the sensor 1101 of this embodiment, the first insulating layer 1051 contains an insulating cured resin. The first insulating layer 1051 containing the insulating cured resin can be formed by applying a curable resin composition to the first surface 1202 of the substrate 1200 and curing the coating by irradiating the coating with active energy rays.

[0346] Then, if Fig.29 As shown, in Fig.28 A second insulating layer 1052 is further stacked on the first insulating layer 1051 of the substrate 1200. The second insulating layer 1052 has working electrode second openings 1521, 1521 of a shape that encloses the entirety of the working electrode first openings 1511, 1511 at a position overlapping with the working electrode first openings 1511, 1511 of the first insulating layer 1051, a reference electrode second opening 1522 of a shape that encloses the entirety of the reference electrode first opening 1512 at a position overlapping with the reference electrode first opening 1512, and a counter electrode second opening 1523 of a shape that matches the counter electrode first opening 1513 at a position overlapping with the counter electrode first opening 1513. The second insulating layer 1052 further includes a front end opening 1061 and a flow path 1062 in a portion between the counter electrode 1330 and the front end portion 1201 of the substrate 1200 when viewed in a plan view from the thickness direction T1 of the substrate 1200 .

[0347] As described above, in a particularly preferred embodiment of the sensor 1101 of this embodiment, the second insulating layer 1052 includes an insulating sheet. The second insulating layer 1052 including the insulating sheet may further include a bonding layer including an adhesive or a bonding agent disposed on one surface of the insulating sheet. The second insulating layer 1052 including the insulating sheet may be formed by attaching the insulating sheet to the first insulating layer 1051 via the bonding layer.

[0348] Then, if Fig.30 As shown, the reagent layer 1315 is arranged in the first openings 1511 and 1511 of the working electrode of the first insulating layer 1051 , and the silver / silver chloride layer 1322 is arranged in the first opening 1512 of the reference electrode of the first insulating layer 1051 .

[0349] Then, if Fig.31 As shown, working electrode protection films 1316, 1316 are arranged in the working electrode second openings 1521, 1521 of the second insulating layer 1052 to form working electrodes 1310, 1310, and reference electrode protection film 1326 is arranged in the reference electrode second opening 1522 of the second insulating layer 1052 to form reference electrode 1320. It should be noted that Fig.30 and Fig.31The order of forming the layers is not particularly limited, and the layers may be formed in another order.

[0350] <Further Embodiment of Sensor 1101>

[0351] Reference Figures 41 to 47 , a further preferred embodiment of the sensor 1101 disclosed in the second embodiment is described.

[0352] Fig.41 In the sensor 1101 according to the embodiment shown, the substrate 1200 includes:

[0353] A main body 1210 including a front end portion 1201 and a first end portion 1211 on the opposite side of the front end portion 1201 and provided with a detection electrode 1300;

[0354] A connecting portion 1220 including a second end portion 1222 connected to the first end portion 1211 of the main body portion 1210 and a third end portion 1223 on the opposite side of the second end portion 1222 and extending from the second end portion 1222 to the third end portion;

[0355] The base end portion 1230 includes a fourth end portion 1234 connected to the third end portion 1223 of the connecting portion 1220, and

[0356] The connection portion 1220 of the substrate 1200 includes, near the third end portion 1223 , a bent portion 1225 in which the substrate 1200 is bent so that the first surface 1202 side of the substrate 1200 protrudes.

[0357] It should be noted that Fig.41 , Fig.42 , Fig.46 as well as Fig.47 , a top view of the sensor 1101 is shown before the substrate 1200 is bent at the bending portion 1225 .

[0358] like Fig.41 As shown, the wiring 1005 is preferably arranged on the first surface 1202 of the substrate 1200 from the main body 1210 through the connecting portion 1220 to the base end 1230. Fig.41 As shown, the terminal 1006 is preferably arranged on the first surface 1202 of the base end portion 1230, and the wiring 1005 is electrically connected to one of the detection electrodes 1300 on the first surface 1202 of the main body 1210 and one of the terminals 1006 on the first surface 1202 of the base end portion 1230, respectively.

[0359] Fig.41The substrate 1200 of the sensor 1101 shown is bent at the bent portion 1225 in such a manner that the first surface 1202 on which the detection electrode 1300 and the like are arranged protrudes. The substrate 1200 bent at the bent portion 1225 has a shape in which the main body 1210 and the connecting portion 1220, which are closer to the second end 1222 than the bent portion 1225, protrude relative to the base end 1230. Fig.43 and Fig.44 In the illustrated embodiment, the main body 1210 and the connecting portion 1220 of the substrate 1200 , portions closer to the second end 1222 than the bent portion 2125 , protrude substantially perpendicularly to the base end 1230 of the substrate.

[0360] The substrate 1200 is bent at the bending portion 1225 of the connecting portion 1220. Fig.41 In the sensor 1101 shown, Fig.43 and Fig.44 As shown, even if the base end portion 1230 of the substrate 1200 is not tilted (for example, in Fig.43 and Fig.44 The base end portion 1230 of the substrate 1200 is held in a horizontal direction as shown in the figure), and the main body 1210 of the substrate 1200 provided with the detection electrode 1300 can be oriented in a manner such that the front end portion 1201 is the lower end and immersed in the liquid sample X1 for the measurement of the test substance.

[0361] Fig.41 The sensor 1101 shown is as Fig.42 As shown, it can be used in the form of a plurality of connected and integrated multi-sensors 1105. Fig.42 The multiple sensor 1105 shown is formed by integrating the base end portions 1230 of the substrate 1200 of four sensors 1101 .

[0362] for Fig.41 For the sensor 1101 shown, the insulating layer 1050 includes: a main body portion 1210 disposed on the first surface 1202 of the substrate 1200, and a second insulating layer 1052 including a first insulating sheet in an area of ​​a portion of the connecting portion 1220 that is closer to the second end portion 1222 than the bending portion 1225. Here, the portion of the connecting portion 1220 that is closer to the second end portion 1222 than the bending portion 1225 refers to: a portion of the connecting portion 1220 that extends from one end of the second end portion 1222 of the bending portion 1225 to the second end portion 1222. Fig.41In the illustrated sensor 1101, the insulating layer 1050 further includes a first insulating layer 1051 containing an insulating cured resin formed on the entire first surface 1202 of the substrate 1200. The second insulating layer 1052 including the first insulating sheet includes a bonding layer (not shown) containing a binder or adhesive in addition to the first insulating sheet, and the second insulating layer 1052 is attached to the above-mentioned region of the first surface 1202 of the substrate 1200 from the bonding layer side via the first insulating layer 1051. Fig.41 In the sensor 1101 shown, since the portion immersed in the liquid sample X1 is entirely covered by the second insulating layer 1052 including the first insulating sheet, the necessary insulation between the detection electrode 1300 and the wiring 1005 is achieved, and it is easy to form the front end opening 1061 and the flow path 1062 in the first insulating sheet of the second insulating layer 1052, it is preferred.

[0363] Fig.41 The insulating layer 1050 of the sensor 1101 shown includes a non-insulating region 1055 that does not contain an insulating sheet in the region of the bent portion 1225 of the connecting portion 1220 on the first surface 1202 of the substrate 1200. Fig.44 and Fig.45 , the effect of this structure is explained.

[0364] Fig.44 The main body 1210 including the tip 1201 of the substrate 1200 is immersed in the liquid sample X1. Fig.41 A schematic side view of the sensor 1101 is shown. Fig.44 As shown, in the sensor 1101, the substrate 1200 is bent at the bent portion 1225 of the connecting portion 1220 to the following state: the first surface 1202 side protrudes, and the main body 1210 and the portion of the connecting portion 1220 closer to the second end 1222 side than the bent portion 1225 are substantially perpendicular to the base end 1230. Fig.41 and Fig.44In the sensor 1101 shown, the insulating layer 1050 includes: a first insulating layer 1051 containing an insulating curing resin and covering the entire first surface 1202 of the substrate 1200; and a second insulating layer 1052 containing a first insulating sheet and covering the main body 1210 and the region of the portion of the connecting portion 1220 closer to the second end 1222 than the bent portion 1225 in the first surface 1202 of the substrate 1200 via the first insulating layer 1051. The insulating layer 1050 includes a non-insulating region 1055 that does not contain an insulating sheet in the region of the bent portion 1225 of the connecting portion 1220 in the first surface 1202 of the substrate 1200 and contains the first insulating layer 1051 containing an insulating curing resin. In the present embodiment, the second insulating layer 1052 containing the first insulating sheet is flat and does not deform. Therefore, the substrate 1200 is not affected by the contraction or extension caused by the deformation of the first insulating sheet, and the main body 1210 and the connecting portion 1220 closer to the second end 1222 than the bent portion 1225 maintain a flat shape. Fig.44 As shown, the main body 1210 of the substrate 1200 of the sensor 1101 can be held in the liquid sample X1 in a vertical posture with the front end 1201 at the bottom, and the detection electrode 1300 can normally detect the test substance.

[0365] on the other hand, Fig.45 The second insulating layer 1052 including the first insulating sheet is formed on the entire region including the bent portion 1225 of the first surface 1202 of the substrate 1200, and has the same Fig.41 and Fig.44 A sensor 1151 of the comparative example has the same structure as the sensor 1101 shown in FIG. In the sensor 1151 of the comparative example, the second insulating layer 1052 including the insulating sheet on the first surface 1202 of the substrate 1200 is extended by bending at the bending portion 1225 of the substrate 1200. In order to shrink the extended second insulating layer 1052 including the insulating sheet, the main body 1210 and the connecting portion 1220 of the substrate 1200 are subjected to the following Fig.45 The main body 1210 of the warped substrate 1200 is tilted upward toward the first surface 1202 on which the detection electrode 1300 is disposed. Fig.45 As shown, the contact between the liquid sample X1 and the detection electrode 1300 on the first surface 1202 of the main body 1210 is hindered, and the measurement accuracy of the test substance by the detection electrode 1300 is reduced. Fig.41 and Fig.44In the illustrated sensor 1101 , the insulating layer 1050 includes the non-insulating region 1055 in the region on the bent portion 1225 of the first surface 1202 of the substrate 1200 , thereby solving this problem of the sensor 1151 of the comparative example.

[0366] based on Fig.46 ,right Fig.41 and 44 Still another embodiment of the sensor 1101 is illustrated. Fig.46 In the sensor 1101 shown, the insulating layer 1050 includes: a main body portion 1210 arranged on the first surface 1202 of the substrate 1200, and a second insulating layer 1052a including a first insulating sheet in an area of ​​a portion of the connecting portion 1220 closer to the second end portion 1222 side than the bent portion 1225; and a second insulating layer 1052b including a second insulating sheet in an area of ​​the base end portion 1230 arranged on the first surface 1202 of the substrate 1200. Here, the first insulating sheet of the second insulating layer 1052a and the second insulating sheet of the second insulating layer 1052b are separated in an area of ​​the bent portion 1225 of the connecting portion 1220 on the first surface 1202 of the substrate 1200. Fig.46 In the sensor 1101 shown, the insulating layer 1050 further includes a first insulating layer 1051 including an insulating cured resin formed on the entire first surface 1202 of the substrate 1200. The insulating layer 1050 includes a non-insulating region 1055 including the first insulating layer 1051 without an insulating sheet in the region of the bent portion 1225 of the connection portion 1220 on the first surface 1202 of the substrate 1200. The second insulating layer 1052a including the first insulating sheet and the second insulating layer 1052b including the second insulating sheet are respectively arranged in the above-mentioned region from the first insulating layer 1051 to the first surface 1202 of the substrate 1200. According to this embodiment, the reduction in detection accuracy caused by the warping of the substrate 1200 of the sensor 1101 can be suppressed, and the insulation between the wiring 1005 and the terminal 1006 on the base end portion 1230 of the substrate 1200 can be improved.

[0367] based on Fig.47 ,right Fig.46 Another embodiment of the sensor 1101 is described below. Fig.47In the sensor 1101 shown, the first insulating sheet of the second insulating layer 1052a and the second insulating sheet of the second insulating layer 1052b are separated in the region of the bent portion 1225 of the connection portion 1220 on the first surface 1202 of the substrate 1200 via a cutout 1056 formed in a direction intersecting the direction in which the connection portion 1220 extends. In the present embodiment, the insulating layer 1050 includes a second insulating layer 1052c including a plurality of third insulating sheets separated from the second insulating layers 1052a and 1052b via the cutout 1056 in the region of the bent portion 1225 of the connection portion 1220 on the first surface 1202 of the substrate 1200. According to this embodiment, the reduction in detection accuracy due to the warping of the substrate 1200 of the sensor 1101 can be suppressed, and the insulation between the wirings 1005 on the bent portion 1225 of the connection portion 1220 of the substrate 1200 can be improved.

[0368] <Sensor unit 900>

[0369] Reference Fig.51 , Fig.52 as well as Fig.53 The sensor assembly 900 will be described.

[0370] The illustrated sensor assembly 900 may be used to position the main body 1210 of the substrate 1200 including the detection electrodes 1300 of the sensor 1101 in each well of a 24-well plate 925 .

[0371] Sensor assembly 900 Fig.51 As shown, it has: a lower support plate 957, 6 sets of multi-connected sensors 1105 (see Fig.42 ), an upper support plate 959 equipped with a port 961 for providing additives, and a gasket 960.

[0372] In this embodiment, the multiple sensors 1105 are as follows: Fig.42 As shown in FIG. 1 , four sensors 1101 are connected at the base end 1230 of the substrate 1200 to form an integrated structure. The first surface 1202 side of each of the four sensors 1101 on the substrate 1200 protrudes at the bent portion 1225 of the connecting portion 1220 of the substrate 1200, and the main body 1210 and the portion of the connecting portion 1220 closer to the second end 1222 than the bent portion 1225 are bent to be perpendicular to the base end 1230. Fig.51 As shown, in the multiple sensor 1105 having four sensors 1101, six sets of lower support plates 957 are installed.

[0373] The lower support plate 957 has through holes 957b at positions corresponding to the main body 1210 and the connecting portion 1220 of the substrate 1200 of each of the 24 sensors 1101 included in the 6 sets of multiple sensors 1105. The main body 1210 and the connecting portion 1220 of the substrate 1200 of each of the 24 sensors 1101 are inserted into each through hole 957b of the lower support plate 957. When the sensor 1101 is inserted into the through hole 957b of the lower support plate 957, as shown in FIG. Fig.52 As shown, the upper surface of the lower support plate 957 is engaged with the lower surface of the base end portion 1230 of the substrate 1200 of the sensor 1101. In addition, a first engaging portion 957d is provided in a portion of the inner peripheral wall of the through hole 957b of the lower support plate 957 that engages with the inner side of the bent portion 1225 of the connecting portion 1220 of the substrate 1200 of the sensor 1101, which supports the bent portion 1225 of the connecting portion 1220 of the substrate 1200 from below.

[0374] On the other hand, on the lower side surface of the upper support plate 959 , pin-shaped second engaging portions 959 a are provided to be inserted into the 24 through holes 957 b of the lower support plate 957 .

[0375] When the six multi-connected sensors 1105 are sandwiched between the lower support plate 957 and the upper support plate 959, as shown in FIG. Fig.52 As shown, the bent portion of the connection portion 1220 of the substrate 1200 of the sensor 1101 is supported by being engaged with the upper surface of the first engaging portion 957d of the lower support plate 957 and the lower surface of the second engaging portion 959a of the upper support plate 959. Fig.52 As shown, the first engaging portion 957d of the lower support plate 957 has an upper surface curved portion shape including a curved surface on the upper surface. In addition, the second engaging portion 959a of the upper support plate 959 has a lower surface curved portion shape including a curved surface on the lower surface. With this structure, in the sensor 1101, the front end portion 1201 of the substrate 1200 becomes the lower end, and the main body 1210 of the substrate 1200 and the portion of the connecting portion 1220 closer to the front end side than the bent portion 1225 are supported to be in a vertical state. As a result, the posture of the main body of the substrate 1200 of the sensor 1101 relative to the liquid sample accommodated in each hole of the orifice plate can be stabilized, and the detection accuracy based on the sensor 1101 can be improved.

[0376] Finally, if Fig.51 As shown, a gasket 960 having a through hole is overlapped and arranged on the upper surface of the upper support plate 959, thereby completing the sensor assembly 900.

[0377] The sensor assembly 900 can be combined with other components to form an adapter assembly 920. Fig.53 As shown, the following are placed in order from the bottom: an adapter bottom (culture container installation portion) 924 , a well plate (culture container) 925 , an adapter top 926 , and a sensor assembly 900 .

[0378] In this embodiment, the well plate 925 has 24 wells of 4×6.

[0379] The adapter top 926 is provided to adjust the height of the orifice plate 925, and different adapter tops 926 can be used according to the height of the orifice plate 925. This is to adjust the height relationship between the sensor assembly 900 and the orifice plate 925 when the sensor assembly 900 is placed on the adapter top 926.

