A reagent layer containing conductive carbon filler, a sensor having the reagent layer, and a method for forming the reagent layer.

Abstract

The present invention addresses the problem of providing: a reagent layer for preparing an electrochemical sensor which can detect an analyte with high sensitivity and / or is excellent in durability and preferable for measurement (continuous monitoring) over a long period; and a method for forming same. The reagent layer according to the present invention comprises a conductive carbon filler (a), an anionic dispersant (b), and a cationic mediator (c). The method for forming the reagent layer according to the present invention involves: (1) a step for preparing a reagent liquid including (a) a conductive carbon filler, (b) an anionic dispersant, and (c) a cationic mediator; (2) a step for applying the reagent liquid to a reagent layer forming part; and (3) a step for drying the applied reagent liquid to form a reagent layer.

Description

[Technical Field]

[0001] The present invention relates to a reagent layer and a sensor containing a conductive carbon filler. More specifically, the present invention relates to a conductive carbon filler dispersant as an agent for improving the dispersibility of the conductive carbon filler in an aqueous solvent, a reagent layer containing the dispersant, the conductive carbon filler and a cationic mediator, a sensor equipped with the reagent layer, and a method for forming the reagent layer. [Background technology]

[0002] Conventionally, sensors that measure analytes in a sample by reacting them with proteins are known. Examples of such sensors include enzyme-based electrochemical sensors, such as glucose sensors made using glucose oxidoreductase and, if necessary, redox mediators (oxidox substances that mediate electron transport) or redox polymers (polymers to which redox mediators are attached via linkers, etc.). Glucose sensors are used, for example, for self-testing of blood glucose levels. Traditionally, it was common to collect a small amount of blood as a sample, but in recent years, implantable electrochemical glucose sensors that are implanted in the body to continuously measure glucose in the blood or interstitial space have also been developed. Glucose sensors are also used to measure glucose in samples outside of biological materials, such as in culture media. Such glucose sensors generally measure the glucose concentration in a sample continuously or semi-continuously over long periods, such as several days to several weeks.

[0003] In recent years, conductive carbon fillers with high specific surface area, such as nanocarbon materials, have been used in electrodes for glucose sensors, typified by electrochemical glucose sensors, and in biofuel cells, with the aim of achieving higher sensitivity and higher output by improving the specific surface area of ​​the electrodes. For example, Patent Document 1 discloses a sensor made from a carbon black dispersion using hydroxypropyl cellulose, and Patent Document 2 discloses an enzyme-immobilized electrode using ethyl cellulose as a binder and carbon particles. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2021-082394 [Patent Document 2] WO2013 / 065581 publication [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] Generally, to form a reagent layer containing conductive carbon filler and redox mediator or redox polymer, and optionally further containing oxidoreductase, etc., on a working electrode (enzyme electrode), a reagent solution is prepared by dispersing these necessary components in an aqueous solvent, applying the reagent solution to the desired site, and drying it. However, conductive carbon filler is generally highly hydrophobic, making it difficult to disperse in aqueous solvents, and aggregation due to van der Waals forces is prone to occur, making it difficult to maintain a dispersed state. If a reagent layer is formed using a reagent solution in which the conductive carbon filler is not sufficiently dispersed, problems such as a significant decrease in the detection sensitivity of the analyte and poor durability in long-term measurements (continuous monitoring) in humid environments such as in vivo or in culture media may occur. It should be noted that oxidoreductase should be dissolved in an aqueous solvent, and it is not appropriate to use organic solvents as solvents for preparing the reagent solution.

[0006] In one aspect, the present invention aims to provide a reagent layer and a method for forming the same for creating an electrochemical sensor capable of detecting analytes with high sensitivity. In another aspect, the present invention aims to provide a reagent layer and a method for forming the same for creating a durable electrochemical sensor suitable for long-term measurement (continuous monitoring). [Means for solving the problem]

[0007] The inventors of the present application have found that in a reagent solution prepared preferably under specific conditions using a conductive carbon filler and a cationic redox mediator or redox polymer (cationic mediator) and an anionic dispersant, aggregation and precipitation of the conductive carbon particles do not occur and the dispersion state is good. A reagent layer formed using such a reagent solution can adsorb the cationic mediator closely onto an electrode. Therefore, the electrochemical sensor has improved detection sensitivity for an analyte and is excellent in durability for continuous monitoring, and thus the present invention has been completed.

[0008] That is, the present invention includes at least the following matters. [Item 1] A reagent layer containing a conductive carbon filler (a), an anionic dispersant (b), and a cationic mediator (c). [Item 2] The reagent layer according to Item 1, wherein the anionic dispersant (b) is a polymer having a weight average molecular weight of 70,000 or less. [Item 3] The reagent layer according to Item 1, wherein the anionic dispersant (b) is a polymer having a carboxy group and / or a sulfo group in a side chain. [Item 4] The reagent layer according to Item 3, wherein the anionic dispersant (b) is a polymer containing at least one selected from the group consisting of units derived from acrylic acid, units derived from maleic acid, and units derived from styrene sulfonic acid. [Item 5] The reagent layer according to Item 1, wherein the cationic mediator (c) is a compound in which a redox mediator compound (c1) and a cationic polymer (c2) are bonded via a linker part (c3) as required. [Item 6] The reagent layer according to Item 5, wherein the cationic polymer (c2) has a quaternary ammonium cation group. [Item 7] The reagent layer according to Item 1, wherein the conductive carbon filler (a) is carbon black. [Item 8] The reagent layer according to item 1, further comprising an oxidoreductase (e) for oxidizing or reducing the analyte. [Section 9] The reagent layer according to item 8, wherein the oxidoreductase (e) is of the coenzyme-bound form. [Section 10] The reagent layer according to item 8, wherein the oxidoreductase (e) is crosslinked with the cationic polymer (c2). [Section 11] An electrochemical sensor for detecting or quantifying analytes, comprising a working electrode, a counter electrode, and a reagent layer according to any one of items 1 to 10. [Section 12] The electrochemical sensor according to item 11, further comprising a reference electrode. [Section 13] Furthermore, the electrochemical sensor according to claim 11, further comprising a protective film covering at least the reagent layer. [Section 14] (1) A step of preparing a reagent solution containing a conductive carbon filler (a), an anionic dispersant (b), and a cationic mediator (c), (2) A step of applying the reagent solution to the reagent layer forming area, (3) A step of drying the applied reagent solution to form a reagent layer. A method for forming a reagent layer, including [a specific component]. [Section 15] The method for forming a reagent layer according to item 14, wherein the pH of the reagent solution is 8.0 or less. [Section 16] The method for forming a reagent layer according to item 15, wherein the concentration of metal ions in the reagent solution is 200 mM or less. [Section 17] The method for forming a reagent layer according to item 16, wherein the metal ion in the reagent solution is an alkali metal ion and its concentration is less than 100 mM. [Section 18] The method for forming a reagent layer according to claim 14, wherein the anionic dispersant (b) is a polymer comprising at least one selected from the group consisting of acrylic acid-derived units, maleic acid-derived units, and styrene sulfonic acid-derived units, having a weight-average molecular weight of 70,000 or less. [Section 19] The method for forming a reagent layer according to item 14, wherein the cationic mediator (c) is a compound in which a redox mediator compound (c1) and a cationic polymer (c2) having a quaternary ammonium cationic group are bonded via a linker portion (c3) as needed. [Effects of the Invention]

[0009] The present invention makes it possible to effectively disperse conductive carbon fillers in reagent solutions containing aqueous solvents. Furthermore, by using such a reagent solution (carbon dispersion) containing conductive carbon fillers, it becomes possible to manufacture highly practical electrochemical sensors that achieve improved sensitivity and durability (for example, the ability to maintain responsiveness during measurement in a humid environment) while still containing conductive carbon fillers.

[0010] In this invention, it is possible to determine whether the conductive carbon material is "dispersed" (including "redispersed") or "aggregated" (including "aggregated / precipitated") in the reagent solution by visual inspection in most cases. For example, if the conductive carbon material is "aggregated," the floating or precipitated aggregates can be easily identified visually.

[0011] Centrifugation can also be used to distinguish between the dispersion and aggregation of conductive carbon fillers. For example, when a reagent solution in which conductive carbon fillers are "dispersed" is centrifuged, no precipitate of conductive carbon fillers is formed. However, when a reagent solution in which conductive carbon fillers are "aggregated" is centrifuged, aggregates precipitate, separating the conductive carbon filler layer from a clear aqueous solution phase. The centrifugation conditions can be, for example, 10,000 × g for 5 minutes, but can be appropriately adjusted depending on the embodiment, taking into consideration, for example, the type and concentration of conductive carbon fillers. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 is a plan view of a sensor in one embodiment of the present invention. Figure 1(A) shows the entire sensor, and Figure 1(B) shows a magnified view of the tip portion of the sensor. [Figure 2] Figure 2 is a cross-sectional view of the sensor in a specific part of Figure 1(B). Figure 2(A) is a cross-sectional view taken along the arrow AA in Figure 1(B). Figure 2(B) is a cross-sectional view taken along the arrow BB in Figure 1(B). Figure 2(C) is a cross-sectional view taken along the arrow CC in Figure 1(B). [Figure 3] Figure 3 is a top view showing another example of the front side (the side having the working electrode and the reference electrode) of a sensor in one embodiment of the present invention. [Figure 4] Figure 4 is a cross-sectional view along the line A-A' in Figure 3. [Figure 5] Figure 5 is a cross-sectional view along the B-B' line in Figure 4. [Figure 6] Figure 6 is a cross-sectional view along the C-C' line in Figure 4. [Figure 7] Figure 7 is a plan view of a sensor according to one embodiment of the present invention. Figure 7(A) shows the electrode pattern before the film (insulating resist film) is formed, and Figure 7(B) shows the electrode pattern after the film is formed. [Figure 8]Figure 8 shows images of the recovered solution samples, derived from the various reagent solution samples prepared in Test Example 1, reflecting the dispersion effect of carbon black. [A] Recovered solution samples corresponding to reagent solution samples 1-1 to 1-8. [B] Recovered solution samples corresponding to reagent solution samples 1-9 to 1-11. [Figure 9] Figure 9 shows the measurement results regarding the sensor responsiveness in Test Example 6. [A] Current response value results. [B] Cyclic voltammetry results. [Figure 10] Figure 10 shows the measurement results regarding the durability of the sensor in Test Example 7. [A] Results for a sensor made using poly(acrylic acid). [B] Results for a sensor made using hydroxypropyl cellulose. [Explanation of symbols]

[0013] 11. Sensor (Probe) 21 circuit boards 22 electrodes 22a Working electrode 22b Reference pole 22c opposite pole 23 Reagent layer 24 Silver / Silver Chloride Layer 25 film X1 Sensor Head Direction of insertion of the X2 sensor into the body X3 The opposite region, not covered by film. Uncovered area of ​​the X4 sensor head. X5 Region where the reagent layer is not formed X6 Area where the reagent layer and electrode (working electrode) are not formed (trimmed off). 101 Sensor 111 Insulating substrate 112 Conductive Thin Film 112a Working electrode area 112b Reference pole area 112c Counter-polar region 113 Groove 114 Working electrode 115 Reference pole 116a Insulating resist film (top surface) 116b Insulating resist film (bottom surface) 117 Opposite 118 Reagent layer 119 Protective film 121 Sensing Unit 122 Terminal section 201 Sensor 202 Sensing Department 203 Terminal section 204 Notch 210 circuit boards 220 electrode 221 First working electrode 221a Exposed region in the sensing part of the first working electrode 221b Exposed region at the terminal portion of the first working electrode 222 Second working electrode 222a Exposed region in the sensing part of the second working electrode 222b Exposed region at the terminal portion of the second working electrode 223 Reference pole 223a Exposed area in the sensing section of the reference pole 223b Exposed area at the terminal portion of the reference pole 224 Opposite poles 224a Exposed area in the opposite sensing section 224b Exposed area at the terminal portion of the opposite pole 225 Groove 230 Film (insulating resist film) [Modes for carrying out the invention]

[0014] — Reagent layer — The reagent layer of the present invention comprises a conductive carbon filler (a), an anionic dispersant (b), and a cationic mediator (c). The reagent layer of the present invention is a layer that can be formed by a formation method using a carbon dispersion as described later, i.e., a reagent solution in which the conductive carbon filler (a) is dispersed, and preferably a layer in which the conductive carbon filler (a), anionic dispersant (b), and cationic mediator (c) are in a uniform state.

[0015] (a) Conductive carbon filler The "conductive carbon filler" in this invention is not limited to a specific type, and various conductive carbon fillers can be used depending on the embodiment of the invention (e.g., reagent layer, electrochemical sensor application), effects, etc. Conductive carbon materials of various shapes such as spherical (particulate), flake, fibrous, and porous can be used as conductive carbon fillers. Conductive carbon filler (a) may be of any one type, or two or more types may be used in combination (for example, mixed).

