Reagent composition, sensor, and method for producing the same

Abstract

To provide a biosensor. And to avoid the generation of precipitates in the production of a composition for a sensor using a ruthenium compound.SOLUTION: There is provided a method for manufacturing a biosensor containing an oxidoreductase, a ruthenium compound, and an electron transfer accelerator. The present invention also provides a method for producing a composition for a biosensor by first adding and dissolving an oxidoreductase in powder form in a solution, and then mixing a ruthenium compound in powder form into the solution in which the oxidoreductase is dissolved.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to a method for producing a biosensor that includes an electron transfer facilitator, an oxidoreductase, and a metal complex compound. [Background technology]

[0002] Patent Document 1 discloses a sensor using two mediators, 1-methoxy-5-methylphenazinium methylsulfate (1-methoxyPMS, mPMS) and hexaammineruthenium(III). In the sensor, the ruthenium compound is preferably contained in the inorganic gel layer, and mPMS is contained in the enzyme layer.

[0003] Patent Document 2 discloses a sensor using two mediators, 1-methoxy-5-methylphenazinium ethyl sulfate (mPES) and hexaammineruthenium(III). In the sensor, the ruthenium compound is preferably contained in the inorganic gel layer, and the mPES is contained in the enzyme layer.

[0004] Patent Documents 1 and 2 disclose that oxidoreductases such as glucose dehydrogenase and lactate dehydrogenase react with a substrate, reduce mPMS or mPES, and then reduce a ruthenium compound, transferring electrons to an electrode for detection as a current value. The oxidoreductase is contained in an enzyme layer. In such sensors, the mediator that first receives electrons from the enzyme, such as mPMS or mPES, is coexisting with the oxidoreductase in the same layer (enzyme layer) where it is easily accessible. In contrast, the ruthenium compound is thought to be located in a different layer adjacent to the enzyme layer. When multiple types of mediators are present in the same layer, electrons are not necessarily transferred from the first mediator to the second mediator, and then transferred from the second mediator to the electrode. Conversely, electrons may be transferred back to the first mediator, then transferred to the second mediator, and then transferred to the electrode. This may complicate reaction kinetic analysis. Furthermore, this may affect the sensor's response time and performance. To avoid such concerns, when multiple types of mediators are used, it is generally considered that the first mediator is placed in the enzyme layer and the second mediator is placed in a different layer.

[0005] Numerous documents, including patent applications and manufacturer's manuals, are cited herein. The disclosures of these documents, while not considered relevant to the patentability of this invention, are hereby incorporated by reference in their entirety. More particularly, all referenced documents are hereby incorporated by reference to the same extent as if each individual document were specifically and individually indicated to be incorporated by reference. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2013-083634 (Patent No. 5584740) [Patent Document 2] JP 2018-054555 (Patent No. 6773507) Summary of the Invention [Problem to be solved by the invention]

[0007] The present inventors attempted to develop a glucose sensor with an electrode that uses FADGDH as the enzyme and mPMS and a ruthenium complex as mediators. First, powdered ruthenium was prepared in solution. Next, powdered phosphate buffer and powdered mPMS as the mediator were added and dissolved in the prepared solution. Powdered FADGDH was then added to the prepared solution. As a result, they encountered the problem of precipitate formation when the enzyme powder was added to the ruthenium complex solution. The same problem occurred even when the FADGDH product used was changed to a different FADGDH product. Therefore, it was believed that the precipitation was not a problem specific to the FADGDH product used.

[0008] When an electrode was fabricated using the supernatant of the solution containing the precipitate and used to measure glucose concentration, linear current values ​​were only obtained within a limited concentration range. Therefore, it was thought that it would be difficult to develop a glucose sensor without resolving the precipitate problem. The precipitate was recovered and its absorption spectrum was measured, and the waveform matched that of a ruthenium complex solution. The possibility that the precipitate was an enzyme was considered, but the absorption spectrum of the precipitate exhibited a waveform different from that of a solution containing only the enzyme. Therefore, the inventors identified the precipitate as a ruthenium complex.

[0009] Therefore, an object of the present invention is to provide an enzyme sensor containing a ruthenium compound, which avoids precipitation of the ruthenium compound and has improved production efficiency, and a method for producing the same. To the best of the inventors' knowledge, there have been no reports of precipitation occurring in conventional enzyme sensors that use a ruthenium compound as a mediator. Therefore, this is a new problem. [Means for solving the problem]

[0010] As a result of extensive research, the present inventors have found that, instead of mixing the respective reagents constituting the reagent composition in powder form and then adding water to dissolve them, a homogeneous composition can be prepared without causing precipitation of ruthenium compounds by dissolving the oxidoreductase in advance and adding the enzyme solution to the other components. Furthermore, they have found that a sensor prepared using this composition can quantify glucose up to a high concentration range compared to a sensor prepared without using such a method, and have completed the present invention, which includes this as one embodiment.

[0011] The present disclosure encompasses the following embodiments. [1] (i) mixing and dissolving a powdered oxidoreductase with a solution; and (ii) After step (i), a step of mixing and dissolving the oxidoreductase solution and the ruthenium compound powder. A method for producing a composition for an oxidoreductase sensor, comprising: [2] The method according to embodiment 1, wherein the oxidoreductase is added to a final concentration of 20 to 80 mg / mL. [3] The method according to embodiment 1 or 2, wherein the ruthenium compound is mixed to a final concentration of 180 to 500 mM. [4] (iii) After step (i), the method further comprises a step of mixing the solution in which the oxidoreductase is dissolved with a second electron transfer promoter powder to dissolve the second electron transfer promoter, or mixing the solution in which the second electron transfer promoter is dissolved with the second electron transfer promoter solution; 4. The method of claim 1, 2 or 3, wherein the second electron transfer promoter is selected from the group consisting of phenazine methosulfate (PMS), 1-methoxy PMS (mPMS), and 1-methoxy-5-ethylphenazinium ethyl sulfate (mPES). [5] The method according to embodiment 4, wherein the second electron transfer promoter is mixed to a final concentration of 0.1 to 100 mM. [6] The method according to any one of embodiments 1 to 5, wherein the oxidoreductase is flavin-dependent glucose dehydrogenase (FADGDH), amadoriase, lactate dehydrogenase, or sarcosine oxidase. [Effects of the Invention]

[0012] According to one embodiment of the present disclosure, it is possible to provide a biosensor that combines an electron transfer promoter, an oxidoreductase, an electrode, and a metal complex compound without causing precipitation of a ruthenium compound, and a method for manufacturing the biosensor. Also, according to one embodiment of the present disclosure, it is possible to provide a glucose sensor that combines an electron transfer promoter, GDH, an electrode, and a metal complex compound without causing precipitation of a ruthenium compound, and a method for manufacturing the glucose sensor. [Brief explanation of the drawings]

[0013] [Figure 1] 1 shows a flow chart of a conventional dissolution procedure. [Figure 2] 1 shows a flow chart of the dissolution procedure of the present invention and comparative examples. [Figure 3] This is a photograph of a precipitate (sediment). [Figure 4] The results of chronoamperometry measurements at 300 mM Ru are shown. [Figure 5] The results of chronoamperometry measurements at 150 mM Ru are shown. [Figure 6] The results of chronoamperometry measurements at 30 mM Ru are shown. DETAILED DESCRIPTION OF THE INVENTION

[0014] In one embodiment, the present disclosure provides a biosensor containing an electron transfer promoter, an oxidoreductase, a metal complex compound, and an electrode. Depending on the type of oxidoreductase used, the biosensor can measure a specific compound that can be recognized as a substrate by the oxidoreductase. For example, if flavin-dependent glucose dehydrogenase (FADGDH) is used as the oxidoreductase, a glucose sensor containing FADGDH can be provided, and glucose can be measured using this.

