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 biosensor comprising an electron transfer promoter, an oxidoreductase, and a metal complex compound. This relates to a method for producing the above. [Background technology]

[0002] Patent Document 1 discloses 1-methoxy-5-methylphenazinium methyl sulfate (1-methylphenazinium methyl sulfate). Two mediators, hexaammineruthenium(III) and hexaammine PMS (mPMS), were used. The present invention discloses a sensor in which the ruthenium compound is preferably an inorganic gel. The cellulose layer contains mPMS, and the enzyme layer contains mPMS.

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

[0004] In Patent Documents 1 and 2, glucose dehydrogenase, lactate dehydrogenase, etc. After the oxidoreductase reacts with the substrate, it reduces mPMS and mPES, followed by ruthenium oxidation. The method disclosed in this publication is to reduce the compound and transfer electrons to an electrode, thereby detecting the current value. In such sensors, mPMS and mPES are used. The mediator that first accepts electrons from the enzyme is located in the same layer that is accessible to the redox enzyme. The ruthenium compounds are present in a different form adjacent to the enzyme layer. If multiple types of mediators exist in the same layer, After the electrons are transferred from the first mediator to the second mediator, the second mediator does not necessarily Instead of electrons being transferred from the mediator to the electrode, they are transferred back to the first mediator. The electrons are transferred, then transferred again to a second mediator, and then transferred to the electrode. This may complicate the analysis of reaction kinetics. There is a concern that this may affect the response time and performance of the sensor. When multiple types of mediators are used, the first mediator is placed on the enzyme layer. However, it will be common to place the second mediator in a different layer.

[0005] Numerous documents are cited throughout this specification, including patent applications and manufacturer's manuals. The disclosure of these documents is not considered relevant to the patentability of this invention, but The entire contents of all references are incorporated herein by reference. More particularly, all references are incorporated herein by reference. as if each individual document were specifically and individually indicated to be incorporated by reference. The disclosure of which is hereby 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 used FADGDH as the enzyme and mPMS and ruthenium as the mediators. We have been working on the development of a glucose sensor with an electrode that uses a ruthenium complex. Ruthenium in the form of powder was then added to the prepared solution. Powdered mPMS was added as a buffer and a mediator and dissolved. Powdered FADGDH was added to the prepared solution. When the enzyme powder was added, a precipitate formed, which was a problem. The same problem occurred when the product was changed to another FADGDH product. This issue was not believed to be specific to FADGDH products.

[0008] An electrode was made using the supernatant of the solution from which the precipitate had formed, and was used to measure glucose concentration. The linearity of the current value was only obtained in a limited concentration range. It was thought that it would be difficult to develop a glucose sensor without resolving this problem. The absorbance spectrum of the solution was measured and found to be identical to that of the ruthenium complex solution. The possibility that the precipitate was an enzyme was considered, but the absorption spectrum of the precipitate was consistent with that of the enzyme alone. The absorbance spectrum of the liquid showed a waveform different from that of the precipitate. It was identified as a ruthenium complex.

[0009] Therefore, the present invention relates to an enzyme sensor containing a ruthenium compound, and the precipitation of the ruthenium compound. The present invention provides a sensor containing a ruthenium compound and a method for manufacturing the same, which avoids the above-mentioned problems and improves production efficiency. To the best of the inventors' knowledge, no ruthenium compound has been used as a mediator. There have been no reports of precipitates occurring in conventional enzyme sensors that use This is a new challenge. [Means for solving the problem]

[0010] As a result of extensive research, the present inventors have discovered that the reagent composition is a mixture of powdered reagents. Instead of dissolving the enzyme by adding water afterwards, the enzyme solution is dissolved in advance. By adding the liquid to other components, ruthenium compounds do not precipitate and a uniform composition is achieved. Furthermore, the sensor produced using the composition exhibits the following properties: Compared with sensors fabricated without such methods, the quantitative determination of glucose concentrations up to high levels was possible. The present inventors have found that this is possible, 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. Process A method for producing a composition for an oxidoreductase sensor, comprising: [2] Embodiment 1, in which the oxidoreductase is added to a final concentration of 20 to 80 mg / mL. The manufacturing method described in [3] An embodiment in which a ruthenium compound is mixed to a final concentration of 180 to 500 mM. 3. The manufacturing method according to 1 or 2. [4] (iii) After step (i), the solution in which the oxidoreductase is dissolved and a second electron transfer promoter powder are mixed. or a second electron transfer promoter in a state where the second electron transfer promoter is dissolved in the solution. further comprising the step of mixing an accelerator solution; The second electron transfer promoter is phenazine methosulfate (PMS), 1-methoxy PM S (mPMS), and 1-methoxy-5-ethylphenazinium ethyl sulfate (m 4. The method of claim 1, 2 or 3, wherein the polymer is selected from the group consisting of: PES. [5] The second electron transfer promoter is mixed to a final concentration of 0.1 to 100 mM. The manufacturing method according to embodiment 4. [6] The oxidoreductase is flavin-dependent glucose dehydrogenase (FADGDH), 1. The enzyme of embodiment 1 is madoriase, lactate dehydrogenase, or sarcosine oxidase. 6. The method for producing a semiconductor device according to any one of claims 1 to 5. [Effects of the Invention]

