JP2026022017A - Reagent composition, sensor and method for producing same - Google Patents

Abstract

[Problem] To provide a biosensor. Also, to avoid the occurrence of precipitates in the production of a sensor composition using a ruthenium compound. [Solution] To provide a method for producing a biosensor that includes an oxidoreductase, a ruthenium compound, and an electron transfer promoter. Also provided is a method for producing a biosensor composition by first adding and dissolving a powdered oxidoreductase in a solution, and then mixing a powdered ruthenium compound into the solution in which the oxidoreductase has dissolved. [Selected Figure] None

Description

The present disclosure relates to a method for producing a biosensor that includes an electron transfer promoter, an oxidoreductase, and a metal complex compound.

Patent Document 1 discloses a sensor that uses 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.

Patent Document 2 discloses a sensor that uses 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 mPES is contained in the enzyme layer.

Patent Documents 1 and 2 disclose that oxidoreductases such as glucose dehydrogenase and lactate dehydrogenase react with a substrate, reduce mPMS or mPES, then reduce a ruthenium compound, and transfer electrons to an electrode, resulting in 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 contained in the same layer (enzyme layer) where it is easily accessible to the oxidoreductase. 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 again to the second mediator, and then transferred to the electrode. This raises concerns that reaction kinetic analysis may become complicated. It is also feared that this may affect the sensor's response time and performance. To avoid such concerns, when using multiple types of mediators, it is generally considered to be the case that the first mediator is placed in the enzyme layer and the second mediator is placed in a different layer.

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

JP 2013-083634 A (Patent No. 5584740) JP 2018-054555 (Patent No. 6773507)

The inventors undertook the development of a glucose sensor with an electrode that uses FADGDH as the enzyme and mPMS and a ruthenium complex as the mediator. First, powdered ruthenium was prepared in solution. Next, powdered phosphate buffer and powdered mPMS as the mediator were added and dissolved in this prepared solution. Next, powdered FADGDH was added to this 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.

When an electrode was made using the supernatant of the solution from which the precipitate had formed and used to measure glucose concentration, linear current values were only obtained within a limited concentration range. Therefore, it was thought that developing a glucose sensor would be difficult without resolving the precipitate issue. When the precipitate was recovered and its absorption spectrum measured, the waveform matched that of a ruthenium complex solution. It was considered possible that the precipitate was an enzyme, 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.

The present invention therefore aims to provide an enzyme sensor containing a ruthenium compound that avoids precipitation of the ruthenium compound and has improved production efficiency, as well as a method for producing the same. To 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.

After extensive research, the inventors discovered that instead of mixing the individual reagents that make up the reagent composition in powder form and then adding water to dissolve them, a uniform composition can be prepared without causing ruthenium compound precipitation by dissolving the oxidoreductase in advance and then adding the enzyme solution to the other components. Furthermore, they discovered that sensors prepared using this composition are capable of quantifying glucose up to high concentrations compared to sensors prepared without this method, and completed the present invention, which incorporates this as one embodiment.

The present disclosure encompasses the following embodiments.
[1] (i) mixing and dissolving a powdered oxidoreductase with a solution; and
(ii) A method for producing a composition for an oxidoreductase sensor, which comprises, after step (i), a step of mixing and dissolving the oxidoreductase solution and the ruthenium compound powder.
[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 powder of a second electron transfer promoter 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;
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.

According to one embodiment of the present disclosure, a biosensor that combines an electron transfer promoter, an oxidoreductase, an electrode, and a metal complex compound without causing precipitation of the ruthenium compound, and a method for manufacturing the biosensor, can be provided. Furthermore, according to one embodiment of the present disclosure, a glucose sensor that combines an electron transfer promoter, GDH, an electrode, and a metal complex compound without causing precipitation of the ruthenium compound, and a method for manufacturing the glucose sensor can be provided.

1 shows a flow chart of a conventional dissolution procedure. 1 shows a flow chart of the dissolution procedure of the present invention and comparative examples. 1 is a photograph of a precipitate (sediment). The results of chronoamperometry measurements at 300 mM Ru are shown. The results of chronoamperometry measurements at 150 mM Ru are shown. The results of chronoamperometry measurements at 30 mM Ru are shown.

In certain embodiments, 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 the oxidoreductase can recognize as a substrate. For example, if flavin-dependent glucose dehydrogenase (FADGDH) is used as the oxidoreductase, a glucose sensor containing FADGDH can be provided, which can be used to measure glucose.