[0015] Unless otherwise specified, the term "biosensor" as used herein refers to a sensor comprising an electron transfer promoter, an electrode, an oxidoreductase, and a metal complex compound. This can be used as a sensor for a substrate recognized by the oxidoreductase. In one embodiment, the electrode comprises an electrode portion having a working electrode and a counter electrode. In another embodiment, the electrode comprises an electrode portion having a working electrode, a counter electrode, and a reference electrode. The electrode may be, for example, a triode electrode or a printed electrode. In one embodiment, the electrode portion may be disposed on an insulating substrate.

[0016] As used herein, a mediator refers to a compound that participates in electron transfer. For example, in a system using an electrode, the mediator receives electrons from an oxidoreductase to become a reduced form, and then transfers electrons to the electrode to return to an oxidized form. From this perspective, a compound that functions as a mediator can also be called an electron mediator. In this specification, these terms are synonymous.

[0017] Electron transfer promoter Herein, a compound that promotes the transfer of electrons from an oxidoreductase to an electrode is referred to as an "electron transfer promoter." An "electron transfer promoter" can be used in a biosensor equipped with an oxidoreductase, such as a glucose sensor, to promote the generation of a response current depending on the concentration of a target compound when added. In certain embodiments, the electron transfer promoter can modify the function of a mediator. Herein, the expression "modifying the function of a mediator" refers to the fact that, for a mediator that transfers no or very little electrons from an oxidoreductase to an electrode in the absence of the electron transfer promoter, electrons are transferred from the oxidoreductase to an electrode in the presence of the electron transfer promoter. mPMS and mPES, as described in Patent Documents 1 and 2, can be considered electron transfer promoters. Herein, the electron transfer promoter may be referred to as a mediator.

[0018] Examples of electron transfer promoters include phenazine methosulfate and its derivatives, specifically 1-methoxy-5-methylphenazinium methylsulfate (mPMS) and 1-methoxy-5-methylphenazinium ethylsulfate (mPES), thionine and its derivatives, specifically 3-amino-7-(2,3,4,5,6-pentahydroxyhexanamido)-5-phenothiazinium, Azure C, Azure A, methylene blue, toluidine blue, phenylenediamine and its derivatives, specifically N,N,N',N'-tetramethylphenylenediamine, 2,4-Diaminodiphenylamine, etc.

[0019] The content of electron transfer promoter per biosensor is not limited as long as it is an amount that allows the measurement of the specified component to be measured, and may be, for example, 10 pmol to 1000 nmol, 10 pmol to 100 nmol, 10 pmol to 60 nmol, 10 pmol to 10 nmol, 10 pmol to 1 nmol, 40 to 900 pmol, 50 to 500 pmol, or for example, 100 to 300 pmol, but is not limited to these.

[0020] The concentration of the electron transfer promoter in the reagent composition to be applied to the biosensor is not limited as long as it is a concentration that allows the measurement of the specified component to be measured, and may be, for example, 0.01 to 500 mM, 0.05 to 100 mM, 0.05 to 50 mM, 0.05 to 10 mM, 1 to 5 mM, for example, 0.5 to 5 mM, but is not limited to these.

[0021] When the content of electron transfer promoter per biosensor is 5 nmol, the content of FADGDH is, in certain embodiments, 1 to 100 U, 1 to 10 U, 1 to 6 U, for example, 1 to 4 U. In certain embodiments, the content of FADGDH is, in certain embodiments, 0.1 to 100 μg, 1 to 50 μg, for example, 1 to 20 μg. In certain embodiments, the content of other oxidoreductases per sensor is, in certain embodiments, 0.1 to 1000 μg, 1 to 500 μg, 1 to 50 μg, for example, 1 to 20 μg. In certain embodiments, the content of the metal complex compound is an amount that reaches a saturation concentration, or may be 5 to 50 μg, 10 to 40 μg, for example, 15 to 25 μg. The same applies to other oxidoreductases.

[0022] Generally, electron transfer promoters and mediators, such as PMS, are unstable in solution. Therefore, it is preferable to dissolve them in solution, apply them to the electrode quickly, and store them in a dry state. When preparing a solution containing multiple electron transfer promoters or mediator reagents, it is preferable to mix them in powder form and then dissolve them, as this shortens the time they remain in solution, rather than preparing and mixing individual solutions.

[0023] The concentration of the oxidoreductase in the reagent composition to be applied to the biosensor is not limited as long as it is a concentration that allows the measurement of the specified component to be measured, and may be, for example, but is not limited to, 0.001 to 1000 mg / mL, 0.01 to 500 mg / mL, 0.05 to 300 mg / mL, 0.1 to 400 mg / mL, 1 to 500 mg / mL, or for example, 10 to 100 mg / mL.

[0024] Metal complex compounds Examples of metal complex compounds include, but are not limited to, ruthenium compounds and osmium compounds. In one embodiment, the content of the metal complex compound per glucose sensor is not limited as long as it is an amount that allows measurement of a predetermined component to be measured, and can be an amount typically used in a glucose sensor, for example, 0.1 μg to 100 mg, 1 μg to 50 mg, 1 μg to 10 mg, 1 μg to 1 mg, 1 to 100 μg, 5 to 50 μg, 10 to 40 μg, or for example, 15 to 25 μg.

[0025] The concentration of the metal complex compound in the reagent composition to be applied to the biosensor is not limited as long as it is a concentration that allows the measurement of the specified component to be measured, and may be, for example, but is not limited to, 0.01 to 1000 mM, 0.05 to 800 mM, 0.05 to 700 mM, 1 to 700 mM, 5 to 600 mM, e.g., 10 to 500 mM.

[0026] Ruthenium Compounds The ruthenium compound may be a ruthenium compound used in conventional glucose sensors or biosensors, or an equivalent thereof that will be developed in the future. In some embodiments, the ruthenium compound may be present in the reaction system as an oxidized ruthenium complex. The ruthenium complex is not particularly limited. In some embodiments, the ruthenium complex may be a compound represented by the following general formula: [Ru(NH3)5X]n + (wherein X is NH3, CN, pyridine, a halogen ion, nicotinamide, or H2O). The halogen ion is Cl. - , F - , Br - , I - In the formula, n + represents the valence of the oxidized ruthenium(III) complex, which is determined by the type of X. For example, if X is NH3, the compound is a hexaammineruthenium complex compound, and the halogen is Cl. - If so, it is hexaammineruthenium chloride.