[0016] Examples of conductive carbon fillers include carbon black, graphite powder, porous carbon materials, and nanocarbon materials. Specific examples of carbon black include furnace black, thermal black, acetylene black, Ketjen black, and channel black. Specific examples of graphite powder include pyrolytic graphite and spheroidal graphite. Specific examples of porous carbon materials include not only activated carbon powder or activated carbon fibers with mesopores of 2-50 nm, but also carbon materials with interconnected mesopores. Specific examples of nanocarbon materials include single-walled carbon nanotubes, multi-walled carbon nanotubes, graphene, fullerenes, carbon nanohorns, and carbon nanocoils. The size of the conductive carbon filler (a) should be within an appropriate range. For example, if the conductive carbon filler is spherical (particulate), its average particle size (e.g., the average value measured for a predetermined number of particles using an electron microscope) is generally in the range of 10 nm to 20 μm. The BET specific surface area of ​​the conductive carbon filler is, for example, 10 m². 2 It is 1 / g or more, preferably 30m 2 The amount is 1 / g or more. The conductive carbon filler (a) in the present invention is preferably a spherical (particulate) carbon filler, such as carbon black.

[0017] (b) Anionic dispersants In the present invention, "anionic dispersant" refers to a compound that has the essential function of dispersing the conductive carbon filler (a) in a reagent solution and is negatively charged as a net charge of the entire molecule (i.e., anionic). The anionic dispersant (b) may be used alone or in combination of two or more (for example, mixed together).

[0018] However, "anionic surfactants" and "anionic polysaccharides" do not fall under the category of anionic dispersant (b) in the present invention. Examples of anionic surfactants include methylnaphthalene sulfonic acid formalin condensate salt (e.g., sodium methylnaphthalene sulfonic acid formalin condensate, product name "Demol MS" (Kao)), naphthalene sulfonic acid formalin condensate salt (e.g., sodium β-naphthalene sulfonic acid formalin condensate, product name "Demol N" (Kao)), alkylene maleic acid copolymer salt (e.g., sodium diisobutylene-maleic anhydride copolymer, product name "Demol EP" (Kao)), sodium dodecyl sulfate, sodium cholate, and sodium deoxycholate. Examples of anionic polysaccharides include anionic cellulose derivatives such as carboxymethylcellulose, anionic guar gums such as carboxymethylated guar gum, and xanthan gums. Such anionic surfactants or anionic polysaccharides are not used as the anionic dispersant (b) of the present invention.

[0019] In a particular embodiment of the present invention, the electrochemical sensor has a protective film covering the reagent layer formed on the sensor (working electrode). In such an embodiment, in order to prevent the anionic dispersant (b) from flowing out of the protective film (into living organisms, culture media, etc.), the sensor (working electrode) may be immersed in an aqueous solvent beforehand after the protective film is formed to elute and remove the anionic dispersant (b) from the reagent layer. In such a process, depending on the relationship between the pore size of the protective film and the molecular weight, for example, the anionic dispersant (b) bound to the conductive carbon filler (a) may not elute (leach out of the protective film) from the reagent layer (leach out of the protective film), but the anionic dispersant (b) not bound to the conductive carbon filler (a) may elute (leach out of the protective film). The aqueous solvent used in this process can be the same as the aqueous solvent used to prepare the reagent solution in the method for forming the reagent layer of the present invention, which will be described later.

[0020] For example, an "anionic polymer" (excluding anionic surfactants and anionic polysaccharides) that has the essential function of dispersing a conductive carbon filler (a) in a reagent solution and is negatively charged as the net charge of the polymer as a whole (anionic) can be used as an anionic dispersant (b) in the present invention. The anionic polymer may have any "chain structure," such as linear, branched, or comb-like, and the "chain structure" may contain one or more ring structures derived from cyclic compounds (aromatic hydrocarbon rings, non-aromatic hydrocarbon rings, aromatic heterocycles, non-aromatic heterocycles, etc.). The main chain (the relatively long polymer portion in the chain structure) and side chains (the relatively short polymer portion in the chain structure) of the anionic polymer generally consist mainly of carbon atoms and may contain at least one heteroatom selected from the group consisting of nitrogen atoms, oxygen atoms, and sulfur atoms; in other words, there may be bonds containing heteroatoms such as ether bonds, thioether bonds, and amide bonds in between. Anionic polymers generally have multiple (many) negatively charged functional groups (anionic functional groups) in their side chains, and may also have anionic functional groups at one or both ends of their main chain. Various anionic polymers having anionic functional groups in their side chains and / or ends are known, and it is possible to purchase those with desired properties, prepare them by modifying suitable polymers, or synthesize them using suitable monomers. Anionic polymers may be homopolymers (single polymers), copolymers (polymers) or polymers in which they are bonded and / or mixed, and may also be random polymers, block polymers, or graft polymers.

[0021] Examples of anionic functional groups include carboxyl groups which may have substituents, preferably unsubstituted carboxyl groups, and which react in aqueous solvents with -COO - A group that produces -SO3 in an aqueous solvent, or a sulfo group which may have substituents, preferably an unsubstituted sulfo group, and which produces -SO3 - The groups that produce this are listed below.

[0022] Examples of anionic polymers include ethylene-based polymers having anionic functional groups in their side chains (and terminals). Examples of ethylene-based polymers include homopolymers and copolymers synthesized from one or more monomers selected from monomers containing ethylenically polymerizable carbon-carbon double bonds, such as vinyl groups (CH2=CH-), allyl groups (CH2=CH-CH2-), acryloyl groups (CH2=CH-C(O)-), and methacryloyl groups (CH2=C(CH3)-C(O)-), as well as their modifications (for example, those obtained by saponifying vinyl acetate units to produce vinyl alcohol, or those subjected to treatments that impart hydrophilicity). Examples of monomers containing ethylenic carbon-carbon double bonds include ethylene, propylene, butadiene, isobutene, tetrafluoroethylene, vinyl alcohol, vinyl acetate, vinyl chloride, vinylidene chloride, styrene, methylstyrene, allylamine, diallylamine, diallyldimethylammonium chloride, acrylic acid, methacrylic acid, methyl acrylate (also known as methyl acrylate), methyl methacrylate (also known as methyl methacrylate), butyl acrylate (also known as butyl acrylate), butyl methacrylate (also known as butyl methacrylate), hydroxyethyl methacrylate, and acrylonitrile.

[0023] In one embodiment of the present invention, the anionic dispersant (b) is a polymer comprising at least one selected from the group consisting of acrylic acid-derived units, maleic acid-derived units, and styrene sulfonic acid-derived units, that is, a homopolymer or copolymer synthesized using at least one monomer selected from the group consisting of acrylic acid, maleic acid, and styrene sulfonic acid (e.g., 4-styrene sulfonic acid). The copolymer may also be synthesized using at least one monomer selected from the group consisting of acrylic acid, maleic acid, and styrene sulfonic acid (e.g., 4-styrene sulfonic acid) along with monomers other than at least one selected from the group consisting of acrylic acid, maleic acid, and styrene sulfonic acid (e.g., 4-styrene sulfonic acid).

[0024] In one embodiment of the present invention, the anionic dispersant (b) is a polymer having a carboxyl group and / or a sulfo group in its side chain as an anionic functional group.

[0025] Examples of preferred polymers for the anionic dispersant (b) of the present invention, which can be used in either of the two embodiments described above, include poly(acrylic acid), poly(styrene sulfonic acid), poly(styrene sulfonic acid-co-maleic acid), poly(ethylene oxide)-b-poly(acrylic acid), and poly(styrene)-b-poly(acrylic acid).

[0026] The degree of polymerization, weight-average molecular weight, and other properties and characteristics of the anionic polymer used as an anionic dispersant (b) can be adjusted according to the embodiments and effects of the present invention and the type of anionic polymer. The degree of polymerization of the anionic polymer is usually 50 or higher. The average molecular weight of the anionic polymer, typically the weight-average molecular weight, is, for example, 1,000 or higher, preferably 5,000 or higher. The upper limit of the weight-average molecular weight of the anionic polymer is not particularly limited, but is, for example, 100,000 or less, preferably 70,000 or less.

[0027] The weight-average molecular weight and molecular weight distribution of anionic polymers can be measured by known methods depending on the type of anionic polymer; for example, gel permeation chromatography (GPC) can be used. Furthermore, when purchasing and using commercially available anionic polymers, the values ​​indicated in the catalog (e.g., as "Mw" or "MW") can be considered as the weight-average molecular weight.

[0028] (c) Cationic mediator In this invention, "cationic mediator" refers to a compound that has the essential function of a redox mediator (redox mediator compound) itself, or a compound that contains a part derived from such a redox mediator compound, and which is positively charged (cationic) as a net charge of the entire molecule. "Redox mediator" refers to an oxidation-reduction substance that mediates electron transfer, for example, a substance that carries out electron transfer resulting from the oxidation-reduction reaction of an analyte by an oxidoreductase. Only one type of cationic mediator may be used, or two or more types may be used in combination.

[0029] In a preferred embodiment of the present invention, the cationic mediator (c) is a compound having a structure in which a "redox mediator compound" (c1) and a "cationic polymer" (c2) are bonded together via a "linker portion" (c3) as needed. The "cationic mediator" in the present invention, that is, the "redox mediator," "cationic polymer," and "linker" that constitute it Department This is not limited to a specific type, and various types can be used, taking into consideration the embodiments, effects, etc., of the present invention.

[0030] (c1) Redox mediator compound The "redox mediator compound" in this invention is not limited to a specific type, and various redox mediators or derivatives thereof can be used, taking into consideration the embodiments of the invention (such as reagent layers and electrochemical sensors), their effects, etc.

[0031] Examples of redox mediator compounds include phenazine compounds, phenothiazine compounds, osmium complexes, ruthenium complexes, quinone compounds, and ferrocene compounds.

[0032] In one embodiment of the present invention, the redox mediator compound is a phenazine-based compound or a phenothiazine-based compound. The phenazine-based compound and the phenothiazine-based compound have a negative redox potential (vs. Ag / AgCl·saturated KCl) (lower than 0 V), and are not affected by easily oxidizable compounds such as ascorbic acid (vitamin C) and uric acid, which are contaminants for electrochemical measurements contained in the sample, for example, contained in a biological sample or a culture medium sample and are not analytes. Therefore, they can be said to be preferable redox mediators.

[0033] The phenazine-based compound and the phenothiazine-based compound each refer to a compound having a phenazine skeleton or a phenothiazine skeleton represented by the following general formulas (1) and (2) and capable of functioning as a redox mediator. For example, in the phenothiazine-based compound represented by the general formula (2), when R 3 is a substituted amino group represented by the formula -NR 31 R 32 (wherein both R 31 and R 32 are substituents, or one of them is a substituent and the other is a hydrogen atom), the phenothiazine-based compound becomes a compound that is a cationic mediator alone and has the resonance structures of the following general formulas (2-1) and (2-2).

[0034]

Chemical formula

[0035]

Chemical formula

[0036] In the general formulas (1) and (2), R 1 ~R 9Each of these independently represents one of the groups or atoms described in (i) to (ix') below (however, (ix') is limited to cases where the cationic mediator (c) is a compound having a structure in which a "redox mediator compound" (c1) and a "cationic polymer" (c2) are linked via a "linker moiety" (c3) as necessary, that is, when (ix') reacts with a specific reactive group of the cationic polymer (c2) or a specific reactive group of the linker compound as a specific reactive group of a phenazine-based compound or phenothiazine-based compound as the redox mediator compound (c1) (details will be described later).): (i) Hydrogen atom; (ii) halogen atom; (iii) A hydroxyl group which may have a substituent; (iv) an amino group which may have a substituent; (v) A saturated or unsaturated hydrocarbon group which may have substituents (e.g., C 1-15 Alkyl alkyl group, preferably C 1-6 alkyl group); (vi) an acyl group which may have substituents (e.g., C 1-6 Acyl group, i.e., C 1-6 Alkyl-carbonyl group; (vii) A phenyl group which may have a substituent; (viii) Quaternary ammonium cation group; (ix) an activated esterified carboxyl group (e.g., a carboxyl group activated with N-hydroxysuccinimide (NHS)); (ix') Carboxy group.

[0037] The substituents that the substituents in (iii) to (vii) above may have include, for example, (a) a halogen atom, (b) a hydroxyl group, (c) an amino group, and (d) a linear or branched saturated or unsaturated hydrocarbon group (for example, C 1-15 Alkyl alkyl group, preferably C 1-6 Alkyl alkyl groups, more C 1-3 (Alkyl group), (e)Acyl group (e.g., C 1-6 Acyl group, preferably C 1-3Examples of groups include (f) acyl group, (g) guanidino group, (h) mesyl group, (i) phenyl group, (j) formyl group (aldehyde group), (k) epoxy group, (l) maleimide group, (m) activated esterified carboxyl group, (m') carboxyl group, and (n) oxyethylene group. However, (m') is limited to cases where the cationic mediator (c) is a compound having a structure in which a "redox mediator compound" (c1) and a "cationic polymer" (c2) are linked via a "linker moiety" (c3) as needed, that is, (m') reacts with a specific reactive group of the cationic polymer (c2) or a specific reactive group of the linker compound as a specific reactive group of the phenazine-based compound or phenothiazine-based compound as the redox mediator compound (c1) (details will be described later). The substituents that the substituents in (iii) to (vii) above may have may be any group consisting of one of (a) to (n) above, or may be a group consisting of two or more groups selected from (a) to (n), for example, a group such as (d) a linear or branched saturated or unsaturated hydrocarbon group further substituted with (c) an amino group, (i) a thiol group, (j) a formyl group, (k) an epoxy group, (l) a maleimide group, (m) an activated esterified carboxyl group, or (n) an oxyethylene group. Those skilled in the art can carry out the present invention by selecting chemically appropriate substituents from (a) to (n) for each of (iii) to (vii) above, or by selecting and linking two or more chemically appropriate substituents from (a) to (n). The number of substituents that each of the above (iii) to (vii) may have is not particularly limited; for example, there may be one, two, or three substituents, and multiple substituents may be bonded to a single atom (for example, the carbon atom of an alkyl group, the nitrogen atom of an amino group).

