Reagent layer including conductive carbon filler, sensor having reagent layer, and method for forming 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

Reagent layer containing conductive carbon filler, sensor having said reagent layer, and method for forming said reagent layer

[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 a conductive carbon filler in an aqueous solvent, a reagent layer containing the dispersant, a conductive carbon filler, and a cationic mediator, a sensor including the reagent layer, and a method for forming the reagent layer.

[0002] Conventionally, sensors that measure analytes in a sample by reacting proteins with the analyte have been known. Examples of such sensors include electrochemical sensors using enzymes, such as glucose sensors fabricated using glucose oxidoreductase and, optionally, a redox mediator (a redox substance that mediates electron transfer) or a redox polymer (a polymer to which a redox mediator is bound via a linker, etc.). Glucose sensors are used, for example, for self-testing blood glucose levels. While traditionally, samples were typically obtained by collecting a small amount of blood, in recent years, implantable electrochemical glucose sensors have been developed that are implanted in vivo to continuously measure glucose in blood or interstitial space. Glucose sensors are also used to measure glucose in samples other than biological materials, such as culture media. Such glucose sensors typically 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 a large specific surface area, such as nanocarbon materials, have been used in glucose sensors, such as electrochemical glucose sensors, and electrodes used in biofuel cells, in order to achieve higher sensitivity and higher output by improving the specific surface area of ​​the electrode. 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.

[0004] JP 2021-082394 A

[0005] Typically, to form a reagent layer containing a conductive carbon filler and a redox mediator or redox polymer, and optionally a redox enzyme, 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. However, conductive carbon fillers are generally difficult to disperse in aqueous solvents due to their strong hydrophobicity, and maintaining a dispersed state is difficult due to their tendency to aggregate due to van der Waals forces. If a reagent layer is formed using a reagent solution in which the conductive carbon filler is not sufficiently dispersed, problems arise, such as a significant decrease in analyte detection sensitivity and poor durability during long-term measurements (continuous monitoring) in humid environments such as in vivo or in culture media. Note that it is appropriate to dissolve redox enzymes in an aqueous solvent, and using organic solvents as the solvent for preparing the reagent solution is not appropriate.

[0006] In one aspect, the present invention aims to provide a reagent layer for producing an electrochemical sensor capable of detecting an analyte with high sensitivity, and a method for forming the same. In another aspect, the present invention aims to provide a reagent layer for producing an electrochemical sensor with excellent durability and suitable for long-term measurements (continuous monitoring), and a method for forming the same.

[0007] The present inventors have discovered that in a reagent solution prepared, preferably under specific conditions, using a conductive carbon filler, a cationic redox mediator or redox polymer (cationic mediator), and an anionic dispersant, the conductive carbon particles do not aggregate or precipitate and remain well dispersed; and that a reagent layer formed using such a reagent solution can tightly adsorb the cationic mediator onto the electrode, thereby improving the analyte detection sensitivity of the electrochemical sensor and providing it with excellent durability for continuous monitoring, which led to the completion of the present invention.

[0008] That is, the present invention encompasses at least the following: [Item 1] A reagent layer comprising 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 unit selected from the group consisting of an acrylic acid-derived unit, a maleic acid-derived unit, and a styrenesulfonic acid-derived unit. [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, optionally via a linker moiety (c3). [Item 6] The reagent layer according to Item 5, wherein the cationic polymer (c2) has a quaternary ammonium cationic 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) that oxidizes or reduces an analyte. [Item 9] The reagent layer according to Item 8, wherein the oxidoreductase (e) is a coenzyme-bound oxidoreductase. [Item 10] The reagent layer according to Item 8, wherein the oxidoreductase (e) is crosslinked with the cationic polymer (c2). [Item 11] An electrochemical sensor for detecting or quantifying an analyte, comprising a working electrode, a counter electrode, and the reagent layer according to any one of Items 1 to 10. [Item 12] The electrochemical sensor according to Item 11, further comprising a reference electrode. [Item 13] The electrochemical sensor according to Item 11, further comprising a protective film covering at least the reagent layer. [Item 14] A method for forming a reagent layer, comprising: (1) preparing a reagent solution containing a conductive carbon filler (a), an anionic dispersant (b), and a cationic mediator (c), (2) applying the reagent solution to a portion where a reagent layer is to be formed, and (3) drying the applied reagent solution to form a reagent layer. [Item 15] The method for forming a reagent layer according to Item 14, wherein the pH of the reagent solution is 8.0 or less.[Item 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. [Item 17] The method for forming a reagent layer according to Item 16, wherein the metal ions in the reagent solution are alkali metal ions, and the concentration thereof is less than 100 mM. [Item 18] The method for forming a reagent layer according to Item 14, wherein the anionic dispersant (b) is a polymer having a weight-average molecular weight of 70,000 or less and containing at least one unit selected from the group consisting of an acrylic acid-derived unit, a maleic acid-derived unit, and a styrene sulfonic acid-derived unit. [Item 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, optionally via a linker moiety (c3).

[0009] The present invention makes it possible to satisfactorily disperse a conductive carbon filler in a reagent solution containing an aqueous solvent. Furthermore, by using such a reagent solution (carbon dispersion) in which the conductive carbon filler is dispersed, it becomes possible to fabricate an electrochemical sensor that is highly practical and that achieves high sensitivity and improved durability (for example, the ability to maintain responsiveness during measurement in a humid environment) even while containing the conductive carbon filler.

[0010] In the present invention, it is possible to visually determine whether the conductive carbon material is "dispersed" (including "re-dispersed") or "aggregated" (including "aggregation / precipitation") in the reagent solution in most cases. For example, if the conductive carbon material is "aggregated," floating or precipitated aggregates can be easily confirmed visually.

[0011] Centrifugation can also be used to determine the dispersion and aggregation of the conductive carbon filler. For example, when a reagent solution in which the conductive carbon filler is "dispersed" is centrifuged, no precipitation of the conductive carbon filler occurs. However, when a reagent solution in which the conductive carbon filler is "aggregated" is centrifuged, the aggregates precipitate and the solution separates into a layer of conductive carbon filler and a transparent aqueous solution phase. The centrifugation conditions can be, for example, 10,000 × g for 5 minutes, but can also be adjusted appropriately depending on the embodiment, taking into account, for example, the type and concentration of the conductive carbon filler.

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

[0013] LIST OF REFERENCE SIGNS 11 Sensor (probe) 21 Substrate 22 Electrode 22a Working electrode 22b Reference electrode 22c Counter electrode 23 Reagent layer 24 Silver / silver chloride layer 25 Film X1 Sensor head X2 Insertion direction of sensor into living body X3 Area of ​​counter electrode not covered with film X4 Area of ​​sensor head not covered with film X5 Area where reagent layer is not formed X6 Area where reagent layer and electrode (working electrode) are not formed (trimmed) 101 Sensor 111 Insulating substrate 112 Conductive thin film 112a Working electrode area 112b Reference electrode area 112c Counter electrode area 113 Groove 114 Working electrode 115 Reference electrode 116a Insulating resist film (upper surface) 116b Insulating resist film (lower surface) 117 Counter electrode 118 Reagent layer 119 Protective film 121 Sensing section 122 Terminal section 201 Sensor 202 Sensing section 203 Terminal section 204 Notch section 210 Substrate 220 Electrode 221 First working electrode 221a Exposed area in sensing section of first working electrode 221b Exposed area in terminal section of first working electrode 222 Second working electrode 222a Exposed area in sensing section of second working electrode 222b Exposed area in terminal section of second working electrode 223 Reference electrode 223a Exposed area in sensing section of reference electrode 223b Exposed area in terminal section of reference electrode 224 Counter electrode 224a Exposed area in sensing section of counter electrode 224b Exposed area in terminal section of counter electrode 225 Groove 230 Film (insulating resist film)

[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 can be formed by a forming method using a carbon dispersion liquid, as described below, i.e., a reagent liquid in which the conductive carbon filler (a) is dispersed, and is preferably a layer in which the conductive carbon filler (a), anionic dispersant (b), and cationic mediator (c) are uniformly contained.

[0015] (a) Conductive Carbon Filler The "conductive carbon filler" in the present invention is not limited to a specific type, and various conductive carbon fillers can be used taking into consideration the embodiment of the present invention (such as the use of a reagent layer or an electrochemical sensor), the action and effect, etc. As the conductive carbon filler, conductive carbon materials of various shapes such as spherical (particulate), scaly, fibrous, porous, etc. can be used. As the conductive carbon filler (a), only one type may be used, 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 spherical graphite. Specific examples of porous carbon materials include activated carbon powder or activated carbon fiber having mesopores of 2 to 50 nm, as well as carbon materials having interconnected pores where the mesopores are connected. 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) may be within an appropriate range. For example, when the conductive carbon filler is spherical (particulate), its average particle diameter (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 m ２ / g or more, preferably 30m ２ The conductive carbon filler (a) in the present invention is preferably a spherical (particulate) carbon filler, such as carbon black.

[0017] (b) Anionic Dispersant In the present invention, the term "anionic dispersant" refers to a compound that has the essential function of dispersing the conductive carbon filler (a) in a reagent solution and that has a negative net charge (anionic) across the molecule. The anionic dispersant (b) may be used alone or in combination of two or more (e.g., mixed).