[0027] oxidoreductase Examples of oxidoreductases include various oxidoreductases classified into EC group 1, such as glucose oxidase, glucose dehydrogenase, amadoriase (also called fructosyl peptide oxidase or fructosyl amino acid oxidase), peroxidase, galactose oxidase, bilirubin oxidase, pyruvate oxidase, D- or L-amino acid oxidase, amine oxidase, cholesterol oxidase, choline oxidase, xanthine oxidase, sarcosine oxidase, D- or L-lactate oxidase (LOD), ascorbate oxidase, cytochrome oxidase, alcohol dehydrogenase, cholesterol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, and fructose dehydrogenase (FDH). , sorbitol dehydrogenase, D- or L-lactate dehydrogenase, malate dehydrogenase, glycerol dehydrogenase, 17B hydroxysteroid dehydrogenase, estradiol 17B dehydrogenase, D- or L-amino acid dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, 3-hydroxysteroid dehydrogenase, diaphorase, catalase, glutathione reductase, cytochrome b5 reductase, adrenoxin reductase, adrenodoxin reductase, nitrate reductase, phosphate dehydrogenase, bilirubin oxidase, laccase, polyamine oxidase, formate dehydrogenase, pyranose oxidase, pyranose dehydrogenase, tauropine dehydrogenase, and the like. Examples of coenzymes for the above enzymes include nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide (FAD), and pyrroloquinoline quinone. The activity of the above-listed oxidoreductases can be measured using various substrates, for example, by the method described in "Methods in Enzymology" (vols. 1-602). The origin of the above-listed oxidoreductases (e.g., glucose dehydrogenase, LOD, amadoriase, etc.) is not particularly limited, and those derived from prokaryotes, eukaryotes, microorganisms, fungi, plants, or animals can be used.Any known oxidoreductase can be used as these various oxidoreductases, and various commercially available enzyme products can also be used.

[0028] In certain embodiments, the oxidoreductase may be FADGDH. As used herein, FADGDH refers to flavin adenine dinucleotide-dependent glucose dehydrogenase or flavin adenine dinucleotide-binding glucose dehydrogenase. In certain embodiments, commercially available FADGDH may be used. In other embodiments, a variant of a commercially available FADGDH or its equivalent may be used. As used herein, Mucor type FADGDH refers to a wild-type FADGDH of the genus Mucor and / or a variant thereof, and includes both the wild-type and the variant unless otherwise specified. As used herein, Botryotinia type FADGDH refers to a wild-type FADGDH of the genus Botryotinia and / or a variant thereof, and includes both the wild-type and the variant unless otherwise specified. As used herein, Aspergillus-type FADGDH refers to wild-type FADGDH of the genus Aspergillus and / or its variants, and includes both the wild-type and its variants unless otherwise specified. As used herein, Penicillium-type FADGDH refers to wild-type FADGDH of the genus Penicillium and / or its variants, and includes both the wild-type and its variants unless otherwise specified. As used herein, Circinella-type FADGDH refers to wild-type FADGDH of the genus Circinella and / or its variants, and includes both the wild-type and its variants unless otherwise specified.

[0029] Examples of Mucor microorganisms include, but are not limited to, Mucor prainii, Mucor javanicus, Mucor circinelloides f. circinelloides, Mucor guilliermondii, Mucor hiemalis f. silvaticus, Mucor subtilissimus, and Mucor dimorphosporus. Examples of Botryotinia microorganisms include, but are not limited to, Botryotinia fuckeliana. Examples of Aspergillus microorganisms include, but are not limited to, Aspergillus oryzae, Aspergillus sojae, Aspergillus niger, and Aspergillus terreus. Organisms of the genus Penicillium include, but are not limited to, Penicillium sclerotiorum, Penicillium janthinellum, and Penicillium paneum.

[0030] As used herein, a variant of a Mucor wild-type FADGDH refers to a mutant having an amino acid sequence in which amino acid residues in the amino acid sequence of the wild-type FADGDH are substituted, and having FAD-dependent glucose dehydrogenase activity. In a specific embodiment, the variant has low substrate specificity for maltose and galactose, and high substrate specificity for glucose. The number of substituted amino acid residues can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1 to 10, 1 to 5, 1 to 3, or e.g., 1 or 2.

[0031] As used herein, a variant of wild-type FADGDH from Mucor prainii refers to a variant having a sequence in which amino acid residues in the amino acid sequence of the wild-type FADGDH have been substituted and having FAD-dependent glucose dehydrogenase activity. In a specific embodiment, the variant has low reactivity toward maltose, xylose, and galactose, and high reactivity toward glucose. The number of substituted amino acid residues can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1 to 10, 1 to 5, 1 to 3, or e.g., 1 or 2.

[0032] For example, when the enzyme used is a variant of wild-type FADGDH from the genus Botryothinia, the variant refers to a variant having a sequence in which amino acid residues in the amino acid sequence of the wild-type FADGDH are substituted and having FAD-dependent glucose dehydrogenase activity. In certain embodiments, the variant has low reactivity toward maltose and galactose and high reactivity toward glucose. The number of substituted amino acid residues can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1 to 10, 1 to 5, 1 to 3, or e.g., 1 to 2.

[0033] For example, when the enzyme used is a variant of wild-type FADGDH from Botryotinia fuckeliana, the variant refers to a variant having a sequence in which amino acid residues in the amino acid sequence of the wild-type FADGDH are substituted and having FAD-dependent glucose dehydrogenase activity. In certain embodiments, the variant has low reactivity toward maltose and galactose and high reactivity toward glucose. The number of substituted amino acid residues can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1 to 10, 1 to 5, 1 to 3, or e.g., 1 or 2.

[0034] For example, when the enzyme used is a variant of wild-type FADGDH of the genus Aspergillus, the variant refers to a variant having a sequence in which amino acid residues in the amino acid sequence of the wild-type FADGDH are substituted and having FAD-dependent glucose dehydrogenase activity. In certain embodiments, the variant has low reactivity toward maltose and galactose and high reactivity toward glucose. The number of substituted amino acid residues can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1 to 10, 1 to 5, 1 to 3, or e.g., 1 or 2.

[0035] For example, when the enzyme used is a variant of wild-type FADGDH of the genus Penicillium, the variant refers to a variant having a sequence in which amino acid residues in the amino acid sequence of the wild-type FADGDH are substituted and having FAD-dependent glucose dehydrogenase activity. In certain embodiments, the variant has low reactivity toward maltose and galactose and high reactivity toward glucose. The number of substituted amino acid residues can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 1 to 10, 1 to 5, 1 to 3, or e.g., 1 to 2.