[0012] According to the present disclosure, in one embodiment, a ruthenium compound is not precipitated and electron transfer is not performed. A biosensor and its biosensor which combine a promoter, an oxidoreductase, an electrode and a metal complex compound. According to the present disclosure, a method for manufacturing an ion sensor can be provided. The ruthenium compounds do not precipitate, and the electron transfer promoter, GDH, electrodes, and metal complexes are To provide a glucose sensor in which compounds are combined and a method for manufacturing the glucose sensor can be done. [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 certain embodiments, the present disclosure provides a method for producing a compound comprising: an electron transfer facilitator; an oxidoreductase; a metal complex compound; and an electrode. The biosensor is a biosensor containing the oxidoreductase used. Depending on the type, it is possible to measure a specific compound that the oxidoreductase can recognize as a substrate. For example, flavin-dependent glucose dehydrogenase (FAD) By using FADGDH, a glucose sensor having FADGDH can be provided. Glucose can be measured.

[0015] In this specification, unless otherwise specified, a biosensor is a sensor comprising an electron transfer promoter, an electrode, an acid, This refers to a sensor that contains a redox enzyme and a metal complex compound. In one embodiment, the electrode can be used as a sensor for a substrate to be detected. In another embodiment, the electrode includes an electrode portion having a working electrode, a counter electrode, and a reference electrode. The electrode may be, for example, a tripolar electrode or a printed electrode. In this embodiment, the electrode portion may be disposed on an insulating substrate.

[0016] In this specification, the term "mediator" refers to a compound that participates in electron transfer, for example, using an electrode. In this system, the mediator receives electrons from the redox enzyme to become reduced, and then the mediator is transferred to the electrode. From this perspective, the compound that functions as a mediator These terms are used interchangeably in this specification. do.

[0017] Electron transfer promoter As used herein, a compound that promotes the transfer of electrons from an oxidoreductase to an electrode is referred to as an "electron The "electron transfer promoter" is a biosensor equipped with an oxidoreductase, e.g. For example, it can be used as a glucose sensor, and when a target compound is added, the concentration of the target compound is determined. In one embodiment, the electron transfer promoter is a medium. In the present specification, the electron transfer promoter can "modify the function of the mediator." "Modify the function" means to improve the ability of the oxidoreductase to transfer electrons to the electrode in the absence of an electron transfer promoter. For mediators that do not or hardly undergo oxidation reduction in the presence of an electron transfer promoter, This refers to the transfer of electrons from the original enzyme to the electrode. PMS and mPES can be considered as electron transfer promoters. The agent that promotes delivery is sometimes called a mediator.

[0018] Examples of electron transfer promoters include phenazine methosulfate and its derivatives, specifically 1-Methoxy-5-methylphenazinium methyl sulfate (mPMS) and 1-Methoxy-5-methylphenazinium methyl sulfate (mPMS) are Toxic-5-methylphenazinium ethyl sulfate (mPES), thionine and its Derivatives, specifically 3-amino-7-(2,3,4,5,6-pentahydroxyhexane Amido)-5-phenothiazinium, Azure C, Azure A, Methylene Blue, Tolui Gin blue, phenylenediamine and its derivatives, specifically N,N,N',N'-tetraphenylene Examples include methylphenylenediamine and 2,4-Diaminodiphenylamine. It can be obtained.

[0019] The content of electron transfer promoter per biosensor is determined by the amount of the specific component to be measured. There are no limitations as long as the amount is measurable, and examples include 10 pmol to 1000 nmol, 10 pmol l~100nmol, 10pmol~60nmol, 10pmol~10nmol, 10 pmol to 1 nmol, 40 to 900 pmol, 50 to 500 pmol, e.g., 100 to It may be, but is not limited to, 300 pmol.