Unless otherwise specified, the term "biosensor" 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 three-electrode electrode or a printed electrode. In one embodiment, the electrode portion may be disposed on an insulating substrate.

As used herein, a mediator refers to a compound that participates in electron transfer. For example, in a system that uses an electrode, the mediator receives electrons from an oxidoreductase to become reduced, and then donates electrons to the electrode to return to its 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.

Electron Transfer Enhancer In this specification, a compound that enhances the transfer of electrons from an oxidoreductase to an electrode is referred to as an "electron transfer enhancer." An "electron transfer enhancer" can be used in a biosensor equipped with an oxidoreductase, such as a glucose sensor, and enhances the generation of a response current depending on the concentration of a target compound when added. In certain embodiments, the electron transfer enhancer can modify the function of a mediator. In this specification, the expression "modifying the function of a mediator" refers to the fact that, for a mediator that does not or barely transfers electrons from an oxidoreductase to an electrode in the absence of the electron transfer enhancer, electrons are transferred from the oxidoreductase to an electrode in the presence of the electron transfer enhancer. mPMS and mPES, as described in Patent Documents 1 and 2, can be considered as electron transfer enhancers. In this specification, the electron transfer enhancer may also be referred to as a mediator.

Examples of electron transfer accelerators 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.

The amount of electron transfer promoter contained per biosensor is not limited as long as it is an amount that allows the measurement of the specific 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.

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

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. For other oxidoreductases, the content 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.

In general, electron transfer promoters and mediators, like 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, rather than preparing individual solutions and mixing them, as this will shorten the time they remain in solution.

The concentration of the oxidoreductase in the reagent composition applied to the biosensor is not limited as long as it is a concentration that allows the measurement of the specific component to be measured, and may be, for example, but 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 10 to 100 mg/mL.

Metal Complex Compounds Examples of metal complex compounds include, but are not limited to, ruthenium compounds and osmium compounds. In certain embodiments, the content of the metal complex compound per glucose sensor is not limited as long as it is an amount that allows the measurement of a predetermined component to be measured, and can be an amount typically used in glucose sensors, such as 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.

The concentration of the metal complex compound in the reagent composition applied to the biosensor is not limited as long as it is a concentration that allows the specific component to be measured to be measured, and can be, for example, but 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, or 10 to 500 mM.

Ruthenium Compound 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 an embodiment, the ruthenium compound may be present in the reaction system as an oxidized ruthenium complex. The ruthenium complex is not particularly limited. In an embodiment, the ruthenium complex may be a compound represented by the following general formula:
[Ru(NH ₃ )5X]n ⁺
(In the formula, X is NH ₃ , CN, pyridine, a halogen ion, nicotinamide, or H ₂ O. Examples of halogen ions include Cl ^- , F ^- , Br ^- , and 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 NH ₃ , the compound is a hexaammineruthenium complex compound, and if the halogen is Cl ^- , the compound is hexaammineruthenium chloride.

Oxidoreductases 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), ascorbic acid 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, but are not limited to these. Coenzymes for the above enzymes include nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide (FAD), pyrroloquinoline quinone, etc. 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 oxidoreductases can be used as these various oxidoreductases, and various commercially available enzyme products can also be used.

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 its 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 its variant unless otherwise specified. As used herein, Aspergillus-type FADGDH refers to a wild-type FADGDH of the Aspergillus genus and/or a variant thereof, and includes both the wild-type and its variants unless otherwise specified. As used herein, Penicillium-type FADGDH refers to a wild-type FADGDH of the Penicillium genus and/or a variant thereof, and includes both the wild-type and its variants unless otherwise specified. As used herein, Circinella-type FADGDH refers to a wild-type FADGDH of the Circinella genus and/or a variant thereof, and includes both the wild-type and its variants unless otherwise specified.

Microorganisms of the genus Mucor 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. Microorganisms of the genus Botryothinia include, but are not limited to, Botryothinia fuckeliana. Microorganisms of the genus Aspergillus include, but are not limited to, Aspergillus oryzae, Aspergillus sojae, Aspergillus niger, and Aspergillus terreus. Microorganisms of the genus Penicillium include, but are not limited to, Penicillium sclerotiorum, Penicillium janthinellum, and Penicillium paneum.

As used herein, a variant of a wild-type FADGDH of the genus Mucor 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 which has FAD-dependent glucose dehydrogenase activity. In certain embodiments, 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, or 1 to 3, e.g., 1 to 2.