[0018] However, "anionic surfactants" and "anionic polysaccharides" do not fall under the category of the anionic dispersant (b) of the present invention. Examples of anionic surfactants include methylnaphthalenesulfonic acid formalin condensate salts (e.g., methylnaphthalenesulfonic acid formalin condensate sodium salt, product name "Demol MS" (Kao)), naphthalenesulfonic acid formalin condensate salts (e.g., β-naphthalenesulfonic acid formalin condensate sodium salt, product name "Demol N" (Kao)), alkylene maleic acid copolymer salts (e.g., diisobutylene-maleic anhydride copolymer sodium salt, 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 certain embodiments of the present invention, the electrochemical sensor has a protective film covering a reagent layer formed on the sensor (working electrode). In such embodiments, to prevent the anionic dispersant (b) from leaking outside the protective film (in vivo, in a culture medium, etc.), the sensor (working electrode) may be immersed in an aqueous solvent after the protective film is formed to dissolve and remove the anionic dispersant (b) from the reagent layer. In such a treatment, depending on the relationship between the pore size and molecular weight of the protective film, for example, the anionic dispersant (b) bonded to the conductive carbon filler (a) may not / is unlikely to be eluted from the reagent layer (eluted outside the protective film), while the anionic dispersant (b) not bonded to the conductive carbon filler (a) may be eluted from the reagent layer (eluted outside the protective film). The aqueous solvent used in this treatment may be the same as the aqueous solvent used to prepare the reagent solution used in the method for forming the reagent layer of the present invention described below.

[0020] For example, an "anionic polymer" (excluding those corresponding to anionic surfactants and anionic polysaccharides) that has the essential function of dispersing a conductive carbon filler (a) in a reagent solution and that has a negative net charge (anionic) for the entire polymer can be used as the anionic dispersant (b) in the present invention. The anionic polymer may have a "chain structure" that is linear, branched, or comb-like, and the "chain structure" may contain one or more ring structures derived from a cyclic compound (e.g., an aromatic hydrocarbon ring, a non-aromatic hydrocarbon ring, an aromatic heterocycle, a non-aromatic heterocycle, etc.). The main chain (the relatively long portion of the chain structure) and side chain (the relatively short portion of the chain structure) of the anionic polymer are generally primarily carbon atoms, and may contain at least one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur atoms. In other words, a heteroatom-containing bond such as an ether bond, a thioether bond, or an amide bond may be present along the chain. Anionic polymers generally have multiple (numerous) negatively charged functional groups (anionic functional groups) on their side chains, and may also have anionic functional groups at one or both ends of the main chain. A variety of anionic polymers having anionic functional groups on their side chains and / or ends are known, and they can be purchased, prepared by modifying an appropriate polymer, or synthesized using an appropriate monomer. Anionic polymers may be homopolymers, copolymers, or polymers in which these are bonded and / or mixed, and may also be random polymers, block polymers, or graft polymers.

[0021] The anionic functional group is, for example, a carboxy group which may have a substituent, preferably an unsubstituted carboxy group, which is —COO in an aqueous solvent. － and a sulfo group which may have a substituent, preferably an unsubstituted sulfo group, which forms -SO ３ － Examples of groups that generate the following include:

[0022] Anionic polymers include, for example, ethylene-based polymers having anionic functional groups in the side chains (and terminals). Ethylene-based polymers include those having ethylenic carbon-carbon double bonds capable of radical polymerization, such as vinyl groups (CH ２ ═CH—), allyl group (CH ２ =CH-CH ２ -), acryloyl group (CH ２ ═CH—C(O)—), methacryloyl group (CH ２ =C(CH ３ )-C(O)-), and modified products thereof (for example, those in which vinyl acetate-derived units are converted into vinyl alcohol by saponification, or those treated to impart hydrophilicity). Examples of monomers containing an ethylenic carbon-carbon double bond 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 containing at least one unit selected from the group consisting of an acrylic acid-derived unit, a maleic acid-derived unit, and a styrenesulfonic acid-derived unit, i.e., a homopolymer or copolymer synthesized using at least one monomer selected from the group consisting of acrylic acid, maleic acid, and styrenesulfonic acid (e.g., 4-styrenesulfonic acid). Note that the copolymer may be synthesized using at least one monomer selected from the group consisting of acrylic acid, maleic acid, and styrenesulfonic acid (e.g., 4-styrenesulfonic acid) together with at least one monomer other than acrylic acid, maleic acid, and styrenesulfonic acid (e.g., 4-styrenesulfonic acid).

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

[0025] Preferred polymers for the anionic dispersant (b) of the present invention, which can be used in either of the above two embodiments, include, for example, poly(acrylic acid), poly(styrenesulfonic acid), poly(styrenesulfonic 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, other properties, characteristics, etc. of the anionic polymer used as the anionic dispersant (b) can be adjusted depending on the embodiment and effects of the present invention and the type of anionic polymer. The degree of polymerization of the anionic polymer is usually 50 or more. The average molecular weight, typically the weight-average molecular weight, of the anionic polymer is, for example, 1,000 or more, preferably 5,000 or more. 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 an anionic polymer can be measured by known means depending on the type of anionic polymer, such as gel permeation chromatography (GPC). When a commercially available anionic polymer is purchased and used, the numerical value shown in the catalog or the like (e.g., as "Mw" or "M.W.") can be regarded as the weight-average molecular weight.

[0028] (c) Cationic Mediator In the present invention, the term "cationic mediator" refers to a compound (redox mediator compound) that has the essential function of a redox mediator, or a compound that contains a moiety derived from such a redox mediator compound and has a positive net charge (cationic) throughout the molecule. The term "redox mediator" refers to a redox substance that mediates electron transfer, such as a substance responsible for transferring electrons generated by the redox reaction of an analyte catalyzed 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, if necessary, via a "linker moiety" (c3). The "cationic mediator" of the present invention, i.e., the "redox mediator," "cationic polymer," and "linker moiety" that constitute it, are not limited to a specific type, and various types can be used taking into consideration the embodiment, effects, and the like of the present invention.

[0030] (c1) Redox mediator compound The "redox mediator compound" in the present invention is not limited to a specific type, and various redox mediators or derivatives thereof can be used in consideration of the embodiment of the present invention (reagent layer, use of electrochemical sensor, etc.), action and effect, etc.

[0031] Examples of the redox mediator compound 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. Phenazine-based compounds and phenothiazine-based compounds are preferred redox mediators because they have a negative oxidation-reduction potential (vs. Ag / AgCl·saturated KCl) (lower than 0 V) ​​and are less susceptible to the influence of impurities contained in samples that affect electrochemical measurements, such as easily oxidized compounds contained in biological samples or culture medium samples that are not used as analytes, such as ascorbic acid (vitamin C) and uric acid.

[0033] The phenazine-based compound and the phenothiazine-based compound refer to compounds having a phenazine skeleton or a phenothiazine skeleton, respectively, 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), R ３ is the formula -NR ３１ R ３２ A substituted amino group represented by the formula: ３１ and R ３２ Both are substituents, or one is a substituent and the other is a hydrogen atom.) When this is the case, the phenothiazine compound has the resonance structures of the following general formulas (2-1) and (2-2), and is a compound that can function as a cationic mediator by itself.

[0034]

[0035]

[0036] In general formulas (1) and (2), R １ ~R ９each independently represents any one of the groups or atoms (i) to (ix') below (provided that (ix') applies only when the cationic mediator (c) is a compound having a structure in which a "redox mediator compound" (c1) and a "cationic polymer" (c2) are bonded, if necessary, via a "linker portion" (c3), that is, when (ix') reacts with the specific reactive group of the cationic polymer (c2) or the specific reactive group of the linker compound as the specific reactive group (details will be described later) of the phenazine compound or phenothiazine compound as the redox mediator compound (c1): (i) a hydrogen atom; (ii) a halogen atom; (iii) a hydroxy 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 a substituent (for example, C １－１５ Alkyl group, preferably C １－６ (vi) an acyl group (e.g., C １－６ Acyl group, i.e., C １－６ (vii) a phenyl group which may have a substituent; (viii) a quaternary ammonium cation group; (ix) a carboxy group which has been activated as an ester (for example, a carboxy group which has been activated as an ester with N-hydroxysuccinimide (NHS)); (ix') a carboxy group.

[0037] Examples of the substituents that the above-mentioned (iii) to (vii) may have include: (a) a halogen atom, (b) a hydroxy group, (c) an amino group, and (d) a linear or branched, saturated or unsaturated hydrocarbon group (e.g., C １－１５ Alkyl group, preferably C １－６ alkyl group, more preferably C １－３ alkyl groups), (e) acyl groups (e.g., C １－６ Acyl groups, preferably C １－３Examples of the group containing at least one of the following: (a) acyl group, (b) guanidino group, (c) mesyl group, (d) phenyl group, (e) thiol group, (f) formyl group (aldehyde group), (g) epoxy group, (h) maleimide group, (i) activated esterified carboxy group, (m') carboxy group, and (n) oxyethylene group. However, (m') is applicable only when the cationic mediator (c) is a compound having a structure in which a "redox mediator compound" (c1) and a "cationic polymer" (c2) are bonded, if necessary, via a "linker portion" (c3), that is, when (m') serves as a specific reactive group (described in detail below) of a phenazine compound or phenothiazine compound as the redox mediator compound (c1) and reacts with a specific reactive group of the cationic polymer (c2) or a specific reactive group of the linker compound. The substituents that the above substituents (iii) to (vii) may have may be groups composed solely of any of the above (a) to (n), or groups composed of two or more selected from the above (a) to (n), such as (c) an amino group, (i) a thiol group, (j) a formyl group, (k) an epoxy group, (l) a maleimide group, (m) an activated esterified carboxy group, or (n) a group such as (d) a linear or branched, saturated or unsaturated hydrocarbon group further substituted with an oxyethylene group. Those skilled in the art can practice the present invention by selecting chemically appropriate substituents from (a) to (n) for each of the above (iii) to (vii), 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, and may be, for example, one, two, or three, and multiple substituents may be bonded to one atom (for example, a carbon atom in an alkyl group or a nitrogen atom in an amino group).