[0038] Furthermore, the quaternary ammonium cation group (viii) is -R A -N + (R B )(R C )(R D A base represented by (wherein R in the formula) A , RB , R C and R D Each of these may have substituents (e.g., C 1-15 Alkyl alkyl group, preferably C 1-6 This refers to an alkyl group. Furthermore, the (n) oxyethylene group is similar to the oxyethylene group described in relation to the (c3) linker portion (linker-like hydrophilic portion) (for example, a polyethylene glycol chain (PEG chain)).

[0039] R 1 ~R 9 At least one of these may be a group for positively charging the redox mediator compound. For example, a phenazine compound or a phenothiazine compound may have R in order to make the compound positively charged (cationic) on its own. 1 ~R 9 At least one of these is an (iv) optionally substituted amino group, preferably an unsubstituted amino group, which in an aqueous solvent is -NH3 + The device may have a group that produces (viii) a quaternary ammonium cation group, or a positively charged functional group (hereinafter referred to as a "cationic functional group").

[0040] Furthermore, if the cationic mediator (c) is a compound having a structure in which a redox mediator compound (c1) and a cationic polymer (c2) are bonded via a linker portion (c3) as needed, then R 1 ~R 9 At least one of these may be a reactive group having bonding properties with a cationic polymer (c2) or a linker portion (c3) used as needed, or a linker compound for forming it (hereinafter referred to as the "specific reactive group of the redox mediator compound (c1)," although it may also be simply referred to as the "specific reactive group" when it is clear from the context that it is of the redox mediator compound (c1)).

[0041] R 1 ~R 9At least one of these may be any other group relating to the performance as a redox mediator or a group relating to the manufacture of the reagent layer. For example, a phenazine compound or a phenothiazine compound may be R to improve hydrophilicity. 1 ~R 9 At least one of these may be a functional group known as a "hydrophilic group," such as (iii) a substituted hydroxyl group, preferably an unsubstituted hydroxyl group; (iv) a substituted amino group, preferably an unsubstituted amino group; (viii) a quaternary ammonium cation group; or a group derived from such a hydrophilic group that is hydrophilic (preferably a hydrophilic group substituted with an (n) oxyethylene group or other hydrophilic group); or (v) a substituted saturated or unsaturated hydrocarbon group; (vi) a substituted acyl group; or (vii) a substituted phenyl group, of which the group as a whole is hydrophilic due to having an (n) oxyethylene group or other hydrophilic group as a substituent.

[0042] In this invention, "hydrophilic" refers to a property that has a high affinity for water and dissolves or miscible to a degree that can achieve the desired effect with water or other polar solvents, such as a solvent for reacting a redox mediator compound with a cationic polymer when synthesizing a cationic mediator, or a solvent for dissolving a cationic mediator when forming a predetermined layer in an electrochemical sensor. Examples of polar solvents include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-1-propanol, 2-methyl-2-propanol, formic acid, acetic acid, tetrahydrofuran, acetone, dioxane, methyl ethyl ketone, ethyl acetate, acetonitrile, dimethylformamide, and dimethyl sulfoxide.

[0043] (c2) Cationic polymer "Cationic polymer" is a general term for polymers that have a positive charge as a whole. The cationic polymer (c2) in this invention has a structure that can support a redox mediator compound (c1), that is, it has groups that can react with the groups of the redox mediator compound (c1) itself or the groups of the linker portion (c3) used as needed, and can be applied to a desired site, for example, the working electrode, to form a reagent layer. The type of cationic polymer is not particularly limited, and one type may be used alone, or two or more types may be used in combination.

[0044] Cationic polymers may have linear, branched, or comb-like "chain structures," and these "chain structures" may contain one or more cyclic structures derived from cyclic compounds (aromatic hydrocarbon rings, non-aromatic hydrocarbon rings, aromatic heterocycles, non-aromatic heterocycles, etc.). The main chain and side chains of cationic polymers (c2) generally consist mainly of carbon atoms and may contain at least one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur atoms; in other words, there may be bonds containing heteroatoms, such as ether bonds, thioether bonds, and amide bonds. Cationic polymers (c2) generally have multiple (many) positively charged functional groups (cationic functional groups) in their side chains, and may also have cationic functional groups at one or both ends of the main chain. Furthermore, from the viewpoint of enabling the support of multiple (many) redox mediator compounds (c1) on a single cationic polymer (c2) via a linker moiety (c3) as needed, it is appropriate for the cationic polymer (c2) to have reactive groups on its side chains that have bonding affinity to groups of the redox mediator compound (c1) itself or to the linker moiety (c3) used as needed, or to groups of the linker compound used to form it (referred to herein as "specific reactive groups of the cationic polymer (c2)," although it may also be simply called "specific reactive groups" when it is clear from the context that they belong to the cationic polymer (c2)). In addition, one or both ends of the main chain may also have specific reactive groups. Various cationic polymers (c2) having cationic functional groups and specific reactive groups on their side chains and / or ends are known, and it is possible to purchase those with desired properties, produce them by modifying appropriate polymers, or synthesize them using appropriate monomers. The cationic polymer (c2) may be a homopolymer, a copolymer, or a polymer in which they are bonded and / or mixed, and may also be a random polymer, a block polymer, or a graft polymer.

[0045] Cationic polymers (C2) include, for example, ethylene-based polymers, imine-based polymers, and amino acid-based polymers, which have cationic functional groups and specific reactive groups in their side chains (and terminals). Also included in cationic polymers (C2) are proteins and polypeptides that have natural amino acid sequences or modified (substituted, deleted, added, etc.) amino acid sequences, which can be described as polymers with amino acids as monomers (however, conceptually, they are distinguished from artificially synthesized amino acid-based polymers), and which have cationic functional groups and specific reactive groups in their side chains (and terminals). Polysaccharide-based polymers that originally have specific reactive groups and cationic functional groups, or have had them introduced, are also included in cationic polymers (C2).

[0046] Examples of ethylene-based polymers include homopolymers and copolymers synthesized from one or more monomers selected from monomers containing ethylenically active carbon-carbon double bonds capable of radical polymerization, such as vinyl groups (CH2=CH-), allyl groups (CH2=CH-CH2-), acryloyl groups (CH2=CH-C(O)-), and methacryloyl groups (CH2=C(CH3)-C(O)-), as well as their modified products (for example, those obtained by saponifying vinyl acetate units to produce vinyl alcohol, or those subjected to treatments that impart hydrophilicity). Examples of monomers containing ethylenically active carbon-carbon double bonds include ethylene, propylene, butadiene, isobutene, tetrafluoroethylene, vinyl alcohol, vinyl acetate, vinyl chloride, vinylidene chloride, styrene, methylstyrene, allylamine, diallylamine, diallyldimethylammonium chloride, acrylic acid, methacrylic acid, methyl acrylate (also known as methyl acrylate), methyl methacrylate (also known as methyl methacrylate), butyl acrylate (also known as butyl acrylate), butyl methacrylate (also known as butyl methacrylate), hydroxyethyl methacrylate, and acrylonitrile. In addition, (meth)acrylic polymers such as copolymers of methyl methacrylate and hydroxyethyl methacrylate, copolymers of butyl methacrylate and hydroxyethyl methacrylate, and poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate), which are generally known as biocompatible polymers, and polyester polymers such as polyethylene terephthalate are also preferred ethylene-based polymers.

[0047] Preferred ethylene-based polymers as cationic polymers (c2) include, for example, polyallylamine hydrochloride, allylamine hydrochloride-diallylamine hydrochloride copolymer, and allylamine-diallyldimethylammonium chloride copolymer. (Meth)acrylic polymers having quaternary ammonium cations, amino groups, etc. in their side chains (and terminals) are also preferred ethylene-based polymers. More specifically, a single polymer containing constituent units derived from 2-aminoethyl methacrylate, (vinylbenzyl)trimethylammonium chloride, methacryloylcholinchloride, etc. Solitary Compounds or copolymers, such as poly(diallyldimethylammonium chloride), poly(allylamine hydrochloride), allylamine hydrochloride-diallylamine hydrochloride copolymer, and allylamine-diallyldimethylammonium chloride copolymer, can preferably be used as cationic polymers (C2).

[0048] Examples of imine polymers include poly(ethyleneimine). Poly(ethyleneimine) has side chain structures such as -(CH2)2-NH2, -(CH2)2-NH-(CH2)2-NH2, and -(CH2)2-N((CH2)2-NH2)2, and the amino group -NH2 in this structure becomes a specific reactive group of the cationic polymer (c2), which can be bonded to a redox mediator compound (c1) or linker moiety (c3) having a corresponding appropriate specific reactive group (e.g., an activated esterified carboxyl group).

[0049] Examples of amino acid-based polymers include poly(L-lysine), poly(L-arginine), and poly(L-ornithine). Poly(L-lysine) has a -(CH2)4-NH2 structure in its side chain. Poly(L-arginine) has a -(CH2)2-NH-C(=NH)-NH2 structure in its side chain. The amino group (-NH2) (or guanidino group (-NH-C(=NH)-NH2)) contained in the side chains of these amino acid-based polymers becomes a specific reactive group of the cationic polymer (c2), and can bond to a redox mediator compound (c1) or linker moiety (c3) having a corresponding appropriate specific reactive group (e.g., an activated esterified carboxyl group). Note that the amino acid-based polymer may also form a salt, such as poly(L-arginine hydrochloride).

[0050] Examples of polysaccharide polymers include cellulose derivatives such as chitosan. Chitosan is a polysaccharide obtained by hydrolyzing chitin and has an amino group in its side chain (sugar structure). The amino groups contained in the side chain of such polysaccharide polymers become specific reactive groups of the cationic polymer (c2), and can bond with a redox mediator compound (c1) or linker moiety (c3) having a corresponding appropriate specific reactive group (e.g., an activated esterified carboxyl group).

[0051] The degree of polymerization, weight-average molecular weight, and other properties and characteristics of the cationic polymer (C2) can be adjusted according to the embodiments and effects of the present invention and the type of cationic polymer (C2). The degree of polymerization of the cationic polymer (C2) is usually 100 or higher. The average molecular weight of the cationic polymer (C2), typically the weight-average molecular weight, is usually 10,000 or higher, preferably 50,000 or higher, and more preferably 100,000 or higher. The upper limit of the weight-average molecular weight of the cationic polymer (C2) is not particularly limited, but is usually less than 10,000,000, and preferably less than 1,000,000.

[0052] The weight-average molecular weight and molecular weight distribution of cationic polymers (c2) can be measured by known methods depending on the type of cationic polymer (c2), such as gel permeation chromatography (GPC). Furthermore, when purchasing and using commercially available cationic polymers (c2), the value indicated in the catalog (e.g., as "Mw" or "MW") can be considered the weight-average molecular weight.

[0053] (c3) Linker section The linker portion (c3) is a structure that mediates the bonding between the redox mediator compound (c1) and the cationic polymer (c2), and is present as needed. Generally, the linker portion (c3) is a chain-like structure in which the portion excluding the pre-reaction reactive groups or post-reaction bonding structures at both ends consists mainly of carbon atoms and may contain at least one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur atoms. In other words, it is a chain-like structure in which bonds containing heteroatoms, such as ether bonds, thioether bonds, and amide bonds, may be present along the chain. The "chain-like structure" of the linker portion (c3) may be linear or branched. Furthermore, the "chain-like structure" may contain one or more ring structures derived from cyclic compounds (aromatic hydrocarbon rings, non-aromatic hydrocarbon rings, aromatic heterocycles, non-aromatic heterocycles, etc.).

[0054] In one embodiment of the present invention, the linker portion (c3) also functions as a part (hydrophilic portion) for improving the hydrophilicity of the cationic mediator (c), and can be called a "linker-like hydrophilic portion." When the linker portion (c3) is a linker-like hydrophilic portion, from the viewpoint of hydrophilicity, it is preferable that it is linear and does not contain any ring structures derived from the cyclic compound.

[0055] In a preferred embodiment of the present invention, the linker-like hydrophilic portion is given by formula: -(OC2H4) qIt contains an oxyethylene group represented by -. In the formula, q represents, for example, an integer from 1 to 80, preferably an integer from 3 to 36. Note that oxyethylene chains represented by this formula in which q is relatively large are generally called "polyethylene glycol chains (PEG chains)".