[0380] The orifice plate 925 has several types including a general-purpose product, and the adapter top 926 can be used according to the type.

[0381] In the sensor assembly 900 arranged on the adapter top 926, four legs (support bodies) 940 provided on the lower surface side thereof are inserted into positioning holes 942 provided in the adapter bottom 924 as the culture container setting part through through holes 941 of the adapter top 926 below.

[0382] In the adapter assembly 920 including the sensor assembly 900 and the orifice plate 925 formed in this manner, the main body 1210 of the substrate 1200 including the detection electrode 1300 of the sensor 1101 is held in a stable posture within each hole of the orifice plate 925 .

[0383] Although not shown in the figure, in the adapter assembly 920, the terminal 1006 on the base end portion 1230 of the substrate 1200 of the sensor 1101 included in the sensor assembly 900 can be connected to the electrochemical measurement unit 1111 (see FIG. 11) of the external analysis unit 1102 through the through hole of the gasket 960 of the sensor assembly 900. Fig.48 That is, the adapter assembly 920 having the sensor assembly 900 can be combined with the external analysis unit 1102 and the control unit 1104 to form Fig.48 Analytical device 1100 is shown.

[0384] <Second disclosed embodiment>

[0385] As the second disclosed embodiment or comparative example, the following five sensors were prepared. It should be noted that in the sensors S1 to S5 used in the following experiments, the reagent layers 1315, 1315 of the working electrodes 1310, 1310 and the working electrode protective films 1316, 1316 and the reference electrode protective film 1326 of the reference electrode 1320 were not provided.

[0386] Sensor S1

[0387] Sensor S1 is Fig.49A A comparative example of the structure shown in FIG. 1 . The sensor S1 has a first insulating layer 1051 and a second insulating layer 1052 including an insulating sheet stacked on the entire first surface 1202 of the substrate 1200. In the sensor S1, the front end opening 1061 and the flow path 1062 are not formed, and the portion between the counter electrode 1330 and the front end 1201 of the first surface 1202 of the substrate 1200 is also covered by the insulating layer 1050 including the first insulating layer 1051 and the second insulating layer 1052. That is, the sensor S1 does not have the non-insulating region 1055 of the insulating layer 1050 for preventing the substrate 1200 from warping, and the front end opening 1061 and the flow path 1062 for guiding the liquid sample to the upper surface 1331 of the counter electrode 1330.

[0388] Sensor S2

[0389] Sensor S2 is Fig.49B A comparative example of the structure shown in FIG. 1 is provided in the sensor S2. In the sensor S2, instead of the second insulating layer 1052 covering the entire sensor S1, a second insulating layer 1052a including a first insulating sheet and a second insulating layer 1052b including a second insulating sheet are provided, and a non-insulating region 1055 including the first insulating layer 1051 but not including an insulating sheet is provided on the bent portion 1225 of the connection portion 1220 of the substrate 1200. The second insulating layer 1052a is provided at a portion of the connection portion 1220 of the substrate 1200 closer to the front end side than the bent portion 1225, and the second insulating layer 1052b is provided at the base end 1230 of the substrate 1200. That is, in the sensor S2, the non-insulating region 1055 of the insulating layer 1050 for preventing the substrate 1200 from warping is provided, but the front end opening 1061 and the flow path 1062 for guiding the liquid sample to the upper surface 1331 of the counter electrode 1330 are not provided.

[0390] Sensor S3

[0391] Sensor S3 is Fig.49C The structure of the sensor 1101 is shown in FIG. 1 . In the sensor S3, Fig.46The sensor 1101 shown in the figure similarly has a non-insulating region 1055 of the insulating layer 1050 for preventing warping of the substrate 1200, and a front end opening 1061 and a flow path 1062 formed only in the second insulating layer 1052 (see Fig.36 ).

[0392] Sensor S4

[0393] Sensor S4 is Fig.49D The sensor S4 is an embodiment of the sensor 1101 having the structure shown in FIG. 1 . The second insulating layer 1052a including the first insulating sheet and the second insulating layer 1052b including the second insulating sheet of the insulating layer 1050 in the sensor S3 are replaced with the second insulating layer 1052 including the insulating sheet as a whole and as a whole covering the first surface 1202 of the substrate 1200, and the portion 1515 (see FIG. 1 ) between the counter electrode 1330 of the first insulating layer 1051 and the front end portion 1201 of the substrate 1200 is replaced with the second insulating layer 1052a including the first insulating sheet and the second insulating layer 1052b including the second insulating sheet. Fig.36 , Fig.49C ) is peeled off, thereby forming the front end opening 1061 and the flow path 1062. That is, in the sensor S4, the non-insulating region 1055 of the insulating layer 1050 for preventing the warping of the substrate 1200 is not provided, but the front end opening 1061 and the flow path 1062 formed in the first insulating layer 1051 and the second insulating layer 1052 are provided (see Fig.38 ).

[0394] Sensor S5

[0395] Sensor S5 is Fig.49E The sensor S5 is an embodiment of the sensor 1101 having the structure shown in FIG. 1. The sensor S5 is a sensor S3 in which a portion 1515 (see FIG. 1515 ) between the counter electrode 1330 and the front end portion 1201 of the substrate 1200 is formed. Fig.36 , Fig.49C ) is peeled off to form the front end opening 1061 and the flow path 1062. That is, the sensor S5 has the non-insulating region 1055 of the insulating layer 1050 for preventing the warping of the substrate 1200, and the front end opening 1061 and the flow path 1062 formed in the first insulating layer 1051 and the second insulating layer 1052 (see Fig.38 ).

[0396] Materials used in manufacturing the above-mentioned five sensors are shown.

[0397] (Substrate)

[0398] As an insulating substrate, a Figures 49A to 49E The substrate 1200 has a shape shown in FIG. 1 , includes a main body portion 1210 , a connecting portion 1220 , and a base end portion 1230 , and is made of polyethylene terephthalate and has a thickness of 188 μm.

[0399] The main body 1210 of the substrate 1200 is formed into a rectangular shape with a horizontal width of 11 mm and a vertical width of 4.2 mm.

[0400] After the insulating layer 1050 is stacked to complete the sensor, the connecting portion 1220 of the substrate 1200 is bent at the bending portion 1225 so that it protrudes toward the first surface 1202 side, and the main body 1210 and the connecting portion 1220 that are closer to the front end side than the bending portion 1225 are perpendicular to the base end portion 1230.

[0401] (Conductive layer)

[0402] On the first surface 1202 of the substrate 1200, a carbon conductive layer with a thickness of 5 μm is formed by applying a carbon paste and heating. Fig. 27 and Fig.46 The working electrode conductive layers 1311, 1311, the reference electrode conductive layer 1321, the counter electrode (counter electrode conductive layer) 1330, the wiring 1005, and the terminal 1006 are of the shapes shown in the figure.

[0403] (1st insulation layer)

[0404] On the first surface 1202 of the substrate 1200 provided with a carbon conductive layer, Fig.28 As shown in FIG. 1 , a first insulating layer 1051 having a thickness of 5 μm is stacked, which covers the first surface 1202 of the substrate 1200 and the carbon conductive layer, and the portion above the carbon conductive layer. The first insulating layer 1051 is formed by applying a curable resin composition (negative resist composition) that is a curable resin that is cured by active energy ray irradiation to produce insulating properties, and irradiating the coating film with active energy rays. Appropriate masking is performed to form working electrode first openings 1511, 1511, reference electrode first opening 1512, and counter electrode first opening 1513 in the first insulating layer 1051.

[0405] The silver / silver chloride layer 1322 is disposed in the reference electrode first opening 1512 of the first insulating layer 1051 .

[0406] (Second insulation layer)

[0407] The second insulating layer 1052 is arranged in a predetermined region on the first surface 1202 of the substrate 1200 on which the carbon conductive layer and the first insulating layer 1051 are arranged. The second insulating layer 1052 is attached to the predetermined region on the first insulating layer 1051 arranged on the first surface 1202 of the substrate 1200 via a bonding layer, and the second insulating layer 1052 is a laminate of a polyethylene terephthalate sheet (insulating sheet) having a thickness of 50 μm and a bonding layer containing an adhesive having a thickness of 10 μm and a surface water-repellent treatment.

[0408] The working electrode second openings 1521, 1521 and the reference electrode second opening 1522 are each formed into a circle with a diameter of 2.0 mm. The counter electrode second opening 1523 is formed into a rectangle with a horizontal width of 2.1 mm and a vertical width of 1.8 mm in the main body 1210.

[0409] In the following experiments, sensors S1 to S5 were used which did not have reagent layers 1315 , 1315 of working electrodes 1310 , 1310 and working electrode protection films 1316 , 1316 , and reference electrode protection film 1326 of reference electrode 1320 .

[0410] A 24-well plate having cylindrical wells with a depth of 17.40 mm, a bottom diameter of 16.26 mm, and a hole diameter of 15.62 mm was prepared. 0.85 mL, 0.94 mL, 1.0 mL, or 1.2 mL of RPMI medium supplemented with 500 μM ascorbic acid (hereinafter referred to as "liquid sample X1") was added to each well of the 24-well plate.

[0411] For sensors S1 to S5, Figures 43-45 As shown, the base end portion 1230 of the substrate 1200 is held horizontally with the first surface 1202 facing upward, and the main body 1210 of the substrate 1200 is immersed in each amount of the liquid sample X1 in the well with the front end 1201 at the bottom.

[0412] The voltage difference between the working electrodes 1310, 1310 (excluding the reagent layer and the protective film) of each of the sensors S1 to S5 and the reference electrode 1320 (excluding the protective film) was controlled to 500 mV by a constant potential meter, and the current value between each of the two working electrodes 1310, 1310 and the counter electrode 1330 was measured over time until 0.7 hours later. In addition, in order to confirm whether the liquid sample X1 is in contact with the counter electrode 1330, the voltage of the counter electrode 1330 is measured over time.

[0413] In order to promote the contact between each detection electrode 1300 and the liquid sample X1, the measurement is performed while the 24-well plate is vibrated after a certain period of time has passed since the start of the measurement.

[0414] FIG50 shows the measurement results of the current values ​​of the two working electrodes 1310, 1310 when four volumes of liquid samples are used for each of the sensors S1 to S5. Fig.50A The results for sensor S1 are shown, Fig.50B The results for sensor S2 are shown, Fig.50C The results for sensor S3 are shown, Fig.50D The results for sensor S4 are shown, Fig.50E The results for sensor S5 are shown.

[0415] When no vibration is applied, the sensor S1 of the comparative example (refer to Fig.49A ) In all volumes of liquid sample X1, the current value of the working electrode bottomed out and no normal current value was measured. When vibration was applied, normal current values ​​were measured on the working electrode in 1.2mL, 1.0mL, and 0.94mL of liquid sample X1, but in 0.85mL of liquid sample X1, the current value of the working electrode bottomed out and normal measurement could not be performed.

[0416] When no vibration is applied, the sensor S2 of the comparative example (refer to Fig.49B ) In the 1.2mL, 1.0mL, and 0.85mL liquid samples X1, the current value of the working electrode bottomed out and no normal current value was measured, but in the 0.94mL liquid sample X1, a normal current value was measured on the working electrode. When vibration was applied, normal current values ​​were measured on the working electrode in all volumes of the liquid sample X1.

[0417] Sensor S3 of the embodiment (see Fig.49C ) Regardless of whether vibration is applied or not, normal current values ​​are measured at the working electrode in all volumes of liquid sample X1.

[0418] Sensor S4 of the embodiment (see Fig.49D ) When no vibration was applied, normal current values ​​were measured on the working electrode in 1.2mL, 1.0mL, and 0.94mL of the liquid sample X1, and the current value of the working electrode in the 0.85mL liquid sample bottomed out and could not be measured normally. When vibration was applied, normal current values ​​were measured on the working electrode regardless of the volume of the liquid sample X1.

[0419] Sensor S5 of the embodiment (see Fig.49E ) Regardless of whether vibration is applied or not, normal current values ​​are measured at the working electrode in all volumes of liquid sample X1.

[0420] The above results are summarized in the following table.

[0421] [Table 1]

[0422]

[0423] F: Unable to measure the working electrode current value

[0424] A: Successfully measured the working electrode current value

[0425] () indicates the results without vibration

[0426] The values ​​in ( ) are the results when vibration is applied.

[0427] It was confirmed that no matter which sensor was used, when the current value of the working electrode hit the bottom and could not be measured normally, the voltage value of the counter electrode also hit the bottom, and when the current value of the working electrode was normal, the voltage value of the counter electrode was also normal (data not shown in the figure). This means that in order to perform normal measurements through the sensor, it is necessary to make the counter electrode fully contact with the liquid sample. In addition, based on the above-mentioned current value measurement results, it was confirmed that by forming a front end opening and a flow path in the insulating layer, the liquid sample can be guided to the upper surface of the counter electrode, and the contact obstruction between the counter electrode and the liquid sample can be suppressed, and by suppressing the warping of the substrate, the contact obstruction can be further suppressed.

[0428] <Supplementary Notes Regarding the Second Publication>

[0429] In a conventional cell culture analysis device including a sensor for analyzing a cell culture fluid, the sensor is fixed to a through-hole portion provided in a base body, and a lead wire for extracting a signal is connected to the sensor.

[0430] For example, Patent Document 2 (US Pat. No. 9,170,255) discloses a cell culture analysis apparatus having a cassette that is engaged with a plate on which a plurality of cell culture containers are provided.

[0431] The cell culture analysis device of Patent Document 2 has sensors for measuring the inside of each culture container, and a plurality of openings for inserting these sensors are provided on the box, and the sensors are connected to fiber cables in each opening. Furthermore, these fiber cables are connected to an external control unit.

[0432] The sensor of the cell culture analysis device described in Patent Document 2 has the following problems.

[0433] That is, in the cell culture analysis apparatus disclosed in Patent Document 2, it is difficult to accurately position each sensor inserted into a plurality of culture containers.

[0434] Therefore, the immersion depth of the sensor inserted into the culture container in the culture medium may vary, which may reduce the measurement accuracy. In particular, when the immersion depth of the sensor in the culture medium is insufficient, the liquid sample cannot fully contact the detection electrode of the sensor, which may reduce the measurement accuracy.

[0435] The second disclosed object is to provide a sensor in which, when the sensor is immersed in a liquid sample, contact obstruction of the liquid sample with respect to a detection electrode of the sensor is suppressed, and a sensor module including the sensor.

[0436] Therefore, in this specification, as a second disclosure, a sensor and a sensor module described in any one of the following Supplements 1 to 11 are disclosed.

[0437] (Note 1)

[0438] A sensor is used while immersed in a liquid sample, comprising:

[0439] an insulating substrate including a front end portion immersed in the liquid sample;

[0440] a detection electrode disposed near the front end portion on the first surface of the substrate and including a working electrode, a counter electrode, and a reference electrode;

[0441] A wiring arranged on the first surface of the substrate and connected to the detection electrode;

[0442] an insulating layer disposed on the first surface of the substrate and formed so as to cover at least a portion of the detection electrode and the wiring;

[0443] a front end opening formed in the insulating layer, communicating with the upper surface of the counter electrode and opening on the front end side of the substrate; and

[0444] A flow path is formed in the insulating layer, connects the front end opening and the upper surface of the counter electrode, and guides the liquid sample from the front end opening to the upper surface of the counter electrode.

[0445] (Note 2)

[0446] The sensor according to Supplementary Note 1, wherein the insulating layer comprises: a counter electrode opening portion formed at a position overlapping with the counter electrode when viewed from above in the thickness direction of the substrate and penetrating in the thickness direction;

[0447] The upper surface of the counter electrode is exposed through the counter electrode opening.

[0448] (Note 3)

[0449] The sensor according to Supplementary Note 1 or 2, wherein the insulating layer comprises: when viewed from above in the thickness direction of the substrate,

[0450] A working electrode opening formed at a position overlapping with the working electrode and penetrating in the thickness direction; and

[0451] A reference electrode opening is formed at a position overlapping with the reference electrode and penetrates in the thickness direction.

[0452] The working electrode includes a working electrode protection film disposed in the working electrode opening.

[0453] The reference electrode includes a reference electrode protection film disposed in the reference electrode opening.

[0454] (Note 4)

[0455] The sensor according to any one of Appendixes 1 to 3, wherein the insulating layer comprises:

[0456] a first insulating layer disposed on the first surface of the substrate and comprising an insulating cured resin;

[0457] a second insulating layer disposed on the first insulating layer and including an insulating sheet,

[0458] The front end opening and the flow path are formed in the second insulating layer.

[0459] (Note 5)

[0460] The sensor according to Supplementary Note 4, wherein the front end opening and the flow path are further formed in the first insulating layer.

[0461] (Note 6)

[0462] The sensor according to any one of Supplementary Notes 1 to 5, wherein the flow path has an expansion portion that expands the flow path width toward the front end opening.