[0038] The quaternary ammonium cation group (viii) is -R Ａ -N ＋ (R Ｂ )(R Ｃ )(R Ｄ ) (wherein R Ａ , R Ｂ, R Ｃ and R Ｄ are each a saturated hydrocarbon group which may have a substituent (e.g., C １－１５ Alkyl group, preferably C １－６ The (n) oxyethylene group is the same as the oxyethylene group (e.g., polyethylene glycol chain (PEG chain)) described in relation to the (c3) linker portion (linker-like hydrophilic portion).

[0039] R １ ~R ９ At least one of R may be a group for positively charging the redox mediator compound. For example, a phenazine-based compound or a phenothiazine-based compound may have R １ ~R ９ (iv) an amino group which may have a substituent, preferably an unsubstituted amino group, which is —NH ３ ＋ or (viii) a positively charged functional group (referred to as a "cationic functional group" in this specification) such as a quaternary ammonium cationic group.

[0040] In addition, when 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, R １ ~R ９ At least one of the groups may be a reactive group capable of bonding to the cationic polymer (c2), or the linker portion (c3) used as needed, or a group carried by a linker compound for forming the linker portion (c3) (referred to herein as the "specific reactive group of the redox mediator compound (c1)", although when it is clear from the context that it is that of the redox mediator compound (c1), it may also be simply referred to as the "specific reactive group").

[0041] R １ ~R ９At least one of R may be other groups related to the performance as a redox mediator or to the preparation of the reagent layer. For example, phenazine-based compounds or phenothiazine-based compounds may contain R １ ~R ９ At least one of the above may be (iii) a hydroxy group which may have a substituent, preferably an unsubstituted hydroxy group, (iv) an amino group which may have a substituent, preferably an unsubstituted amino group, (viii) a quaternary ammonium cation group, or other functional group which is so-called a "hydrophilic group", or a group derived from such a hydrophilic group and has hydrophilicity (preferably, (n) a hydrophilic group substituted with an oxyethylene group or other hydrophilic group), or (v) a saturated or unsaturated hydrocarbon group which may have a substituent, (vi) an acyl group which may have a substituent, or (vii) a phenyl group which may have a substituent, which has an oxyethylene group or other hydrophilic group as a substituent, thereby making the group as a whole hydrophilic.

[0042] In the present invention, "hydrophilic" refers to a property of having a high affinity for water and being soluble or miscible to a degree that enables the intended purpose to be achieved in 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 having a net positive charge across the entire polymer. The cationic polymer (c2) in the present invention may be any polymer as long as it has a structure capable of supporting the redox mediator compound (c1), i.e., a group capable of reacting with a group possessed by the redox mediator compound (c1) itself or a group possessed by the linker portion (c3) used as needed, and can be applied to a desired site, for example, on a working electrode, to form a reagent layer. The type of cationic polymer is not particularly limited, and any one type may be used alone, or two or more types may be used in combination.

[0044] The cationic polymer may have a "chain structure" such as a linear, branched, or comb-like structure, and the "chain structure" may contain one or more ring structures derived from a cyclic compound (such as an aromatic hydrocarbon ring, a non-aromatic hydrocarbon ring, an aromatic heterocycle, or a non-aromatic heterocycle). The main chain and side chains of the cationic polymer (c2) are generally primarily carbon atoms and may contain at least one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur atoms. In other words, they may contain a bond containing a heteroatom, such as an ether bond, a thioether bond, or an amide bond. The cationic polymer (c2) generally has multiple (a large number of) positively charged functional groups (cationic functional groups) in its side chains and may further have a cationic functional group at one or both ends of the main chain. Furthermore, from the viewpoint of enabling one molecule of cationic polymer (c2) to support multiple (large numbers of) redox mediator compounds (c1), if necessary via a linker portion (c3), it is appropriate for the cationic polymer (c2) to have a reactive group (referred to herein as a "specific reactive group of the cationic polymer (c2)," although it may also be simply referred to as a "specific reactive group" when the context clearly indicates that it is that of the cationic polymer (c2)) in its side chain, which reactive group has the ability to bond with a group possessed by the redox mediator compound (c1) itself or a group possessed by the linker portion (c3) used as needed or a linker compound for forming it. Furthermore, one or both ends of the main chain may also have a specific reactive group. Various cationic polymers (c2) having cationic functional groups and specific reactive groups in their side chains and / or ends are known, and it is possible to purchase one having the desired properties, prepare it by modifying an appropriate polymer, or synthesize it using an appropriate monomer. The cationic polymer (c2) may be any of a homopolymer, a copolymer, and a polymer in which these are combined and / or mixed, and may also be any of a random polymer, a block polymer, and a graft polymer.

[0045] Examples of cationic polymers (c2) include 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). Cationic polymers (c2) also include proteins and polypeptides that can be considered polymers with amino acids as monomers (however, conceptually, they are distinguished from artificially synthesized amino acid polymers), have natural amino acid sequences or modified (substituted, deleted, added, etc.) amino acid sequences, and have cationic functional groups and specific reactive groups in their side chains (and terminals). Polysaccharide-based polymers that originally have or have introduced specific reactive groups and cationic functional groups are also included in cationic polymers (c2).

[0046] The ethylene-based polymer includes a radically polymerizable ethylenic carbon-carbon double bond, such as a vinyl group (CH ２ ═CH—), allyl group (CH ２ =CH-CH ２ -), acryloyl group (CH ２ ═CH—C(O)—), methacryloyl group (CH ２ =C(CH ３)-C(O)-), and modified products thereof (for example, those in which vinyl acetate-derived units are converted into vinyl alcohol by saponification, or those treated to impart hydrophilicity). Examples of monomers containing an ethylenic carbon-carbon double bond 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 called methyl acrylate), methyl methacrylate (also called methyl methacrylate), butyl acrylate (also called butyl acrylate), butyl methacrylate (also called butyl methacrylate), hydroxyethyl methacrylate, and acrylonitrile. Further, (meth)acrylic polymers such as a copolymer of methyl methacrylate and hydroxyethyl methacrylate, a copolymer of butyl methacrylate and hydroxyethyl methacrylate, and poly(2-methacryloyloxyethylphosphorylcholine-co-n-butyl methacrylate), which are generally known as biocompatible polymers, and polyester polymers such as polyethylene terephthalate are also preferred as ethylene-based polymers.

[0047] Examples of ethylene-based polymers preferred as the cationic polymer (c2) include polyallylamine hydrochloride, allylamine hydrochloride-diallylamine hydrochloride copolymer, and allylamine-diallyldimethylammonium chloride copolymer. (Meth)acrylic polymers having quaternary ammonium cations, amino groups, or the like in their side chains (and terminals) are also preferred ethylene-based polymers. More specifically, homopolymers or copolymers containing structural units derived from 2-aminoethyl methacrylate, (vinylbenzyl)trimethylammonium chloride, methacryloylcholine chloride, or the like, as well as poly(diallyldimethylammonium chloride), poly(allylamine hydrochloride), allylamine hydrochloride-diallylamine hydrochloride copolymer, and allylamine-diallyldimethylammonium chloride copolymer are preferred for use as the cationic polymer (c2).

[0048] An example of an imine polymer is poly(ethyleneimine). Poly(ethyleneimine) has a -(CH ２ ) ２ -NH ２ , -(CH ２ ) ２ -NH-(CH ２ ) ２ -NH ２ , -(CH ２ ) ２ -N((CH ２ ) ２ -NH ２ ) ２ and the amino group in the structure: -NH ２ serves as a specific reactive group of the cationic polymer (c2), and can be bonded to a redox mediator compound (c1) or a linker portion (c3) having a corresponding appropriate specific reactive group (e.g., an activated esterified carboxy group).

[0049] Examples of amino acid polymers include poly(L-lysine), poly(L-arginine), and poly(L-ornithine). Poly(L-lysine) has -(CH ２ ) ４ -NH ２Poly(L-arginine) has the structure -(CH ２ ) ２ -NH-C(=NH)-NH ２ The amino group contained in the side chain of these amino acid polymers is: -NH ２ (or guanidino group: -NH-C(=NH)-NH ２ ) or the like serves as a specific reactive group of the cationic polymer (c2), and can be bonded to a redox mediator compound (c1) or a linker part (c3) having a corresponding appropriate specific reactive group (e.g., an activated esterified carboxy group). The amino acid polymer may 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 amino groups in its side chains (in the sugar structure). The amino groups contained in the side chains of such polysaccharide polymers serve as specific reactive groups for the cationic polymer (c2), and can be bonded to a redox mediator compound (c1) or a linker portion (c3) having a corresponding appropriate specific reactive group (e.g., an activated esterified carboxy group).

[0051] The degree of polymerization, weight-average molecular weight, other properties, characteristics, etc. of the cationic polymer (c2) can be adjusted depending on the embodiment 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 more. The average molecular weight, typically the weight-average molecular weight, of the cationic polymer (c2) is usually 10,000 or more, preferably 50,000 or more, and more preferably 100,000 or more. The upper limit of the weight-average molecular weight of the cationic polymer (c2) is not particularly limited, but is usually less than 10,000,000, preferably less than 1,000,000.