[0056] In one embodiment of the present invention, the linker-like hydrophilic portion is given by formula:-(CH2) p It contains a hydrocarbon chain represented by -. In one embodiment of the present invention, the main chain of the linker-like hydrophilic portion is the above formula:-(CH2) p A hydrocarbon chain represented by - and the above formula: -(OC2H4) q It includes both oxyethylene chains represented by -. The value of p in the formula representing the hydrocarbon chain can be appropriately adjusted to balance with the value of q in the formula representing the oxyethylene chain, taking into account the hydrophilicity of the linker-like hydrophilic portion.

[0057] The linker portion (c3) can be derived from a compound (hereinafter referred to as the "linker compound") having at least two reactive groups (hereinafter referred to as the "specific reactive groups of the linker compound," however, when it is clear from the context that they belong to the linker compound) that are compatible with the group (specific reactive group) of the redox mediator compound (c1) and the group (specific reactive group) of the cationic polymer (c2). For example, by reacting the linker compound with the redox mediator compound (c1), and then reacting the linker compound in a state where it is bound to the redox mediator compound (c1) (in other words, the "redox mediator compound (c1)-linker compound reaction product") with the cationic polymer (c2), a cationic mediator (c) is produced as a compound in which the redox mediator compound (c1) and the cationic polymer (c2) are bound via the linker portion (c3). Furthermore, by reacting a linker compound with a cationic polymer (c2), and then reacting the linker compound in its bonded state with the cationic polymer (c2) (in other words, the "cationic polymer (c2)-linker compound reaction product") with a redox mediator compound (c1), a cationic mediator (c) is produced as a compound in which the redox mediator compound (c1) and the cationic polymer (c2) are bonded via a linker portion (c3).

[0058] <Specific reactive groups> In the present invention, various known reactive groups can be used as the specific reactive groups of the redox mediator compound (c1), the cationic polymer (c2), and the linker compound, and various combinations are possible. The predetermined reactive groups in the present invention may be bonded together by covalent bonds or by non-covalent bonds (e.g., electrostatic interactions), but from the viewpoint of bond stability, for example, it is preferable that they be bonded together by covalent bonds.

[0059] In the present invention, the specific reactive group is preferably at least one selected from the group consisting of "carboxyl group or its active ester, amino group, thiol group, formyl group (aldehyde group), epoxy group, and maleimide group" (hereinafter referred to as the "preferred specific reactive group group"). The amino group, thiol group, formyl group (aldehyde group), epoxy group, and maleimide group as preferred specific reactive groups correspond to the R in general formulas (1) and (2) of the redox mediator compound (c1), respectively. 1 ~R 9 Substituents (iii) to (vii) as shown correspond to substituents (c), (i), (j), (k), and (l), which are exemplified as substituents that substituents (iii) to (vii) may further have, and the carboxyl group and its active ester correspond to substituents (m') and (m), respectively. Table 1 shows the groups that each reactant in the preferred specific reactant group can react with. If a reactant in the preferred specific reactant group is selected as one of the specific reactants in the present invention, the group that reacts with it may be another reactant in the preferred specific reactant group (underlined in the table), or a reactant not in the preferred specific reactant group (not underlined in the table, or not included in the table).

[0060] [Table 1]

[0061] In a preferred embodiment of the present invention, the specific reactive group of the redox mediator compound (c1) or the specific reactive group of the linker compound and the specific reactive group of the cationic polymer (c2) are such that one is "reactive group A" in Table 1 and the other is "reactive group B". Similarly, in a preferred embodiment of the present invention, the specific reactive group of the redox mediator compound (c1) and the specific reactive group of the linker compound are such that one is "reactive group A" in Table 1 and the other is "reactive group B".

[0062] In a more preferred embodiment of the present invention, the specific reactive group of the redox mediator compound (c1) or the specific reactive group of the linker compound and the specific reactive group of the cationic polymer (c2) are either an amino group or a carboxyl group or its active ester. Similarly, in a more preferred embodiment of the present invention, the specific reactive group of the redox mediator compound (c1) and the specific reactive group of the linker compound are either an amino group or a carboxyl group or its active ester. For example, when using a redox mediator compound (c1) having an activated esterified carboxyl group as a specific reactive group; a linker compound having an amino group as a first specific reactive group and an activated esterified carboxyl group (e.g., an NHS activated esterified carboxyl group) as a second specific reactive group; and a cationic polymer (c2) having an amino group as a specific reactive group, first a linker-like hydrophilic portion-introduced redox mediator compound is obtained by the reaction (first amide bond) between the activated esterified carboxyl group of the redox mediator compound (c1) and the amino group of the linker compound, and then a cationic mediator (c) is obtained by the reaction (second amide bond) between the activated esterified carboxyl group of the obtained linker-like hydrophilic portion-introduced redox mediator compound and the amino group of the cationic polymer (c2).

[0063] The reagent layer of the present invention is typically positioned on the working electrode in the electrochemical sensor of the present invention. Only one type of reagent layer may be used, or two or more types may be used in combination (for example, two or more reagent layers with different compositions (types and / or contents) of conductive carbon filler (a), anionic dispersant (b), and cationic mediator (c) may be laminated).

[0064] The reagent layer may contain an oxidoreductase of a type corresponding to the analyte, such as in an electrochemical sensor taking the form of a biosensor, or it may not contain an oxidoreductase, such as in the case of an electrochemical sensor other than a biosensor, or a sensor other than an electrochemical sensor (e.g., an optical sensor).

[0065] The reagent layer may optionally contain components other than the conductive carbon filler (a), anionic dispersant (b), and cationic mediator (c). Such optional components include, for example, an "oxidoreductase" (d).

[0066] (d) Oxidoreductase Oxidoreductase (d) refers to an enzyme capable of oxidizing (including dehydrogenating) or reducing the analyte targeted by the electrochemical sensor, etc. The electrochemical sensor of the present invention typically takes the form of an electrochemical sensor (biosensor) containing oxidoreductase in the reagent layer, but is not limited thereto. It can also take the form of an electrochemical sensor other than a biosensor, or a sensor other than an electrochemical sensor (e.g., an optical sensor), which does not contain oxidoreductase in the reagent layer. Depending on the application of the electrochemical sensor, etc., it is possible to select whether or not the reagent layer contains oxidoreductase (d), and what type of oxidoreductase (d) is included, depending on the type of analyte.

[0067] Examples of oxidoreductases (d) include oxidase enzymes (glucose oxidase (Gox), lactate oxidase, pyruvate oxidase, cholesterol oxidase, amino acid oxidase, glutamate oxidase, fructosyl amino acid oxidase, alcohol oxidase, ascorbate oxidase, fructosyl peptide oxidase, bilirubin oxidase, aldehyde oxidase, etc.) and dehydrogenase enzymes (glucose dehydrogenase (GDH), lactate dehydrogenase, pyruvate dehydrogenase, amino acid dehydrogenase, glutamate dehydrogenase, 3-hydroxybutyrate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase). Oxidoreductases (d) may be used individually or in combination of two or more as needed.

[0068] In one embodiment of the present invention, oxidoreductase (d) is coenzyme-bound. join Examples of type (d) oxidoreductase include pyrroloquinoline quinone (PQQ)-bound GDH and flavin adenine dinucleotide (FAD)-bound GDH. In embodiments where glucose is used as an analyte, FAD-bound GDH is preferred from the viewpoint of low reactivity to maltose (for example, the enzyme activity for maltose can be 5% or less, preferably 3% or less, when the enzyme activity for glucose is 100%). Examples of FAD-bound GDH include those derived from Aspergillus species (e.g., Oryzae, Tereus) and Mucor species.

[0069] In one embodiment of the present invention, the oxidoreductase (d) may be crosslinked with a cationic mediator (c), particularly with a cationic polymer (c2) comprising it. For example, the oxidoreductase (d) and the cationic mediator (c), particularly the cationic polymer (c2), can be crosslinked by using a crosslinking agent (e). Examples of crosslinking agents (e) include glutaraldehyde. The crosslinking agent (e) is a reactive group (e.g., an amino group) of the protein oxidoreductase (d) and a reactive group of the cationic mediator (c), for example, a reactive group of the cationic polymer (c2) that did not react with the redox mediator compound (c1) or the linker moiety (c2), or the redox mediator compound (c1) or the linker moiety (c 3By reacting with each of the reactive groups (e.g., amino groups) that are of a different type than those used for the reaction with ), the oxidoreductase (d) and the cationic mediator (c) can be linked via the crosslinking agent (e). By forming a larger molecular weight complex through such crosslinking, the outflow of the oxidoreductase (d) and the cationic mediator (c), particularly the redox mediator compound (c1) constituting them, from outside the protective membrane can be further suppressed. When the crosslinking agent (e) is used, the oxidoreductases (d) may also be linked to each other via the crosslinking agent (e) by reacting with the reactive groups that each of the two molecules of oxidoreductase (d) possesses. That is, when the crosslinking agent (e) is used, in the reagent layer, the oxidoreductase (d) may be crosslinked with the cationic mediator (c), particularly the cationic polymer (c2), and at the same time, the oxidoreductases (d) may also be crosslinked with each other.

[0070] — Method for forming the reagent layer — The method for forming a reagent layer of the present invention comprises at least the following steps (1), and usually further (2) and (3): (1) A step of preparing a reagent solution containing a conductive carbon filler (a), an anionic dispersant (b), and a cationic mediator (c) (this may be referred to as the "reagent solution preparation step" in this specification); (2) The step of applying the reagent solution to the reagent layer formation site (which may be referred to as the "apply step" in this specification); (3) A step of drying the applied reagent solution to form a reagent layer (which may be referred to as the "reagent layer formation step" in this specification).

[0071] (1) Reagent solution preparation process The reagent solution preparation step (1) is a step of preparing a reagent solution that comprises the main components constituting the reagent layer, namely at least a conductive carbon filler (a), an anionic dispersant (b), and a cationic mediator (c), and may optionally also contain other components, such as an oxidoreductase (d) or a crosslinking agent (e).

[0072] Reagent solutions can generally be prepared by adding the necessary components from (a) to (e) above to an aqueous solvent (water, or a mixed solvent of water and a water-compatible solvent). The aqueous solvent can be adjusted to have an appropriate pH by adding pH adjusters, buffers (liquids), or other compounds as needed.

[0073] In one embodiment of the present invention, the pH of the reagent solution is 8.0 or less. By using a buffer solution in an amount such that the pH of the reagent solution becomes 8.0 or less, the effect of improving the dispersibility of the conductive carbon filler (a) is more easily achieved. If the reagent solution contains an oxidoreductase (d), it is appropriate to set the pH of the reagent solution within a range in which the activity of the oxidoreductase (d) is maintained.

[0074] The pH of reagent solutions is usually adjusted using buffer solutions. Various buffer solutions can be used, including, for example, acetate buffer (acetic acid and sodium acetate), phosphate buffer, citrate buffer, citrate-phosphate buffer, Tris buffer (tris-hydroxymethylaminomethane, also known as trometamol), Bis-Tris buffer (bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane), HEPES buffer (2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid), MES (2-morpholinoethanesulfonic acid monohydrate), MOPS (3-morpholinopropanesulfonic acid), and PBS (phosphate-buffered saline, generally sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate). By using an appropriate buffer solution, the pH of the reagent can be set to the desired value (range).

[0075] In one embodiment of the present invention, the concentration of metal ions in the reagent solution is 200 mM or less. When the concentration of metal ions in the reagent solution is high, depending on the type of metal ion, the dispersibility of the conductive carbon filler may be inhibited, and aggregation may occur. Therefore, it is preferable to have a low concentration of metal ions in the reagent solution. When using a compound containing metal ions (buffer) or a buffer solution prepared thereby to adjust the pH of the reagent solution, it is preferable to calculate the concentration of metal ions in the reagent solution based on the amount of buffer or buffer solution required to achieve the desired pH, and to select a buffer or buffer solution that can keep the concentration at 200 mM or less. When using a compound (buffer) that does not contain metal ions or a buffer solution prepared thereby, the concentration of metal ions in the reagent solution can naturally be kept at 200 mM or less. When the metal salt is an alkali metal ion, the concentration of metal ions in the reagent solution is preferably less than 100 mM, for example, 80 mM or less.

[0076] Since metal ions in the reagent solution originate from various substances used in its preparation, the composition of the reagent solution, as well as the types and amounts of each component, can be adjusted to ensure that the concentration of metal ions remains within an appropriate range. For example, since metal ions in the reagent solution may originate from the buffer solution, the composition of the buffer solution used to adjust the pH of the reagent solution (the types and concentrations of compounds contained in the buffer solution) should be appropriate. Furthermore, since metal ions in the reagent solution may originate from substances that form salts as anionic dispersants (b), the amount of such salt-forming anionic dispersants (b) should be taken into consideration as needed.

[0077] The order in which the components in the reagent solution are added can be adjusted as appropriate, but it is preferable to carry out the steps (1-1) and (1-2) below in that order. (1-1) A step of dispersing a conductive carbon filler (a) in an aqueous solution containing at least an anionic dispersant (b) to obtain a dispersion. (1-2) Adding a cationic mediator (c) to the obtained dispersion to obtain a reagent solution.