[0036] In certain embodiments, the oxidoreductase may be an amadoriase. As used herein, amadoriase refers to a flavin adenine dinucleotide-dependent fructosyl peptide oxidase, a flavin adenine dinucleotide-dependent fructosyl amino acid oxidase, or a flavin adenine dinucleotide-binding fructosyl peptide oxidase or a flavin adenine dinucleotide-binding fructosyl amino acid oxidase. In certain embodiments, commercially available amadoriase may be used. In other embodiments, a variant of a commercially available amadoriase or an equivalent thereof may be used. In the present specification, the origin of amadoriase is not particularly limited, and examples thereof include those derived from the genera Coniochaeta, Eupenicillium, Pyrenochaeta, Arthrinium, Curvularia, Neocosmospora, Cryptococcus, Phaeosphaeria, Aspergillus, Emericella, Ulocladium, Penicillium, Fusarium, Achaetomiella, and Acaetomium. The term "amadoriase" refers to an amadoriase derived from the genus Haetomium, Thielavia, Chaetomium, Gelasinospora, Microascus, Leptosphaeria, Ophiobolus, Pleospora, Coniochaetidium, Pichia, Debaryomyces, Corynebacterium, Agrobacterium, or Arthrobacter, and unless otherwise specified, includes both wild-type and modified forms thereof.

[0037] In certain embodiments, the oxidoreductase may be lactate oxidase. As used herein, lactate oxidase refers to flavin adenine mononucleotide-dependent lactate oxidase or flavin adenine mononucleotide-binding lactate oxidase. In certain embodiments, commercially available lactate oxidase may be used. In other embodiments, a variant of a commercially available lactate oxidase or an equivalent thereof may be used. As used herein, the origin of lactate oxidase is not particularly limited, and refers to lactate oxidase derived from, for example, the genus Aerococcus, Streptococcus, Pediococcus, or Enterococcus, and includes both wild-type and variant forms thereof unless otherwise specified.

[0038] In certain embodiments, the oxidoreductase may be lactate dehydrogenase. As used herein, lactate dehydrogenase refers to flavin adenine mononucleotide-dependent lactate dehydrogenase or flavin adenine mononucleotide-binding lactate dehydrogenase. In certain embodiments, commercially available lactate dehydrogenase may be used. In other embodiments, a variant of a commercially available lactate dehydrogenase or its equivalent may be used. As used herein, the origin of lactate dehydrogenase is not particularly limited, and refers to, for example, lactate dehydrogenase derived from the genus Pichia, Ogataea, or Candida, and includes both wild-type and variant forms thereof unless otherwise specified.

[0039] In certain embodiments, the oxidoreductase may be sarcosine oxidase. As used herein, sarcosine oxidase refers to flavin adenine dinucleotide-dependent sarcosine oxidase or flavin adenine dinucleotide-binding sarcosine oxidase. In certain embodiments, commercially available sarcosine oxidase may be used. In other embodiments, a variant of a commercially available sarcosine oxidase or an equivalent thereof may be used. As used herein, the origin of the sarcosine oxidase is not particularly limited, and examples include sarcosine oxidases derived from the genera Bacillus, Corynebacterium, Cylindrocarpon, Pseudomonas, and Arthrobacter. Unless otherwise specified, both wild-type and variants thereof are included.

[0040] In certain embodiments, FADGDH may include recombinant FADGDH having a tag sequence for enzyme purification, a peptide sequence, a signal sequence, a cleavage recognition sequence, and / or cleavage residues of these sequences added to the N-terminus or C-terminus of its amino acid sequence, and having FAD-dependent glucose dehydrogenase activity.

[0041] In one embodiment, the content of FADGDH per glucose sensor can be, but is not limited to, for example, 0.1 to 50 U, 0.5 to 20 U, 1 to 10 U, e.g., 1 to 5 U. In this specification, the enzyme unit U of FADGDH is defined as the amount of enzyme that converts 1 μmol of glucose in 1 minute at 37°C.

[0042] In this specification, the content of FADGDH per glucose sensor refers, in a specific embodiment, to the amount of FADGDH used in a glucose sensor having one electrode system with a working electrode and a counter electrode. In another embodiment, the content refers to the amount of FADGDH contained in a reagent layer disposed on one electrode system. In another embodiment, the content refers to the amount of FADGDH incorporated into a reagent so as to be included in the reaction system when a sample is added.

[0043] In one embodiment, the LOD content per lactate sensor can be, but is not limited to, for example, 0.1 to 50 U, 0.5 to 20 U, 1 to 10 U, or e.g., 1 to 5 U. In this specification, the enzyme unit U of LOD is defined as the amount of enzyme that oxidizes 1 μmol of lactate in 1 minute at 37°C.

[0044] In this specification, the content of LOD per lactate sensor refers, in certain embodiments, to the amount of LOD used in a lactate sensor having one electrode system with a working electrode and a counter electrode. In another embodiment, the content refers to the amount of LOD contained in a reagent layer disposed on one electrode system. In another embodiment, the content refers to the amount of LOD incorporated into a reagent so as to be included in the reaction system when a sample is added.

[0045] In certain embodiments, the biosensor has a size that is typically used for measuring samples. For example, in certain embodiments, a glucose sensor has a size that is typically used for samples that may contain glucose, such as blood. The sample, e.g., a blood sample, added to the sensor may be, for example, 0.1 to 2 μL, 0.2 to 1 μL, or 0.2 to 0.5 μL. The sensor can be designed depending on the volume of the sample or reaction system.

[0046] Reagent Layer In certain embodiments, the biosensor of the present disclosure includes an electron transfer promoter, an electrode, an oxidoreductase, and a metal complex compound. In certain embodiments, the biosensor has an electrode and a reagent layer, and the reagent layer includes an electron transfer promoter, an oxidoreductase, and a metal complex compound. In certain embodiments, the electrode includes an electrode portion having a working electrode and a counter electrode. In certain embodiments, the electrode portion may be disposed on an insulating substrate. In certain embodiments, the reagent layer may be disposed on the electrode portion.

[0047] In certain embodiments, the present disclosure provides a method for manufacturing a biosensor, comprising: adding a metal complex compound and an electron transfer promoter in powder form to a solution containing an oxidoreductase, or mixing the metal complex compound and the electron transfer promoter in powder form, then adding a solution containing the oxidoreductase and mixing them, and applying the resulting solution to an electrode when all the reagents have dissolved, to form a reagent layer. Unlike a method in which the oxidoreductase is mixed with other reagent components in powder form rather than in solution and then dissolved, this method of forming a reagent layer by adding a metal complex compound and an electron transfer promoter in powder form to a solution containing the oxidoreductase, or mixing the metal complex compound and the electron transfer promoter in powder form to a solution containing the oxidoreductase, then adding a solution containing the oxidoreductase and mixing them, and applying the resulting solution to an electrode when all the reagents have dissolved, can obtain a reagent composition in a well-dissolved state without forming a precipitate, and when this is subsequently applied to an electrode to fabricate a biosensor, it enables the quantification of glucose up to a high concentration range.