[0463] (Note 7)

[0464] The sensor according to any one of Supplementary Notes 1 to 6, wherein the substrate comprises:

[0465] a main body including the front end portion and a first end portion on the opposite side to the front end portion and provided with the detection electrode;

[0466] a connecting portion including a second end portion connected to the first end portion of the main body portion and a third end portion on the opposite side of the second end portion and extending from the second end portion to the third end portion; and

[0467] a base end portion including a fourth end portion connected to the third end portion of the connecting portion,

[0468] The connection portion of the substrate includes a bent portion formed by bending the substrate in the vicinity of the third end portion so that the first surface side protrudes.

[0469] (Note 8)

[0470] According to the sensor described in Appendix 7, the insulating layer includes a first insulating sheet, and the first insulating sheet is arranged on the main body portion in the first surface of the substrate and an area of ​​the connecting portion that is closer to the second end side than the bent portion.

[0471] (Note 9)

[0472] The sensor according to Supplementary Note 8, wherein the insulating layer has a non-insulating region that does not include an insulating sheet and is located in a region of the bent portion of the connecting portion on the first surface of the substrate.

[0473] (Note 10)

[0474] The sensor according to Supplementary Note 8 or 9, wherein the insulating layer further includes a second insulating sheet disposed on the region of the base end portion of the first surface of the substrate,

[0475] The first insulating sheet and the second insulating sheet are separated in a region of the bent portion of the connecting portion on the first surface of the substrate.

[0476] (Note 11)

[0477] A sensor assembly comprising: the sensor according to any one of Supplementary Notes 1 to 10; and

[0478] A support member is provided which engages with the substrate of the sensor and supports an engaging portion of the sensor so that the front end portion of the substrate is at a lower end.

[0479] <Third disclosed embodiment>

[0480] Hereinafter, an embodiment of the sensor involved in the third disclosure will be described. In this embodiment, detailed descriptions beyond the necessary level are sometimes omitted. For example, detailed descriptions of matters that are already sufficiently known and repeated descriptions of substantially the same configurations are sometimes omitted. This is to avoid the following description from becoming unnecessarily lengthy, thereby making it easier for those skilled in the art to understand. It should be noted that for the parts of the configuration of the sensor involved in the third disclosure that are marked with the same symbols as the structures in the sensor involved in the first disclosure, reference can be made to the description of the corresponding structures in the sensor involved in the first disclosure.

[0481] In addition, the applicant provides the drawings and the following description in order to enable those skilled in the art to fully understand the third disclosure, and does not intend to limit the matters described in the following “Supplementary Notes Related to the Third Disclosure” by these drawings and the following description.

[0482] <Materials>

[0483] Examples of materials that can be used in the sensor disclosed in the third aspect of this specification will be described.

[0484] When the insulating substrate is provided in the sensor disclosed in the third aspect of the present specification, the material thereof is not particularly limited, and for example, the same material as that of the insulating substrate of the sensor disclosed in the first aspect of the present specification can be used.

[0485] The conductive layer possessed by the working electrode, reference electrode and counter electrode of the sensor disclosed in the third aspect of this specification is a layer containing conductive materials such as carbon and gold. The conductive layer can be manufactured by forming a layer of the conductive material as described above on the surface of the substrate using a sputtering method, an evaporation method, a screen printing method, etc. The conductive layer can be processed into a specified pattern using a laser trimming method as needed. In the third disclosure, the conductive layer of the working electrode is sometimes referred to as the working electrode conductive layer, and the conductive layer of the reference electrode is sometimes referred to as the reference electrode conductive layer. In the third disclosure, regarding the conductive layer of the counter electrode, since the conductive layer itself constitutes the counter electrode, it is only referred to as the counter electrode. A more preferred embodiment of the conductive material constituting the working electrode conductive layer and the counter electrode is described below.

[0486] The wiring of the sensor disclosed in the third aspect of the present specification may be made of the same conductive material as the conductive layer.

[0487] The sensor disclosed in the third aspect of this specification can be used to detect a specified test substance (analyte) in the liquid sample by being immersed in a liquid sample. The liquid sample is preferably a liquid sample containing water as a solvent. As the liquid sample, a cell culture fluid and a liquid sample prepared using blood obtained from a living body can be cited. For examples of test substances, as described in the first aspect of this specification regarding the test substance, one or more selected from glucose and lactic acid are particularly preferred. As the liquid sample, a liquid sample containing cells, especially living cells, is preferred, and a cell culture fluid is particularly preferred.

[0488] The working electrode of the sensor disclosed in the third aspect of the present specification comprises: a reagent layer disposed on the working electrode conductive layer and containing a reagent participating in the redox reaction of the test substance. The reagent can be appropriately selected according to the test substance. As the reagent, a combination of an oxidoreductase and a mediator (electron transfer substance) can be included, or an oxidoreductase can be included. The oxidoreductase can contain a coenzyme.

[0489] Examples of the oxidoreductase include oxidase and dehydrogenase. Specific examples of the oxidoreductase are as described in the first disclosure of the present specification regarding the sensor, and glucose oxidase, lactate oxidase, glucose dehydrogenase, or lactate dehydrogenase is particularly preferred.

[0490] In addition, the medium is not particularly limited, and examples thereof are as described in the sensor disclosed in the first aspect of this specification.

[0491] The reagent layer of the working electrode of the sensor disclosed in the third aspect of this specification may further include components such as a buffer, a hydrophilic polymer compound, a conductive carbon filler, and a crosslinking agent in addition to the above reagents. Examples of the hydrophilic polymer compound and the conductive carbon filler are as described in the sensor disclosed in the first aspect of this specification.

[0492] The working electrode of the sensor disclosed in Article 3 of this specification may further have a protective film. The protective film on the working electrode is a film that can prevent or inhibit the reagent contained in the reagent layer from leaking out of the protective film and can permeate the test substance present outside the protective film. The protective film having such a property preferably contains a polymer compound. As the polymer compound contained in the protective film, there can be cited a polymer compound containing 4-vinylpyridine in the structural unit and a polymer compound containing a cation exchange functional group. As a polymer compound containing a cation exchange functional group, a polymer compound having proton conductivity and the like can be exemplified. As a cation exchange functional group, an anionic functional group can be exemplified.

[0493] Examples of the polymer compound containing 4-vinylpyridine in a structural unit and the polymer compound having proton conductivity are as described in the sensor disclosed in the first aspect of the present specification.

[0494] The protective film provided in the working electrode of the sensor disclosed in the third aspect of the present specification may be a laminate of two or more protective films, and examples thereof are as described in connection with the sensor disclosed in the first aspect of the present specification.

[0495] Another preferred example of the protective film provided in the working electrode of the sensor disclosed in the third aspect of the present specification is a protective film comprising a polymer compound containing 4-vinylpyridine as a structural unit.

[0496] The reference electrode of the sensor disclosed in the third aspect of the present specification may include a protective film. The protective film of the reference electrode may be made of the above-mentioned material for the protective film of the working electrode.

[0497] The counter electrode of the sensor disclosed in the third aspect of the present specification may be covered with a protective film. Preferred embodiments of the protective film covering the counter electrode will be described later.

[0498] In the sensor disclosed in the third aspect of this specification, the electrode including the working electrode and the counter electrode disposed on the insulating substrate, except for the portion in contact with the liquid sample, is preferably covered with an insulating layer. The insulating layer may include an insulating resin. The insulating layer may be a multilayer structure of two or more layers. The insulating layer of the multilayer structure may include, for example: a first insulating layer disposed on the insulating substrate and the conductive layer, and a second insulating layer disposed on the first insulating layer. Preferred embodiments of the materials constituting the first insulating layer and the second insulating layer are as described in the description of the sensor involved in the first disclosure.

[0499] <Overview of sensor 300>

[0500] A sensor 300 according to an embodiment of the third disclosure will be described with reference to the drawings.

[0501] The sensor 300 of this embodiment is as follows Figures 54-57As shown, it has: an insulating substrate 2, a first working electrode 10a, a second working electrode 10b, a reference electrode 20 and a counter electrode 60 arranged on the first surface 2a of the substrate 2, and a wiring 50 electrically connected to the first working electrode 10a, the second working electrode 10b, the reference electrode 20 and the counter electrode 60, respectively. The sensor 300 of this embodiment includes two working electrodes, but in other embodiments not shown, the working electrode can be only one, or can be more than three. In the following description, when the first working electrode 10a and the second working electrode 10b are not distinguished, they are sometimes described as working electrodes 10a, 10b. In addition, the first working electrode 10a, the second working electrode 10b, the reference electrode 20 and the counter electrode 60 are sometimes collectively described as electrodes. In addition, the sensor 300 of this embodiment is a three-pole sensor including a working electrode, a reference electrode and a counter electrode as electrodes, but it can also be a two-pole sensor that does not contain a reference electrode and only includes a working electrode and a counter electrode. Although not shown in the figure, the reference electrode and / or the counter electrode may also be disposed on a substrate different from the substrate on which the working electrode is disposed.

[0502] Sensor 300 and sensor 1 of the first disclosure Fig.21 The contents shown can be used similarly to immerse the sample in a liquid sample X to detect a predetermined test substance in the liquid sample X. Specific examples of the liquid sample and the test substance are described in the column of <Material>.

[0503] The working electrodes 10a, 10b of the sensor 300 are as follows Fig.56 , 57 As shown, it comprises working electrode conductive layers 11a and 11b and reagent layers 15a and 15b containing reagents participating in the redox reaction of the test substance in the liquid sample. Preferred embodiments of the reagents are as described in the column of <Materials>.

[0504] When the reagent layers 15a, 15b of the working electrodes 10a, 10b of the sensor 300 contain a reagent that oxidizes the test substance in the liquid sample, electrons move from the test substance to the working electrode conductive layers 11a, 11b under the condition that a specified voltage is applied to the electrodes of the sensor 300. Similarly, when the reagent layers 15a, 15b contain a reagent that reduces the test substance in the liquid sample, electrons move from the working electrode conductive layers 11a, 11b to the test substance. Since the amount of electrons that move depends on the concentration of the test substance, the concentration of the test substance in the liquid sample or the concentration change can be measured based on the current value or the change in the current value flowing through the working electrodes 10a, 10b of the sensor 300.

[0505] The sensor 300 can constitute an analysis device for analyzing a substance to be tested in a liquid sample. Fig.24 The analysis device 100 shown in the figure may be an analysis device in which the sensor 1 according to the first disclosure is replaced with the sensor 300 according to the third disclosure, and includes the sensor 300, the analysis unit 102, and the control unit 104. The functions of the analysis device are as follows. Fig.24 The analysis device 100 shown is as described in the description.

[0506] <Structure of sensor 300>

[0507] For the structure of the sensor 300 according to the third disclosure, refer to Fig.54 and as its cross-sectional view Fig.55 , 56 It should be noted that the structure of the reference electrode 20 of the sensor 300 involved in the third disclosure is the same as the structure of the reference electrode 20 of the sensor 1 involved in the first disclosure. Fig. 22 The description of the reference electrode 20 of the sensor 1 according to the first disclosure is used as the description of the reference electrode 20 of the sensor 300 according to the third disclosure.

[0508] The sensor 300 includes: an insulating substrate 2, working electrode conductive layers 11a and 11b arranged on the first surface 2a of the substrate 2, a counter electrode 60, a reference electrode conductive layer 21, and wiring 50. It should be noted that in the embodiment of the sensor 300 involved in the third disclosure shown in the figure, the entire counter electrode 60 is formed by a conductive layer. In order to specify the portion of the conductive layer in the counter electrode, it can also be referred to as a "counter electrode conductive layer" or a "conductive layer of the counter electrode". On the first surface 2a of the substrate 2, a first insulating layer 3 is further arranged, and on the first insulating layer 3, a second insulating layer 4 is further arranged. The height of the upper surface of the first insulating layer 3 from the first surface 2a of the substrate 2 is higher than the height of the working electrode conductive layers 11a and 11b, the counter electrode 60, the reference electrode conductive layer 21 and the wiring 50 from the first surface 2a of the substrate 2. Therefore, a portion of the first insulating layer 3 is disposed on the working electrode conductive layers 11 a and 11 b , the counter electrode 60 , the reference electrode conductive layer 21 , and the wiring 50 .

[0509] like Fig.55As shown, the first insulating layer 3 includes a first opening 302 for the counter electrode formed at a position overlapping with a portion of the counter electrode 60 and penetrating along the thickness direction T of the substrate 2. Furthermore, the second insulating layer 4 includes a second opening 402 for the counter electrode formed at a position overlapping with the entire first opening 302 for the counter electrode of the first insulating layer 3 and penetrating along the thickness direction T of the substrate 2. The counter electrode 60 is exposed to the outside through the first opening 302 for the counter electrode and the second opening 402 for the counter electrode. A portion 60a of the counter electrode 60 that contacts the liquid sample when the sensor 300 is immersed in the liquid sample is located at the bottom surface of the recess formed by the first opening 302 for the counter electrode.

[0510] like Fig.56 , 57 As shown, the working electrodes 10a and 10b include working electrode conductive layers 11a and 11b, and reagent layers 15a and 15b disposed on the working electrode conductive layers 11a and 11b and containing reagents participating in the redox reaction of the test substance. Preferred embodiments of the reagents in the reagent layers 15a and 15b are as described in the column of <Materials>.

[0511] Similar to the first disclosure, in order to distinguish the "reagent layer 15a" of the first working electrode 10a from the "reagent layer 15b" of the second working electrode 10b, the former is sometimes expressed as the "first reagent layer 15a" of the first working electrode 10a, and the latter is expressed as the "second reagent layer 15b" of the second working electrode 10b. In addition, the reagent contained in the reagent layer 15a of the first working electrode 10a is sometimes expressed as the "first reagent", and the reagent contained in the second reagent layer 15b of the second working electrode 10b is sometimes expressed as the "second reagent". In a preferred embodiment, the sensor 300 includes a plurality of working electrodes 10a, 10b, and the first reagent contained in the first reagent layer 15a of the first working electrode 10a of the plurality of working electrodes 10a, 10b is different from the second reagent contained in the second reagent layer 15b of the second working electrode 10b different from the first working electrode 10a. In the sensor 300 involved in this preferred embodiment, since the first reagent can participate in the redox reaction of the first test substance in the liquid sample, and the second reagent can participate in the redox reaction of the second test substance in the liquid sample that is different from the first test substance, it can be used to detect multiple test substances in the liquid sample, including the first test substance and the second test substance.

[0512] like Fig.56 , 57As shown, the working electrodes 10a and 10b may have protective films 16a and 16b disposed on the reagent layers 15a and 15b. The preferred embodiment of the protective films 16a and 16b is as described in the column of <Material>. In the example shown in the figure, a first protective film 16a formed of one layer is disposed on the first reagent layer 15a, and a protective film 16b including a second protective film 16ba and a third protective film 16bb is disposed on the second reagent layer 15b, but the present invention is not limited to this example, and the protective films 16a and 16b may have the same structure or other structures not shown in the figure.

[0513] like Fig.56 , 57 As shown, the first insulating layer 3 includes working electrode first openings 303a and 303b formed at positions overlapping with parts of the working electrode conductive layers 11a and 11b, respectively, and extending through the thickness direction T of the substrate 2. Furthermore, the second insulating layer 4 includes working electrode second openings 403a and 403b formed at positions overlapping with parts that entirely enclose the working electrode first openings 303a and 303b of the first insulating layer 3, and extending through the thickness direction T of the substrate 2. The working electrodes 10a and 10b are exposed to the outside through the working electrode first openings 303a and 303b and the working electrode second openings 403a and 403b. The portions 11a1 and 11b1 of the working electrode conductive layers 11a and 11b that come into contact with the liquid sample when the sensor 300 is immersed in the liquid sample are located at the bottom surface of the recess formed by the working electrode first openings 303a and 303b. In addition, the reagent layers 15a and 15b are enclosed in the first openings 303a and 303b of the working electrodes, and the outer peripheries of the reagent layers 15a and 15b are defined by the inner peripheries of the first openings 303a and 303b of the working electrodes. The protective films 16a and 16b are enclosed in the second openings 403a and 403b of the working electrodes, and the outer peripheries of the protective films 16a and 16b are defined by the inner peripheries of the second openings 403a and 403b of the working electrodes.

[0514] <Suppression of hydrogen peroxide generation by sensor 300>

[0515] The sensor 300 involved in the third disclosure is characterized in that when the sensor 300 is immersed in a liquid sample and an electrochemical measurement is performed on the test substance in the liquid sample, the hydrogen peroxide concentration in the liquid sample can be maintained at less than 15 μM during the measurement by suppressing the generation of hydrogen peroxide during the measurement and / or reducing the concentration of the generated hydrogen peroxide. Hydrogen peroxide is known to be toxic to cells. Therefore, when an electrochemical sensor is used to continuously measure the test substance in a liquid sample containing cells such as a cell culture fluid for a long period of time or more, it is desirable to suppress the increase in the hydrogen peroxide concentration during the measurement.