[0078] The concentrations of the conductive carbon filler (a), the anionic dispersant (b), the cationic mediator (c), and the oxidoreductase (d) used as needed in the reagent solution, or the ratio of their concentrations, can be appropriately adjusted considering the embodiments, effects, etc., of the present invention.

[0079] The concentration of conductive carbon filler (a) in the reagent solution is, for example, within the range of 0.1 to 30 w / v% (mg / mL).

[0080] The concentration of the anionic dispersant (b) in the reagent solution is, for example, within the range of 0.01 to 30 w / v% (mg / mL).

[0081] In the reagent solution preparation step (1), a treatment (sometimes referred to herein as "dispersion treatment") may be performed to improve the dispersibility of the conductive carbon filler (a) in the reagent solution by applying physical energy to the reagent solution. The dispersion treatment may be performed, for example, between step (1-1) and step (1-2), or alternatively, or in addition to, after step (1-2) (before the next apply step (2)).

[0082] The means (methods and conditions, apparatus used, etc.) for supplying "physical energy" are not particularly limited, as long as they can improve the dispersibility of the conductive carbon filler (a) in the reagent solution. In a typical embodiment of the present invention, sonication is used as the above means, but other means can also be used. The conditions for sonication (output, time, etc.) can be adjusted as appropriate. Apparatus for sonication can also be selected as appropriate; for example, an ultrasonic homogenizer can be used.

[0083] It is preferable to add the oxidoreductase (d) at the stage after the dispersion treatment is completed in the reagent solution preparation step (1) so as not to be affected by physical energy. It is preferable to add the crosslinking agent (e), such as glutaraldehyde, for crosslinking the oxidoreductase (d) with the cationic mediator (c) or crosslinking the oxidoreductases (d) with each other, after adding the oxidoreductase (d).

[0084] Furthermore, when using a compound in which a redox mediator compound (c1) and a cationic polymer (c2) are bonded via a linker portion (c3) as necessary, as the cationic mediator (c), it is preferable to improve the dispersibility of the conductive carbon filler (a) by dispersing the cationic mediator (c) through a dispersion treatment, for example, by applying physical energy to the reagent solution through sonication, before adding it.

[0085] (2) Apply process The apply step (2) is a step in which the reagent solution obtained in the reagent solution preparation step (1) is applied (dropped, coated, etc.) to at least a part of the reagent layer formation site, such as the working electrode.

[0086] The means for "applying" (methods and conditions, apparatus used, etc.) are not particularly limited as long as they can form a reagent layer from the reagent solution, and means similar to the reagent solution application step in conventional reagent layer formation methods (or electrochemical sensor manufacturing methods) can be employed. In a typical embodiment of the present invention, the application step is performed by dropping an appropriate amount of the reagent solution obtained in the reagent solution preparation step (1) onto at least a part of the working electrode (the part that forms the reagent layer). The area to be applied and the amount of reagent solution can be adjusted according to the desired thickness of the reagent layer.

[0087] (3) Reagent layer formation process Reagent layer formation process ( 3 ) is a step in which the reagent solution applied to the reagent layer formation site (for example, at least a part of the working electrode) by the apply step (2) is dried to form a coating layer.

[0088] The means for "drying" (methods and conditions, apparatus used, etc.) are not particularly limited as long as they can form a (preferably uniform) reagent layer from the applied reagent solution, and means similar to those used in the conventional reagent solution drying process in the manufacture of electrochemical sensors can be employed. In a typical embodiment of the present invention, the reagent layer formation process is performed by leaving the working electrode to which the reagent solution has been applied at room temperature, thereby forming the reagent layer.

[0089] — Electrochemical Sensor — The electrochemical sensor for detecting or quantifying analytes according to the present invention has a working electrode, a counter electrode, and a reagent layer of the present invention as described above, disposed on the working electrode, and optionally further has a protective film covering at least the reagent layer.

[0090] The "protective film" is a membrane-like component that prevents or suppresses the leakage of substances contained in the reagent layer (conductive carbon filler (a), cationic mediator (c), oxidoreductase (d) used as needed, etc.) into the environment outside the protective film (in living organisms, in culture media, etc.). The protective film covering the reagent layer should preferably have permeable pores so that analytes present in the environment outside the protective film can come into contact with the reagent layer. Such a protective film is preferably formed, for example, when the electrochemical sensor is used for continuous measurement, but may not be formed when the electrochemical sensor is used for one-off measurement.

[0091] Since the working electrode (probe equipped with it), which has a reagent layer formed thereon, is used by being inserted into a living body or immersed in a culture medium, it is preferable that the protective film covering its surface has biocompatibility such that proteins and cells do not or do not easily adsorb to it, and is generally formed from a biocompatible polymer having such properties. Examples of biocompatible polymers include copolymers of methyl methacrylate and hydroxyethyl methacrylate, copolymers of butyl methacrylate and hydroxyethyl methacrylate, and poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate).

[0092] A protective film can be formed by preparing a solution containing raw materials for forming the protective film (protective film solution), immersing the area where the protective film is to be formed, for example, the area of ​​the working electrode where at least the reagent layer is formed, immersing it, removing it, and drying it, and repeating such a process multiple times as necessary.

[0093] The electrochemical sensor of the present invention may further include a reference electrode as needed. That is, the electrochemical sensor of the present invention can be a two-electrode type consisting of a working electrode and a counter electrode, or a three-electrode type consisting of a working electrode, a counter electrode and a reference electrode.

[0094] In one embodiment of the present invention, the electrochemical sensor can be fabricated as an implantable electrochemical sensor, for example, as a biosensor for Continuous Glucose Monitoring (CGM) for self-monitoring of blood glucose, which continuously or semi-continuously measures glucose concentration in blood or interstitial fluid, for example, over several days to several weeks. On the other hand, in one embodiment of the present invention, the electrochemical sensor can also be fabricated as a non-implantable electrochemical sensor, for example, as a biosensor for continuously or semi-continuously measuring the concentration of glucose in a culture medium, etc.

[0095] Hereinafter, embodiments of the electrochemical sensor of the present invention when it is embedded will be described with reference to the drawings. However, the drawings and the following description are illustrative, and it should be understood that the technical scope of the present invention is not limited to the embodiments disclosed therein, but includes various modified embodiments to the extent that the problems of the present invention can be solved and the effects of the present invention can be achieved.

[0096] In the following description, the surfaces facing the front of the page in Figures 1(A) and (B), Figure 3, and Figure 7, and the surfaces facing upwards in Figures 2(A) to (C) and Figures 4 to 6 will be referred to as the "top surface," the surfaces facing away from the page in Figures 1(A) and (B), Figure 3, and Figure 7, and the surfaces facing downwards in Figures 2(A) to (C) and Figures 4 to 6 will be referred to as the "bottom surface," and the surfaces facing either left or right in Figures 1(A) and (B), Figures 2(A) to (C), Figures 5 to 7, and the surfaces facing either the front or back of the page in Figure 4 will be referred to as the "side surfaces." Furthermore, the dimension in the vertical direction of the paper in Figure 1(A), that is, the dimension parallel to arrow X2 indicating the direction of sensor insertion into the body, as well as the dimension in the vertical direction of the paper in Figures 3 and 7, is sometimes called "length," while the dimension in the horizontal direction of the paper in Figure 1(A), that is, the dimension perpendicular to arrow X2, as well as the dimension in the vertical direction of the paper in Figures 3 and 7, is sometimes called "width."

[0097] Figure 1 is a plan view of sensor 11 in one embodiment of the present invention. Figure 1(A) shows the entire sensor 11. Figure 1(B) shows a magnified view of the tip portion (sensing portion) of sensor 11 shown in Figure 1(A). Sensor 11 is suitable for constructing an implantable biosensor system, for example, for use in self-testing of blood glucose levels, in which case sensor 11 can be inserted into the body as a protruding portion (probe) from a main body (not shown). Sensor 11 can also be used, for example, in constructing a system for measuring the concentration of analytes in a culture medium.

[0098] As shown in Figure 1(A), the region X1 (head) of the sensor 11 is housed in a main body (not shown), and the tip portion (sensing portion) of the sensor 11 protrudes from the main body. Arrow X2 indicates the insertion direction when, for example, the sensor 11 is inserted into a living organism. The dimensions of the sensing portion are, for example, a length of 20 to 3 mm, preferably 10 to 3 mm, and a width of, for example, 1 to 50 μm, preferably 500 to 50 μm.

[0099] In Figure 1, the sensor 11 comprises a substrate 21, electrodes 22, a reagent layer 23, a silver / silver chloride layer (sometimes called a reference layer) 24, and a film 25. The electrodes 22 are uniformly formed on the substrate 21 and include a working electrode 22a, a reference electrode 22b, and a counter electrode 22c. The working electrode 22a and the reference electrode 22b are physically and electrically separated by groove A1, and the reference electrode 22b and the counter electrode 22c are physically and electrically separated by groove A2. The reagent layer 23 is formed on the working electrode 22a. The silver / silver chloride layer 24 is formed on the reference electrode 22b. The film 25 covers the upper surface of the sensor 11 except for parts of the electrodes 22 (such as the portion of region X4 of the head region X1 and the portion of region X3 of the counter electrode 22c) and parts of the reagent layer 23 (by providing openings to expose them). The exposed region X4 of the electrodes 22 is connected to the circuit of the main body 11.

[0100] Furthermore, as shown by region X5 in Figure 1(B), it is preferable that the reagent layer 23 is not formed at the tip of the sensor 11 (over a predetermined distance from the tip). In other words, it is preferable that the reagent layer 23 is formed away from the tip of the sensor 11. This is because it is possible to suppress the reagent layer 23 from peeling off (lifting) from the sensor 11 when the sensor 11 is inserted into a living organism.

[0101] Figure 2(A) is a cross-sectional view taken along the line AA in Figure 1(B). In the portion of the sensor 11 where the reagent layer 23 is formed, the substrate 21, electrode 22 (working electrode 22a), and reagent layer 23 are stacked in this order. In portions 11a and 11b, the electrode 22 (working electrode 22a), reagent layer 23, and film 25 are not stacked (they have been trimmed), and the substrate 21 is exposed.

[0102] Figure 2(B) is a cross-sectional view taken along the BB arrow in Figure 1(B). In the portion to the right of groove A1, where the silver / silver chloride layer 24 is formed, the substrate 21, electrode 22 (reference electrode 22b), silver / silver chloride layer 24, and film 25 are laminated. In the portion to the left of groove A1, which is physically and electrically separated from the reference electrode 22b, the substrate 21, electrode 22 (working electrode 22a), and film 25 are laminated. The side surface of the silver / silver chloride layer 24 (right side surface in Figure 2(B)) is exposed and the film 25 is not placed thereon. In this embodiment, the upper surface of the silver / silver chloride layer 24 is covered with the film 25, but it may also be exposed and not covered with the film 25.

[0103] Figure 2(C) is a cross-sectional view taken along the CC arrow in Figure 1(B). The substrate 21 and electrode 22 (counter electrode 22c) are laminated in the portion to the right of groove A2. The upper surface of the counter electrode 22c is not covered by the film 25 and is exposed. The substrate 21, electrode 22 (reference electrode 22b), and film 25 are laminated in the portion sandwiched between grooves A1 and A2. The substrate 21, electrode 22 (working electrode 22a), and film 25 are laminated in the portion to the left of groove A1.

[0104] The substrate 21 is typically a sheet-like synthetic resin. The material of the substrate 21 is not particularly limited as long as it is a resin material such as a plastic material (synthetic resin) that has at least one of the following characteristics: flexibility, ease of processing, and heat resistance. A typical example of such a resin material for the substrate 21 is polyethylene terephthalate (PET), but other general-purpose plastics such as polyethylene, polypropylene, and polyethylene naphthalate can also be used. Furthermore, if high heat resistance is required, polyimide is preferred.

[0105] The electrode 22 is a thin film (thin layer) formed on the substrate 21. The material of the electrode 22 is not particularly limited as long as it is a metal or carbon material that has conductivity and stability (e.g., oxidation resistance or salt resistance). Gold is a typical example of such an electrode 22 material, but platinum, palladium, and carbon are also examples. If the working electrode 22a (reagent layer 23) is formed on one of the upper and lower surfaces of the sensor 11 and the counter electrode 22c is formed on the other, different electrode materials may be used for each.

[0106] The working electrode 22a is given a potential (relative to the reference electrode 22b) sufficient to oxidize the mediator reduced by the reaction of an analyte (e.g., glucose) by an oxidoreductase. The glucose concentration is measured by monitoring the current flowing between the working electrode 22a and the counter electrode 22c. The reagent layer 23 is formed on the upper surface of the working electrode 22a at the tip of the sensor 11.