As used herein, a variant of wild-type FADGDH from Mucor prainii refers to a variant that has a sequence in which amino acid residues in the amino acid sequence of the wild-type FADGDH have been substituted and that has FAD-dependent glucose dehydrogenase activity. In certain embodiments, 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 to 2.

For example, when the enzyme used is a variant of a wild-type FADGDH of the genus Botryothinia, the variant has a sequence in which amino acid residues in the amino acid sequence of the wild-type FADGDH have been substituted, and has FAD-dependent glucose dehydrogenase activity. In certain embodiments, the variant has low reactivity to maltose and galactose and high reactivity to 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.

For example, when the enzyme used is a variant of the wild-type FADGDH of Botryotinia fuckeliana, the variant has a sequence in which amino acid residues in the amino acid sequence of the wild-type FADGDH have been substituted, and has FAD-dependent glucose dehydrogenase activity. In certain embodiments, the variant has low reactivity to maltose and galactose and high reactivity to 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.

For example, when the enzyme used is a variant of a wild-type FADGDH of the genus Aspergillus, the variant has a sequence in which amino acid residues in the amino acid sequence of the wild-type FADGDH have been substituted, and has 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.

For example, when the enzyme used is a variant of a wild-type FADGDH of the genus Penicillium, the variant has a sequence in which amino acid residues in the amino acid sequence of the wild-type FADGDH have been substituted, and has FAD-dependent glucose dehydrogenase activity. In certain embodiments, the variant has low reactivity to maltose and galactose, and high reactivity to 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.

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 amadoriases may be used. In other embodiments, variants of commercially available amadoriases or their equivalents may be used. In the present specification, the origin of amadoriase is not particularly limited, and examples thereof include those derived from the genus Coniochaeta, the genus Eupenicillium, the genus Pyrenochaeta, the genus Arthrinium, the genus Curvularia, the genus Neocosmospora, the genus Cryptococcus (C), and the like. The genus Pseudomonas ryptococcus, the genus Phaeosphaeria, the genus Aspergillus, the genus Emericella, the genus Ulocladium, the genus Penicillium, the genus Fusarium, the genus Achaetomiella, the genus Acaetomium, The genus Haetomium, the genus Thielavia, the genus Chaetomium, the genus Gelasinospora, the genus Microascus, the genus Leptosphaeria, the genus Ophiobolus, the genus Pleospora, the genus Coniocetidium It refers to amadoriase derived from the genus Chaetidium, Pichia, Debaryomyces, Corynebacterium, Agrobacterium, or Arthrobacter, and unless otherwise specified, includes both wild-type and modified forms thereof.

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 modified version 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 modified versions thereof unless otherwise specified.

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 an equivalent thereof 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.

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 modified version 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 modified versions thereof are included.

In certain embodiments, the 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.

In one embodiment, the content of FADGDH per glucose sensor can be, for example, but not limited to, 0.1 to 50 U, 0.5 to 20 U, 1 to 10 U, or 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.

In this specification, the FADGDH content per glucose sensor refers, in certain embodiments, 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 placed on one electrode system. In another embodiment, the content refers to the amount of FADGDH formulated in a reagent so as to be included in the reaction system when a sample is added.

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

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 placed on one electrode system. In another embodiment, the content refers to the amount of LOD incorporated into a reagent so that it is included in the reaction system when a sample is added.

In certain embodiments, the biosensor has a size that is typical for use with measurement samples. For example, in certain embodiments, a glucose sensor has a size that is typical for use with 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 according to the volume of the sample or reaction system.

Reagent Layer In certain embodiments, the biosensor of the present disclosure comprises 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 comprises an electron transfer promoter, an oxidoreductase, and a metal complex compound. In certain embodiments, the electrode comprises 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.

In certain embodiments, the present disclosure provides a method for manufacturing a biosensor, which includes 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 and mixing a solution containing an oxidoreductase, and applying the resulting solution to an electrode when all the reagents have dissolved. Unlike methods 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 an oxidoreductase, or mixing the metal complex compound and the electron transfer promoter in powder form, then adding and mixing a solution containing an oxidoreductase, and applying the resulting solution to an electrode when all the reagents have dissolved, can obtain a well-dissolved reagent composition without forming a precipitate, which, when applied to an electrode to fabricate a biosensor, enables the quantification of glucose up to high concentrations.

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

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

In one embodiment, the present disclosure provides a system for measuring the concentration of a target compound in a sample, comprising a biosensor, means for applying a voltage to electrodes of the biosensor, and means for measuring 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.