[0048] In an embodiment, a buffer may be added to a solution containing the oxidoreductase, the metal complex compound, and the electron transfer promoter. The buffer may be added to a solution containing the oxidoreductase, or may be mixed in powder form with the metal complex compound and the electron transfer promoter.

[0049] The temperature at which the oxidoreductase, metal complex compound, and electron transfer promoter are dissolved is preferably 10 to 40° C. If the mixture is stored at 4° C. or below, the metal complex compound may precipitate, and if the mixture is stored at 40° C. or above for a long period of time, the oxidoreductase may be inactivated.

[0050] In one embodiment, the present disclosure provides a system for measuring the concentration of a target compound in a sample, the system including a biosensor, a means for applying a voltage to electrodes of the biosensor, and a means for measuring a current. The means for applying a voltage may include a contact portion that can come into contact with the electrodes and a power source (e.g., a DC power source). The system of the present disclosure may include a potentiostat or a galvanostat. In one embodiment, the biosensor may be a glucose sensor. Also, in one embodiment, the target compound in the sample may be glucose. That is, in one embodiment, the present disclosure provides a method for measuring a glucose concentration using a glucose concentration measurement system. In one embodiment, the biosensor may be a lactate sensor. Also, in one embodiment, the target compound in the sample may be lactate. That is, in one embodiment, the present disclosure provides a method for measuring a lactate concentration using a lactate concentration measurement system.

[0051] In one embodiment, the present disclosure provides a method for measuring the concentration of a target compound, comprising contacting a sample that may contain the target compound with an oxidoreductase, applying a voltage to an electrode, and measuring a response current in the presence of a metal complex compound and an electron transfer promoter. The applied voltage is not particularly limited, but when a ruthenium compound is used as the metal complex compound, it can be, for example, 10 to 1,000 mV, 10 to 800 mV, 50 to 500 mV, or 0 to 100 mV. Unless otherwise specified, the potential described herein is that when a silver-silver chloride reference electrode is used.

[0052] The concentration of a target compound can be measured by contacting the sample with an oxidoreductase, maintaining the sample without applying a potential for a certain period of time, and then applying a voltage. Alternatively, the voltage can be applied simultaneously with the contact. The period during which the sample is maintained without applying a potential can be longer than 0 seconds and shorter than 1 minute, for example, 1 to 30 seconds, for example, 1 to 10 seconds. For example, the oxidoreductase can be FADGDH and the target compound can be glucose. Alternatively, the oxidoreductase can be LOD and the target compound can be lactic acid. Similarly, for the various oxidoreductases classified into EC Group 1 listed above, substrates recognized by these oxidoreductases can be used as target compounds, and systems for measuring the concentrations of such target compounds are provided.

[0053] In certain embodiments, the reagent layer may further include buffers, surfactants, inorganic compounds, and other ingredients.

[0054] Buffers include, but are not limited to, phosphate buffers, amine buffers, and buffers having a carboxyl group. Examples of amine buffers include Tris, ACES, CHES, CAPSO, TAPS, CAPS, Bis-Tris, TAPSO, TES, Tricine, and ADA. Examples of buffers having a carboxyl group include acetic acid-sodium acetate buffer, malic acid-sodium acetate buffer, malonic acid-sodium acetate buffer, and succinic acid-sodium acetate buffer. Buffers may be used alone or in combination.

[0055] Surfactants include, but are not limited to, nonionic, anionic, cationic, and amphoteric surfactants. Amphoteric surfactants include, but are not limited to, carboxybetaine, sulfobetaine, and phosphobetaine. Sulfobetaines include, but are not limited to, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate), CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate), and alkylhydroxysulfobetaines.

[0056] In some embodiments, the inorganic compound may be a layered inorganic compound, including those conventionally used in glucose sensors and equivalents thereof developed in the future. Examples of inorganic compounds include, but are not limited to, swellable clay minerals with ion exchange capacity, synthetic smectites such as smectite, bentonite, synthetic fluorine mica, vermiculite, synthetic hectorite, and synthetic saponite; swellable synthetic micas including synthetic fluorine mica; synthetic micas including Na-type mica; and combinations thereof.

[0057] In some embodiments, the reagent layer may include an enzyme layer. In some embodiments, the enzyme layer may include FADGDH. In some embodiments, the enzyme layer including FADGDH may include additives such as sodium polyacrylate, trehalose, or glucomannan.

[0058] The reagent layer may be a single layer or a multilayer structure. Each layer may have one or more components. In one embodiment, the reagent layer may be an inorganic gel layer with an enzyme layer containing FADGDH deposited thereon. The reagent layer may be disposed on the electrode in a dry state.

[0059] The sample to be measured may be a biological sample (eg, blood, body fluid, urine, etc.) or other liquid sample.

[0060] In one embodiment, the glucose sensor has a working electrode containing FADGDH, a counter electrode, and optionally a reference electrode. The working electrode can be a carbon electrode, a gold electrode, a platinum electrode, or the like. FADGDH may or may not be immobilized on the electrode. The counter electrode can be a conventional electrode such as a platinum electrode or Pt / C. The reference electrode can be a conventional electrode such as an Ag / AgCl electrode. FADGDH can be immobilized using a crosslinking reagent, encapsulation in a polymer matrix, coating with a dialysis membrane, photocrosslinkable polymers, conductive polymers, or redox polymers. Alternatively, FADGDH can be immobilized in a polymer or adsorbed onto an electrode together with an electron mediator, such as ferrocene or its derivatives, or a combination of these methods. Typically, FADGDH is immobilized on a carbon electrode using glutaraldehyde, followed by treatment with a reagent containing an amine group to block the glutaraldehyde. Other redox enzymes can also be immobilized in the same manner.

[0061] Methods for manufacturing glucose sensors are known in the art, for example, Liu, et. al., Anal. Chem. 2012, 84, 3403-3409 and Tsujimura, et. al., J. Am. Chem. Soc. 2014, 136, 14432-14437 (both of which are incorporated herein by reference in their entirety).

[0062] In one embodiment, the glucose sensor may include printed electrodes. In this case, the electrodes may be formed on an insulating substrate. Specifically, the electrodes may be formed on the substrate by printing techniques such as photolithography, screen printing, gravure printing, or flexography. Materials that can be used to form the insulating substrate include, for example, silicon, glass, ceramic, polyvinyl chloride, polyethylene, polypropylene, polyester, etc. Materials that are highly resistant to various solvents or chemicals may be used.