[0516] According to the sensor 300 , the detection sensitivity of the test substance does not significantly decrease, and the concentration of hydrogen peroxide in the liquid sample can be maintained at a low concentration of less than 15 μM during the measurement period.

[0517] Here, "electrochemical measurement of the test substance in the above-mentioned liquid sample" means, for example: when the sensor 300 immersed in the liquid sample is connected to a constant potential instrument, a predetermined potential is applied to the working electrodes 10a and 10b with reference to the reference electrode 20, the current between the working electrodes 10a and 10b and the counter electrode 60 is detected, and the test substance is measured based on the detected current. The electrochemical measurement using the sensor 300 is typically performed continuously for a period of several hours or more or several days, for example, more than 1 hour, preferably more than 72 hours (3 days), more preferably more than 96 hours (4 days), further preferably more than 168 hours (7 days), and most preferably more than 240 hours (10 days). There is no particular upper limit to the above period, for example, it is within 336 hours (14 days). According to the sensor 300, the hydrogen peroxide concentration in the liquid sample during this period can be maintained at less than 15 μM. The hydrogen peroxide concentration of the liquid sample during the above period is more preferably maintained at less than 10 μM.

[0518] In a preferred embodiment of the sensor 300 having the above-mentioned features, the counter electrode 60 contains a catalyst for decomposing hydrogen peroxide in the portion 60a in contact with the liquid sample. It is believed that the generation of hydrogen peroxide is mainly caused by the oxygen (O 2 ) is reduced to hydrogen peroxide (H 2 O 2 ). In particular, in the measurement of the test substance that undergoes an oxidation reaction on the working electrodes 10a and 10b, the generation of hydrogen peroxide on the counter electrode 60 may become a problem. As the measurement of the test substance that undergoes an oxidation reaction on the working electrodes 10a and 10b, there can be mentioned the measurement using the working electrodes 10a and 10b having the reagent layers 15a and 15b containing oxidases, dehydrogenases, etc. that participate in the oxidation of the test substance. According to the sensor 300 having the counter electrode 60 containing the catalyst that decomposes hydrogen peroxide in the portion 60a that contacts the liquid sample, during the measurement, due to the oxygen (O 2 ) is reduced to water (H 2O), so the generation of hydrogen peroxide is suppressed. In addition, among the reagents contained in the reagent layers 15a and 15b of the working electrodes 10a and 10b, there are reagents (such as oxidase and dehydrogenase) that are accompanied by a reaction that generates hydrogen peroxide during measurement. According to this embodiment, even if hydrogen peroxide is generated on the working electrodes 10a and 10b, it can be decomposed into water by the above-mentioned catalyst contained in the counter electrode 60, so the increase in the concentration of hydrogen peroxide in the liquid sample being measured can be suppressed.

[0519] In the third disclosure, the catalyst may be, for example, a catalyst containing a metal. A catalyst containing a metal is preferred because it has high stability compared to an organic catalyst such as catalase. As a catalyst containing a metal, a catalyst containing one or more selected from platinum, nickel, palladium, iron, manganese and tungsten is preferred, and a catalyst containing platinum is particularly preferred. These catalysts containing a metal have high activity in decomposing hydrogen peroxide, and are therefore preferred, and among them, a catalyst containing platinum is particularly preferred from the viewpoint of catalyst activity and stability.

[0520] In the third disclosure, the catalyst may be contained in the portion of the counter electrode that contacts the liquid sample, or may be contained only in the portion that contacts the liquid sample, or may be contained in the entire counter electrode. In addition, the catalyst may be contained in the counter electrode in the form of a film that covers at least the surface of the portion that contacts the liquid sample, or may be contained in the form of a conductive material that is dispersed in the entire counter electrode or at least in the portion that contacts the liquid sample.

[0521] In the third disclosure, examples of counter electrodes in which the above-mentioned catalyst is contained in the form of a film covering the surface of the part in contact with the liquid sample include: a counter electrode obtained by forming a film of the above-mentioned metal that can be used as a catalyst on the surface of the part in contact with the liquid sample by sputtering, plating or the like; a counter electrode obtained by applying a dispersion of particles containing the above-mentioned catalyst in a liquid medium to the surface of the part in contact with the liquid sample and drying it. Among them, the counter electrode obtained by using a dispersion of particles containing the above-mentioned catalyst in a liquid medium can be prepared by a simple method such as coating and printing, so it is preferred. As the particles of the above-mentioned catalyst, particles of the above-mentioned metal can be mentioned, and platinum particles are particularly preferred. As platinum particles, platinum nanoparticles with an average particle size of less than 1 μm, for example, less than 800 nm, preferably less than 600 nm are preferred.

[0522] In the third disclosure, as an example of a counter electrode containing the above-mentioned catalyst in a state of being dispersed in a conductive material constituting the whole of the counter electrode or at least the part in contact with the liquid sample, there can be cited: a counter electrode obtained by forming the whole or at least the part in contact with the liquid sample using a paste or liquid composition containing particles of the above-mentioned catalyst and particles of the conductive material. The counter electrode of this mode can include the above-mentioned catalyst in the counter electrode while forming the counter electrode on the substrate, so it can be produced by a small number of steps. As the particles of the above-mentioned catalyst, particles of the above-mentioned metal can be cited, and platinum particles are particularly preferred. The preferred mode of the platinum particles is as described above.

[0523] In the third disclosure, the counter electrode may contain carbon, gold or the like as a conductive material, and particularly preferably contains carbon as a conductive material. As carbon, conductive carbon materials such as glassy carbon, carbon black, graphite, diamond-like carbon, graphene, carbon nanotubes, and fullerenes may be used.

[0524] In the third disclosure, the content of the catalyst in the electrode can be appropriately set according to the type and activity of the catalyst. For example, when the entire electrode or at least the portion in contact with the liquid sample contains carbon as a conductive material and platinum particles as the catalyst, the platinum particles can be contained in an amount of, for example, 0.5% by weight or more, preferably 1% by weight or more, more preferably 3% by weight, and particularly preferably 5% by weight or more relative to the carbon. The upper limit is not particularly limited. From the perspective of cost, the platinum particles can be contained in an amount of 50% by weight or less (the ratio of the platinum particles to the carbon is, for example, 0.5% by weight or more and 50% by weight or less), further preferably 30% by weight or less, and most preferably 10% by weight or less. By setting the ratio of carbon to platinum particles within this range, the increase in the concentration of hydrogen peroxide in the liquid sample during the measurement period can be suppressed.

[0525] Although not shown in the figure, in the third disclosure, the surface of the counter electrode can also be further covered by a protective film. The protective film covering the counter electrode preferably contains a polymer compound having a cation exchange functional group, and particularly preferably contains a polymer compound having a cation exchange functional group on the side chain. As the polymer compound having a cation exchange functional group here, a polymer compound containing a structural unit having a sulfonic acid group can be cited, preferably a polymer compound containing a perfluorinated compound having a sulfonic acid group on the side chain in the structural unit, particularly preferably a copolymer of tetrafluoroethylene and perfluoro-2-(2-fluorosulfonylethoxy)propyl vinyl ether, and most preferably Nafion (registered trademark). Anions such as chloride ions and organic acid ions that may be contained in the liquid sample may have an adverse effect on the activity of catalysts such as platinum catalysts that decompose hydrogen peroxide. By covering the counter electrode with a protective film containing a polymer compound having a cation exchange functional group, the contact of anions with the counter electrode can be prevented, and the activity reduction of the above-mentioned catalyst can be suppressed.

[0526] On the other hand, in the sensor 300 involved in the present disclosure, it is preferred that at least the portion 11a1, 11b1 of the working electrode conductive layer 11a, 11b of the working electrode 10a, 10b that contacts the liquid sample does not contain a catalyst that decomposes hydrogen peroxide as a whole. When the working electrode conductive layer 11a, 11b of the working electrode 10a, 10b of the sensor 300 contains the above-mentioned catalyst, especially a catalyst containing platinum, there is a possibility that the background noise during measurement increases and the current response value decreases. As the working electrode conductive layer 11a, 11b or the portion 11a1, 11b1 that does not contain a catalyst that decomposes hydrogen peroxide, a layer of a conductive material such as carbon, gold, or a portion thereof that does not contain a catalyst that decomposes hydrogen peroxide can be cited.

[0527] The working electrode conductive layers 11a and 11b of the working electrodes 10a and 10b more preferably satisfy the following conditions: using a three-electrode electrolysis cell having the working electrode conductive layers 11a and 11b as working electrodes, a silver-silver chloride (saturated KCl) electrode as a reference electrode, and a platinum electrode as a counter electrode, when a potential in the range of -0.2 V to 0.3 V (relative to the silver-silver chloride (saturated KCl) electrode) is applied to the working electrode conductive layers in phosphate buffered saline, the current value measured is ±5.5 nA / mm 2The following. Since the sensor 300 having working electrode conductive layers 11a and 11b that meet this condition has a very small background current when measuring the test substance and has high sensor responsiveness, it is preferred. As such working electrode conductive layers 11a and 11b, carbon conductive layers containing carbon can be cited, and more preferably, carbon conductive layers that do not contain catalysts such as platinum-containing catalysts that decompose hydrogen peroxide. The carbon here can use the materials exemplified with respect to the conductive material of the counter electrode 60. It should be noted that the current value is ±5.5nA / mm 2 The following means that the portion of the working electrode conductive layer 11a, 11b used as the working electrode in contact with the phosphate buffered saline solution is 1 mm in the vertical direction. 2 The absolute value of the current value of the projected area is 5.5 nA or less. The current value using the above-mentioned three-electrode electrolysis cell can be specifically measured according to the procedure described in the above-mentioned <3rd Publication / Experiment 5>.

[0528] The inventors of the present invention further discovered that in the sensor 300 of the present disclosure, in the mode in which the portion 60a of the counter electrode 60 in contact with the liquid sample includes a catalyst containing platinum, an unexpected effect such as a negative current with a large absolute value can flow at a low potential. Therefore, the counter electrode 60 including a catalyst containing platinum in the portion 60a in contact with the liquid sample can efficiently function as a counter electrode with a smaller area than a counter electrode not including a catalyst containing platinum, and thus can contribute to the reduction of the size of the sensor. Therefore, in the sensor 300 of the present disclosure, the projected area of ​​the counter electrode 60 can be preferably set to 250% or less, more preferably to 100% or less, preferably to 3% or more (the projected area of ​​the counter electrode is, for example, 3% or more and 250% or less relative to the projected area of ​​the working electrode conductive layer), and more preferably to 10% or more. In addition, the sensor 300 having the counter electrode 60 including a catalyst containing platinum in the portion 60a in contact with the liquid sample is also applicable to the case where a plurality of working electrodes 10a and 10b are provided as shown in the figure. It should be noted that the projection area of ​​the counter electrode 60 refers to the projection area of ​​the portion 60a in contact with the liquid sample (equivalent to the bottom surface of the concave portion formed by the first opening 302 of the counter electrode) in the vertical direction. The projection area of ​​the working electrode conductive layer 11a and 11b refers to the projection area of ​​the portions 11a1 and 11b1 in contact with the liquid sample (equivalent to the bottom surface of the concave portion formed by the first opening 303a and 303b of the working electrode) in the vertical direction, and when there are a plurality of working electrodes 10a and 10b, it refers to the total area thereof.

[0529] <Third disclosed embodiment>

[0530] In Experiments 1 to 7 of the sensor according to the third disclosure, as examples and comparative examples of the sensor according to the third disclosure, Fig.54 The structure of the sensor 300 is shown.

[0531] The characteristics of the sensor used in each experiment are described in each of Experiments 1 to 7. First, the materials and manufacturing methods common to the sensors used in each experiment are described in outline.

[0532] (Substrate)

[0533] As the insulating substrate 2, the same as in the first disclosed embodiment is used. Fig.54 The shape shown in the figure is a substrate of 188 μm in thickness made of polyethylene terephthalate.

[0534] (Conductive layer)

[0535] On the first surface 2a of the insulating substrate 2, a carbon conductive layer with a thickness of 5 μm was formed by applying the prescribed carbon paste described in Experiments 1 to 7 and heating at 140°C for 1 hour: Figure 1 The working electrode conductive layers 11 a and 11 b , the reference electrode conductive layer 21 and the counter electrode (counter electrode conductive layer) 60 are of the same shape, and the wirings 50 electrically connected to them respectively.

[0536] (1st insulation layer)

[0537] Next, on the first surface 2a of the substrate 2 provided with the carbon conductive layer, a first insulating layer 3 is formed of a fluororesin containing a copolymer of vinylidene fluoride and hexafluoropropylene, covering the first surface 2a of the substrate 2 and the carbon conductive layer, and having a thickness of 5 μm on the carbon conductive layer. The method for manufacturing the first insulating layer 3 is as described in the first disclosed embodiment.

[0538] The first working electrode opening 303a on the first working electrode conductive layer 11a of the first insulating layer 3 was formed into a circle with a diameter of 1.2 mm, and the first working electrode opening 303b on the second working electrode conductive layer 11b was formed into a circle with a diameter of 1.4 mm. The first reference electrode opening 301 was formed into a circle with a diameter of 1.1 mm, and the first counter electrode opening 302 was formed into a rectangle with a size of 1.8 mm x 2.1 mm.

[0539] (Second insulation layer)

[0540] Next, a composition containing a polyester resin and a fluorine-based surface modification additive containing a perfluoroalkyl group in a solvent was applied onto the first insulating layer 3 and heated at 140° C. for 1 hour to laminate the second insulating layer 4 having a thickness of 40 μm.

[0541] The working electrode second openings 403a and 403b of the second insulating layer 4 on the working electrode conductive layers 11a and 11b are respectively formed into a circle with a diameter of 2 mm. The reference electrode second opening 401 is formed into a circle with a diameter of 2 mm, and the counter electrode second opening 402 is formed into a rectangle with a size of 1.8 mm x 2.1 mm.

[0542] (First working electrode (for glucose measurement) reagent layer)

[0543] In the first opening 303a of the working electrode of the first insulating layer 3 on the first working electrode conductive layer 11a, a 0.4 μL droplet of the first liquid composition A containing sodium phosphate buffer (pH 7.4), carbon black dispersion, polymer-bonded mediator, glucose oxidase and a cross-linking agent in water is formed and dried to form a first reagent layer 15a containing glucose oxidase and the mediator.

[0544] (Second working electrode (for lactic acid measurement) reagent layer)

[0545] In the first opening 303b of the working electrode of the first insulating layer 3 of the second working electrode conductive layer 11b, a 0.6 μL droplet of the second liquid composition A containing a carbon black dispersion, hydroxypropyl cellulose, a polymer-bonded medium, lactate oxidase, polyimidazole, poly-L-lysine and a cross-linking agent in water is formed and dried to form a second reagent layer 15b containing lactate oxidase and the medium.

[0546] (Protective film for the first working electrode (for glucose measurement))

[0547] The following reagents were mixed in ethanol to the following final concentrations, and the mixture was allowed to react for about 1 hour to prepare a first liquid composition B (P4VP-tBuMA polymer dispersion).

[0548] P4VP-tBuMA (Mn of poly-4-vinylpyridine: 74,000, Mn of poly-tert-butyl methacrylate: 87,000, Mw / Mn: 1.16, manufactured by NARD), final concentration: 5.72 wt%

[0549] PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ~1000, manufactured by Sigma-Aldrich), final concentration 0.9 wt%

[0550] In the working electrode second opening 403a of the second insulating layer 4 on the first working electrode 10a, a 0.65 μL droplet of the first liquid composition B is formed and dried, and then a further 0.65 μL droplet of the first liquid composition B is formed and dried to form a first protective film 16a.

[0551] (Second working electrode (for lactic acid measurement) protective film)

[0552] To 23093.67 mg of a 21.5 wt% Nafion (registered trademark) dispersion (manufactured by Sigma-Aldrich), 6472.79 mg of ethanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 1226.46 mg of a 5 mol / L sodium hydroxide aqueous solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added to adjust the pH of the dispersion (neutralization treatment of the cation exchange group), and the precipitate was dissolved using a vortex mixer to prepare 30792.92 mg of a 16.12 wt% Nafion (registered trademark) dispersion. The obtained 16.12 wt% Nafion (registered trademark) dispersion was used as the second liquid composition B. The second liquid composition B contains Nafion (registered trademark) in a solvent containing a lower alcohol.

[0553] A 0.60 μL droplet of the second liquid composition B was formed in the working electrode second opening 403 b of the second insulating layer 4 on the second working electrode 10 b and dried to form a second protective film 16 ba .

[0554] Next, the same composition as the first liquid composition B (P4VP-tBuMA polymer dispersion) was used as the third liquid composition B for the following treatment. That is, a 0.65 μL droplet of the third liquid composition B was formed in the second opening 403b of the working electrode of the second insulating layer 4 on which the second protective film 16ba was formed, and dried to form the third protective film 16bb. In this way, a protective film 16b in which the third protective film 16bb was stacked on the second protective film 16ba was formed on the second working electrode 10b.