In certain embodiments, the present disclosure provides a method for measuring the concentration of a compound to be measured, comprising contacting a sample that may contain the compound to be measured with an oxidoreductase, applying a voltage to an electrode, and measuring the 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 may be, for example, 10 to 1000 mV, 10 to 800 mV, 50 to 500 mV, or 0 to 100 mV. Unless otherwise specified, the potential described herein is the potential when a silver-silver chloride reference electrode is used.

The concentration of the target compound can be measured by contacting the sample with the oxidoreductase, holding 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 time for holding the sample without applying a potential can be greater than 0 seconds and less 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 as EC Group 1 listed above, the substrates recognized by these oxidoreductases can be used as target compounds, and systems for measuring the concentrations of such target compounds are provided.

In some embodiments, the reagent layer may further include buffers, surfactants, inorganic compounds, and other ingredients.

Buffers include, but are not limited to, phosphate buffers, amine buffers, and buffers containing a carboxyl group. Examples of amine buffers include Tris, ACES, CHES, CAPSO, TAPS, CAPS, Bis-Tris, TAPSO, TES, Tricine, and ADA. Examples of buffers containing 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.

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.

In some embodiments, the inorganic compound may be a layered inorganic compound, and may be one that has been used in glucose sensors in the past or an equivalent thereof that will be developed in the future. Examples of inorganic compounds include, but are not limited to, swellable clay minerals with ion exchange capacity, smectite, bentonite, synthetic fluorine mica, synthetic smectites such as vermiculite, synthetic hectorite, and synthetic saponite; swellable synthetic micas including synthetic fluorine micas; synthetic micas including Na-type mica, and combinations thereof.

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.

The reagent layer may have a single layer structure or a multi-layer 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 layered thereon. The reagent layer may be disposed on the electrode in a dry state.

The sample to be measured may be a biological sample (e.g., blood, body fluid, urine, etc.) or other liquid sample.

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. When immobilizing FADGDH, immobilization methods include using a crosslinking reagent, encapsulation in a polymer matrix, coating with a dialysis membrane, photocrosslinkable polymers, conductive polymers, and 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 can be used. Typically, FADGDH is immobilized on a carbon electrode using glutaraldehyde, and then treated with a reagent containing an amine group to block the glutaraldehyde. Other redox enzymes can also be immobilized in a similar manner.

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 (the entire contents of both are incorporated herein by reference).

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 may constitute 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.

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.

Glucose sensors can be used in combination with measuring devices that include a means for applying a predetermined voltage for a fixed period of time, a means for measuring the electrical signal transmitted from the biosensor, and a means for converting the electrical signal into the concentration of the substance being measured. The same applies to biosensors, including lactate sensors.

The electrochemical measurement methods disclosed herein can be amperometric, potentiometric, or coulometric. In certain embodiments, the electrochemical measurement measures the current value when a reduced electron carrier becomes oxidized by the application of a potential.

In some embodiments, the methods of the present disclosure do not involve medical procedures. In some embodiments, the methods of the present disclosure do not involve diagnostic procedures 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. These 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.

The present disclosure will be further explained below using examples and comparative examples. However, the present disclosure should not be construed as being limited to the following examples.

[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 monohydrogen phosphate 33.6 mg (Fujifilm Wako Pure Chemical Industries, Ltd.), and monopotassium dihydrogen phosphate 22.6 mg (Fujifilm Wako Pure Chemical Industries, Ltd.), all in powder form, were mixed together, and 2.0 mL of ultrapure water was added and mixed at 25°C. However, 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.

Next, a test was conducted with some 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.

The resulting precipitate was collected, washed with ultrapure water, and then redissolved. Absorption spectroscopy was then performed. Peaks were observed near 210 nm and 270 nm, indicating that the precipitate was a ruthenium complex.

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. When the precipitate was collected and weighed in a 2 mL system, it was found to be 69.6 mg, representing 37% Ru. This confirmed that the precipitation that occurs when 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 in which only the enzyme was dissolved was measured, a waveform different from the absorption spectrum of the precipitate was observed. Therefore, it was confirmed that the precipitate was not an enzyme.

Next, further tests were conducted by changing 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 in this phenomenon was observed whether mPMS, dipotassium monohydrogen phosphate, or monopotassium dihydrogen phosphate had been added in the previous step.

Next, tests were conducted with different Ru concentrations. First, 155 mg of powdered Ru was weighed out, to which 1.0 mL of ultrapure water was added and 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 monohydrogen phosphate, and 11.3 mg of monopotassium dihydrogen phosphate were added in powder form to this Ru solution, in that order, and mixed. All components dissolved well without forming any precipitates. Finally, 60 mg of powdered FADGDH-AA was added to this mixed solution, and precipitates were observed.