[0063] Glucose concentration can be measured as follows: A buffer solution is placed in a thermostatic cell and maintained at a constant temperature. A metal complex compound (e.g., a ruthenium compound) and an electron transfer promoter are used for electron transfer. FADGDH is used as the oxidoreductase. A carbon electrode is used as the working electrode, with a counter electrode (e.g., a platinum electrode) and a reference electrode (e.g., an Ag / AgCl electrode). A constant voltage is applied to the carbon electrode, and after the current becomes steady, a sample containing glucose is added and the increase in current is measured. The glucose concentration in the sample can be calculated according to a calibration curve created using glucose solutions of standard concentrations.

[0064] The glucose sensor can be used in combination with a measuring device that includes a means for applying a predetermined voltage for a fixed period of time, a means for measuring the electrical signal transmitted from the biosensor, a means for converting the electrical signal into the concentration of the substance to be measured, etc. The same applies to biosensors including lactate sensors.

[0065] The electrochemical measurements of the present disclosure can be amperometric, potentiometric, or coulometric. In certain embodiments, the electrochemical measurements measure the current flow as a reduced electron carrier is converted to an oxidized state by the application of a potential.

[0066] In some embodiments, the methods of the present disclosure do not involve medical intervention. In some embodiments, the methods of the present disclosure do not involve diagnostic intervention by a physician. In some embodiments, the methods of the present disclosure may aid in the diagnosis of diabetes. In some embodiments, the methods of the present disclosure may be used for monitoring blood glucose levels, which do not require the judgment of a physician. In some embodiments, the methods of the present disclosure do not involve prior art. In some embodiments, the methods of the present disclosure exclude the methods described in Japanese Patent Application Publication No. 2013-083634. In some embodiments, the methods of the present disclosure exclude the methods described in Japanese Patent Application Publication No. 2018-054555.

[0067] The present disclosure will be further described below using examples and comparative examples, but the present disclosure should not be construed as being limited to the following examples. [Example]

[0068] [Comparative Example 1] Glucose dehydrogenase (Mucorum spp., product name: FADGDH-AA, manufactured by Kikkoman Biochemifa Corporation) 120 mg, hexaammineruthenium chloride(III) (Tokyo Chemical Industry Co., Ltd., hereafter referred to as Ru) 186 mg, mPMS 0.68 mg (Dojindo Laboratories), dipotassium phosphate monobasic 33.6 mg (Fujifilm Wako Pure Chemical Industries, Ltd.), and monopotassium phosphate dibasic 22.6 mg (Fujifilm Wako Pure Chemical Industries, Ltd.), all in powder form, were mixed, and 2.0 mL of ultrapure water was added. After stirring at 25°C, the mixture did not dissolve well, and a significant precipitate was observed (Figure 3). The precipitate was recovered and weighed to be 56.2 mg.

[0069] Next, a test was conducted with slight modifications to the mixing process. First, 93 mg of powdered Ru was weighed out, and 1.0 mL of ultrapure water was added. The mixture was mixed at 25°C or 37°C. The powder dissolved completely (final Ru concentration: 300 mM). Next, 0.34 mg of mPMS, 16.8 mg of dipotassium monohydrogen phosphate, and 11.3 mg of monopotassium dihydrogen phosphate were added to the Ru solution in powder form, in that order, and mixed. All components dissolved well without precipitates. Finally, 60 mg of powdered FADGDH-AA was added to the mixed solution, and precipitates were immediately observed. A similar precipitate was observed when the weight of FADGDH-AA was increased to 20 mg.

[0070] The resulting precipitate was collected, washed with ultrapure water, and then redissolved. The absorption spectrum was measured. Peaks were observed around 210 nm and 270 nm, indicating that the precipitate was a ruthenium complex.

[0071] When the same test was performed using Aspergillus-derived FADGDH (manufactured by BBI) instead of FADGDH-AA, the formation of a similar precipitate was observed. The precipitate was collected and weighed in a 2 mL system, yielding 69.6 mg, representing 37% Ru. This confirmed that the precipitation generated by mixing and dissolving each component in powder form is not a phenomenon specific to the specific enzyme FADGDH-AA. Furthermore, when the absorption spectrum of a solution containing only the enzyme was measured, a waveform different from that of the precipitate was observed. Therefore, it was confirmed that the precipitate was not an enzyme.

[0072] Next, we further tested the dissolution order. 93 mg of powdered Ru was weighed out and dissolved in 1.0 mL of ultrapure water. 60 mg of powdered FADGDH-AA was then added to this solution, and a precipitate was immediately observed. In other words, precipitation occurred simply by adding the enzyme powder to the Ru solution, and no difference was observed in this phenomenon whether mPMS, dipotassium phosphate, or monopotassium phosphate had been added beforehand.

[0073] Next, tests were conducted with different Ru concentrations. First, 155 mg of powdered Ru was weighed out, and 1.0 mL of ultrapure water was added. The mixture was then mixed at 25°C or 37°C. The powder dissolved completely (final Ru concentration: 500 mM). Next, 0.34 mg of mPMS, 16.8 mg of dipotassium phosphate monobasic, and 11.3 mg of monopotassium phosphate monobasic were added to the Ru solution in powder form, in that order, and mixed. All components dissolved well without precipitates. Finally, 60 mg of powdered FADGDH-AA was added to the mixed solution, and precipitates were observed.

[0074] Further tests were conducted with different Ru concentrations. First, 56 mg of powdered Ru was weighed out, and 1.0 mL of ultrapure water was added. The mixture was then mixed at 25°C or 37°C. The entire powder dissolved well (final Ru concentration: 180 mM). Next, 0.34 mg of mPMS, 16.8 mg of dipotassium phosphate monobasic, and 11.3 mg of monopotassium phosphate monobasic were added to the Ru solution in powder form, in that order, and mixed. All components dissolved well without precipitates. Finally, 60 mg of powdered FADGDH-AA was added to the mixed solution, and precipitates were observed.

[0075] Without wishing to be bound by any particular theory, one hypothesis for the significant precipitation observed when powdered oxidoreductase was added to a Ru solution, or when both powdered oxidoreductases were mixed with Ru and dissolved, is that electrons flowed from FADGDH to Ru for some reason, causing Ru to convert from hexaammineruthenium chloride(III) to hexaammineruthenium chloride(II), which then precipitated. Hexaammineruthenium chloride(II) is known to be only slightly soluble in water. On the other hand, hexaammineruthenium chloride(III) is highly soluble in water. Furthermore, because the reagents used contained no glucose, it is highly unlikely that an enzymatic reaction occurred during dissolution.

[0076] Comparative Example 2 Amadoriase (product name: FPOX-CE, manufactured by Kikkoman Biochemifa Corporation) was used instead of FADGDH-AA. 93 mg of powdered Ru was weighed out, and 1.0 mL of ultrapure water was added. Mixing at 25°C or 37°C resulted in complete dissolution of the powder. Next, 0.34 mg of mPMS, 16.8 mg of dipotassium monohydrogen phosphate, and 11.3 mg of monopotassium dihydrogen phosphate were added in powder form to the Ru solution, and mixing resulted in complete dissolution of all components without precipitates. Finally, 60 mg of powdered FPOX-CE was added to the mixed solution, resulting in the formation of precipitates.