[0052] The weight-average molecular weight and molecular weight distribution of the cationic polymer (c2) can be measured by known means depending on the type of cationic polymer (c2), for example, gel permeation chromatography (GPC) can be used. When a commercially available cationic polymer (c2) is purchased and used, the numerical value shown in the catalog or the like (for example, as "Mw" or "M.W.") can be regarded as the weight-average molecular weight.

[0053] (c3) Linker Moiety The linker moiety (c3) is a structure that is present as needed and serves to mediate the bond between the redox mediator compound (c1) and the cationic polymer (c2). The linker moiety (c3) generally has a chain structure that, excluding the reactive groups at both ends before the reaction or the bond structure after the reaction, is mainly composed 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, it is a moiety having a chain structure that may contain a bond containing a heteroatom, such as an ether bond, a thioether bond, or an amide bond. The "chain structure" of the linker moiety (c3) may be linear or branched. The "chain structure" may also contain one or more ring structures derived from a cyclic compound (e.g., an aromatic hydrocarbon ring, a non-aromatic hydrocarbon ring, an aromatic heterocycle, or a non-aromatic heterocycle).

[0054] In one embodiment of the present invention, the linker portion (c3) also functions as a site (hydrophilic portion) for improving the hydrophilicity of the cationic mediator (c), and can also be called a "linker-like hydrophilic portion." When the linker portion (c3) is a linker-like hydrophilic portion, it is preferable that it be linear and contain no ring structures derived from cyclic compounds, from the viewpoint of hydrophilicity.

[0055] In one preferred embodiment of the present invention, the linker-like hydrophilic moiety has the formula: -(OC ２ H ４ ) ｑIn the formula, q represents, for example, an integer of 1 to 80, preferably an integer of 3 to 36. Oxyethylene chains represented by this formula in which q is somewhat large are generally called "polyethylene glycol chains (PEG chains)" or the like.

[0056] In one embodiment of the present invention, the linker-like hydrophilic moiety has the formula: -(CH ２ ) ｐ In one embodiment of the present invention, the main chain of the linker-like hydrophilic moiety comprises a hydrocarbon chain represented by the above formula: -(CH ２ ) ｐ - and a hydrocarbon chain represented by the formula: -(OC ２ H ４ ) ｑ The value of p in the formula representing the hydrocarbon chain can be appropriately adjusted to be balanced with the value of q in the formula representing the oxyethylene chain, taking into consideration the hydrophilicity of the linker-like hydrophilic moiety.

[0057] The linker moiety (c3) can be derived from a compound (referred to herein as a "linker compound") having at least two reactive groups (referred to herein as the "specific reactive groups of the linker compound," although they may also be simply referred to as the "specific reactive groups" when the context clearly indicates that they belong to the linker compound) that can bond with the groups (specific reactive groups) of the redox mediator compound (c1) and the cationic polymer (c2). For example, a linker compound is reacted with the redox mediator compound (c1), and then the linker compound bound to the redox mediator compound (c1) (in other words, the "redox mediator compound (c1)-linker compound reactant") is reacted with the cationic polymer (c2), thereby producing a cationic mediator (c) as a compound in which the redox mediator compound (c1) and the cationic polymer (c2) are bound via the linker moiety (c3). Alternatively, a linker compound may be reacted with a cationic polymer (c2), and then the linker compound bound to the cationic polymer (c2) (in other words, the "cationic polymer (c2)-linker compound reactant") may be reacted with a redox mediator compound (c1), to produce a cationic mediator (c) as a compound in which the redox mediator compound (c1) and the cationic polymer (c2) are bound via the linker moiety (c3).

[0058] <Specific Reactive Group> As the specific reactive group in the present invention, i.e., the specific reactive group of the redox mediator compound (c1), the specific reactive group of the cationic polymer (c2), and the specific reactive group of the linker compound, various known reactive groups can be adopted and various combinations can be made. The specific reactive groups in the present invention may be bonded to each other by a covalent bond or by a non-covalent bond (e.g., electrostatic interaction), but are preferably bonded by a covalent bond, for example, from the viewpoint of bond stability.

[0059] The specific reactive group in the present invention is preferably at least one selected from the group consisting of a carboxy group or its activated ester, an amino group, a thiol group, a formyl group (aldehyde group), an epoxy group, and a maleimide group (referred to as a "preferred specific reactive group" in this specification). The preferred specific reactive groups are an amino group, a thiol group, a formyl group (aldehyde group), an epoxy group, and a maleimide group, each of which corresponds to R in the general formulas (1) and (2) relating to the redox mediator compound (c1). １ ~R ９ These correspond to the substituents (c), (i), (j), (k), and (l) given as examples of substituents that the substituents (iii) to (vii) in (1) may further have, and a carboxy group and its activated ester form correspond to the substituents (m') and (m), respectively. Groups with which each of the reactive groups included in the preferred group of specific reactive groups can react are shown in Table 1. When a reactive group included in the preferred group of specific reactive groups is selected as one of the specific reactive groups in the present invention, the group that reacts with it may be another reactive group included in the preferred group of specific reactive groups (those underlined in the table), or it may be a reactive group not included in the preferred group of specific reactive groups (those not underlined in the table or those not included in the table).

[0060]

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

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

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

[0064] The reagent layer may contain a type of "oxidoreductase" corresponding to the analyte, such as in an electrochemical sensor in the form of a biosensor, or it may not contain an oxidoreductase, such as in an electrochemical sensor other than a biosensor, or in the form of 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), the anionic dispersant (b), and the cationic mediator (c). Examples of such optional components include an "oxidoreductase" (d).

[0066] (d) Oxidoreductase The oxidoreductase (d) refers to an enzyme that can oxidize (including dehydrogenation) or reduce the analyte that is the target of an electrochemical sensor or the like. The electrochemical sensor of the present invention typically takes the form of an electrochemical sensor (biosensor) that contains an oxidoreductase in the reagent layer, but is not limited thereto. It can also take the form of an electrochemical sensor other than a biosensor that does not contain an oxidoreductase in the reagent layer, or a sensor other than an electrochemical sensor (e.g., an optical sensor). Depending on the application of the electrochemical sensor or the like, it can be selected whether the reagent layer contains an oxidoreductase (d), and depending on the type of analyte, it can be selected what type of oxidoreductase (d) to contain.

[0067] Examples of the oxidoreductase (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). Any one of the oxidoreductases (d) may be used alone, or two or more of them may be used in combination as necessary.

[0068] In one embodiment of the present invention, the oxidoreductase (d) is a coenzyme-bound type. Examples of coenzyme-reduced oxidoreductase (d) include pyrroloquinoline quinone (PQQ)-bound GDH and flavin adenine dinucleotide (FAD)-bound GDH. In an embodiment using glucose as an analyte, FAD-bound GDH is preferred from the viewpoint of low reactivity with maltose, which is not an analyte (for example, the enzymatic activity with maltose can be 5% or less, preferably 3% or less, when the enzymatic activity with glucose is taken as 100%). Examples of FAD-bound GDH include those derived from the genus Aspergillus (such as oryzae and Terreus) and those derived from the genus Mucor.

[0069] In one embodiment of the present invention, the oxidoreductase (d) may be crosslinked with the cationic mediator (c), particularly the cationic polymer (c2) constituting the oxidoreductase (d). For example, the oxidoreductase (d) can be crosslinked with the cationic mediator (c), particularly the cationic polymer (c2), using a crosslinker (e). Examples of the crosslinker (e) include glutaraldehyde. The crosslinker (e) reacts with a reactive group (e.g., an amino group) of the oxidoreductase (d), which is a protein, and a reactive group (e.g., an amino group) of the cationic mediator (c), for example, a reactive group that has not reacted with the redox mediator compound (c1) or the linker portion (c2), or a reactive group of a different type from that used to react with the redox mediator compound (c1) or the linker portion (c2), thereby bonding the oxidoreductase (d) and the cationic mediator (c) via the crosslinker (e). By forming a complex with a larger molecular weight through such crosslinking, it is possible to further suppress the outflow of the oxidoreductase (d) and the cationic mediator (c), particularly the redox mediator compound (c1) constituting them, from the protective film. When a crosslinking agent (e) is used, the crosslinking agent (e) may react with the reactive groups of each of the two molecules of the oxidoreductase (d), thereby bonding the oxidoreductases (d) together. That is, when the crosslinking agent (e) is used, the oxidoreductases (d) may be crosslinked with the cationic mediator (c), particularly the cationic polymer (c2), and at the same time, the oxidoreductases (d) may be crosslinked with each other in the reagent layer.

[0070] -Method of Forming Reagent Layer- The method of forming a reagent layer of the present invention includes at least the following step (1), and usually further includes steps (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) (sometimes referred to herein as a "reagent solution preparation step"); (2) a step of applying the reagent solution to a reagent layer formation site (sometimes referred to herein as an "application step"); (3) a step of drying the applied reagent solution to form a reagent layer (sometimes referred to herein as a "reagent layer formation step").

[0071] (1) Reagent Solution Preparation Step The reagent solution preparation step (1) is a step of preparing a reagent solution that contains the main components constituting the reagent layer, i.e., at least a conductive carbon filler (a), an anionic dispersant (b), and a cationic mediator (c), and may further contain other components, such as an oxidoreductase (d) and a crosslinking agent (e), as needed.