[0107] The silver / silver chloride layer 24 is formed on the upper surface of the reference electrode 22b at the tip of the sensor 11, if necessary. In this embodiment, the working electrode 22a, the reference electrode 22b and the counter electrode 2 are configured to achieve more accurate measurements. 2 An example of a three-electrode configuration consisting of c is shown, but it does not include the reference electrode 22b, and instead includes the working electrode 22a and counter electrode 2 2 It is also possible to use a two-electrode configuration consisting of c (for example, most SMBG (self-monitoring of blood glucose) devices currently on the market use such a two-electrode configuration). The reference electrode can be a silver / silver chloride electrode with a silver / silver chloride (Ag / AgCl) layer 24 formed on it, as shown in this embodiment, or it can be a hydrogen electrode or one with a mercury-containing layer, such as a Calomel electrode.

[0108] The film 25 is an insulating sheet-like member formed (laminated) on predetermined portions of the electrodes 22 (working electrode 22a, reference electrode 22b, and counter electrode 22c) formed on the substrate 21, and on the silver / silver chloride layer 24. The thickness of the film 25 is usually 1 μm to 150 μm, preferably 3 μm to 50 μm, and more preferably 5 μm to 30 μm. The film 25 has openings in portions corresponding to the reagent layer 23 at the tip of the sensor 11 and in portions corresponding to a part of the counter electrode 22c, so that the reagent layer 23 and the counter electrode 22c in those portions are exposed.

[0109] The film 25 can be, for example, a sheet of the same resin material as the substrate 21 with an adhesive sheet (e.g., acrylic, rubber, or hot-melt) attached to it. The resin material sheet may be the same resin material as the substrate 21, or it may be a sheet of a different resin material. The adhesive sheet alone may be used as the film 25. It is also possible to use a layer formed from a thermal or photoplastic resist film or resist ink as the film 25.

[0110] The reagent solution for forming the reagent layer 23 can be applied by dropping it onto the surface of the electrode 22 (working electrode 22a) through an opening in the corresponding part of the film 25. From the viewpoint of workability in such a process, it is preferable that the contact angle (α) of the reagent solution with respect to the surface of the film 25 is higher than the contact angle (β) of the reagent solution with respect to the opening of the film 25, i.e., the surface of the exposed working electrode 22a, and the larger the difference (α-β), the better. For example, it is preferable that α is 90° or more and β is 50° or less. The "contact angle" referred to here is the "static contact angle" measured by the "θ / 2 method". Even if the material forming the film 25 and / or the material forming the exposed working electrode 22a do not satisfy the above contact angle conditions, it is possible to satisfy the above contact angle conditions by performing an appropriate surface treatment, for example, a water-repellent treatment on the film 25 and a hydrophilic treatment on the working electrode 22a.

[0111] Although not shown in Figures 1(A) and (B), at least the portion of the sensor 11 including the reagent layer 23 may be covered with a protective film. In other words, a protective film may be formed (laminated) on the upper surface of the reagent layer 23 in Figure 2(B). The reagent layer will be described later with reference to Figures 4 to 6 in which it is illustrated.

[0112] The method for manufacturing the sensor 11 is not particularly limited, and any method that can be appropriately selected and used to manufacture a sensor 11 having the above-described configuration in a predetermined location can be used. For example, the sensor 11 can be manufactured by performing the following steps (i) to (viii): (i) A step of forming (stacking) electrodes 22 on the upper surface of the substrate 21; (ii) A step of forming grooves A1 and A2 that physically and electrically separate the electrode 22 into three regions: the working electrode 22a, the reference electrode 22b, and the counter electrode 22c; (iii) A step of forming a silver / silver chloride layer 24 on the upper surface of the electrode 22 (reference electrode 22b); (iv) A step of forming (laminating) a film 25 on the upper surface of the electrode 22 and the silver / silver chloride layer 24; (v) A step of forming a reagent layer 23 on the upper surface of the electrode 22 (working electrode 22a); (vi) A step of removing part (region X6) of the reagent layer 23 and the electrode 22; (vii) The process of cutting out the sensor 11 from the substrate 21; (viii) Step of forming a protective film.

[0113] Regarding step (i) above, the method for forming (laminating) the electrode 22 can be appropriately selected and adjusted, taking into consideration the combination of materials for the electrode 22 and the substrate 21. For example, if the substrate 21 is made of a synthetic resin such as PET and the electrode 22 is made of metal, the electrode 22 made of the metal material can be formed on the surface of the substrate 21 by vapor deposition (including sputtering), but other methods such as printing, plating, and spin coating can also be used. If the substrate 21 is made of a synthetic resin such as PET and the electrode 22 is made of carbon, for example, the electrode 22 made of carbon can be formed by printing carbon paste on the surface of the substrate 21. Note that the substrate 21 in this step does not need to have the shape of the sensor 11 in advance, and a substrate 21 with dimensions larger than the sensor 11 can be used so that the sensor 11 can be cut out in a later step (vii).

[0114] For step (ii) described above, laser trimming can be used as a means for forming grooves A1 and A2.

[0115] Regarding step (iii) above, the method for forming the silver / silver chloride layer 24 can be appropriately selected and adjusted, taking into consideration the combination of materials for the silver / silver chloride layer 24 and the electrode 22. For example, the silver / silver chloride layer 24 can be formed on the upper surface of the electrode 22 by printing or coating it using a silver / silver chloride paste (ink) on the upper surface of the electrode 22, which is made of metal or carbon, by a screen printing method, an inkjet method, etc., and then drying it. Alternatively, the silver / silver chloride layer 24 can also be formed by printing, coating, plating, etc., silver (Ag) on ​​the upper surface of the electrode 22, and then chlorinating the surface.

[0116] Regarding step (iv) above, the method for forming (laminating) the film 25 can be appropriately selected and adjusted, taking into consideration the combination of materials for the film 25, the electrode 22, and the silver / silver chloride layer 24. For example, an opening corresponding to the dimensions of the reagent layer 23 (at least larger than the dimensions of the reagent layer 23) can be formed in a film 25 consisting of a laminate of a resin sheet and an adhesive sheet, or an adhesive sheet alone. Such a film 25 can be placed on the upper surface of the electrode 22 (working electrode 22a) such that its opening surrounds the portion of the electrode 22 that forms the reagent layer 23, and then fixed with an adhesive sheet. Alternatively, a film 25 having a predetermined opening can also be formed (laminated) by removing the portion at the predetermined position and dimensions as described above using a resist film or resist ink.

[0117] Furthermore, the film 25 is not formed on the upper surface of a portion (region X3) of the counter electrode 22c of the electrode 22, leaving the counter electrode 22c exposed. Therefore, for example, after forming the film including the upper surface of the counter electrode 22c as described above, a notched opening may be formed in the film 25 by cutting or the like.

[0118] In step (v) above, for example, a film 25 having a predetermined opening and a pre-prepared reagent solution are used to apply the reagent solution to at least a portion of the working electrode 22a by dropping it onto the opening portion of the film 25 formed (laminated) in step (iv) above. Then, by drying the applied reagent solution, a reagent layer 23 is formed on the upper surface of the working electrode 22a.

[0119] The opening of the film 25 may have dimensions and a shape that allow for the formation of a reagent layer with a width greater than the width of the sensor 11 (the tip portion shown in Figure 1(B)). In this case, the reagent layer formed from the applied reagent solution, which is wider than the width of the sensor 11, is shaped to have a predetermined width and shape by the following step (vi).

[0120] In step (vi) above, the removal (trimming) of the reagent layer 23 and electrode 22 is performed at the widthwise end of the sensor 11 (tip portion) over a predetermined length in the longitudinal direction of the sensor 11 (e.g., the direction of insertion into a living organism). By trimming in such a step (vi), after forming the reagent layer over a certain area, an appropriate portion (preferably a uniform portion) is selected to form a reagent layer of a predetermined area aligned between sensors. In addition, in the next step (vii), the sensor 11 can be cut out from the substrate 21 along its outer shape without breaking the formed reagent layer. The method for removing the reagent layer 23 and electrode 22 can be appropriately selected and adjusted considering the material of the reagent layer 23 (composition of the reagent solution) and the material of the electrode 22, etc. For example, the reagent layer 23 and electrode 22 can be removed by laser trimming.

[0121] Regarding step (vii) described above, the method for cutting out (cutting) the sensor 11 from the substrate 21 can be appropriately selected and adjusted considering the material of the substrate 21 and the shape of the sensor to be cut out. For example, if the material of the substrate 21 is resin, general cutting techniques can be used.

[0122] The cutting position includes the portion trimmed by step (vi). For example, the cutting position can be near the center line of the bottom of the recess created by laser trimming. In other words, the cutting is performed at a position slightly away from the trimmed reagent layer 23 and working electrode 22a.

[0123] Step (viii) described above can refer to the method for forming a protective film as described in relation to the electrochemical sensor of the present invention, and can also be appropriately selected and adjusted considering the combination of the protective film material, i.e., the composition of the protective film solution and the material of the part to be covered (mainly the material of the reagent layer 23, i.e., the composition of the reagent solution), the shape and area of ​​the part to be covered, etc. For example, the working electrode 22a (at least the part on which the reagent layer 23 is formed) can be immersed in the protective film solution, and then removed and dried to cover that part with a protective film.

[0124] Figure 3 is a plan view showing a sensor 101 in another embodiment of the present invention. The sensor 101 consists of a sensing section (a tip portion inserted into a living organism or immersed in a culture medium) 121 and a terminal section 122 for electrically connecting to the internal circuitry of a main body (not shown).

[0125] Figure 4 shows a cross-sectional view of the sensor 101 along the A-A' cutting line in Figure 3. Conductive thin films 112 are provided on both sides of the insulating substrate 111. On one side (front side) of the insulating substrate 111, the conductive thin film 112 is separated into an working electrode region 112a and a reference electrode region 112b by forming grooves 113 that reach the surface of the insulating substrate 111 through laser drawing, thereby electrically insulating it.

[0126] The upper surface of the insulating substrate 111 is covered with an insulating resist film 116a having an opening for forming a reference electrode 115 at a predetermined position in the reference electrode region 112b, and the lower surface of the insulating substrate 111 is covered with an insulating resist film 116b. On the other hand, Conductive layer thin film 112 Up to a certain distance from the edge, neither the top nor bottom surface is covered with insulating resist films 116a and 116b. The front region 112a becomes the working electrode 114, and the back region 112c becomes the counter electrode 117. The reagent layer 118 is formed on the working electrode 114.

[0127] Figure 5 shows a cross-sectional view along the B-B' line in Figure 4, and Figure 6 shows a cross-sectional view along the C-C' line in Figure 4. As shown in Figure 5, along the B-B' line, the protective film 119, reagent layer 118, working electrode 114, insulating substrate 111, counter electrode 117, and protective film 119 are formed in order from the top surface to the bottom surface (in the direction of the arrow in the figure). Also, as shown in Figure 6, along the C-C' line, the protective film 119, reference electrode 115 (insulating resist film 116a), working electrode region 112a, insulating substrate 111, counter electrode region 112c, insulating resist film 116b, and protective film 119 are formed in order from the top surface to the bottom surface (in the direction of the arrow in the figure). Note that the working electrode region 112a and counter electrode region 112c shown in Figure 6 do not function as a working electrode and counter electrode because they have insulating resist films 116a and 116b on their upper sides.

[0128] Figure 7 is a plan view showing a sensor 201 in another embodiment of the present invention. The sensor 201 includes a sensing unit 202 with a configuration for electrochemically measuring analytes, a terminal unit 203 with a configuration for electrically connecting to the internal circuitry of a main body (not shown), and a notch 204 provided near the terminal unit. The sensor 201 is a suitable embodiment for constructing a system for measuring analytes in a culture medium, for example, the sensing unit 202 having a structure that allows it to be immersed in a culture medium with a small amount of liquid, and the notch 204 having a structure suitable for installing the sensor 201 in a culture vessel. Figure 7(A) shows the electrode pattern before the film (insulating resist film) 230 is formed, and Figure 7(B) shows the electrode pattern after the film 230 is formed.

[0129] As shown in Figure 7(A), the sensor 201 has electrodes 220 formed on a substrate 210, and the electrodes 220 are physically and electrically separated by grooves 225 into a first working electrode 221, a second working electrode 222, a reference electrode 223, and a counter electrode 224. As shown in Figure 7(B), by covering with a film 230, the first working electrode 221 has an exposed area 221a in the sensing section 202 and an exposed area 221b in the terminal section 203. Similarly, the second working electrode 222, the reference electrode 223, and the counter electrode 224 also have exposed areas 222a, 223a, and 224a in the sensing section 202 and exposed areas 222b, 223b, and 224b in the terminal section 203, respectively. The sensing unit 202 includes the exposed areas 221a of the first working electrode and 222a of the second working electrode, arranged side-by-side to maximize their area, as well as the exposed area 223a of the reference electrode and the exposed area 224a of the counter electrode. Different types of reagent layers (not shown) for measuring different analytes can be formed on the exposed areas 221a of the first working electrode and 222a of the second working electrode. The exposed areas 221b, 222b, 223b, and 224b of the terminal portions 203 of the first working electrode, second working electrode, reference electrode, and counter electrode can be electrically connected to the main body (not shown) by, for example, electrode pads (not shown). [Examples]

[0130] The materials used in this embodiment (including the test example) are as follows: (a) Conductive carbon filler (a-1) Carbon Black…Sigma-Aldrich "05-1530"

[0131] (b) Anionic dispersants (b-1A) Poly(acrylic acid) 100,000...Sigma-Aldrich "523925", Average Mw~100,000 (b-1B) Poly(acrylic acid) 25,000... Fujifilm Wako Pure Chemical Corporation "162-18581", average molecular weight: approximately 25,000 (b-1C) Poly(acrylic acid) 5,000... Fujifilm Wako Pure Chemical Corporation "165-18571", average molecular weight: approximately 5,000 (b-2A) Poly(styrene sulfonate sodium salt)...Sigma-Aldrich "81609", Average Mw~16,800 (b-2B) Poly(4-styrene sulfonate sodium)...Sigma-Aldrich "243051", Average Mw~70,000 (b-3) Poly(4-styrenesulfonic acid-co-maleic acid) sodium salt...Sigma-Aldrich "434558", Average Mw~20,000 (b-4) Poly(ethylene oxide)-b-poly(acrylic acid)...Polymer Source "P6348-EOAA", Mn(POA-b-PAA):2-b-2.4(10 3 g / mol) (b-5) Poly(styrene)-b-poly(acrylic acid)...Polymer Source "P2397-SAA", Mn(PS-b-PAA):1.5-b-44(10 3 g / mol)

[0132] (Non-b) Non-anionic dispersants (Non-b-1) Hydroxypropylcellulose... NISSO HPC, Nippon Soda Co., Ltd.