[0077] Comparative Example 3 A test was performed using sarcosine oxidase (Kikkoman Biochemifa Corporation, product name: SOD-EP) instead of FADGDH-AA. 93 mg of powdered Ru was weighed out, and 1.0 mL of ultrapure water was added. Mixing at 25°C or 37°C resulted in complete dissolution of the powder. Next, 0.34 mg of mPMS, 16.8 mg of dipotassium monohydrogen phosphate, and 11.3 mg of monopotassium dihydrogen phosphate were added to the Ru solution in powder form, in that order, and mixed. All components dissolved without precipitate. Finally, 60 mg of powdered SOD-EP was added to the mixed solution, resulting in the formation of precipitates.

[0078] Comparative Example 4 The test was performed using lactate dehydrogenase (product name: LDH-E, manufactured by Kikkoman Biochemifa Corporation) instead of FADGDH-AA. 93 mg of powdered Ru was weighed out, and 1.0 mL of ultrapure water was added. The mixture was then mixed at 25°C or 37°C, resulting in complete dissolution of the powder. Next, 0.34 mg of mPMS, 16.8 mg of dipotassium monohydrogen phosphate, and 11.3 mg of monopotassium dihydrogen phosphate were added to the Ru solution in powder form, in that order, and mixed. All components dissolved without precipitate. Finally, 60 mg of powdered LDH-E was added to the mixed solution, resulting in the formation of precipitates.

[0079] [Example 1] On the other hand, the following test was conducted by dissolving the oxidoreductase enzymes first, rather than mixing them in powder form. First, 60 mg of powdered FADGDH-AA was weighed out and dissolved in 1.0 mL of ultrapure water. The enzyme solution dissolved satisfactorily. Next, 93 mg of powdered Ru was added to the enzyme solution, which dissolved satisfactorily without forming any precipitates. Next, 0.34 mg of powdered mPMS, 16.8 mg of powdered dipotassium phosphate monobasic, and 11.3 mg of powdered monopotassium phosphate dibasic were added in that order and mixed. The enzymes dissolved without forming any precipitates.

[0080] We also performed a test in which the order of adding the enzyme solutions was changed. First, 93 mg of Ru, 0.34 mg of mPMS, 16.8 mg of dipotassium hydrogen phosphate, and 11.3 mg of monopotassium hydrogen phosphate were mixed in powder form, and then 1.0 mL of a solution in which 60 mg of FADGDH-AA had been dissolved in ultrapure water was added and mixed. In this case, too, the enzymes dissolved well without forming any precipitates.

[0081] From the above results, it was confirmed that when preparing a solution containing an oxidoreductase, a metal complex compound, and an electron transfer promoter, slight differences in the process of dissolving these components can cause the ruthenium compound to precipitate, resulting in precipitation, and this has been a technical issue in the fabrication of biosensors. Furthermore, it was newly discovered that one effective way to solve this phenomenon is to dissolve the oxidoreductase powder in advance before mixing.

[0082] As will be shown in the following examples, the industrial importance of solving the problem of "preventing precipitation of ruthenium compounds" disclosed in the present invention lies in the fact that this phenomenon poses a major challenge to providing a biosensor capable of accurate measurements. Specifically, when mixing and preparing a reagent composition as a pre-process before applying the reagent composition to an electrode to fabricate a biosensor, there is a step of preparing a solution in which various components are dissolved. If precipitation occurs during this process, it is expected that the ruthenium concentration in the composition to be applied will become very non-uniform. Furthermore, this can lead to the removal of a large amount of ruthenium compound from the reagent composition as a precipitate during the step of passing the reagent composition through a filter to remove solids before application.

[0083] In this way, if the amount of ruthenium compound in the reagent composition for electrode preparation is reduced to an extent that exceeds expectations, it will ultimately be possible to load only an amount significantly reduced compared to the initial concentration set to exhibit the desired performance on the biosensor, which may result in a deterioration in the quantitative measurement of glucose, which is the original purpose of the biosensor. At the laboratory level in prototyping reagent compositions, those skilled in the art usually weigh each reagent separately, dissolve them separately, mix them, and apply them to the sensor. It is thought that even those skilled in the art have not noticed such problems in the manufacturing process, such as directly mixing powders and dissolving them all at once.

[0084] If such precipitation occurs on a very small scale during a short preparation operation, it may be possible to deal with it by taking this into account, for example, by adjusting the initial blend amounts. However, if a similar method is used in actual industrial production on a certain scale or larger, in the case of a mediator or electron transfer promoter that is unstable in solution, such as PMS, the mediator may deteriorate over the time that passes from mixing and preparation to application and drying, which could result in a deterioration of sensor performance.

[0085] As disclosed in Patent Documents 1 and 2, rather than mixing all of the components, it is technically possible to place a different mediator in each of multiple layers, and to repeat the mixing and preparation of the reagents and dissolving and applying them separately for each layer. In this case, it is conceivable that precipitation can be prevented by intentionally placing the oxidoreductase and Ru in different layers. In fact, such a method has been commonly used in prior art, and there are almost no known disclosures of producing sensors with a single layer, and the issue of precipitation of ruthenium compounds as described above has not even been brought to light.

[0086] This time, the inventors intended to fabricate a single-layer sensor, and in that case, considering that it would be desirable to shorten as much as possible the time that the mediator and electron transfer promoter are in liquid form, they thought it would be preferable to mix the various reagents as powders and then turn them into a solution. However, by doing so, the problem of precipitation of the ruthenium compound became apparent for the first time, and they further discovered that this problem could be solved by adding the ruthenium compound to an enzyme solution, or by adding an aqueous solution containing an enzyme as a solution in which the ruthenium compound is dissolved.

[0087] In this example, the liquid volume was varied in the range of 0.5 to 2 mL, and the same phenomenon was observed regardless of the liquid volume. Therefore, it is believed that the liquid volume does not affect whether or not precipitation occurs.

[0088] [Example 2] Based on the findings of Example 1, we verified whether glucose could be quantified using a sensor fabricated as a single layer containing FADGDH-AA, Ru, and mPMS by chronoamperometry using printed electrodes. Specifically, 100 μL of a solution containing FADGDH-AA at a final concentration of 17.8 U / mL, 300 mM Ru, and 1 mM mPMS in PBS was applied to the printed electrode. The printed electrode was connected to a carbon working electrode (12.6 mm 2 The device was connected to an ALS 814D electrochemical analyzer (BAS) using a dedicated connector (Drop Sense, DRP-CAC) and screen-printed electrodes (DRP-110, Drop Sense). Glucose was added to a final concentration of 10-30 mM glucose, and the current was measured 30 seconds after the start of chronoamperometry. Figure 4 shows the current concentration-dependent increase up to 30 mM glucose, demonstrating high linearity.