[0072] The reagent solution can generally be prepared by adding the necessary components (a) to (e) to an aqueous solvent (water or a mixed solvent of water and a water-compatible solvent). The aqueous solvent can be adjusted to an appropriate pH by adding a pH adjuster, a buffer (liquid), other compounds, etc., 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 that makes the pH of the reagent solution 8.0 or less, the effect of improving the dispersibility of the conductive carbon filler (a) is easily achieved. Note that, when the reagent solution contains an oxidoreductase (d), it is appropriate that the pH of the reagent solution be set within a range in which the activity of the oxidoreductase (d) is maintained.

[0074] The pH of a reagent solution is usually adjusted using a buffer. Various buffers can be used, including acetate buffer (acetic acid and sodium acetate), phosphate buffer, citrate buffer, citrate phosphate buffer, Tris buffer (trishydroxymethylaminomethane, 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 containing sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate). The pH of a reagent can be adjusted to a desired value (range) by using an appropriate buffer (buffering agent).

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

[0076] Since the metal ions in the reagent solution are derived from various substances used in preparing the reagent solution, the composition of the reagent solution and the type and amount of each component can be adjusted so that the metal ion concentration is within an appropriate range. For example, since the metal ions in the reagent solution may be derived from a buffer solution, the composition of the buffer solution used to adjust the pH of the reagent solution (the type and concentration of compounds contained in the buffer solution) may be appropriate. Furthermore, since the metal ions in the reagent solution may be derived from an anionic dispersant (b) that forms a salt, the amount of the anionic dispersant (b) that forms such a salt may also be taken into consideration, as necessary.

[0077] The order of addition of the components contained in the reagent solution can be adjusted as appropriate, but it is preferable to perform the steps in the order of the following steps (1-1) and (1-2): (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, and (1-2) a step of 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 to one another, can be appropriately adjusted taking into consideration the embodiment, effects, and the like of the present invention.

[0079] The concentration of the 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 as a "dispersion treatment" in this specification) for improving the dispersibility of the conductive carbon filler (a) in the reagent solution may be carried out by applying physical energy to the reagent solution. The dispersion treatment may be carried out, for example, between the step (1-1) and the step (1-2), or alternatively or in addition, after the step (1-2) (before the subsequent application step (2)).

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

[0083] The oxidoreductase (d) is preferably added after the dispersion treatment in the reagent solution preparation step (1) so as not to be affected by physical energy. The crosslinking agent (e), such as glutaraldehyde, for crosslinking the oxidoreductase (d) with the cationic mediator (c) or between the oxidoreductases (d) is preferably added after the addition of the oxidoreductase (d).

[0084] Furthermore, when a compound in which a redox mediator compound (c1) and a cationic polymer (c2) are bonded, if necessary, via a linker portion (c3), is used as the cationic mediator (c), it is preferable to add the cationic mediator (c) after improving the dispersibility of the conductive carbon filler (a) by applying physical energy to the reagent solution through a dispersion treatment, for example, ultrasonic treatment, etc.

[0085] (2) Applying Step The applying step (2) is a step of applying (dropping, coating, etc.) the reagent solution obtained in the reagent solution preparing step (1) to the reagent layer forming site, for example, at least a part of the working electrode.

[0086] The means for "applying" (the method and its conditions, the apparatus used, etc.) are not particularly limited as long as they are capable of forming a reagent layer from the reagent solution, and any means similar to the reagent solution application step in conventional methods for forming a reagent layer (or methods for manufacturing an electrochemical sensor) can be used. In a typical embodiment of the present invention, the applying step is performed by dripping an appropriate amount of the reagent solution obtained in the reagent solution preparation step (1) onto at least a portion of the working electrode (the portion where the reagent layer will be formed). The area to be applied and the amount of reagent solution can be adjusted depending on the desired thickness of the reagent layer.

[0087] (3) Reagent Layer Forming Step The reagent layer forming step (4) is a step of drying the reagent solution applied to the reagent layer forming portion (e.g., at least a part of the working electrode) in the applying step (2) to form a coating layer.

[0088] The means for "drying" (the method and conditions, the apparatus used, etc.) is not particularly limited as long as it can form a (preferably uniform) reagent layer from the applied reagent solution, and a means similar to the drying step of a reagent solution in the manufacture of conventional electrochemical sensors can be adopted. In a representative embodiment of the present invention, the reagent layer forming step 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 an analyte of 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 if necessary, further has a protective film covering at least the reagent layer.

[0090] The "protective film" is a membrane-like member for preventing or suppressing leakage of substances contained in the reagent layer (such as the conductive carbon filler (a), the cationic mediator (c), and the oxidoreductase (d) used as needed) into the environment outside the protective film (such as in a living body or in a culture medium). The protective film that covers the reagent layer suitably has permeable holes within it 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] Because a working electrode (or a probe equipped with the working electrode) on which a reagent layer is formed is used by inserting it into a living body or immersing it in a culture medium, the protective film covering its surface should be biocompatible, meaning that proteins and cells do not or are unlikely to 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-methacryloyloxyethylphosphorylcholine-co-n-butyl methacrylate).

[0092] The protective film can be formed by preparing a solution (protective film solution) containing raw materials for forming the protective film, immersing the area where you want to form the protective film, for example, the area of ​​the working electrode where at least the reagent layer is formed, in the solution, lifting it out, and drying it, and repeating this 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 may be a two-electrode type composed of a working electrode and a counter electrode, or a three-electrode type composed 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, a biosensor for continuous glucose monitoring (CGM) for self-monitoring of blood glucose, which measures the glucose concentration in blood or interstitial fluid continuously or semi-continuously, 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, a biosensor for continuously or semi-continuously measuring the concentration of glucose or the like in a culture medium.

[0095] Hereinafter, an embodiment of the electrochemical sensor of the present invention when it is an implantable type will be described with reference to the drawings. However, it should be understood that the drawings and the following description are merely illustrative, and the technical scope of the present invention is not limited to the disclosed embodiments, but includes various modified embodiments within the scope of solving the problems of the present invention and achieving the effects of the present invention.

[0096] In the following description, the surfaces facing the front of the paper in FIGS. 1(A) and (B), 3, and 7, and the surfaces facing the top of the paper in FIGS. 2(A) to (C), and 4 to 6, will be referred to as the "top surface," the surfaces facing the back of the paper in FIGS. 1(A) and (B), 3, and 7, and the surfaces facing the bottom of the paper in FIGS. 2(A) to (C), and 4 to 6 will be referred to as the "bottom surface," and the surfaces facing either the left or right of the paper in FIGS. 1(A) and (B), 2(A) to (C), and 5 to 7, and the surfaces facing either the front or back of the paper in FIG. 4 will be referred to as the "side surface." In addition, the dimension in the vertical direction of the paper of Figure 1(A), i.e., the direction parallel to arrow X2 indicating the insertion direction of the sensor into the living body, and the dimension in the vertical direction of the paper of Figures 3 and 7 may be referred to as "length," and the dimension in the horizontal direction of the paper of Figure 1(A), i.e., the direction perpendicular to arrow X2, and the dimension in the vertical direction of the paper of Figures 3 and 7 may be referred to as "width."

[0097] FIG. 1 is a plan view of a sensor 11 according to one embodiment of the present invention. FIG. 1(A) shows the entire sensor 11. FIG. 1(B) shows an enlarged view of the tip portion (sensing portion) of the sensor 11 shown in FIG. 1(A). The sensor 11 is suitable for constructing an implantable biosensor system that is attached to a living body for self-testing of blood glucose levels, for example. In this case, the sensor 11 can be inserted into the living body as a protruding portion (probe) from a main body (not shown). The sensor 11 can also be used when constructing a system for measuring the concentration of an analyte in a culture medium, for example.

[0098] 1A, 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 inserting the sensor 11 into a living body. 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 1 to 50 μm, preferably 500 to 50 μm.

[0099] In FIG. 1 , the sensor 11 includes a substrate 21, an electrode 22, a reagent layer 23, a silver / silver chloride layer (sometimes referred to as a reference layer) 24, and a film 25. The electrode 22 is uniformly formed on the substrate 21 and includes 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 a groove A1, while the reference electrode 22b and the counter electrode 22c are physically and electrically separated by a 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 top surface of the sensor 11 except for a portion of the electrode 22 (e.g., region X4 of the head region X1 and region X3 of the counter electrode 22c) and the reagent layer 23 (by providing openings that expose them). The exposed region X4 of the electrode 22 is connected to the circuitry of the main body 11.

[0100] 1B, 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, by doing so, it is possible to prevent the reagent layer 23 from peeling off (turning up) from the sensor 11 when the sensor 11 is inserted into a living body.

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

[0102] 2(B) is a cross-sectional view taken along the arrows BB in FIG. 1(B). In the portion to the right of the groove A1 where the silver / silver chloride layer 24 is formed, the substrate 21, the electrode 22 (reference electrode 22b), the silver / silver chloride layer 24, and the film 25 are laminated. In the portion to the left of the groove A1, which is physically and electrically separated from the reference electrode 22b, the substrate 21, the electrode 22 (working electrode 22a), and the film 25 are laminated. The side surface of the silver / silver chloride layer 24 (the right side surface in FIG. 2(B)) is exposed without the film 25 disposed 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 without being covered with the film 25.