[0133] (c) Cationic mediator (c-1) Polymer-bound PNT1... This has a structure in which a phenothiazine-based compound NHS form (PNT-70) as a redox mediator compound (c1) is bonded to a cationic polymer (c2) which is a copolymer of 2-aminoethyl methacrylate hydrochloride, (4-vinylphenyl)methaneamine, and methacryloylcholinchloride (hereinafter referred to as "polymer C"). (c-2) Polymer-bound PNT2... This has a structure in which a phenothiazine compound (PNT-70) as a redox mediator compound (c1) and poly(L-lysine) hydrochloride (ALAMANDA POLYMERS "PLKC800") (hereinafter referred to as "PLL") as a cationic polymer (c2) are bonded together. The synthesis methods for PNT-70, polymer C, polymer-bound PNT1, and polymer-bound PNT2 are as follows.

[0134] [Synthesis Example 1] Synthesis of PNT-70

[0135] (1) Synthesis of sulfonic acid fragments [ka]

[0136] The above reaction synthesized a sulfonic acid fragment.

[0137] (2) Carbon Synthesis of acid fragments [ka]

[0138] Carboxylic acid fragments were synthesized by the two-step reaction described above, in acetonitrile and tetrahydrofuran (THF).

[0139] (3) Synthesis of PNT-34 [ka]

[0140] The sulfonic acid fragments and carboxylic acid fragments synthesized as described above were suspended in MeOH / H2O, and 50% Ag2CO3 / Celite was added in portions over 15 minutes at an internal temperature of approximately 47°C. After addition, the mixture was stirred for 2.5 hours at an internal temperature of approximately 68°C. After cooling to room temperature, the mixture was filtered using Celite filtration, and the filtrate was concentrated. The residue was purified multiple times by silica gel column chromatography to obtain PNT-34.

[0141] (4) Synthesis of the condensate (PNT-68) [ka]

[0142] Under an Ar atmosphere, PNT-34 was dissolved in dichloromethane, and amino-PEG12-t-butyl ester and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl) were added. The mixture was then stirred at room temperature for 3 hours. After concentrating the reaction mixture, the condensate (PNT-68) was obtained by purifying it three times by silica gel column chromatography.

[0143] (5) Synthesis of the deprotector (PNT-69) [ka]

[0144] The condensate (PNT-68) obtained in (4) was dissolved in dichloromethane, trifluoroacetic acid (TFA) was added, and the mixture was stirred at room temperature for 3 hours. After concentrating the reaction mixture, the deprotected product (PNT-69) was obtained by azeotropic reaction several times with toluene.

[0145] (6) Synthesis of PNT-69NHS compound (PNT-70) [ka]

[0146] The deprotected compound (PNT-69) was dissolved in dichloromethane, and N-hydroxysuccinimide (NHS) and EDCI·HCl were added. The mixture was then stirred at room temperature for 4 hours. The reaction mixture was purified by silica gel column chromatography to obtain PNT-69NHS (PNT-70), in which the carboxyl groups at the end of the PEG chain (PEG12) were activated (NHS esterified).

[0147] [Synthesis Example 2] Synthesis of Polymer C [ka]

[0148] First, 2-aminoethyl methacrylate hydrochloride, (4-vinylphenyl)methaneamine, and methacloylcholinchloride were prepared. Then, 0.97 mmol of 2-aminoethyl methacrylate hydrochloride, 0.97 mmol of (4-vinylphenyl)methaneamine, 11.97 mmol of methacloylcholinchloride, 0.08 mmol of V-50, and 10.49 g of ethanol were placed in a four-necked flask. The same procedure as for polymer A was then carried out to obtain polymer C. Polymer C corresponds to a polymer with a molar ratio of n:m:l = 21.2:8.1:70.7, as calculated from the results of 1H-NMR. Furthermore, as measured by gel permeation chromatography (GPC), the number-average molecular weight Mn of the polymer was 37,774, and the weight-average molecular weight Mw was 214,367.

[0149] [Synthesis Example 3] Synthesis of Polymer-Bound PNT1 PNT-69NHS (PNT-70) was dissolved in MilliQ water to a concentration of 21.05 mg / mL (Solution (1)). As a high molecular weight polymer, polymer C was dissolved in MilliQ water to a concentration of 15 mg / mL (Solution (2)). Next, WSC (DOJINDO W001) was dissolved in MilliQ water to a concentration of 20 mg / mL (Solution (3)). 40 μL of Solution (1), 140.3 μL of Solution (2), and 383.4 μL of Solution (3) were mixed with 96 μL of separately prepared 250 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.0), and the total volume was adjusted with MilliQ water to 1200 μL. The mixture was then allowed to react at room temperature with stirring for approximately 20 hours. Subsequently, the solution was collected after several ultrafiltrations using a centrifugal ultrafiltration filter (Amicon Ultra-4 50k; Merck Millipore) to remove low molecular weight particles.

[0150] [Synthesis Example 4] Synthesis of Polymer-Bound PNT2 PNT-69NHS (PNT-70) was dissolved in MilliQ water to a concentration of 15 mg / mL (Solution (1)). As a high molecular weight polymer, poly(L-lysine) hydrochloride (ALAMANDA POLYMERS PLKC800) was dissolved in MilliQ water to a concentration of 10 mg / mL (Solution (2)). Next, WSC (DOJINDO W001) was dissolved in MilliQ water to a concentration of 20 mg / mL (Solution (3)). 43.7 μL of Solution (1), 51.3 μL of Solution (2), and 479.3 μL of Solution (3) were mixed with 96 μL of separately prepared 250 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 6.0), and the total volume was adjusted with MilliQ water to 1200 μL. The mixture was then allowed to react at room temperature with stirring for approximately 20 hours. Subsequently, the solution was collected after several ultrafiltrations using a centrifugal ultrafiltration filter (Amicon Ultra-4 50k; Merck Millipore) to remove low molecular weight particles.

[0151] (d) Oxidoreductase (d-1) FAD-dependent glucose dehydrogenase... GDH GLD1: Manufactured by BBI International

[0152] (e) Crosslinking agent (e-1) Glutaraldehyde... 25% glutaraldehyde solution (Fujifilm Wako Pure Chemical Corporation)

[0153] (f) Buffer solution (f-1)Bis-Tris: (Bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane)... Manufactured by Dojin Chemical Co., Ltd. (f-2A)Tris-HCl pH7.5…Invitrogen UltraPure 1M Tris-HCl pH7.5, ThermoFisherSCIENTIFIC (f-2B) Tris-HCl pH8.0…Invitrogen UltraPure 1M Tris-HCl pH8.0, ThermoFisherSCIENTIFIC (f-2C)Tris-HCl pH8.5…1M Tris-HCl (pH8.5), Fujifilm Wako Pure Chemical Industries (Manufacturer: Nippon Gene Co., Ltd.)

[0154] (g) Metal salts Sodium chloride (NaCl)... Fujifilm Wako Pure Chemical Corporation (Product code: 191-01665) Potassium chloride (KCl)...Nacalai Tesque Co., Ltd. (Product code: 28514-75) Sodium bromide (NaBr)... Fujifilm Wako Pure Chemical Corporation (Product code: 192-09412) Potassium bromide (KBr)... Fujifilm Wako Pure Chemical Corporation (Product code: 164-03472) Magnesium bromide (MgBr2) hexahydrate... Fujifilm Wako Pure Chemical Industries, Ltd. (Product code: 138-09192, purity 99.9%) Potassium sulfate (K2SO4)... Fujifilm Wako Pure Chemical Industries, Ltd. (Product code: 169-13552, purity 99.9%)

[0155] [Test Example 1] Dispersion Test <Preparation of reagent solution samples> Reagent solution samples were prepared by adding carbon black (a-1) to aqueous solutions of various anionic dispersants (b) at concentrations of 1 mg / mL or 2 mg / mL, according to the formulations shown in Table 2, to achieve a carbon concentration of 20 mg / mL, and then treating the mixture with an ultrasonic homogenizer for at least 3 minutes.

[0156] [Table 2]

[0157] <Filtering of reagent solution> The reagent solution sample obtained as described above was treated in an ultrasonic bath for approximately 10 minutes before use. The resulting redispersed solution sample was filtered through a 0.8 μm filter (ADVANTEC, DISMIC 25CS080AN), and the recovered filtrate was diluted 10-fold with Milli-Q water to obtain the recovered solution sample.

[0158] Images of the recovered solution samples are shown in Figure 8. The lighter the color of the recovered solution sample, the less carbon black (a-1) has passed through the filter and the more aggregated carbon black (a-1) has been captured by the filter, meaning that the dispersibility is low. However, it has been shown that all of the anionic dispersants (b) in the reagent solution (recovered solution) samples 1-1 to 1-11 have the effect of dispersing the conductive carbon filler (a) in the aqueous solvent, to varying degrees. Furthermore, from the results of reagent solution (recovered solution) samples 1-1 to 1-3, in which poly(acrylic acid) with different average molecular weights was used as the anionic dispersant (b) at the same concentration (1 mg / mL), it is suggested that when an anionic polymer such as poly(acrylic acid) is used as the anionic dispersant (b), the dispersion effect of the conductive carbon filler (a) decreases as the average molecular weight increases.

[0159] [Test Example 2] pH and Metal Ion Concentration Dependence Test Part 1 Reagent solution samples containing various components at predetermined concentrations were prepared according to the formulations shown in Table 3. The buffer solution used was prepared so that the pH of the reagent solution would reach a predetermined value when used in the predetermined amount. This Test Example 2 compares the dispersibility of reagent solution samples containing metal ions (metal salts) at the same concentration, after adjusting the pH using a buffer solution that does not contain metal ions (metal salts).

[0160] The concentration of polymer-bound PNTs was determined by first diluting the stock solution of each polymer-bound PNT 25-fold and adding 100 μL to a microplate. The absorption spectrum was then measured using a plate reader to confirm the concentration in the stock solution, and the concentration in each reagent sample was adjusted based on this value. The value "equivalent to absorbance 3" indicates that the absorbance at 608 nm is approximately 0.12 when diluted 25-fold. A UV-Star® 96-well F-Boden microplate manufactured by Greiner Bio One was used. A Tecan Infinite® M200 Pro plate reader was used.

[0161] [Table 3]

[0162] The reagent solution samples were prepared as follows: First, a conductive carbon filler (a) was added to an aqueous solution of anionic dispersant (b) and treated with an ultrasonic homogenizer for at least 3 minutes. Then, a buffer solution (f) and a metal salt (g) were added. Next, a cationic mediator (c) was added to the treated solution and treated with an ultrasonic homogenizer for approximately 30 seconds. The presence or absence of aggregation in the obtained reagent solution samples was visually confirmed. The results are shown in Table 3.

[0163] [Test Example 3] pH and Metal Ion Concentration Dependence Test Part 2 Reagent solution samples containing various components at predetermined concentrations were prepared according to the formulations shown in Table 4. This Test Example 3 compares the dispersibility of reagent solution samples containing different concentrations of metal ions (metal salts) after adjusting the pH using a buffer solution that does not contain metal ions (metal salts).

[0164] [Table 4]

[0165] The reagent solution samples were prepared as follows: First, a conductive carbon filler (a) was added to an aqueous solution of anionic dispersant (b) and treated with an ultrasonic homogenizer for at least 3 minutes. Then, a buffer solution (f) and a metal salt (g) were added. Next, a cationic mediator (c) was added to the treated solution and treated with an ultrasonic homogenizer for approximately 30 seconds. The presence or absence of aggregation in the obtained reagent solution samples was visually confirmed. The results are shown in Table 4.

[0166] [Test Example 4] pH and Metal Ion Concentration Dependence Test Part 3 Reagent solution samples containing various components at predetermined concentrations were prepared according to the formulations shown in Table 5. This Test Example 4 compares the dispersibility of reagent solution samples containing different types and concentrations of metal salts (metal ions) after adjusting the pH using a buffer solution that does not contain metal ions (metal salts).