[0089] Next, the same test was performed as above, but with the Ru concentration changed to a final concentration of 30 mM, 150 mM, or 180 mM. As a result, the linearity of the current value upon glucose addition worsened in all cases compared to when the Ru concentration was set to a final concentration of 300 mM (Figures 5 and 6). Table 1 shows the results of a comparison of the current increase upon the addition of 20 mM glucose and the current increase upon the addition of 30 mM glucose. The closer (b) / (a) is to 100%, the better the linearity.

[0090] [Table 1]

[0091] As can be seen from Table 1, as the concentration of the ruthenium compound in the reagent composition decreased, the change in the current value when 30 mM glucose was added minus the current value when 20 mM glucose was added became smaller than the change in the current value when 20 mM glucose was added minus the current value when 10 mM glucose was added. In other words, the linearity of the increase in the current value depending on the glucose concentration deteriorated. Therefore, it was found that the precipitation and removal of the ruthenium compound from the composition deteriorated the sensor performance.

[0092] In Example 1 of Patent Document 1, the amount of hexaammineruthenium chloride (III) per sensor was 20 μg. This is equivalent to the amount used in the GLUCOCARD (trademark) X-SENSOR self-testing glucose kit, which requires approximately 0.6 μL of blood. If the amount of liquid reaching the sensor is 0.3 to 0.4 μL, the hexaammineruthenium chloride (III) concentration is estimated to be 162 to 215 mM. In preparing the composition described above, if the hexaammineruthenium chloride (III) concentration is 30% lower than the expected concentration, the concentration will be 113 to 151 mM, which is likely to result in poor linearity.

[0093] [Example 3] Dissolution tests were conducted with varying concentrations of oxidoreductase. First, 80 mg of powdered FADGDH-AA was weighed and dissolved in 1.0 mL of ultrapure water. The enzyme solution dissolved satisfactorily. 93 mg of powdered Ru was then added to the enzyme solution, which dissolved satisfactorily without forming any precipitates. Next, 0.34 mg of powdered mPMS, 16.8 mg of powdered dipotassium phosphate monobasic, and 11.3 mg of powdered monopotassium phosphate dibasic were added in that order and mixed. The mixture dissolved without forming any precipitates.

[0094] We also conducted tests with different Ru concentrations. First, 60 mg of powdered FADGDH-AA was weighed and dissolved in 1.0 mL of ultrapure water. This resulted in satisfactory dissolution. Next, 155 mg of powdered Ru was added to this enzyme solution, resulting in satisfactory dissolution without any precipitates. Next, 0.34 mg of powdered mPMS, 16.8 mg of powdered dipotassium phosphate monobasic, and 11.3 mg of powdered monopotassium phosphate monobasic were added in that order and mixed. These solutions also dissolved without any precipitates.

[0095] [Example 4] Amadoriase (product name: FPOX-CE, manufactured by Kikkoman Biochemifa Corporation) was used instead of FADGDH-AA. First, 60 mg of powdered FPOX-CE was weighed out and dissolved in 1.0 mL of ultrapure water, resulting in satisfactory dissolution. 93 mg of powdered Ru was then added to this enzyme solution, resulting in satisfactory dissolution without any precipitates. 0.34 mg of powdered mPMS, 16.8 mg of powdered dipotassium phosphate monobasic, and 11.3 mg of powdered monopotassium phosphate dibasic were then added in that order and mixed, resulting in dissolution without any precipitates. No Ru-derived precipitates were observed even after 16 hours at 28°C.

[0096] [Example 5] The test was performed using sarcosine oxidase (Kikkoman Biochemifa Corporation, product name: SOD-EP) instead of FADGDH-AA. First, 60 mg of powdered SOD-EP was weighed out and dissolved in 1.0 mL of ultrapure water, resulting in good dissolution. 93 mg of powdered Ru was then added to this enzyme solution, resulting in good dissolution without any precipitates. Next, 0.34 mg of powdered mPMS, 16.8 mg of powdered dipotassium phosphate monobasic, and 11.3 mg of powdered monopotassium phosphate dibasic were added in that order and mixed, resulting in dissolution without any precipitates. No Ru-derived precipitates were observed even after 16 hours at 28°C.

[0097] [Example 6] The test was performed using lactate dehydrogenase (product name: LDH-E, manufactured by Kikkoman Biochemifa Corporation) instead of FADGDH-AA. First, 60 mg of powdered LDH-E was weighed out and dissolved in 1.0 mL of ultrapure water, which resulted in good dissolution. 93 mg of powdered Ru was then added to this enzyme solution, which resulted in good dissolution without precipitates. Next, 0.34 mg of powdered mPMS, 16.8 mg of powdered dipotassium phosphate monobasic, and 11.3 mg of powdered monopotassium phosphate dibasic were added in this order and mixed, resulting in good dissolution without precipitates.

[0098] When an oxidoreductase (powder) was added to a Ru solution, precipitates were observed. This phenomenon was observed not only for FADGDH, but also for amadoriase and sarcosine oxidase. When the oxidoreductase (powder) was first dissolved in a solution and then Ru (powder) was mixed with the solution, no precipitates were observed. The appearance of precipitates could be avoided by dissolving the enzyme first not only for FADGDH, but also for amadoriase, sarcosine oxidase, and lactate dehydrogenase. Therefore, those skilled in the art will understand that the appearance of precipitates can also be avoided for other types of oxidoreductases by dissolving the enzyme first. [Industrial Applicability]

[0099] The biosensor of the present disclosure can be used in the fields of biochemistry, medicine, and medical science. For example, the glucose sensor of the present disclosure is useful for measuring glucose. For example, the lactate sensor of the present disclosure is useful for measuring lactate.

Claims

1. (i) mixing and dissolving a powdered oxidoreductase with a solution; and (ii) After step (i), a step of mixing and dissolving the oxidoreductase solution and the ruthenium compound powder. A method for producing a composition for an oxidoreductase sensor, comprising:

2. The production method according to claim 1, wherein the oxidoreductase is added to a final concentration of 20 to 80 mg / mL.

3. The method according to claim 1, wherein the ruthenium compound is mixed to a final concentration of 180 to 500 mM.

4. (iii) after step (i), the method further comprises a step of mixing the solution in which the oxidoreductase is dissolved with a second electron transfer promoter powder to dissolve the second electron transfer promoter, or mixing the solution in which the second electron transfer promoter is dissolved with the second electron transfer promoter solution, 2. The method of claim 1, wherein the second electron transfer promoter is selected from the group consisting of phenazine methosulfate (PMS), 1-methoxy PMS (mPMS), and 1-methoxy-5-ethylphenazinium ethyl sulfate (mPES).

5. The method according to claim 4, wherein the second electron transfer promoter is mixed to a final concentration of 0.1 to 100 mM.

6. 2. The method according to claim 1, wherein the oxidoreductase is flavin-dependent glucose dehydrogenase (FADGDH), amadoriase, lactate dehydrogenase, or sarcosine oxidase.

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