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

[0104] The substrate 21 is typically a sheet-shaped 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) having at least one of the characteristics of flexibility, ease of processing, and heat resistance. A typical example of the resin material of the substrate 21 is polyethylene terephthalate (PET), but other examples include general-purpose plastics such as polyethylene, polypropylene, and polyethylene naphthalate. Furthermore, when 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 is conductive and stable (e.g., oxidation-resistant or salt-resistant). A typical example of the material of such an electrode 22 is gold, but other examples include platinum, palladium, and carbon. Note that when a working electrode 22a (reagent layer 23) is formed on one of the upper and lower surfaces of the sensor 11 and a counter electrode 22c is formed on the other, different electrode materials may be used.

[0106] The working electrode 22a is applied with a potential (based on the reference electrode 22b) sufficient to oxidize the mediator reduced by the reaction of an analyte (e.g., glucose) with 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, as needed. In this embodiment, a three-electrode configuration consisting of the working electrode 22a, the reference electrode 22b, and the counter electrode 23c is shown as an example to achieve more accurate measurements, but a two-electrode configuration consisting of the working electrode 22a and the counter electrode 23c without the reference electrode 22b is also possible (for example, such a two-electrode configuration is the mainstream for currently commercially available SMBG (self-monitoring blood glucose monitors)). The reference electrode can be a silver / silver chloride electrode with a silver / silver chloride (Ag / AgCl) layer 24 formed thereon, as shown in this embodiment, or a hydrogen electrode, a calomel electrode, or the like, with a mercury-containing layer formed thereon.

[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 or more and 150 μm or less, preferably 3 μm or more and 50 μm or less, and more preferably 5 μm or more and 30 μm or less. The film 25 has openings in a portion corresponding to the reagent layer 23 at the tip of the sensor 11 and in a portion corresponding to part of the counter electrode 22c, and the reagent layer 23 and the counter electrode 22c are exposed in those portions.

[0109] The film 25 can be, for example, a sheet made of the same resin material as the substrate 21 to which an adhesive sheet (e.g., acrylic, rubber, or hot melt) is attached. The resin sheet may be made of the same resin material as the substrate 21, or a different resin material. An adhesive sheet alone may be used as the film 25. A thermoplastic or photoplastic resist film, or a layer formed from resist ink, may also be used 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 portion of the film 25. From the viewpoint of workability of such a process, the contact angle (α) of the reagent solution with respect to the surface of the film 25 is preferably 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, α is preferably 90° or greater and β is preferably 50° or less. Note that 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 does not satisfy the above-described contact angle condition, it is possible to satisfy the above-described contact angle condition by performing an appropriate surface treatment, for example, by performing at least one of 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 1(B), at least the portion of sensor 11 including 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 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 shown.

[0112] The method for manufacturing the sensor 11 is not particularly limited, and any method that can manufacture the sensor 11 having the above-described configuration at a predetermined location can be selected and used as appropriate. For example, the sensor 11 can be manufactured by carrying out the following steps (i) to (viii): (i) forming (laminating) the electrode 22 on the upper surface of the substrate 21; (ii) 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) forming a silver / silver chloride layer 24 on the upper surface of the electrode 22 (reference electrode 22b); (iv) forming (laminating) a film 25 on the upper surfaces of the electrode 22 and the silver / silver chloride layer 24; (v) forming a reagent layer 23 on the upper surface of the electrode 22 (working electrode 22a); (vi) removing a portion (region X6) of the reagent layer 23 and the electrode 22; (vii) cutting out the sensor 11 from the substrate 21; (viii) forming a protective film.

[0113] In the above step (i), the method for forming (laminating) the electrode 22 can be appropriately selected and adjusted taking into consideration the combination of the material of the electrode 22 and the material of the substrate 21, etc. For example, if the material of the substrate 21 is a synthetic resin such as PET and the material of the electrode 22 is a 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 material of the substrate 21 is a synthetic resin such as PET and the material of the electrode 22 is carbon, the electrode 22 made of carbon can be formed, for example, by printing a 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; a substrate 21 having dimensions larger than the sensor 11 can be used so that the sensor 11 can be cut out in the subsequent step (vii).

[0114] In the above step (ii), the grooves A1 and A2 can be formed by, for example, laser trimming.

[0115] In the above step (iii), the method for forming the silver / silver chloride layer 24 can be appropriately selected and adjusted taking into consideration the combination of the material of the silver / silver chloride layer 24 and the material of the electrode 22, etc. For example, the silver / silver chloride layer 24 can be formed on the upper surface of the electrode 22, which is made of metal or carbon, by printing or applying a silver / silver chloride paste (ink) by screen printing, inkjet printing, or the like, and then drying the paste. Alternatively, the silver / silver chloride layer 24 can be formed by printing, applying, plating, or the like on the upper surface of the electrode 22 with silver (Ag), and then chloridizing the surface.

[0116] In the above step (iv), the method for forming (laminating) the film 25 can be appropriately selected and adjusted taking into consideration the combination of the material of the film 25 with the materials of 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) is formed in the film 25, which is made of a laminate of a resin sheet and an adhesive sheet, or a single adhesive sheet. Such a film 25 is placed on the top surface of the electrode 22 (working electrode 22a) so that the opening surrounds the portion of the electrode 22 that forms the reagent layer 23, and is fixed in place by an adhesive sheet. Alternatively, a film 25 with a predetermined opening can be formed (laminate) by using a resist film or resist ink to remove a portion at a predetermined position and size as described above.

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

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

[0119] The opening in film 25 may have a size and shape that allows the formation of a reagent layer with a width greater than that of sensor 11 (the tip portion shown in FIG. 1(B)). In this case, the reagent layer formed from the applied reagent solution and larger than the width of sensor 11 is shaped to have a predetermined width and shape in the next step (vi).

[0120] In the above step (vi), the removal (trimming) of the reagent layer 23 and the electrodes 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 insertion direction into a living body). After forming the reagent layer over a certain area through trimming in step (vi), an appropriate portion (preferably a uniform portion) can be selected to form a reagent layer of a predetermined uniform area across the sensors. Furthermore, in the next step (vii), the sensor 11 can be cut out from the substrate 21 along its outer shape without cracking the formed reagent layer. The method for removing the reagent layer 23 and the electrodes 22 can be appropriately selected and adjusted taking into account the material of the reagent layer 23 (the composition of the reagent solution) and the material of the electrodes 22, etc. For example, the reagent layer 23 and the electrodes 22 can be removed by laser trimming.

[0121] In the above step (vii), the method for cutting the sensor 11 from the substrate 21 can be appropriately selected and adjusted in consideration of the material of the substrate 21, the shape of the sensor to be cut out, etc. For example, when the material of the substrate 21 is resin, a general cutting technique can be used.

[0122] The cutting position includes the portion trimmed in step (vi), and may be, for example, a position near the center line of the bottom of the recess formed by laser trimming. In other words, the cutting is performed at a position somewhat separated from the trimmed reagent layer 23 and working electrode 22 a.

[0123] The above step (viii) can be performed by referring to the method for forming a protective film described in relation to the electrochemical sensor of the present invention, or can be appropriately selected and adjusted taking into consideration the combination of the material of the protective film, i.e., the composition of the protective film solution, and the material of the portion 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 portion to be covered, etc. For example, by immersing the working electrode 22a (at least the portion where the reagent layer 23 is formed) in the protective film solution, pulling it out, and drying it, that portion can be covered with a protective film.

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

[0125] Fig. 4 shows a cross-sectional view of the sensor 101 taken along the line A-A' in Fig. 3. Conductive thin films 112 are provided on both sides of an insulating substrate 111. The conductive thin film 112 on one side (front side) of the insulating substrate 111 has grooves 113 formed by laser drawing that are deep enough to reach the surface of the insulating substrate 111, and is electrically isolated to separate the sensor into a working electrode region 112a and a reference electrode region 112b.

[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, up to a portion a certain distance away from the end of the sensing portion 121, neither the upper nor lower surfaces are covered with the insulating resist films 116a, 116b, and the region 112a on the front side becomes the working electrode 114, and the region 112c on the back side becomes the counter electrode 117. A reagent layer 118 is formed on the working electrode 114.

[0127] FIG. 5 shows a cross-sectional view taken along the line B-B' in FIG. 4, and FIG. 6 shows a cross-sectional view taken along the line C-C' in FIG. 4. As shown in FIG. 5, along the line B-B', a protective film 119, a reagent layer 118, a working electrode 114, an insulating substrate 111, a counter electrode 117, and a protective film 119 are formed in this order from the top to the bottom (in the direction of the arrows in the figure). Also, as shown in FIG. 6, along the line C-C', a protective film 119, a reference electrode 115 (insulating resist film 116a), a working electrode region 112a, an insulating substrate 111, a counter electrode region 112c, an insulating resist film 116b, and a protective film 119 are formed in this order from the top to the bottom (in the direction of the arrows in the figure). The working electrode region 112a and the counter electrode region 112c shown in FIG. 6 are provided with the insulating resist films 116a and 116b on their upper surfaces, and therefore do not function as a working electrode or a counter electrode.

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

[0129] 7(A), sensor 201 includes electrode 220 formed on substrate 210, and electrode 220 is physically and electrically separated by groove 225 into first working electrode 221, second working electrode 222, reference electrode 223, and counter electrode 224. As shown in FIG. 7(B), by being covered with film 230, first working electrode 221 has exposed region 221a in sensing portion 202 and exposed region 221b in terminal portion 203. Similarly, second working electrode 222, reference electrode 223, and counter electrode 224 have exposed regions 222a, 223a, and 224a in sensing portion 202 and exposed regions 222b, 223b, and 224b in terminal portion 203, respectively. In the sensing unit 202, the exposed region 221a of the first working electrode and the exposed region 222a of the second working electrode are arranged side by side to maximize the area, and the exposed region 223a of the reference electrode and the exposed region 224a of the counter electrode are also arranged. Different types of reagent layers (not shown) for measuring different analytes can be formed on the exposed region 221a of the first working electrode and the exposed region 222a of the second working electrode. Exposed regions 221b, 222b, 223b, and 224b of the terminal portion 203 of the first working electrode, the second working electrode, the reference electrode, and the counter electrode, respectively, can be electrically connected to the main body (not shown) by, for example, electrode pads (not shown).