Furthermore, tests were conducted by varying the Ru concentration. First, 56 mg of powdered Ru was weighed out, to which 1.0 mL of ultrapure water was added and 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 monohydrogen phosphate, and 11.3 mg of monopotassium dihydrogen phosphate were added in powder form to this Ru solution, in that order, and mixed. All components dissolved well without forming any precipitates. Finally, 60 mg of powdered FADGDH-AA was added to this mixed solution, and precipitates were observed.

While not wishing to be bound by any particular theory, one hypothesis as to why significant precipitation occurs when powdered oxidoreductase is added to a Ru solution, or when powdered oxidoreductase and Ru are mixed and dissolved, is that electrons flow from FADGDH to Ru for some reason, causing Ru to convert from hexaammineruthenium chloride (III) to hexaammineruthenium chloride (II), which then precipitates. 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, since the reagents used did not contain any glucose, it is highly unlikely that an enzymatic reaction occurred during dissolution.

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

[Comparative Example 3]
The test was conducted 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 to the solution. The mixture was then mixed at 25°C or 37°C, resulting in complete dissolution of the powder. Subsequently, 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 were dissolved without precipitate. Finally, 60 mg of powdered SOD-EP was added to the mixed solution containing these components, resulting in the formation of precipitates.

[Comparative Example 4]
The test was conducted using lactate dehydrogenase (manufactured by Kikkoman Biochemifa Corporation, product name: LDH-E) instead of FADGDH-AA. 93 mg of powdered Ru was weighed out, and 1.0 mL of ultrapure water was added to the solution. The mixture was then mixed at 25°C or 37°C, resulting in complete dissolution of the powder. Subsequently, 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, and the mixture was mixed. All components were dissolved without precipitate. Finally, 60 mg of powdered LDH-E was added to the mixed solution containing the dissolved components, resulting in the formation of precipitates.

[Example 1]
On the other hand, the following test was conducted by dissolving the oxidoreductase enzyme first and then mixing it, rather than mixing it in powder form. First, 60 mg of powdered FADGDH-AA was weighed out, and 1.0 mL of ultrapure water was added and dissolved, 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 monohydrogen, and 11.3 mg of powdered monopotassium phosphate dihydrogen were added in that order and mixed, resulting in good dissolution without any precipitates.

An experiment was also conducted in which the order in which the enzyme solutions were added was changed. First, 93 mg of Ru, 0.34 mg of mPMS, 16.8 mg of dipotassium monohydrogen phosphate, and 11.3 mg of monopotassium dihydrogen 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. Even in this case, no precipitate was formed and the mixture dissolved well.

These results confirm that when preparing a solution containing an oxidoreductase, metal complex compound, and electron transfer promoter, slight differences in the process of dissolving these components can cause the ruthenium compound to precipitate, resulting in sedimentation, and that this has been a technical issue in biosensor fabrication. Furthermore, it has been newly discovered that one effective way to resolve this phenomenon is to dissolve the oxidoreductase powder in advance before mixing.

As will be demonstrated in the following examples, the industrial importance of solving the problem of "preventing precipitation of ruthenium compounds" disclosed in this invention lies in the fact that this phenomenon poses a major challenge to providing biosensors that can perform accurate measurements. Specifically, when mixing and preparing a reagent composition as a pre-process before applying it 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 uneven. Furthermore, this could lead to a large amount of ruthenium compound being removed as precipitate from the reagent composition when the composition is passed through a filter to remove solids before application.

If the amount of ruthenium compound in the reagent composition for electrode fabrication is reduced to an extent greater than expected, the amount that can be loaded onto the biosensor will be significantly reduced compared to the initial concentration set to achieve the desired performance, which may result in a deterioration of the biosensor's original purpose of quantitatively measuring glucose. When prototyping reagent compositions at the laboratory level, those skilled in the art typically weigh out each reagent separately, dissolve them separately, mix them, and apply them to the sensor. It is believed that even those skilled in the art were unaware of this issue in a manufacturing process in which powders are directly mixed and dissolved all at once.

If this type of precipitation occurs on a very small scale during a short preparation operation, it may be possible to address it by taking this into account and adjusting the initial blend amounts, etc. 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.