[0555] (reference electrode)

[0556] The cross-sectional structure of the reference electrode 20 is similar to that of the sensor disclosed in the first embodiment. Fig. 22 Same, so refer to Fig. 22 The following is an outline of the method for manufacturing the reference electrode 20. A silver-silver chloride paste is applied to the reference electrode conductive layer 21 in the reference electrode first opening 301 of the first insulating layer 3, and heated at 140° C. for 1 hour to form a silver-silver chloride layer 22. Next, a reference electrode protective film 23 is disposed on the silver-silver chloride layer 22 in the reference electrode second opening 401 of the second insulating layer 4 to form the reference electrode 20.

[0557] <Publication 3 / Experiment 1>

[0558] Experiment 1-1: In the above method, a carbon conductive layer was formed using a common carbon paste that does not contain a catalyst such as platinum, and a sensor 300 of Experiment 1-1 was produced. In the sensor of Experiment 1-1, the working electrode conductive layers 11a, 11b, the reference electrode conductive layer 21, and the counter electrode (counter electrode conductive layer) 60 were all formed of a carbon conductive layer that does not contain a catalyst for decomposing hydrogen peroxide.

[0559] Experiment 1-2: After forming a carbon conductive layer using a conventional carbon paste that does not contain a catalyst such as platinum, platinum was vapor-deposited only on the counter electrode 60 before forming the first insulating layer 3, and the sensor 300 of Experiment 1-2 was manufactured according to the above-mentioned steps. In the sensor of Experiment 1-2, in the counter electrode 60, a portion 60a exposed through the counter electrode first opening 302 of the first insulating layer 3 and the counter electrode second opening 402 of the second insulating layer 4 and in contact with the liquid sample includes a carbon conductive layer and a platinum vapor-deposited film stacked on the carbon conductive layer, and the working electrode conductive layers 11a, 11b and the reference electrode conductive layer 21 are formed of a carbon conductive layer that does not contain a catalyst such as platinum.

[0560] As a liquid sample containing glucose and lactic acid as test substances, phosphate buffered saline (PBS) (TAKARA BIO INC. PBS Tablets T9181), glucose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and sodium lactate (manufactured by Sigma-Aldrich) were used to prepare PBS containing 30 mM glucose and 15.6 mM lactic acid and adjusted to pH 7.0.

[0561] The sensor of Experiment 1-1 or Experiment 1-2 was immersed in the above liquid sample, a voltage of 100 mV relative to the reference electrode 20 (Ag / AgCl) was applied to the first working electrode 10a and the second working electrode 10b, and the current value between the first working electrode 10a and the counter electrode 60 and the current value between the second working electrode 10b and the counter electrode 60 were measured for 7 consecutive days. The measurement was performed with N=4.

[0562] The liquid sample after 7 days of measurement was used to prepare a hydrogen peroxide concentration measurement sample according to the ratio shown in the following table. The absorbance of the hydrogen peroxide concentration measurement sample at 666 nm was measured for 5 minutes using a plate reader (at 37° C.).

[0563] [Table 2]

[0564]

[0565] In addition, a hydrogen peroxide concentration measurement sample prepared by mixing a solution containing hydrogen peroxide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) of known concentration was replaced with the above liquid sample in the above table, and the absorbance at 666 nm was measured in the same manner to prepare a calibration curve showing the relationship between hydrogen peroxide concentration and absorbance.

[0566] The hydrogen peroxide concentration in the liquid sample was determined based on the calibration curve. The determined hydrogen peroxide concentration was a value corrected for the evaporation of water from the liquid sample during the 7-day continuous measurement. The results are shown in the following table.

[0567] [Table 3]

[0568]

[0569] Based on the above results, the sensor of Experiment 1-2 having a counter electrode formed by a carbon conductive layer having a platinum vapor-deposited film stacked on the portion in contact with the liquid sample was confirmed to be able to suppress or reduce the production of hydrogen peroxide in the liquid sample during the measurement of the test substance in the liquid sample, compared with the sensor of Experiment 1-1 having a counter electrode formed only by a carbon conductive layer.

[0570] <Publication 3 / Experiment 2>

[0571] Experiment 2-1: Similar to the sensor of Experiment 1-1, a sensor 300 in which all conductive layers including the counter electrode 60 are formed of carbon conductive layers was used as the sensor of Experiment 2-1.

[0572] Experiment 2-2: In the sensor of Experiment 2-1, a platinum nanoparticle dispersion (manufactured by Sigma-Aldrich, platinum particle diameter: 70±6 nm) was dripped onto the surface of the counter electrode 60 exposed through the first counter electrode opening 302 and the second counter electrode opening 402 and dried, and then a 21.5 wt% concentration Nafion (registered trademark) dispersion (manufactured by Sigma-Aldrich) was dripped and dried to form a protective film (not shown), thereby manufacturing a sensor 300 of Experiment 2-2. In the sensor 300 of Experiment 2-2 manufactured in this way, a portion 60a of the counter electrode 60 in contact with the liquid sample was a carbon conductive layer modified with platinum nanoparticles on the surface, and the surface of the portion 60a was covered with a protective film (not shown) containing Nafion (registered trademark) as a polymer compound having a cation exchange functional group.

[0573] The sensor 300 of Experiment 2-1 or Experiment 2-2 was immersed in the liquid sample of the composition described in Experiment 1, and a voltage of 100 mV relative to the reference electrode 20 (Ag / AgCl) was applied to the first working electrode 10a and the second working electrode 10b, and the current value between the first working electrode 10a and the counter electrode 60 and the current value between the second working electrode 10b and the counter electrode 60 were measured for 7 consecutive days. The measurement was performed with N=2.

[0574] The liquid sample after 7 days of measurement was used to prepare a hydrogen peroxide concentration measurement sample according to the ratio shown in the following table. The absorbance of the hydrogen peroxide concentration measurement sample at 666 nm was measured for 5 minutes using a plate reader (at 37° C.).

[0575] [Table 4]

[0576]

[0577] In addition, a hydrogen peroxide concentration measurement sample prepared by mixing a solution containing hydrogen peroxide (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) of known concentration was replaced with the above liquid sample in the above table, and the absorbance at 666 nm was measured in the same manner to prepare a calibration curve of hydrogen peroxide concentration and absorbance.

[0578] The hydrogen peroxide concentration in the liquid sample was determined based on the calibration curve. The determined hydrogen peroxide concentration was a value corrected for the amount of water evaporation from the liquid sample during the 7-day continuous measurement. The results are shown in the following table.

[0579] [Table 5]

[0580]

[0581] Based on the above results, the sensor 300 of Experiment 2-2, which has a counter electrode 60 formed by a carbon conductive layer modified with platinum nanoparticles in the portion 60a in contact with the liquid sample, was confirmed to be able to suppress or reduce the generation of hydrogen peroxide in the liquid sample during the measurement of the test substance in the liquid sample, compared with the sensor 300 of Experiment 2-1, which has a counter electrode 60 formed only by a carbon conductive layer.

[0582] <Publication 3 / Experiment 3>

[0583] Experiment 2-1: Similar to the sensors of Experiments 1-1 and 2-1, a sensor 300 in which all conductive layers including the counter electrode 60 are formed of carbon conductive layers was used as the sensor of Experiment 3-1.

[0584] Experiment 3-2: As a carbon paste, a carbon paste containing carbon (including graphite, carbon black) and platinum particles (average particle size 390 nm) at 1 weight % relative to the above-mentioned carbon is used to form working electrode conductive layers 11a, 11b, reference electrode conductive layer 21 and counter electrode (counter electrode conductive layer) 30, and wiring 50. In addition, the sensor 300 of Experiment 3-2 is manufactured through the above-mentioned steps.

[0585] Experiment 3-3: The sensor 300 of Experiment 3-3 was produced according to the procedure of the sensor of Experiment 3-2, except that a carbon paste containing the carbon and 5 wt % of platinum particles based on the carbon was used as the carbon paste.

[0586] The sensor of Experiment 3-1, Experiment 3-2 or Experiment 3-3 was immersed in the liquid sample of the composition described in Experiment 1, and a voltage of 100 mV relative to the reference electrode 20 (Ag / AgCl) was applied to the first working electrode 10a and the second working electrode 10b, and the current value between the first working electrode 10a and the counter electrode 60 and the current value between the second working electrode 10b and the counter electrode 60 were measured for 7 consecutive days. The measurement was performed with N=4.

[0587] The liquid sample after 7 days of measurement was used to prepare a hydrogen peroxide concentration measurement sample according to the ratio shown in the following table. The absorbance of the hydrogen peroxide concentration measurement sample at 666 nm was measured for 5 minutes using a plate reader (at 37° C.).

[0588] [Table 6]

[0589]

[0590] In addition, a hydrogen peroxide concentration measurement sample prepared by mixing a solution containing hydrogen peroxide (manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.) of known concentration was replaced with the above liquid sample in the above table, and the absorbance at 666 nm was measured in the same manner to prepare a calibration curve of hydrogen peroxide concentration and absorbance.

[0591] The hydrogen peroxide concentration in the liquid sample was determined based on the calibration curve. The determined hydrogen peroxide concentration was a value corrected for the amount of water evaporation from the liquid sample during the 7-day continuous measurement. The results are shown in the following table.

[0592] [Table 7]

[0593]

[0594] Based on the above results, the sensors 300 of Experiments 3-2 and 3-3 having a counter electrode 60 formed by a carbon conductive layer containing platinum particles were compared with the sensor 300 of Experiment 3-1 having a counter electrode 60 formed only by a carbon conductive layer, and confirmed that the generation of hydrogen peroxide in the liquid sample can be suppressed or reduced during the measurement of the test substance in the liquid sample.

[0595] <Publication 3 / Experiment 4>

[0596] In Experiments 3-1, 3-2, and 3-3, the following experiments were conducted to investigate the electrochemical properties of a carbon conductive layer containing no platinum (hereinafter, “carbon electrode without platinum particles”), a carbon conductive layer containing 1 wt % of platinum particles relative to carbon (hereinafter, “carbon electrode containing 1% of platinum particles”), and a carbon conductive layer containing 5 wt % of platinum particles relative to carbon (hereinafter, “carbon electrode containing 5% of platinum particles”) used as the counter electrode 60.

[0597] A three-electrode electrolysis cell was constructed with a carbon electrode without platinum particles, a carbon electrode containing 1% platinum particles, and a carbon electrode containing 5% platinum particles as a working electrode, a silver-silver chloride (saturated KCl) electrode (manufactured by BAS) as a reference electrode, and a platinum electrode as a counter electrode, and each electrode was connected to a potentiostat. The working electrode, counter electrode, and reference electrode were immersed in phosphate buffered saline (PBS) (TAKARA BIOINC. PBS Tablets T9181; 0.14M NaCl, 0.0027M KCl, 0.010M PO 4 3＋ ), a potential in the range of ±0.5 V relative to the reference electrode was applied to the working electrode at a scanning rate of 10 mV / s, and cyclic voltammetry analysis was performed. The obtained cyclic voltammetry diagram is shown in Fig.58 shown.

[0598] The higher the content ratio of platinum particles, the greater the absolute value of the positive current value on the high potential side of the electrode, and the greater the absolute value of the negative current value on the low potential side. It can be considered that the negative current value on the low potential side is caused by the reduction of oxygen in PBS. This suggests that when using an electrode containing platinum particles as a working electrode, the increase in background noise, the decrease in current response value, the influence of external interference such as oxygen concentration depending on the environment becomes greater, and there is a possibility of reduced sensitivity.

[0599] On the other hand, when an electrode containing platinum particles is used as a counter electrode, a negative current with a large absolute value can flow at a low potential (potential with a small absolute value) in the negative (minus) potential region due to the increase in current caused by the reduction of oxygen, etc. Therefore, compared with a carbon electrode that does not contain platinum, an electrode containing platinum particles can efficiently function as a counter electrode with a smaller area, thereby contributing to the reduction of the size of the sensor. In addition, by driving the sensor of the present disclosure using an electrode containing platinum particles as a counter electrode at a low potential in the negative potential region, the burden on the constant potential instrument as a measuring device can also be reduced.

[0600] <Publication 3 / Experiment 5>

[0601] In order to further investigate the electrochemical characteristics of the carbon electrode without platinum particles and the carbon electrode containing 1% of platinum particles used in Experiment 4, the following experiment was conducted.

[0602] A three-electrode electrolysis cell was constructed with either a carbon electrode without platinum particles or a carbon electrode containing 1% platinum particles as a working electrode, a silver-silver chloride (saturated KCl) electrode (manufactured by BAS) as a reference electrode, and a platinum electrode as a counter electrode, and each electrode was connected to a constant potential meter. Amperometric measurement was performed: the working electrode, counter electrode, and reference electrode were immersed in phosphate buffered saline (PBS) (TAKARA BIO INC. PBS Tablets T9181; 0.14M NaCl, 0.0027M KCl, 0.010M PO 4 3＋ ), a potential of 0.3 V or -0.2 V relative to the reference electrode is applied to the working electrode, and the current value is measured over time. The measurement result of the current value per unit area of ​​the working electrode is as follows: Fig.59 shown.

[0603] Fig.59 A shows the measurement results when a carbon electrode without platinum particles was used as the working electrode. Fig.59 B shows the measurement results when a carbon electrode containing 1% of platinum particles is used as a working electrode. It is confirmed that the current value (absolute value) when a carbon electrode without platinum particles is used as a working electrode is significantly smaller than the current value (absolute value) when a carbon electrode containing 1% of platinum particles is used as a working electrode (the scales of the vertical axes of A and B are different). This result shows that as the working electrode conductive layers 11a and 11b, the conductive layer without platinum particles is suitable because the background current is small.

[0604] <Publication 3 / Experiment 6>

[0605] In the sensor of Experiment 2-1 described in Experiment 2 above, all conductive layers including the working electrode conductive layers 11 a and 11 b and the counter electrode 60 were carbon conductive layers that did not contain platinum particles.

[0606] On the other hand, the sensor of Experiment 2-2 is the sensor of Experiment 2-1, wherein the portion 60a of the counter electrode 60 in contact with the liquid sample to be measured is a carbon conductive layer whose surface is modified with platinum nanoparticles, and the working electrode conductive layers 11a and 11b are carbon conductive layers that do not contain platinum particles.

[0607] In this experiment 6, the sensor of experiment 2-1 or experiment 2-2 was immersed in the liquid sample of the composition described in experiment 1, and a voltage of 100 mV relative to the reference electrode 20 (Ag / AgCl) was applied to the first working electrode 10a and the second working electrode 10b, and the current value between the first working electrode 10a and the counter electrode 60 and the current value between the second working electrode 10b and the counter electrode 60 were continuously measured for a predetermined time. The measurement was performed with N=2.

[0608] The measurement results of the current values ​​of the first working electrode 10a and the second working electrode 10b are shown in FIG. Fig.60 A and B. Fig.60 The current value of the first working electrode 10a indicated by A corresponds to the glucose concentration. Fig.60 The current value of the second working electrode 10b shown in B corresponds to the lactic acid concentration. No significant difference was recognized in terms of responsiveness between the sensor of Experiment 2-2 in which the working electrode conductive layers 11a and 11b did not contain platinum particles and only the counter electrode 60 contained platinum particles, and the sensor of Experiment 2-1 in which neither the working electrode conductive layers 11a and 11b nor the counter electrode 60 contained platinum particles.

[0609] <Publication 3 / Experiment 7>

[0610] In the sensor of Experiment 3-1 described in Experiment 3 above, all conductive layers including the working electrode conductive layers 11 a and 11 b and the counter electrode 60 were carbon conductive layers that did not contain platinum particles.

[0611] In the sensor of Experiment 3-2, all conductive layers including the working electrode conductive layers 11 a and 11 b and the counter electrode 60 were carbon conductive layers containing 1 wt % of platinum particles relative to carbon.

[0612] In the sensor of Experiment 3-3, all conductive layers including the working electrode conductive layers 11 a and 11 b and the counter electrode 60 were carbon conductive layers containing 5 wt % of platinum particles relative to carbon.

[0613] In this experiment 7, the sensor of experiment 3-1, experiment 3-2 or experiment 3-3 was immersed in the liquid sample of the composition described in experiment 1, and a voltage of 100 mV relative to the reference electrode 20 (Ag / AgCl) was applied to the first working electrode 10a and the second working electrode 10b, and the current value between the first working electrode 10a and the counter electrode 60 and the current value of the second working electrode 10b were continuously measured for a predetermined time. The measurement was performed with N=2.