[0130] The materials used in the present examples (including test examples) 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 Industries, Ltd. "162-18581", Average molecular weight: approximately 25,000 (b-1C) Poly(acrylic acid) 5,000...Fujifilm Wako Pure Chemical Industries, Ltd. "165-18571", Average molecular weight: approximately 5,000 (b-2A) Poly(styrenesulfonic acid sodium salt)...Sigma-Aldrich "81609", Average Mw: 16,800 (b-2B) Poly(sodium 4-styrenesulfonate)...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 dispersant (Non-b-1) hydroxypropyl cellulose..."NISSO HPC" manufactured by Nippon Soda Co., Ltd.

[0133] (c) Cationic Mediators (c-1) Polymer-bound PNT1: This has a structure in which a phenothiazine compound (PNT-70) in the NHS form as the redox mediator compound (c1) is bound to a copolymer of 2-aminoethyl methacrylate hydrochloride, (4-vinylphenyl)methanamine, and methacryloylcholine chloride as the cationic polymer (c2) (hereinafter referred to as "Polymer C"). (c-2) Polymer-bound PNT2: This has a structure in which a phenothiazine compound (PNT-70) as the redox mediator compound (c1) is bound to poly(L-lysine) hydrochloride (ALAMANDA POLYMERS' "PLKC800") as the cationic polymer (c2) (hereinafter referred to as "PLL"). The synthesis methods of the above 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 fragment

[0136] The sulfonic acid fragment was synthesized by the above reaction.

[0137] (2) Synthesis of sulfonic acid fragment

[0138] The carboxylic acid fragment was synthesized by a two-step reaction in acetonitrile and tetrahydrofuran (THF) as described above.

[0139] (3) Synthesis of PNT-34

[0140] The sulfonic acid fragment and carboxylic acid fragment synthesized as above were dissolved in MeOH / H ２ Suspend the mixture in 0.5% Ag at an internal temperature of about 47°C. ２ CO ３ Celite was added in portions over 15 minutes. After the addition, the mixture was stirred for 2.5 hours at an internal temperature of about 68°C. After allowing the mixture to cool to room temperature, it was filtered through Celite and the filtrate was concentrated. The residue was purified multiple times by silica gel column chromatography to obtain PNT-34.

[0141] (4) Synthesis of condensation product (PNT-68)

[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, followed by stirring at room temperature for 3 hours. The reaction solution was concentrated and then purified three times by silica gel column chromatography to obtain a condensate (PNT-68).

[0143] (5) Synthesis of deprotected product (PNT-69)

[0144] The condensation product (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. The reaction solution was concentrated and then subjected to azeotropic distillation with toluene several times to obtain the deprotected product (PNT-69).

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

[0146] The deprotected form (PNT-69) was dissolved in dichloromethane, and N-hydroxysuccinimide (NHS) and EDCI·HCl were added, followed by stirring at room temperature for 4 hours. The reaction solution was purified by silica gel column chromatography to obtain PNT-69 NHS form (PNT-70), in which the terminal carboxy group of the PEG chain (PEG12) was activated (NHS esterified).

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

[0148] First, 2-aminoethyl methacrylate hydrochloride, (4-vinylphenyl)methanamine, and methacryloylcholine chloride were prepared. Then, 0.97 mmol of 2-aminoethyl methacrylate hydrochloride, 0.97 mmol of (4-vinylphenyl)methanamine, 11.97 mmol of methacryloylcholine chloride, 0.08 mmol of V-50, and 10.49 g of ethanol were added to a four-neck flask. The same treatment as for Polymer A was then carried out to obtain Polymer C. Polymer C corresponded to a polymer with a molar ratio of n:m:l = 21.2:8.1:70.7, calculated from the results of 1H-NMR. Furthermore, measurement by gel permeation chromatography (GPC) revealed that 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 (liquid (1)). Polymer C, a high-molecular-weight polymer, was dissolved in MilliQ water to a concentration of 15 mg / mL (liquid (2)). Next, WSC (DOJINDO W001) was dissolved in MilliQ water to a concentration of 20 mg / mL (liquid (3)). 40 μL of liquid (1), 140.3 μL of liquid (2), 383.4 μL of liquid (3), and 96 μL of a separately prepared 250 mM 2-morpholinoethanesulfonic acid (MES) buffer solution (pH 6.0) were mixed, and the total volume was adjusted to 1200 μL with MilliQ water. The mixture was then allowed to react for approximately 20 hours with stirring at room temperature. Thereafter, ultrafiltration was carried out several times using a centrifugal ultrafiltration filter (Amicon Ultra-4 50k; Merck Millipore) to recover the liquid from which low molecular weight substances had been removed.

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

[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 Industries, Ltd.)

[0153] (f) Buffer Solutions (f-1) Bis-Tris: (Bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane)...Dojindo Laboratories (f-2A) Tris-HCl pH 7.5...Invitrogen UltraPure 1 M Tris-HCl pH 7.5, ThermoFisher SCIENTIFIC (f-2B) Tris-HCl pH 8.0...Invitrogen UltraPure 1 M Tris-HCl pH 8.0, ThermoFisher SCIENTIFIC (f-2C) Tris-HCl pH 8.5...1 M Tris-HCl (pH 8.5), Fujifilm Wako Pure Chemical Industries (manufactured by Nippon Gene Co., Ltd.)

[0154] (g) Metal salts Sodium chloride (NaCl)...FUJIFILM Wako Pure Chemical Industries, Ltd. (product code: 191-01665) Potassium chloride (KCl)...NACALAI TESQUE, INC. (product code: 28514-75) Sodium bromide (NaBr)...FUJIFILM Wako Pure Chemical Industries, Ltd. (product code: 192-09412) Potassium bromide (KBr)...FUJIFILM Wako Pure Chemical Industries, Ltd. (product code: 164-03472) Magnesium bromide (MgBr ２ ) hexahydrate...Fujifilm Wako Pure Chemical Industries, Ltd. (product code: 138-09192, purity 99.9%) Potassium sulfate (K ２ SO ４ ) ... Fujifilm Wako Pure Chemical Industries, Ltd. (Product code: 169-13552, purity 99.9%)

[0155] Test Example 1 Dispersibility Test <Preparation of Reagent Solution Samples> According to the formulations shown in Table 2, carbon black (a-1) was added to an aqueous solution of various anionic dispersants (b) at a concentration of 1 mg / mL or 2 mg / mL so that the carbon concentration was 20 mg / mL, and then the solution was treated with an ultrasonic homogenizer for 3 minutes or more to prepare reagent solution samples.

[0156]

[0157] The reagent solution sample obtained as described above was treated in an ultrasonic bath for about 10 minutes before use. The resulting redispersion 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 a recovered solution sample.

[0158] A photograph of the recovered liquid sample is shown in Figure 8. The lighter the color of the recovered liquid sample, the less carbon black (a-1) passed through the filter and the more aggregated carbon black (a-1) was captured by the filter, meaning poor dispersibility. However, it has been shown that the anionic dispersants (b) in all of the reagent liquid (recovered liquid) samples 1-1 to 1-11 have the effect of dispersing the conductive carbon filler (a) in an aqueous solvent, although to varying degrees. Furthermore, the results of reagent liquid (recovered liquid) 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), suggest that when an anionic polymer such as poly(acrylic acid) is used as the anionic dispersant (b), the dispersing 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. A buffer solution was used that was prepared so that the pH of the reagent solution would be a predetermined value when used in a predetermined amount. Test Example 2 compared the dispersibility of reagent solution samples that all contained the same concentration of metal ions (metal salts) after adjusting the pH using a buffer solution that did not contain metal ions (metal salts).

[0160] The concentration of polymer-bound PNT 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 measured using a plate reader to confirm the concentration in the stock solution, and the concentration of each reagent sample was adjusted based on this value. The value "equivalent to absorbance 3" indicates an absorbance of approximately 0.12 at 608 nm when diluted 25-fold. Here, a UV-Star® 96-Well F-Boden microplate manufactured by Greiner Bio-One was used. Furthermore, an infinite® M200 Pro plate reader manufactured by Tecan was used.

[0161]

[0162] The reagent solution samples were prepared as follows. First, 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, to which buffer solution (f) and metal salt (g) were added. Next, 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 resulting reagent solution samples was visually confirmed. The results are also 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. In Test Example 3, the pH was adjusted using a buffer solution that did not contain metal ions (metal salts), and the dispersibility of reagent solution samples containing different concentrations of metal ions (metal salts) was compared.

[0164]

[0165] The reagent solution samples were prepared as follows. First, 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, to which buffer solution (f) and metal salt (g) were added. Next, 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 resulting reagent solution samples was visually confirmed. The results are also 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. In Test Example 4, the pH was adjusted using a buffer solution that did not contain metal ions (metal salts), and the dispersibility of reagent solution samples containing different types and concentrations of metal salts (metal ions) was compared.