As disclosed in Patent Documents 1 and 2, rather than mixing all the components, it is technically possible to place a different mediator in each of multiple layers, and to repeat the process of dissolving and applying the reagent to each layer separately. In this case, it is conceivable that precipitation can be prevented by intentionally placing the oxidoreductase and Ru in separate layers. In fact, such a method is commonly used in prior art, and there are almost no known examples 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.

In this study, the inventors intended to create a single-layer sensor, and in that case, considering the need to shorten the time that the mediator and electron transfer promoter are in liquid form as much as possible, they thought it would be preferable to mix the various reagents as powders and then create a solution. However, doing so brought to light for the first time the issue of ruthenium compound precipitation, and they further discovered that this issue could be resolved by adding a ruthenium compound to an enzyme solution, or by adding an aqueous solution containing an enzyme as a solution for dissolving the ruthenium compound.

In addition, when the liquid volume in this example was varied between 0.5 and 2 mL, the same phenomenon was observed regardless of the liquid volume, so it is believed that the liquid volume does not affect whether or not precipitation occurs.

[Example 2]
Based on the findings of Example 1, the feasibility of quantifying glucose using a single-layer sensor containing FADGDH-AA, Ru, and mPMS was examined 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 a SCREEN-PRINTED ELECTRODES (DRP-110, manufactured by Drop Sense) with a printed carbon working electrode (12.6 mm ² ) and a printed silver reference electrode. The electrode was connected to an ALS Electrochemical Analyzer 814D (manufactured by BAS) using a dedicated connector (DRP-CAC, manufactured by Drop Sense). The applied voltage was set to +100 mV (vs. Ag/AgCl). Glucose was added to a final concentration of 10-30 mM glucose, and the current value was measured 30 seconds after the start of chronoamperometry. The results are shown in Figure 4. As shown in Figure 4, the current value increased concentration-dependently up to 30 mM glucose, demonstrating high linearity.

Next, the above test was repeated with the Ru concentration changed to a final concentration of 30 mM, 150 mM, or 180 mM. As a result, in all cases, the linearity of the current value upon glucose addition worsened 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 comparing the increase in current value upon addition of 20 mM glucose with the increase in current value upon addition of 30 mM glucose. The closer (b)/(a) is to 100%, the better the linearity.

Table 1 shows that as the concentration of ruthenium compound in the reagent composition decreased, the change in (current value when 30 mM glucose was added) minus (current value when 20 mM glucose was added) became smaller compared to the change in (current value when 20 mM glucose was added) minus (current value when 10 mM glucose was added). In other words, the linearity of the increase in current value depending on glucose concentration deteriorated. Therefore, it was found that the precipitation and removal of ruthenium compound from the composition deteriorates sensor performance.

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™ 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 thought to be 162 to 215 mM. If the hexaammineruthenium chloride (III) concentration is 30% lower than the expected concentration in preparing the composition, the concentration will be 113 to 151 mM, which is likely to result in poor linearity.

[Example 3]
A dissolution test was 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 well. 93 mg of powdered Ru was then added to the enzyme solution, which dissolved well 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 this order and mixed. The mixture dissolved without forming any precipitates.

Tests were also conducted with different Ru concentrations. First, 60 mg of powdered FADGDH-AA was weighed out and dissolved in 1.0 mL of ultrapure water, resulting in good dissolution. 155 mg of powdered Ru was then added to this enzyme solution, resulting in good 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 good dissolution without any precipitates.

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

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

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

When oxidoreductase (powder) was added to the Ru solution, precipitates were observed. This phenomenon was not limited to FADGDH, but also observed 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 oxidoreductase by dissolving the enzyme first.

The biosensors disclosed herein can be used in the fields of biochemistry, medicine, and medical science. For example, the glucose sensor disclosed herein is useful for measuring glucose. For example, the lactate sensor disclosed herein is useful for measuring lactate.

Claims (6)

(i) mixing and dissolving a powdered oxidoreductase with a solution; and
(ii) A method for producing a composition for an oxidoreductase sensor, which comprises, after step (i), a step of mixing and dissolving the oxidoreductase solution and the ruthenium compound powder. The manufacturing method described in claim 1, wherein the oxidoreductase is added to a final concentration of 20 to 80 mg/mL. The manufacturing method described in claim 1, in which the ruthenium compound is mixed to a final concentration of 180 to 500 mM. (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). The manufacturing method described in claim 4, wherein the second electron transfer promoter is mixed to a final concentration of 0.1 to 100 mM. 2. The method according to claim 1, wherein the oxidoreductase is flavin-dependent glucose dehydrogenase (FADGDH), amadoriase, lactate dehydrogenase, or sarcosine oxidase.