[0614] The measurement results of the current values ​​of the first working electrode 10a and the second working electrode 10b are shown in FIG. Fig.61 A and B. Fig.61 The current value of the first working electrode 10a indicated by A corresponds to the glucose concentration. Fig.61 The current value of the second working electrode 10b shown in B corresponds to the lactic acid concentration. Compared with the sensor of Experiment 3-2 in which the working electrode conductive layer 11a, 11b and the counter electrode 60 contain platinum particles at 1% by weight relative to carbon, no significant decrease in the current response value was observed, compared with the sensor of Experiment 3-1 in which the working electrode conductive layer 11a, 11b and the counter electrode 60 do not contain platinum particles. On the other hand, the sensor of Experiment 3-3 in which the working electrode conductive layer 11a, 11b and the counter electrode 60 contain platinum particles at 5% by weight relative to carbon, compared with the sensor of Experiment 3-1 in which the working electrode conductive layer 11a, 11b and the counter electrode 60 do not contain platinum particles, the current response value is significantly reduced. This suggests that the presence of platinum in the working electrode conductive layers 11a, 11b reduces the responsiveness of the sensor.

[0615] <Supplementary Notes Regarding the Third Publication>

[0616] Conventionally, there has been known a sensor including a working electrode and a counter electrode for electrochemically measuring a test substance (allanite) in a liquid sample such as a cell culture medium.

[0617] Such sensors are sometimes used for continuous monitoring of the substance to be tested by being immersed in a liquid sample for several days or more. In this case, in addition to having high detection sensitivity for the substance to be tested, it is also desired that the sensor has a low negative impact (e.g., cytotoxicity) on the cultured cells contained in the liquid sample.

[0618] When a sensor having a working electrode and a counter electrode and a reagent layer including a reagent such as an oxidoreductase and a mediator that participate in the redox reaction of the analyte is used to electrochemically measure the analyte in a liquid sample, it is found that hydrogen peroxide may be generated during long-term measurement. If the concentration of hydrogen peroxide in the liquid sample reaches a certain level or more, it may be toxic to cells.

[0619] Therefore, an object of the third disclosure is to provide a sensor including a working electrode and a counter electrode, which can suppress the generation of hydrogen peroxide during measurement or reduce the generated hydrogen peroxide without significantly reducing the sensitivity.

[0620] When the sensor involved in the third disclosure is used to measure the test substance in a liquid sample such as a cell culture fluid, the generation of hydrogen peroxide during the measurement can be suppressed or the generated hydrogen peroxide can be reduced without significantly reducing the sensitivity, thereby suppressing the increase in the concentration of hydrogen peroxide in the liquid sample. Therefore, the sensor involved in the third disclosure has little negative impact on the cells in the liquid sample and can be appropriately used for long-term continuous monitoring.

[0621] Therefore, in this specification, as the third disclosure, a sensor described in any one or more of the following Supplements 3-1 to 3-16 is disclosed.

[0622] (Appendix 3-1)

[0623] A sensor for measuring a substance to be tested in a liquid sample.

[0624] It comprises: a working electrode including a working electrode conductive layer, and a reagent layer disposed on the working electrode conductive layer and including a reagent participating in the redox reaction of the test substance; and

[0625] Counter electrode,

[0626] When the sensor is immersed in the liquid sample and the test substance in the liquid sample is electrochemically measured, the concentration of hydrogen peroxide in the liquid sample during the measurement can be maintained at less than 15 μM.

[0627] (Appendix 3-2)

[0628] The sensor according to Supplementary Note 3-1, wherein the counter electrode includes a catalyst for decomposing hydrogen peroxide at a portion in contact with the liquid sample.

[0629] (Appendix 3-3)

[0630] The sensor according to Supplementary Note 3-2, wherein the working electrode conductive layer does not contain a catalyst for decomposing hydrogen peroxide in a portion in contact with the liquid sample.

[0631] (Appendix 3-4)

[0632] The sensor according to Appendix 3-2 or 3-3, wherein the catalyst contains a metal.

[0633] (Notes 3-5)

[0634] The sensor according to Supplement 3-4, wherein the metal includes one or more selected from the group consisting of platinum, nickel, palladium, iron, manganese, and tungsten.

[0635] (Appendix 3-6)

[0636] The sensor according to Supplement 3-4, wherein the metal contains platinum.

[0637] (Appendix 3-7)

[0638] The sensor according to Appendix 3-6, wherein the platinum is platinum particles.

[0639] (Appendix 3-8)

[0640] The sensor according to Supplementary Note 3-7, wherein the portion of the counter electrode that contacts the liquid sample contains carbon and 1 wt % or more of the platinum particles relative to the carbon.

[0641] (Appendix 3-9)

[0642] The sensor according to Supplementary Note 3-8, wherein the portion of the counter electrode in contact with the liquid sample contains the carbon and 50 wt % or less of the platinum particles relative to the carbon.

[0643] (Note 3-10)

[0644] A sensor according to any one of Appendixes 3-1 to 3-9, wherein the working electrode conductive layer satisfies the following conditions: when a potential in the range of -0.2 V to 0.3 V (relative to the silver-silver chloride (saturated KCl) electrode) is applied to the working electrode conductive layer in phosphate buffered saline using a three-electrode electrolysis cell having the working electrode conductive layer as a working electrode, a silver-silver chloride (saturated KCl) electrode as a reference electrode, and a platinum electrode as a counter electrode, the measured current value is ±5.5 nA / mm 2 the following.

[0645] (Appendix 3-11)

[0646] The sensor according to Appendix 3-10, wherein the working electrode conductive layer is a carbon conductive layer.

[0647] (Appendix 3-12)

[0648] The sensor according to any one of Supplementary Notes 3-1 to 3-11, wherein a projected area of ​​the counter electrode is 250% or less relative to a projected area of ​​the working electrode conductive layer.

[0649] (Appendix 3-13)

[0650] The sensor according to Supplementary Note 3-12, wherein the projected area of ​​the counter electrode is 100% or less relative to the projected area of ​​the working electrode conductive layer.

[0651] (Appendix 3-14)

[0652] The sensor according to Supplementary Note 3-12 or 3-13, comprising a plurality of the working electrodes.

[0653] (Appendix 3-15)

[0654] The sensor according to any one of Appendixes 3-1 to 3-14, further comprising a protective film covering the counter electrode.

[0655] (Appendix 3-16)

[0656] The sensor according to Appendix 3-15, wherein the protective film includes a polymer compound containing a cation exchange functional group.

[0657] <Fourth disclosed embodiment>

[0658] The following is a description of an embodiment of the sensor involved in the fourth disclosure. In this embodiment, detailed descriptions beyond necessity are sometimes omitted. For example, detailed descriptions of matters that are already sufficiently known and repeated descriptions of substantially the same configurations. This is to avoid the following description from becoming unnecessarily lengthy and to make it easy for those skilled in the art to understand. It should be noted that for the configurations in the sensor involved in the fourth disclosure that are marked with the same symbols as the configurations in the sensor involved in the first disclosure, reference can be made to the description of the corresponding configurations in the sensor involved in the first disclosure.

[0659] In addition, the applicant provides the drawings and the following description in order to enable those skilled in the art to fully understand the fourth disclosure, and does not intend to limit the matters described in the following “Supplementary Notes Related to the Fourth Disclosure” by these.

[0660] <Materials>

[0661] Examples of materials that can be used for the sensor disclosed in the fourth aspect of this specification will be described.

[0662] The material of the insulating substrate used in the sensor disclosed in the fourth aspect of the present specification is not particularly limited, and for example, the same material as that of the insulating substrate of the sensor disclosed in the first aspect of the present specification may be used.

[0663] The conductive layer possessed by the working electrode, reference electrode and counter electrode of the sensor disclosed in the fourth aspect of this specification is a layer containing conductive materials such as carbon, gold, platinum, palladium, etc. The conductive layer can be manufactured by forming a layer of the conductive material as described above on the surface of the substrate using a sputtering method, an evaporation method, a screen printing method, etc. The conductive layer can be processed into a prescribed pattern using a laser trimming method as needed. In the fourth disclosure, the conductive layer of the working electrode is sometimes referred to as the working electrode conductive layer, and the conductive layer of the reference electrode is sometimes referred to as the reference electrode conductive layer. In the fourth disclosure, regarding the conductive layer of the counter electrode, since the conductive layer itself constitutes the counter electrode, it is only referred to as the counter electrode.

[0664] The wiring of the sensor disclosed in the fourth aspect of the present specification may be made of the same conductive material as the conductive layer.

[0665] The sensor disclosed in Article 4 of this specification can be used to detect a specified test substance (analyte) in the liquid sample by being immersed in a liquid sample. The liquid sample is preferably a liquid sample containing water as a solvent. As the liquid sample, a cell culture fluid and a liquid sample prepared using blood obtained from a living body can be cited. For examples of test substances, as described in the sensor disclosed in Article 1 of this specification about the test substance, it is particularly preferred that at least one selected from glucose and lactic acid.

[0666] The working electrode of the sensor disclosed in the fourth aspect of the present specification comprises: a reagent layer disposed on the working electrode conductive layer and containing a reagent participating in the redox reaction of the test substance. The reagent can be appropriately selected according to the test substance. As the reagent, a combination of an oxidoreductase and a mediator (electron transfer substance) can be included, or an oxidoreductase can be included. The oxidoreductase can contain a coenzyme.

[0667] Examples of the oxidoreductase include oxidase and dehydrogenase. Specific examples of the oxidoreductase are as described in the first disclosure of the present specification regarding the sensor, and glucose oxidase, lactate oxidase, glucose dehydrogenase, or lactate dehydrogenase is particularly preferred.

[0668] In addition, the medium is not particularly limited, and examples thereof are as described in the sensor disclosed in the first aspect of this specification.

[0669] The reagent layer of the working electrode of the sensor disclosed in the fourth aspect of this specification may further include components such as a buffer, a hydrophilic polymer compound, a conductive carbon filler, and a crosslinking agent in addition to the above reagents. Examples of the hydrophilic polymer compound and the conductive carbon filler are as described in the sensor disclosed in the first aspect of this specification.

[0670] The working electrode of the sensor disclosed in the fourth aspect of this specification further comprises a first protective film disposed on the reagent layer, and a second protective film disposed on the first protective film. The first protective film and the second protective film are sometimes collectively referred to as protective films. The protective film on the reagent layer can be a film that prevents or inhibits the reagent contained in the reagent layer from leaking out of the protective film and can penetrate the test substance present outside the protective film. The preferred mode of the material constituting the first protective film and the second protective film on the reagent layer having such properties is described later.

[0671] The reference electrode of the sensor disclosed in the fourth aspect of this specification may be provided with a protective film. The protective film of the reference electrode may contain a polymer compound. As such a polymer compound, the polymer compound described later contained in the first protective film or the second protective film disposed on the reagent layer of the working electrode can be exemplified, and the polymer compound contained in the second protective film is particularly preferred.

[0672] In the sensor disclosed in the fourth aspect of this specification, the electrodes including the working electrode and the counter electrode disposed on the insulating substrate, except for the portion in contact with the liquid sample, are preferably covered with an insulating layer. The insulating layer may include an insulating resin. The insulating layer may be a multilayer structure of two or more layers. The insulating layer of the multilayer structure may include, for example: a first insulating layer disposed on the insulating substrate and the conductive layer, and a second insulating layer disposed on the first insulating layer. Preferred embodiments of the materials constituting the first insulating layer and the second insulating layer are as described for the sensor involved in the first aspect.

[0673] <Overview of sensor 400>

[0674] A sensor 400 according to an embodiment of the fourth disclosure will be described with reference to the drawings.

[0675] The sensor 400 of this embodiment is as follows Figures 62-64As shown, it has: an insulating substrate 2, a first working electrode 10a, a second working electrode 10b, a reference electrode 20 and a counter electrode 30 arranged on the first surface 2a of the substrate 2, and wiring 50 electrically connected to the first working electrode 10a, the second working electrode 10b, the reference electrode 20 and the counter electrode 30, respectively. The sensor 400 of this embodiment includes two working electrodes, but in other embodiments not shown, the working electrode may be only one, or may be three or more. In the following description, when the first working electrode 10a and the second working electrode 10b are not distinguished, they are sometimes described as working electrodes 10a, 10b. In addition, the first working electrode 10a, the second working electrode 10b, the reference electrode 20 and the counter electrode 30 are sometimes collectively described as electrodes. In addition, the sensor 400 of this embodiment is a three-pole sensor including a working electrode, a reference electrode and a counter electrode as electrodes, but it may also be a two-pole sensor including only a working electrode and a counter electrode without a reference electrode. Although not shown in the figure, the reference electrode and / or the counter electrode may also be disposed on a substrate different from the substrate on which the working electrode is disposed.

[0676] Sensor 400 and sensor 1 disclosed in the first aspect Fig.21 The contents shown can be used similarly to immerse the sample in a liquid sample X to detect a predetermined test substance in the liquid sample X. Specific examples of the liquid sample and the test substance are described in the column of <Material>.

[0677] The first working electrode 10a of the sensor 400 is as follows Fig.64 As shown, it comprises: a working electrode conductive layer 11a, a reagent layer 15a containing a reagent participating in the redox reaction of the test substance in the liquid sample, a first protective film 18aa arranged on the reagent layer 15a, and a second protective film 18ab arranged on the first protective film 18aa. The preferred form of the reagent is as described in the column of <Materials>. It should be noted that although not shown in the figure, the cross-sectional structure of the second working electrode 10b of the sensor 400 is similar to Fig.64 The cross-sectional structure of the first working electrode 10a shown is substantially the same. Fig.64 In the embodiment, the structure is as follows: the first working electrode 10a is replaced by the second working electrode 10b, the working electrode conductive layer 11a is replaced by the working electrode conductive layer 11, the reagent layer 15a is replaced by the reagent layer 15b, the first protective film 18aa is replaced by the first protective film 18ba, the second protective film 18ab is replaced by the second protective film 18bb, and the protective film 18a is replaced by the protective film 18b.

[0678] When the reagent layers 15a, 15b of the working electrodes 10a, 10b of the sensor 400 contain a reagent that oxidizes the test substance in the liquid sample, electrons move from the test substance to the working electrode conductive layers 11a, 11b under the condition that a specified voltage is applied to the electrodes of the sensor 400. Similarly, when the reagent layers 15a, 15b contain a reagent that reduces the test substance in the liquid sample, electrons move from the working electrode conductive layers 11a, 11b to the test substance. The amount of electrons that move depends on the concentration of the test substance, so based on the current value or the change in the current value flowing through the working electrodes 10a, 10b of the sensor 400, the concentration of the test substance in the liquid sample or the concentration change can be measured.

[0679] The sensor 400 can constitute an analysis device for analyzing a substance to be tested in a liquid sample. Fig.24 The analysis device 100 shown in the figure may be an analysis device in which the sensor 1 of the first disclosure is replaced with the sensor 400 of the fourth disclosure, and includes the sensor 400, the analysis unit 102, and the control unit 104. The functions of the analysis device are as follows. Fig.24 The analysis device 100 shown is as described in the description.

[0680] <Structure of sensor 400>

[0681] For the structure of the sensor 400 according to the fourth disclosure, refer to Fig.62 and as its cross-sectional view Fig.63 and 64 It should be noted that the structure of the reference electrode 20 of the sensor 400 involved in the fourth disclosure is the same as the structure of the reference electrode 20 of the sensor 1 involved in the first disclosure. Fig. 22 The description of the reference electrode 20 of the sensor 1 according to the first disclosure is used as the description of the reference electrode 20 of the sensor 400 according to the fourth disclosure.

[0682] The sensor 400 includes: an insulating substrate 2, working electrode conductive layers 11a and 11b arranged on the first surface 2a of the substrate 2, a counter electrode 30, a reference electrode conductive layer 21, and wiring 50. It should be noted that in the embodiment of the sensor 400 involved in the fourth disclosure shown in FIG., the entire counter electrode 30 is formed by a conductive layer. In order to specify the portion of the conductive layer in the counter electrode, it may also be referred to as a "counter electrode conductive layer" or a "conductive layer of the counter electrode". A first insulating layer 3 is further arranged on the first surface 2a of the substrate 2, and a second insulating layer 4 is further arranged on the first insulating layer 3. The height of the upper surface of the first insulating layer 3 from the first surface 2a of the substrate 2 is higher than the height of the working electrode conductive layers 11a and 11b, the counter electrode 30, the reference electrode conductive layer 21 and the wiring 50 from the first surface 2a of the substrate 2. Therefore, a part of the first insulating layer 3 is arranged on the working electrode conductive layers 11 a and 11 b , the counter electrode 30 , the reference electrode conductive layer 21 , and the wiring 50 .

[0683] like Fig.63 As shown, the first insulating layer 3 includes a first opening portion 302 for the counter electrode formed at a position overlapping with a portion of the counter electrode 30 and penetrating along the thickness direction T of the substrate 2. Furthermore, the second insulating layer 4 includes a second opening portion 402 for the counter electrode formed at a position overlapping with the entire first opening portion 302 for the counter electrode of the first insulating layer 3 and penetrating along the thickness direction T of the substrate 2. The counter electrode 30 is exposed to the outside through the first opening portion 302 for the counter electrode and the second opening portion 402 for the counter electrode. When the sensor 400 is immersed in a liquid sample, the portion of the counter electrode 30 located at the bottom surface of the recess formed by the first opening portion 302 for the counter electrode contacts the liquid sample.