[0167]

[0168] The reagent solution sample was prepared as follows: First, 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, to which buffer solution (f) and metal salt (g) were added. Next, cationic mediator (c) was added to the treated solution and treated with an ultrasonic homogenizer for approximately 30 seconds.

[0169] The resulting reagent solution samples were visually inspected for the presence or absence of aggregation. The results are also shown in Table 5. Regarding the results of Test Examples 2 to 4, the results of Test Example 2 indicated that aggregation may occur when the pH of the reagent solution is high (due to the pH itself, not the concentration of metal salts derived from the buffer used to adjust the pH). However, it was found that within an appropriate pH range depending on the composition of the reagent solution, the dispersibility of the conductive carbon filler can be improved and aggregation can be suppressed. Furthermore, the results of Test Examples 3 and 4 indicated that, although the occurrence of aggregation differs depending on the type of metal ion contained in the reagent solution, aggregation generally tends to occur more easily when the metal ion concentration is high. For example, this suggests that aggregation can be suppressed for many metal ions when the metal ion concentration is less than 100 mM. On the other hand, in the case of magnesium ions, aggregation can sometimes be suppressed even when the concentration is higher than 50 mM (e.g., even at around 200 mM). Therefore, it was found that within an appropriate concentration range depending on the type of metal ion, the dispersibility of the conductive carbon filler can be improved and aggregation can be suppressed.

[0170] Test Example 5 Test of Anionic Dispersant (b) According to the formulations shown in Table 6, reagent solution samples containing various components at predetermined concentrations were prepared.

[0171] The test liquid sample was prepared as follows: First, 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, followed by addition of buffer solution (f). Next, cationic mediator (c) was added to the treated solution and treated with an ultrasonic homogenizer for approximately 30 seconds. Finally, oxidoreductase (d) and crosslinker (e) were added to the treated solution.

[0172] The obtained test liquid samples were visually inspected for the presence or absence of aggregation. The results are also shown in Table 6. Polymer-bound PNT1, synthesized using a predetermined copolymer as the cationic polymer (c), and polymer-bound PNT2, synthesized using PLL as the cationic polymer (c), exhibit 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 observed regardless of the cationic polymer (c).

[0173] Test Example 6: Responsiveness evaluation test <Preparation of reagent solution> Reagent solution samples containing various components at predetermined concentrations were prepared according to the formulations shown in Table 7.

[0174] The test liquid samples were prepared as follows: First, conductive carbon filler (a) was added to an aqueous solution of anionic dispersant (b) or its control, and the mixture was treated with an ultrasonic homogenizer for at least 3 minutes, followed by addition of buffer solution (f). Next, cationic mediator (c) was added to the treated solution, and the mixture was treated with an ultrasonic homogenizer for approximately 30 seconds. Finally, oxidoreductase (d) and crosslinker (e) were added to the treated solution.

[0175] <Preparation of Sensor Electrode> 0.6 μL of each of the reagent solution samples prepared as described above was applied to a carbon electrode prepared by screen printing on an insulating substrate, and dried overnight to obtain a sensor electrode.

[0176] <Preparation of RPMI Medium> To a solution prepared using RPMI-1640 Medium (Sigma-Aldrich R1383), 2-morpholinoethanesulfonic acid (MES, Dojindo Laboratories) and 3-morpholinopropanesulfonic acid (MOPS, Dojindo Laboratories) were added as buffer components to a final concentration of 25 mM, and the pH was adjusted to 7.4. The medium thus obtained was used as "RPMI medium" in the following examples.

[0177] <Sensor evaluation of response> A three-electrode measurement system was used, combining 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) as the reference electrode. Using a potentiostat (manufactured by BAS), the time change in current (current response value) was measured at approximately 37 ° C. in RPMI medium by the amperometric method. Specifically, starting from 100 seconds after the start of measurement, glucose was added every 100 seconds to achieve theoretical values ​​of 3 mM, 15 mM, or 30 mM, and the current response value was measured. The current response value was calculated as an average value from five measurements taken 5 seconds, 10 seconds, 15 seconds, 20 seconds, and 25 seconds before the next glucose addition after the addition of the glucose to be measured. The current value at the final concentration was the average value calculated from measurements taken at five points: 80, 85, 90, 95, and 100 seconds after adding a glucose solution to a glucose concentration of 30 mM. The current value at each glucose concentration was the current value after background correction, in which the current value at a glucose concentration of 0 mM was subtracted.

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

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

[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 protective film: Poly(ter.butyl methacrylate-b-4-vinylpyridine) (manufactured by Polymer Source, hereinafter referred to as "tBuMA4VP") Final concentration: 7.11% (wt / v) Tripropylene glycol methyl ether methacrylate-styrene-4-vinylpyridine random copolymer (manufactured by Nard, hereinafter referred to as "TGMAS4VP") Final concentration: 0.89% (wt / v) Poly(ethylene glycol) diglycidyl ether (manufactured by Sigma-Aldrich, 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 dissolved in ethanol before use. The number-average molecular weight Mn of tBuMA4VP was 87,000 for poly(tert-butyl methacrylate) and 74,000 for poly(4-vinylpyridine). The Mw / Mn of tBuMA4VP was 1.16. TGMAS4VP had a tripropylene glycol methyl ether methacrylate:styrene:4-vinylpyridine ratio of 6.6:20.3:73.0, a number-average molecular weight Mn of 60,704, a weight-average molecular weight Mw of 120,095, and an Mw / Mn of 1.98. The number-average molecular weight Mn of PEGDGE was approximately 1,000. The HEPES buffer solution was prepared using 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (manufactured by Dojindo Chemical Industries, Ltd.).

[0183] <Preparation of Sensor Electrodes> 0.5 μL of the reagent solution sample prepared as described above was applied to a gold electrode prepared by sputtering on an insulating substrate, and dried for 15 minutes. This process was repeated twice, for a total of three applications. The electrode was then dried overnight. This resulted in the formation of a reagent layer corresponding to each reagent solution sample on each gold electrode. Each gold electrode with the reagent layer formed thereon was immersed in the protective film polymer solution prepared as described above, pulled out, and dried, and this process was repeated multiple times to form a protective film on each gold electrode. The sensor electrodes were obtained through the above process.

[0184] <Sensor Evaluation of Durability (Response Maintenance Rate)> A three-electrode measurement system was used, combining 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. Using a potentiostat (manufactured by BAS Corporation), the time change in current was measured at approximately 37°C in RPMI medium by the amperometric method. Specifically, starting 1500 seconds after the start of measurement, glucose was added every 1000 seconds to achieve theoretical values ​​of 5 mM, 15 mM, or 30 mM, and the current response value was continuously measured. After measurement, the electrode was stored in RPMI medium at 37°C. Similar measurements were also performed on the first, second, and third days after storage. The current values ​​at glucose concentrations of 5 mM and 15 mM were calculated as an average of five measurements taken 5 seconds, 10 seconds, 15 seconds, 20 seconds, and 25 seconds before the next glucose addition. The current value at the final concentration is the average value calculated from measurements taken at five points: 980 seconds, 985 seconds, 990 seconds, 995 seconds, and 1000 seconds after adding a glucose solution to a glucose concentration of 30 mM. The current value at each glucose concentration is the current value after background correction, in which the current value at a glucose concentration of 0 mM is subtracted.

[0185] The results are shown in Figure 10. Even after the protective film coating, the sensor using poly(acrylic acid) (reagent solution sample 6-1) showed a higher current response than the sensor using hydroxypropyl cellulose (reagent solution sample 6-6). Furthermore, the sensor using poly(acrylic acid) (reagent solution sample 6-1) also showed a higher sensor durability after three days (current maintenance rate relative to the first day). This is presumably 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).

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

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

5. The reagent layer described in claim 1, wherein the cationic mediator (c) is a compound in which a redox mediator compound (c1) and a cationic polymer (c2) are bonded, optionally via a linker portion (c3).

6. The reagent layer according to claim 5, wherein the cationic polymer (c2) has a quaternary ammonium cationic group.

7. The reagent layer of claim 1, wherein the conductive carbon filler (a) is carbon black.

8. The reagent layer of claim 1, further comprising an oxidoreductase (e) that oxidizes or reduces the analyte.

9. The reagent layer according to claim 8, wherein the oxidoreductase (e) is of a coenzyme-bound type.

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

11. An electrochemical sensor for detecting or quantifying an analyte, comprising a working electrode, a counter electrode, and a reagent layer according to any one of claims 1 to 10.

12. The electrochemical sensor according to claim 11, further comprising a reference electrode.

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

14. A method for forming a reagent layer, comprising: (1) preparing a reagent solution containing a conductive carbon filler (a), an anionic dispersant (b), and a cationic mediator (c); (2) applying the reagent solution to a site where a reagent layer is to be formed; and (3) drying the applied reagent solution to form a reagent layer.

15. The method for forming a reagent layer according to claim 14, wherein the pH of the reagent solution is 8.0 or less.

16. The method for forming a reagent layer according to claim 15, wherein the concentration of metal ions in the reagent solution is 200 mM or less.

17. The method for forming a reagent layer according to claim 16, wherein the metal ions in the reagent solution are alkali metal ions and have a concentration of less than 100 mM.

18. The method for forming a reagent layer according to claim 14, wherein the anionic dispersant (b) is a polymer having a weight-average molecular weight of 70,000 or less and containing at least one unit selected from the group consisting of an acrylic acid-derived unit, a maleic acid-derived unit, and a styrene sulfonic acid-derived unit.

19. A method for forming a reagent layer as described in claim 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, optionally via a linker portion (c3).

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