[0167] [Table 5]

[0168] The reagent solution samples were prepared as follows: First, a conductive carbon filler (a) was added to an aqueous solution of anionic dispersant (b) and treated with an ultrasonic homogenizer for at least 3 minutes, after which a buffer solution (f) and a metal salt (g) were added. Next, a cationic mediator (c) was added to the treated solution and treated with an ultrasonic homogenizer for approximately 30 seconds.

[0169] The presence or absence of aggregation in the obtained reagent solution samples was visually confirmed. The results are shown in Table 5. Regarding the results of Test Examples 2 to 4, first from the results of Test Example 2, it was found that aggregation may occur when the pH of the reagent solution is high (due to the effect of pH itself, not the effect of the concentration of metal salts derived from the buffer solution used to adjust the pH), but aggregation can be suppressed by improving the dispersibility of the conductive carbon filler within an appropriate pH range corresponding to the composition of the reagent solution. Furthermore, from the results of Test Examples 3 and 4, it was found that the occurrence of aggregation differs depending on the type of metal ion contained in the reagent solution, but generally, aggregation tends to occur when the concentration of metal ions is high. For example, it is suggested that aggregation can be suppressed for many metal ions if the concentration of metal ions is less than 100 mM. On the other hand, in some cases, such as with magnesium ions, aggregation can be suppressed even if the concentration is higher than 50 mM (for example, even at around 200 mM). Therefore, it was found that aggregation can be suppressed by improving the dispersibility of the conductive carbon filler within an appropriate concentration range corresponding to the type of metal ion.

[0170] [Test Example 5] Test of anionic dispersant (b) Reagent solution samples containing various components at predetermined concentrations were prepared according to the formulations shown in Table 6. [Table 6]

[0171] The test solution samples were prepared as follows: First, a conductive carbon filler (a) was added to an aqueous solution of anionic dispersant (b) and treated with an ultrasonic homogenizer for more than 3 minutes, after which buffer solution (f) was added. Next, a cationic mediator (c) was added to the treated solution and treated with an ultrasonic homogenizer for approximately 30 seconds, and finally, an oxidoreductase (d) and a crosslinking agent (e) were added to the treated solution.

[0172] The presence or absence of aggregation in the obtained test solution samples was visually confirmed. The results are shown in Table 6. Cationic polymer (c 2The polymer - bound PNT1 synthesized using a predetermined copolymer and the polymer - bound PNT2 synthesized using PLL as the cationic polymer (c 2 ) show differences in the effect of improving the dispersibility of the conductive carbon filler (a) depending on the concentration in the reagent solution and the pH of the reagent solution. However, there are conditions under which the effect of improving the dispersibility of the conductive carbon filler (a) can be recognized for any cationic polymer (c 2 ).

[0173] [Test Example 6] Responsiveness Evaluation Test [Preparation of Reagent Solution According to the formulation shown in Table 7, reagent solution samples containing various components at a predetermined concentration were prepared.

Table 7

[0174] The preparation of the test solution sample was carried out as follows. First, the conductive carbon filler (a) was added to an aqueous solution of the anionic dispersant (b) or its control, and treated with an ultrasonic homogenizer for 3 minutes or more, and then the buffer solution (f) was added thereto. Next, the cationic mediator (c) was added to the treated solution and treated with an ultrasonic homogenizer for about 30 seconds, and finally, the redox enzyme (d) and the cross - linking agent (e) were added to the treated solution.

[0175] [Fabrication of Sensor Electrode 0.6 μL of each reagent solution sample prepared as described above was applied onto a carbon electrode fabricated by screen printing on an insulating substrate, and dried overnight to obtain a sensor electrode.

[0176] [Preparation of RPMI Medium A solution prepared with RPMI-1640 Medium (Sigma-Aldrich, R1383) was to which 2-morpholinoethanesulfonic acid (MES, Dojin Chemical Co., Ltd.) and 3-morpholinopropanesulfonic acid (MOPS, Dojin Chemical Co., Ltd.) were added as buffer components to a final concentration of 25 mM each, and the pH was adjusted to 7.4. The culture medium obtained in this way was used as "RPMI medium" in the following examples.

[0177] <Sensor evaluation regarding responsiveness> Using the sensor electrode prepared as described above as the working electrode, a platinum electrode as the counter electrode, and an Ag / AgCl electrode (saturated KCl) (manufactured by BAS Corporation) as the reference electrode, a three-electrode measurement system was used. A potentiostat (manufactured by BAS Corporation) was used to measure the time change of current (current response value) at approximately 37°C in RPMI medium using the amperometric method. Specifically, glucose was added every 100 seconds starting 100 seconds after the start of measurement to achieve theoretical concentrations of 3 mM, 15 mM, or 30 mM, and the current response value was measured. The current response value was calculated as the average of five measurements taken 5 seconds, 10 seconds, 15 seconds, 20 seconds, and 25 seconds before the next glucose addition, after the glucose to be measured was added. The current value at the final concentration was calculated as the average of five measurements taken 80 seconds, 85 seconds, 90 seconds, 95 seconds, and 100 seconds after the glucose solution was added to achieve a glucose concentration of 30 mM. Note that the current values ​​at each glucose concentration are the current values ​​after background correction processing has been applied, which subtracts the current value at a glucose concentration of 0 mM.

[0178] Furthermore, cyclic voltammetry was performed in RPMI medium using a similar three-electrode measurement system. The scan speed was 10 mV / s. Note that both the current response value and the cyclic voltammetry results are the average of the measurements from the two sensors.

[0179] The results of the current response values are shown in Fig. 9[A], and the results of cyclic voltammetry are shown in Fig. 9[B]. Regarding the current responsiveness, the sensors using each anionic dispersant (anionic polymer) (reagent solution samples 6-1 to 6-5) showed higher responsiveness than the sensor using hydroxypropyl cellulose (reagent solution sample 6-6). Similarly, regarding the results of cyclic voltammetry, higher redox peak values were obtained for the sensors using each anionic dispersant (anionic polymer) than for the sensor using hydroxypropyl cellulose. This is presumably because the mediator could be adsorbed closely onto the electrode by using an anionic dispersant (anionic polymer).

[0180] [Test Example 7] Durability (response maintenance rate) evaluation test <Preparation of reagent solution> In Test Example 7, the reagent solution samples 6-1 and 6-6 in Test Example 6 were prepared again and used.

[0181] <Preparation of polymer solution for protective film> The following reagents were mixed to the following final concentrations to prepare a polymer solution for the protective film. · Poly(tert-butyl methacrylate-b-4-vinylpyridine) (manufactured by Polymer Source Co., Ltd., hereinafter referred to as "tBuMA4VP"). Final concentration: 7.11% (wt / v) · Random copolymer of tripropylene glycol methyl ether methacrylate-styrene-4-vinylpyridine (manufactured by Nard Co., Ltd., hereinafter referred to as "TGMAS4VP"). Final concentration: 0.89% (wt / v) · Poly(ethylene glycol) diglycidyl ether (manufactured by Sigma-Aldrich Co., Ltd., hereinafter referred to as "PEGDGE"). Final concentration: 0.98% (wt / v) · HEPES buffer solution (pH 8.0). Final concentration: 5 mM

[0182] tBuMA4VP, TGMAS4VP, and PEGDGE were used after being dissolved in ethanol. The number-average molecular weight (Mn) of tBuMA4VP is 87,000 for poly(ter.butyl methacrylate) and 74,000 for poly(4-vinylpyridine). The Mw / Mn ratio of tBuMA4VP is 1.16. TGMAS4VP has a tripropylene glycol methyl ether methacrylate:styrene:4-vinylpyridine ratio of 6.6:20.3:73.0, with a number-average molecular weight (Mn) of 60,704, a weight-average molecular weight (Mw) of 120,095, and a Mw / Mn ratio of 1.98. The number-average molecular weight (Mn) of PEGDGE is approximately 1,000. HEPES buffer was prepared using 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (manufactured by Dojin Chemical Co., Ltd.).

[0183] <Fabrication of electrodes for sensors> On a gold electrode fabricated by sputtering on an insulating substrate, 0.5 μL of the reagent solution sample prepared as described above was applied, dried for 15 minutes, and this process was repeated twice for a total of three applications. After that, the electrodes were dried overnight. This formed a reagent layer corresponding to each reagent solution sample on each gold electrode. Each gold electrode with the reagent layer was immersed in the protective polymer solution prepared as described above, removed, and dried, repeating this process multiple times to form a protective film on each gold electrode. Sensor electrodes were obtained through the above process.

[0184] <Sensor evaluation regarding durability (response retention rate)> Using the sensor electrode prepared as described above as the working electrode, a platinum electrode as the counter electrode, and an Ag / AgCl electrode (saturated KCl) (manufactured by BAS Corporation) as the reference electrode, a three-electrode measurement system was used. A potentiostat (manufactured by BAS Corporation) was used to measure the time change of current in RPMI medium at approximately 37°C using the amperometric method. Specifically, glucose was added every 1000 seconds starting 1500 seconds after the start of measurement to theoretical values ​​of 5 mM, 15 mM, or 30 mM, and the current response value was continuously measured. After the measurement, the electrodes were stored in RPMI medium at 37°C. Similar measurements were also performed on the 1st, 2nd, and 3rd day after storage. The current values ​​at glucose concentrations of 5 mM and 15 mM are the average values ​​calculated from five measurement points taken 5 seconds, 10 seconds, 15 seconds, 20 seconds, and 25 seconds before the next glucose addition, starting from the time the glucose to be measured was added. Furthermore, the current value at the final concentration is the average value calculated from five measurements taken at 980 seconds, 985 seconds, 990 seconds, 995 seconds, and 1000 seconds after adding glucose solution to achieve a glucose concentration of 30 mM. The current values ​​at each glucose concentration are the values ​​after background correction processing, which subtracts the current value at a glucose concentration of 0 mM.

[0185] The results are shown in Figure 10. Even after protective film coating, the current response was higher for the sensor using poly(acrylic acid) (reagent solution sample 6-1) than for the sensor using hydroxypropyl cellulose (reagent solution sample 6-6). Furthermore, the sensor durability after 3 days (current retention rate compared to the first day) was also higher for the sensor using poly(acrylic acid) (reagent solution sample 6-1). This is presumed to be because the mediator was more firmly adsorbed onto the electrode.

Claims

1. A reagent layer comprising a conductive carbon filler (a), an anionic dispersant (b), and a cationic mediator (c), A reagent layer in which the cationic mediator (c) is a compound in which a redox mediator compound (c1) and a cationic polymer (c2) having a quaternary ammonium cationic group are bonded via a linker portion (c3) as needed.

2. The reagent layer according to claim 1, wherein the anionic dispersant (b) is a polymer with a weight-average molecular weight of 70,000 or less.

3. The reagent layer according to claim 1, wherein the anionic dispersant (b) is a polymer having a carboxyl group and / or a sulfo group in its side chain.

4. The reagent layer according to claim 3, wherein the anionic dispersant (b) is a polymer comprising at least one selected from the group consisting of acrylic acid-derived units, maleic acid-derived units, and styrene sulfonic acid-derived units.

5. The reagent layer according to claim 1, wherein the conductive carbon filler (a) is carbon black.

6. The reagent layer according to claim 1, further comprising an oxidoreductase (e) for oxidizing or reducing the analyte.

7. The reagent layer according to claim 6, wherein the oxidoreductase (e) is of the coenzyme-bound type.

8. The reagent layer according to claim 6, wherein the oxidoreductase (e) is crosslinked with the cationic polymer (c2).

9. An electrochemical sensor for detecting or quantifying analytes, comprising a working electrode, a counter electrode, and a reagent layer according to any one of claims 1 to 8.

10. The electrochemical sensor according to claim 9, further comprising a reference electrode.

11. Furthermore, the electrochemical sensor according to claim 9, further comprising a protective film covering at least the reagent layer.

12. (1) A step of preparing a reagent solution containing a conductive carbon filler (a), an anionic dispersant (b), and a cationic mediator (c), (2) The step of applying the reagent solution to the reagent layer forming area, (3) A step of drying the applied reagent solution to form a reagent layer. A method for forming a reagent layer, including, A method for forming a reagent layer, wherein the cationic mediator (c) is a compound in which a redox mediator compound (c1) and a cationic polymer (c2) having a quaternary ammonium cationic group are bonded via a linker portion (c3) as needed.

13. The method for forming a reagent layer according to claim 12, wherein the pH of the reagent solution is 8.0 or less.

14. The method for forming a reagent layer according to claim 13, wherein the concentration of metal ions in the reagent solution is 200 mM or less.

15. The method for forming a reagent layer according to claim 14, wherein the metal ion in the reagent solution is an alkali metal ion and its concentration is less than 100 mM.

16. The method for forming a reagent layer according to claim 12, wherein the anionic dispersant (b) is a polymer comprising at least one selected from the group consisting of acrylic acid-derived units, maleic acid-derived units, and styrene sulfonic acid-derived units, having a weight-average molecular weight of 70,000 or less.

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