[0684] like Fig.64 As shown, the working electrodes 10a and 10b include working electrode conductive layers 11a and 11b, and reagent layers 15a and 15b disposed on the working electrode conductive layers 11a and 11b and containing reagents participating in the redox reaction of the test substance. Preferred embodiments of the reagents in the reagent layers 15a and 15b are as described in the column of <Materials>.

[0685] Similar to the first disclosure, in order to distinguish the "reagent layer 15a" of the first working electrode 10a from the "reagent layer 15b" of the second working electrode 10b, the former is sometimes expressed as the "first reagent layer 15a" of the first working electrode 10a, and the latter is expressed as the "second reagent layer 15b" of the second working electrode 10b. In addition, the reagent contained in the reagent layer 15a of the first working electrode 10a is sometimes expressed as the "first reagent", and the reagent contained in the second reagent layer 15b of the second working electrode 10b is sometimes expressed as the "second reagent". In a preferred embodiment, the sensor 400 includes a plurality of working electrodes 10a, 10b, and the first reagent contained in the first reagent layer 15a of the first working electrode 10a of the plurality of working electrodes 10a, 10b is different from the second reagent contained in the second reagent layer 15b of the second working electrode 10b different from the first working electrode 10a. In the sensor 400 involved in this preferred embodiment, since the first reagent can participate in the redox reaction of the first test substance in the liquid sample, and the second reagent can participate in the redox reaction of the second test substance in the liquid sample that is different from the first test substance, it can be used to detect multiple test substances in the liquid sample, including the first test substance and the second test substance.

[0686] like Fig.64 As shown, the working electrodes 10a and 10b include protective films 18a and 18b including first protective films 18aa and 18ba disposed on the reagent layers 15a and 15b, and second protective films 18ab and 18bb ​​disposed on the first protective films 18aa and 18ba.

[0687] like Fig.64 As shown, the first insulating layer 3 includes working electrode first openings 303a and 303b formed at positions overlapping with parts of the working electrode conductive layers 11a and 11b, respectively, and extending through the thickness direction T of the substrate 2. Furthermore, the second insulating layer 4 includes working electrode second openings 403a and 403b formed at positions overlapping with parts that entirely enclose the working electrode first openings 303a and 303b of the first insulating layer 3, and extending through the thickness direction T of the substrate 2. The reagent layers 15a and 15b are enclosed in the working electrode first openings 303a and 303b, and the outer peripheries of the reagent layers 15a and 15b are defined by the inner peripheries of the working electrode first openings 303a and 303b. The first protective film 18aa, 18ba and the second protective film 18ab, 18bb ​​are enclosed in the working electrode second openings 403a, 403b, and the outer peripheries of the first protective film 18aa, 18ba and the second protective film 18ab, 18bb ​​are defined by the inner peripheries of the working electrode second openings 403a, 403b.

[0688] <Features of the First Protective Film and the Second Protective Film of the Sensor 400>

[0689] The sensor 400 involved in the fourth disclosure is characterized in that the first protective films 18aa and 18ba contain a first polymer compound containing a cation exchange functional group, and the second protective films 18ab and 18bb ​​contain a second polymer compound containing a cationic functional group. Even when the sensor having this characteristic is used for continuous monitoring of a test substance for a long period of several days or more while being immersed in a liquid sample, the first protective film and the second protective film will not peel off, and stable measurement can be performed.

[0690] It has long been known that there is a sensor including a protective film for transporting cations such as protons between a reagent layer and a liquid sample, wherein the protective film has the following two-layer structure: a first protective film including a polymer compound containing a cation exchange functional group (e.g., a copolymer compound of tetrafluoroethylene and perfluoro-2-(2-fluorosulfonylethoxy)propyl vinyl ether) disposed on the reagent layer, and a second protective film including a polymer compound containing 4-vinylpyridine in a structural unit (e.g., P4VP-tBuMA (a block copolymer compound of poly-4-vinylpyridine and poly-tert-butyl methacrylate)) disposed further on the first protective film. The inventors of the present invention have found that when the sensor is immersed in a liquid sample and the test substance is continuously electrochemically measured, after a long period of more than 3 days, an abnormal value in which the detected current value changes significantly can be detected, resulting in a phenomenon in which a correct measurement cannot be performed. Moreover, in sensors showing abnormal values, peeling between the first protective film and the second protective film was confirmed.

[0691] Then, the inventors conducted intensive research and found that the above-mentioned problem can be solved by the sensor according to the fourth disclosure having the first protective film and the second protective film having the above-mentioned characteristics. It is considered that the first polymer compound of the first protective film contains a cation exchange functional group (i.e., anionic functional group), and the second polymer compound of the second protective film contains a cationic functional group, so that the first protective film and the second protective film are closely attached to each other at the interface by electrostatic coupling, thereby suppressing peeling.

[0692] In the fourth disclosure, the first polymer compound contained in the first protective film only needs to have a cation exchange functional group, and preferably has a cation exchange functional group on the side chain. As the cation exchange functional group, a sulfonic acid group is preferred. The first polymer compound having a sulfonic acid group on the side chain is preferably a polymer compound containing a perfluorinated compound having a sulfonic acid group on the side chain in the structural unit, more preferably a copolymer compound containing a perfluorinated compound having a sulfonic acid group on the side chain and a perfluorinated compound not having an ionic functional group on the side chain in the structural unit, particularly preferably a copolymer compound of tetrafluoroethylene and perfluoro-2-(2-fluorosulfonylethoxy)propyl vinyl ether, and most preferably Nafion (registered trademark).

[0693] In the fourth disclosure, the second polymer compound contained in the second protective film only needs to contain a cationic functional group, and preferably has a cationic functional group on the side chain. Since the pH of the liquid sample to be analyzed sometimes changes during long-term measurement, the cationic functional group in the second polymer compound is preferably a pH-independent cationic functional group, and particularly preferably a functional group containing a quaternary ammonium cation. The quaternary ammonium cation is preferably derived from the reaction of a tertiary amine and an epoxy group. As the tertiary amine, a pyridyl group can be cited, and in particular, a 4-pyridyl group can be cited.

[0694] The second polymer compound preferably comprises a first unit containing a cationic functional group and a second unit containing a hydrophobic functional group, and the ratio of the second unit relative to the total amount of the structural unit of the second polymer compound is preferably 50 mol% or more. Such a second polymer compound has high hydrophobicity, so even if the sensor involved in the fourth disclosure is immersed in a liquid sample of an aqueous system such as a cell culture fluid for a long time, the second protective film will not swell due to water, and it is easy to inhibit the peeling of the first protective film from the interface. The upper limit of the content of the second unit containing a hydrophobic functional group in the second polymer compound is not particularly limited, for example, the ratio of the second unit to the total amount of the structural unit of the second polymer compound can be 70 mol% or less. As a hydrophobic functional group, a saturated hydrocarbon group having 1 to 10 carbon atoms can be cited. For example, a methacrylate or acrylate derived from a monohydric alcohol containing a saturated hydrocarbon group having 1 to 10 carbon atoms is preferred, and as a specific example, a second unit derived from tert-butyl methacrylate is preferred.

[0695] The second polymer compound including the first unit containing a cationic functional group and the second unit containing a hydrophobic functional group can be obtained, for example, by reacting an uncrosslinked polymer compound including a third unit containing a tertiary amine and the second unit containing a hydrophobic functional group with a crosslinker compound containing two or more epoxy groups for a certain period of time, so that the tertiary amine of the third unit reacts with the epoxy group of the crosslinker compound, thereby converting the third unit into the first unit containing a quaternary ammonium cation. In this case, the epoxy group of the crosslinker compound is preferably reacted in an amount of 6.8 mol% or more, more preferably 23 mol% or less relative to the tertiary amine in the third unit (the epoxy group of the crosslinker compound is, for example, 6.8 mol% or more and 23 mol% or less relative to the tertiary amine in the third unit), so that the obtained second polymer compound has a high density of quaternary ammonium cations, and the electrostatic coupling between the first protective film and the second protective film becomes strong, which is preferred. Here, when calculating the ratio (mol %) of the epoxy group of the cross-linking agent compound to the tertiary amine of the third unit in the uncross-linked polymer compound, the molar number of the tertiary amine can be calculated based on the molecular weight of the monomer of the third unit, and the molar number of the epoxy group can be calculated based on the molecular weight of the cross-linking agent compound (when the cross-linking agent compound is a polymer compound, its number average molecular weight is regarded as a uniform molecular weight).

[0696] As the third unit containing a tertiary amine in the uncrosslinked polymer compound, a structural unit derived from 4-vinylpyridine is preferred.

[0697] As a preferred example of the above-mentioned uncrosslinked polymer compound, a copolymer compound of 4-vinylpyridine and tert-butyl methacrylate can be cited. The copolymer compound is more preferably: relative to the total amount of the third unit derived from 4-vinylpyridine and the second unit derived from tert-butyl methacrylate, preferably contains 50 mol% or more, more preferably 70 mol% or less of the second unit derived from tert-butyl methacrylate (the ratio of the above-mentioned second unit to the total amount of the above-mentioned third unit and the above-mentioned second unit is, for example, 50 mol% or more and 70 mol% or less). The copolymer compound of 4-vinylpyridine and tert-butyl methacrylate is particularly preferably a block copolymer compound. The molecular weight of the above-mentioned uncrosslinked polymer compound is not particularly limited, for example, the number average molecular weight (Mn) can be 150,000 to 500,000.

[0698] The crosslinking agent compound containing two or more epoxy groups is, for example, a linear compound having epoxy groups at both ends, preferably a polyalkylene glycol compound modified with glycidyl groups at both ends, specifically, poly(ethylene glycol) diglycidyl ether. The molecular weight of poly(ethylene glycol) diglycidyl ether is not particularly limited, for example, the number average molecular weight (Mn) may be 200 to 2000.

[0699] <Method of Manufacturing Sensor 400>

[0700] A preferred method for manufacturing the sensor 400 according to the third disclosure, wherein the second protective films 18ab and 18bb ​​include a second polymer compound, the second polymer compound including a first unit including a quaternary ammonium cation derived from a reaction between a tertiary amine and an epoxy group, and a second unit including a hydrophobic functional group, the manufacturing method comprising:

[0701] The first step of obtaining a second protective film-forming composition, wherein:

[0702] By maintaining a mixture of an uncrosslinked polymer compound containing a third unit containing a tertiary amine and a second unit containing a hydrophobic functional group in alcohol, and a crosslinker compound containing two or more epoxy groups under a temperature condition of 20° C. or higher for 6 hours or more, the tertiary amine of the third unit reacts with the epoxy group of the crosslinker compound, thereby converting the third unit into the first unit containing the quaternary ammonium cation, thereby obtaining a second protective film-forming composition containing the second polymer compound containing the first unit and the second unit in alcohol; and

[0703] A second step of covering the first protective films 18aa and 18ba of the sensor component with the second protective film-forming composition and drying the composition to form second protective films 18ab and 18bb ​​containing the second polymer compound, wherein:

[0704] The sensor component has:

[0705] Substrate 2,

[0706] The working electrode conductive layers 11a and 11b are arranged on the substrate 2.

[0707] Reagent layers 15a and 15b disposed on the working electrode conductive layers 11a and 11b, and

[0708] The first protective films 18aa and 18ba are arranged on the reagent layers 15a and 15b.

[0709] According to this method, in the first step, the tertiary amine of the third unit in the uncrosslinked polymer compound and the epoxy group of the crosslinker compound are fully crosslinked to obtain a second polymer compound with a high density of quaternary ammonium cations and a second protective film forming composition. The temperature condition in the first step is more preferably above 30°C, and further preferably above 40°C. The upper limit is preferably less than the boiling point of the alcohol, for example, when ethanol is used as the alcohol, it is preferably less than 78°C. The reaction time in the first step is preferably more than 12 hours, and the upper limit is not particularly limited, for example, it is less than 48 hours (the reaction time in the first step is, for example, more than 12 hours and less than 48 hours). As alcohol, ethanol, methanol, propanol are preferred, and ethanol is particularly preferred. The preferred embodiments of the uncrosslinked polymer compound and the crosslinker compound are as described above.

[0710] In the second step, the second protective film-forming composition is applied or dropped onto the first protective films 18aa and 18ba to cover them, and then dried by volatilizing alcohol as a solvent to form second protective films 18ab and 18bb.

[0711] In this method, since cross-linking is performed in advance in the first step, no cross-linking treatment is required after the second protective films 18ab and 18bb ​​are formed in the second step. Therefore, the risk of damage to the reagent layers 15a and 15b containing reagents such as enzymes due to heat during the cross-linking treatment can be reduced.

[0712] <Fourth disclosed embodiment>

[0713] In Experiments 8 to 10 of the sensor according to the fourth disclosure, as examples and comparative examples of the sensor according to the fourth disclosure, Fig.62 In the embodiment, a sensor 400 having a structure that does not include the second working electrode 10 b and includes only the first working electrode 10 a (hereinafter referred to as “working electrode 10 a ”) as a working electrode is used.

[0714] The characteristics of the sensors used in each experiment are described in Experiments 8 to 10. First, the materials and manufacturing methods common to the sensors used in each experiment are briefly described.

[0715] (Substrate)

[0716] As in the first disclosed embodiment, the Fig.62 A polyethylene terephthalate substrate having a shape shown in the middle figure and a thickness of 188 μm is used as the insulating substrate 2 .

[0717] (Conductive layer)

[0718] On the first surface 2a of the insulating substrate 2, a carbon paste is applied and heated at 140°C for 1 hour to form a carbon conductive layer with a thickness of 5 μm: Figure 1 The working electrode conductive layer 11 a , the reference electrode conductive layer 21 , and the counter electrode (counter electrode conductive layer) 30 are of the same shape, and wirings 50 are electrically connected to them respectively.

[0719] (1st insulation layer)

[0720] Next, a first insulating layer 3 having a thickness of 5 μm on the portion on the carbon conductive layer is laminated on the first surface 2a of the substrate 2 provided with the carbon conductive layer, wherein the first insulating layer 3 is formed of a fluororesin containing a copolymer of vinylidene fluoride and hexafluoropropylene and covers the first surface 2a of the substrate 2 and the carbon conductive layer. The method for manufacturing the first insulating layer 3 is as described in the first disclosed embodiment.

[0721] The working electrode first opening 303a on the working electrode conductive layer 11a of the first insulating layer 3 was formed into a circle with a diameter of 1.2 mm. The reference electrode first opening 301 was formed into a circle with a diameter of 1.1 mm, and the counter electrode first opening 302 was formed into a rectangle with a size of 1.8 mm x 2.1 mm.

[0722] (Second insulation layer)

[0723] Next, a composition containing a polyester resin and a fluorine-based surface modification additive containing a perfluoroalkyl group in a solvent was applied on the first insulating layer 3 and heated at 140° C. for 1 hour to laminate the second insulating layer 4 having a thickness of 40 μm.

[0724] The working electrode second opening 403a of the second insulating layer 4 on the working electrode conductive layer 11a was formed into a circle with a diameter of 2 mm. The reference electrode second opening 401 was formed into a circle with a diameter of 2 mm, and the counter electrode second opening 402 was formed into a rectangle with a size of 1.8 mm x 2.1 mm.

[0725] (Working electrode reagent layer)

[0726] In the first opening 303a of the working electrode of the first insulating layer 3, 0.4 mg of a glucose determination reagent solution containing a sodium phosphate buffer solution (pH 7.4) with a final concentration of 3 mM, a carbon black dispersion with a final concentration of 14 mg / mL (carbon black concentration), a polymer-bonded medium, a FAD-dependent glucose dehydrogenase with a final concentration of 3200 U / mL, and a cross-linking agent with a final concentration of 0.1 mM (concentration of the bonding site) is applied on the working electrode conductive layer 11a and dried to form a reagent layer 15a containing glucose dehydrogenase and a medium.

[0727] (Working electrode protective film)

[0728] In the second opening 403a of the working electrode of the second insulating layer 4, the first protective film 18aa is formed on the reagent layer 15a, and the second protective film 18ab is formed on the first protective film 18aa, thereby forming a protective film 18a having a two-layer structure including the first protective film 18aa and the second protective film 18ab. The specific method for manufacturing the first protective film 18aa and the second protective film 18ab is described in Experiments 8 to 10.

[0729] (reference electrode)

[0730] Since the cross-sectional structure of the reference electrode 20 is similar to that of the sensor disclosed in the first embodiment, Fig. 22 Same, so refer to Fig. 22 The following is an outline of the method for preparing the reference electrode 20. A silver-silver chloride paste is applied to the reference electrode conductive layer 21 in the reference electrode first opening 301 of the first insulating layer 3, and heated at 140° C. for 1 hour to form a silver-silver chloride layer 22. Next, a reference electrode protective film 23 is disposed on the silver-silver chloride layer 22 in the reference electrode second opening 401 of the second insulating layer 4 to form the reference electrode 20.

[0731] <Publication 4 / Experiment 8>

[0732] (Working electrode protective film)

[0733] To 23093.67 mg of a 21.5 wt% Nafion (registered trademark) dispersion (manufactured by Sigma-Aldrich), 6472.79 mg of ethanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 1226.46 mg of a 5 mol / L sodium hydroxide aqueous solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added to adjust the pH of the dispersion (neutralization treatment of the cation exchange group), and the precipitate was dissolved using a vortex mixer to prepare 30792.92 mg of a 16.12 wt% Nafion (registered trademark) dispersion. 0.6 mg of the obtained 16.12 wt% Nafion (registered trademark) dispersion was applied to the reagent layer 15a in the second opening 403a of the working electrode of the second insulating layer 4 and dried to form a first protective film 18aa containing Nafion (registered trademark) as a polymer compound containing a cation exchange functional group.

[0734] Next, the two components shown below were mixed in ethanol to give the final concentrations described, and reacted at room temperature (25°C ± 3°C) for about 1 hour to prepare two second protective film-forming compositions. P4VP-tBuMA is a block copolymer of poly-4-vinylpyridine (P4VP) of a predetermined molecular weight and poly-tert-butyl methacrylate (tBuMA) of a predetermined molecular weight.

[0735] The second protective film-forming composition of Experiment 8-1:

[0736] P4VP-tBuMA (Mn of poly-4-vinylpyridine: 74,000, Mn of poly-tert-butyl methacrylate: 87,000, Mw / Mn: 1.16, manufactured by Polymer Source), final concentration: 5.72 wt%

[0737] PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ~1000, manufactured by Sigma-Aldrich), final concentration 0.91 wt%

[0738] The second protective film-forming composition of Experiment 8-2:

[0739] P4VP-tBuMA (Mn of poly-4-vinylpyridine: 120,000, Mn of poly-tert-butyl methacrylate: 270,000, Mw / Mn: 1.15, manufactured by Polymer Source), final concentration: 5.72 wt%

[0740] PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ~1000, manufactured by Sigma-Aldrich), final concentration 2.0 wt%

[0741] On the first protective film 18aa in the second opening 403a of the working electrode of the second insulating layer 4, 0.65 mg of the second protective film forming composition of Experiment 8-1 or Experiment 8-2 was applied and dried to form the second protective film 18ab of Experiment 8-1 or Experiment 8-2.

[0742] The sensor 400 including the second protective film 18ab of Experiment 8-1 was used as the sensor of Experiment 8-1.

[0743] The sensor 400 including the second protective film 18ab of Experiment 8-2 was used as the sensor of Experiment 8-2.

[0744] As a liquid sample containing the test substance, RPMI-1640 Medium (R1383 manufactured by Sigma-Aldrich), glucose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and sodium lactate (manufactured by Sigma-Aldrich) were used. As the final concentration, 30 mM glucose and 15 mM lactic acid were contained, and the pH was adjusted to 6.5 to prepare the RPMI medium.

[0745] The sensor of Experiment 8-1 or Experiment 8-2 was immersed in the liquid sample, a voltage of 100 mV was applied to the working electrode 10a relative to the reference electrode 20 (Ag / AgCl), and the current value between the working electrode 10a and the counter electrode 30 was continuously measured for about 14 days. The measurement was performed with N=12.

[0746] The current value is measured as follows Fig.65 shown. Fig.65 A shows the measurement results using the sensor of Experiment 8-1, Fig.65 B shows the measurement results using the sensor of Experiment 8-2.

[0747] In a sample of a portion of the sensor of Experiment 8-1 having a second protective film 18ab formed from a second protective film-forming composition comprising P4VP-tBuMA containing P4VP with Mn: 74000 and tBuMA with Mn: 87000 and containing PEGDGE at a final concentration of 0.91 wt %, an abnormally high current value was detected more than 4 days after the start of measurement.

[0748] On the other hand, in the sensor of Experiment 8-2 having a second protective film 18ab formed from a second protective film forming composition comprising P4VP-tBuMA containing P4VP with Mn: 120,000 and tBuMA with Mn: 270,000 and containing PEGDGE at a final concentration of 2.0 wt%, no abnormally high current value was detected even after more than 4 days from the start of the measurement.

[0749] This result suggests that the occurrence of abnormally high current values ​​in long-term measurement can be suppressed by increasing the ratio of the unit derived from tBuMA containing a hydrophobic functional group and the concentration of the cross-linking agent compound in the second protective film 18ab.

[0750] <Publication 4 / Experiment 9>

[0751] As the composition of the second protective film-forming composition, P4VP-tBuMA and PEGDGE were contained in ethanol at the following final concentrations, and two compositions were designed. It should be noted that, regarding the fourth disclosure, in the experiment shown below, the molar number of pyridine derived from P4VP calculated based on the molecular weight of the monomer of P4VP and the molar number of epoxy groups derived from PEGDGE calculated based on the number average molecular weight of PEGDGE (molecular weight of PEGDGE considered uniform) were used in the calculation of the ratio (mol %) of epoxy groups derived from PEGDGE to pyridine derived from P4VP.

[0752] Composition A (10.17 mol % of epoxy groups derived from PEGDGE relative to pyridine derived from 4-vinylpyridine):

[0753] P4VP-tBuMA (Mn of poly-4-vinylpyridine: 120,000, Mn of poly-tert-butyl methacrylate: 270,000, Mw / Mn: 1.15, manufactured by Polymer Source), final concentration: 5.72 wt%

[0754] PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ~1000, manufactured by Sigma-Aldrich), final concentration 0.9 wt%

[0755] Composition B (6.77 mol % of epoxy groups derived from PEGDGE relative to pyridine derived from 4-vinylpyridine):

[0756] P4VP-tBuMA (Mn of poly-4-vinylpyridine: 120,000, Mn of poly-tert-butyl methacrylate: 270,000, Mw / Mn: 1.15, manufactured by Polymer Source), final concentration: 5.72 wt%

[0757] PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ~1000, manufactured by Sigma-Aldrich), final concentration 0.6 wt%

[0758] A mixture containing the components of composition A in ethanol was kept at 40° C. for 16 hours to perform crosslinking in advance, thereby preparing a second protective film-forming composition of Experiment 9-1.

[0759] A mixture containing the components of composition B was stored in ethanol at 40° C. for 16 hours to perform crosslinking in advance, thereby preparing a second protective film-forming composition of Experiment 9-2.

[0760] A mixture containing the components of composition B in ethanol was kept at 53° C. for 16 hours to perform crosslinking in advance, thereby preparing a second protective film-forming composition of Experiment 9-3.

[0761] As the second protective film forming composition, except that the second protective film forming composition of Experiment 9-1, Experiment 9-2, and Experiment 9-3 was used to form the second protective film 18ab of the working electrode 10a, the sensors of Experiment 9-1, Experiment 9-2, and Experiment 9-3 were prepared through the same steps as Experiment 8.

[0762] The sensor of Experiment 9-1, Experiment 9-2 or Experiment 9-3 was immersed in the same liquid sample as that used in Experiment 8, and a voltage of 100 mV was applied to the working electrode 10a relative to the reference electrode 20 (Ag / AgCl), and the current value between the working electrode 10a and the counter electrode 30 was continuously measured for about 14 days. The measurement was performed with N = 8 for Experiment 9-1, N = 12 for Experiment 9-2, and N = 4 for Experiment 9-3.

[0763] The current value is measured as follows Fig.66 shown. Fig.66 A shows the measurement results using the sensor of Experiment 9-1, Fig.66 B shows the measurement results using the sensor of Experiment 9-2, Fig.66 C shows the measurement results using the sensor of Experiment 9-3.

[0764] It is suggested that in a sensor having a second protective film 18ab formed by pre-crosslinking a second protective film-forming composition obtained by mixing P4VP-tBuMA and PEGDGE and reacting them for a certain period of time on a first protective film 18aa containing Nafion (registered trademark) having a cation exchange functional group, the occurrence of abnormally high current values ​​in long-term measurements can be suppressed. In addition, it is suggested that the occurrence of abnormally high current values ​​in long-term measurements can be further suppressed by increasing the temperature during pre-crosslinking.

[0765] <Publication 4 / Experiment 10>

[0766] As the composition of the second protective film-forming composition, P4VP-tBuMA and PEGDGE were contained in ethanol at the following final concentrations, and three compositions were designed.

[0767] Composition C (22.67 mol % of epoxy groups derived from PEGDGE relative to pyridine derived from 4-vinylpyridine):

[0768] P4VP-tBuMA (Mn of poly-4-vinylpyridine: 120,000, Mn of poly-tert-butyl methacrylate: 270,000, Mw / Mn: 1.15, manufactured by Polymer Source), final concentration: 5.72 wt%

[0769] PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ~1000, manufactured by Sigma-Aldrich), final concentration 2.0 wt%

[0770] Composition D (10.17 mol % of epoxy groups derived from PEGDGE relative to pyridine derived from 4-vinylpyridine):

[0771] P4VP-tBuMA (Mn of poly-4-vinylpyridine: 120,000, Mn of poly-tert-butyl methacrylate: 270,000, Mw / Mn: 1.15, manufactured by Polymer Source), final concentration: 5.72 wt%

[0772] PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ~1000, manufactured by Sigma-Aldrich), final concentration 0.9 wt%

[0773] Composition E (6.77 mol % of epoxy groups derived from PEGDGE relative to pyridine derived from 4-vinylpyridine):

[0774] P4VP-tBuMA (Mn of poly-4-vinylpyridine: 120,000, Mn of poly-tert-butyl methacrylate: 270,000, Mw / Mn: 1.15, manufactured by Polymer Source), final concentration: 5.72 wt%

[0775] PEGDGE (poly(ethylene glycol) diglycidyl ether, Mn: ~1000, manufactured by Sigma-Aldrich), final concentration 0.6 wt%

[0776] A mixture containing the components of composition C in ethanol was preliminarily cross-linked by being kept at room temperature (25° C.±3° C.) for about 1 hour, thereby preparing a second protective film-forming composition of Experiment 10-1.

[0777] A mixture containing the components of the composition D in ethanol was previously cross-linked by being kept at 40° C. for 16 hours, thereby preparing a second protective film-forming composition of Experiment 10-2.

[0778] A mixture containing the components of the composition E in ethanol was preliminarily cross-linked by being kept at 53° C. for 16 hours, thereby preparing a second protective film-forming composition of Experiment 10-3.

[0779] As the second protective film forming composition, except using the second protective film forming composition of Experiment 10-1, Experiment 10-2, and Experiment 10-3 to form the second protective film 18ab of the working electrode 10a, the sensors of Experiment 10-1, Experiment 10-2, and Experiment 10-3 were prepared through the same steps as Experiment 8.

[0780] Next, the sensors of Experiment 10-1, Experiment 10-2, and Experiment 10-3 were stored at 60° C. for 14 days in the presence of a desiccant. The purpose of this storage is to accelerate deterioration.

[0781] Each sensor stored at 60°C for 14 days was immersed in the same liquid sample as that used in Experiment 8, a voltage of 100 mV was applied to the working electrode 10a relative to the reference electrode 20 (Ag / AgCl), and the current value between the working electrode 10a and the counter electrode 30 was measured for 10 consecutive days. The measurement was performed with N=4. The current value 24 hours after the start of the measurement (the average of N=4) was recorded as 100%, and the current value measured at the time point thereafter (the average of N=4) was expressed as a relative value.

[0782] The current value is measured as follows Fig.67 shown.

[0783] It was confirmed that the sensors of Experiments 10-2 and 10-3, which had a second protective film 18ab on a first protective film 18aa comprising Nafion (registered trademark) containing a cation exchange functional group, had a smaller change in current value after the temperature acceleration test than the sensor of Experiment 10-1, and the second protective film 18ab was formed using a second protective film-forming composition that was cross-linked in advance by mixing P4VP-tBuMA and PEGDGE and reacting them for more than 6 hours. In addition, it was confirmed that the change in current value after the temperature acceleration test can be further reduced by increasing the temperature during the pre-crosslinking. These results suggest that a sensor with excellent long-term stability can be realized by forming a second protective film 18ab on a first ...

Claims

1. A sensor, characterized in that: A substrate having insulating properties, and a working electrode disposed on the substrate, The working electrode comprises: a conductive layer disposed on the substrate; a first insulating layer at least partially disposed on the conductive layer, the first insulating layer having a first opening extending through the thickness direction and formed at a position overlapping with a portion of the conductive layer when viewed from above in the thickness direction of the substrate, and a water-repellent surface; a second insulating layer disposed on the first insulating layer, the second insulating layer having a second opening extending through the thickness direction and formed at a position overlapping with a portion of the first insulating layer that entirely encloses the first opening when viewed from above in the thickness direction, and a surface that is liquid-repellent to alcohol, a reagent layer disposed in the first opening of the first insulating layer, the reagent layer including an outer periphery defined according to an inner periphery of the first opening of the first insulating layer and a reagent participating in a redox reaction, A protective film is disposed in the second opening of the second insulating layer, the protective film including an outer peripheral edge defined according to an inner peripheral edge of the second opening of the second insulating layer.

2. The sensor according to claim 1, wherein: The surface of the first insulating layer includes a fluororesin.

3. The sensor according to claim 1 or 2, wherein: The surface of the second insulating layer includes a compound containing a perfluoroalkyl group.

4. The sensor according to any one of claims 1 to 3, comprising a plurality of the working electrodes. A first reagent contained in a first reagent layer of a first working electrode included in the plurality of working electrodes and a second reagent contained in a second reagent layer of a second working electrode different from the first working electrode are different from each other.

5. The sensor according to claim 4, wherein: The first reagent contains glucose dehydrogenase or glucose oxidase, The second reagent contains lactate oxidase or lactate dehydrogenase.

6. The sensor according to any one of claims 1 to 5, wherein: The protective film includes a first protective film including a polymer compound containing 4-vinylpyridine in a structural unit.

7. The sensor according to any one of claims 1 to 6, wherein: The protective film includes a second protective film disposed on the reagent layer and a third protective film disposed on the second protective film, the second protective film includes a polymer compound containing a cation exchange functional group, and the third protective film includes a polymer compound containing 4-vinylpyridine in a structural unit. 8 . The sensor according to claim 1 , further comprising a counter electrode and a reference electrode disposed on the substrate.

9. A method, which is a method for manufacturing a sensor according to any one of claims 1 to 8, characterized in that include: forming droplets of a liquid composition A containing the reagent in water in the first opening of the first insulating layer of the substrate provided with the conductive layer, the first insulating layer, and the second insulating layer, and then drying the droplets to form the reagent layer; as well as After the formation of the reagent layer, droplets of the liquid composition B containing protective film components in alcohol are formed in the second opening of the second insulating layer and then dried to form the protective film.

10. The method according to claim 9, wherein: The surface of the first insulating layer includes a fluororesin.

11. The method according to claim 9 or 10, wherein: The surface of the second insulating layer includes a compound containing a perfluoroalkyl group.

12. The method according to any one of claims 9 to 11, wherein: The sensor comprises a plurality of working electrodes. The formation of the reagent layer comprises: forming droplets of a first liquid composition A containing a first reagent in water in the first opening of the first insulating layer of the first working electrode, and then drying the droplets to form a first reagent layer of the first working electrode included in the plurality of working electrodes; and Droplets of a second liquid composition A containing a second reagent different from the first reagent in water are formed in the first opening of the first insulating layer of the second working electrode, and then dried to form a second reagent layer of a second working electrode different from the first working electrode contained in a plurality of the working electrodes.

13. The method according to claim 12, wherein: The first reagent contains glucose dehydrogenase or glucose oxidase, The second reagent contains lactate oxidase or lactate dehydrogenase.

14. The method according to any one of claims 9 to 13, wherein: The protective film includes a first protective film disposed on the reagent layer, the first protective film includes a polymer compound containing 4-vinylpyridine in a structural unit, The forming of the protective film comprises: After the reagent layer is formed, droplets of the first liquid composition B containing the polymer compound containing 4-vinylpyridine in a structural unit in alcohol are formed in the second opening of the second insulating layer and then dried to form the first protective film.

15. The method according to any one of claims 9 to 14, wherein: The protective film comprises: a second protective film disposed on the reagent layer and a third protective film disposed on the second protective film, the second protective film comprises a polymer compound containing a cation exchange functional group, and the third protective layer comprises a polymer compound containing 4-vinylpyridine in a structural unit. The forming of the protective film comprises: After forming the reagent layer, droplets of a second liquid composition B containing the polymer compound having a cation exchange functional group in alcohol are formed in the second opening of the second insulating layer, and then dried to form the second protective film; and After the formation of the second protective film, droplets of the third liquid composition B containing the polymer compound containing 4-vinylpyridine in the structural unit in alcohol are formed in the second opening of the second insulating layer of the second working electrode, and then dried to form the third protective film.

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