[0020] The concentration of the electron transfer promoter in the reagent composition to be applied to the biosensor is determined based on the measurement objective. There are no limitations as long as the concentration is such that the predetermined component can be measured. For example, the concentration may be 0.01 to 500 mM, 0.0 5-100mM, 0.05-50mM, 0.05-10mM, 1-5mM, e.g., 0.5 It can be, but is not limited to, up to 5 mM.

[0021] When the content of electron transfer promoter per biosensor is 5 nmol, FAD In one embodiment, the content of GDH is 1 to 100 U, 1 to 10 U, 1 to 6 U, for example In one embodiment, the content of FADGDH is 0.1 to 100 μL. g, 1 to 50 μg, for example, 1 to 20 μg. In one embodiment, the content per sensor is 0.1 to 1000 μg, 1 to 500 μg. The content of the metal complex compound is, in some embodiments, 1 to 50 μg, for example, 1 to 20 μg. In the form, the amount is the amount that reaches the saturation concentration, or 5 to 50 μg, 10 to 40 μg, e.g. For example, it may be 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 apply the solution to the electrode immediately after dissolving it and store it in a dry state. When preparing a solution containing multiple electron transfer promoters and mediator reagents, The time that the powder remains in solution is longer when mixed and dissolved than when prepared as a powder and then mixed. Short and preferable.

[0023] The concentration of the oxidoreductase in the reagent composition to be applied to the biosensor is determined based on the measurement target. There are no limitations as long as the concentration is such that a specific component can be measured, for example, 0.001 to 1000 mg / mL. , 0.01~500mg / mL, 0.05~300mg / mL, 0.1~400mg / m The concentration may be, but is not limited to, 1 to 500 mg / mL, for example, 10 to 100 mg / mL.

[0024] Metal complex compounds Metal complex compounds include ruthenium compounds and osmium compounds. In one embodiment, the content of the metal complex compound per glucose sensor is The amount is not limited as long as it is an amount that allows the measurement of a specific component to be measured. The amount may be an amount typically used for the drug, for example, 0.1 μg to 100 mg, 1 μg to 50mg, 1μg~10mg, 1μg~1mg, 1~100μg, 5~50μg, 10~ It may be 40 μg, 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 determined based on the measurement target. There are no limitations as long as the concentration allows the predetermined component to be measured, for example, 0.01 to 1000 mM, 0. 0.05-800mM, 0.05-700mM, 1-700mM, 5-600mM, e.g., 1 It can be, but is not limited to, 0 to 500 mM.

[0026] Ruthenium Compounds Ruthenium compounds include those used in conventional glucose sensors and biosensors. nium compounds, or their later developed equivalents, may be used. In this case, ruthenium compounds can exist in the reaction system as oxidized ruthenium complexes. The ruthenium complex is not particularly limited. In one embodiment, the ruthenium complex is The general formula is: [Ru(NH3)5X]n + (wherein X is NH3, CN, pyridine, a halogen ion, nicotinamide, or H2 O). 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, Rogen is Cl - If so, it is hexaammineruthenium chloride.

[0027] oxidoreductase As the oxidoreductase, various oxidoreductases classified into EC group 1, such as glucose glucose dehydrogenase, amadoriase (fructosyl peptide oxidase or fructosyl amino acid oxidase), peroxidase, Galactose oxidase, bilirubin oxidase, pyruvate oxidase, D- or or L-amino acid oxidase, amine oxidase, cholesterol oxidase, Phosphorus oxidase, xanthine oxidase, sarcosine oxidase, D- or L- Lactate oxidase (LOD), ascorbate oxidase, cytochrome oxidase, Alcohol dehydrogenase, cholesterol dehydrogenase, aldehyde dehydrogenase enzymes, aldehyde oxidase, fructose dehydrogenase (FDH), sorbitol lactate dehydrogenase, D- or L-lactate dehydrogenase, malate dehydrogenase glycerol dehydrogenase, 17B hydroxysteroid dehydrogenase, Stradiol 17B dehydrogenase, D- or L-amino acid dehydrogenase, Lyceraldehyde 3-phosphate dehydrogenase, 3-hydroxysteroid dehydrogenase enzymes, diaphorase, catalase, glutathione reductase, cytochrome b5 reductase Adrenoxin reductase, adrenodoxin reductase, nitrate reductase , phosphate dehydrogenase, bilirubin oxidase, laccase, polyamine oxidase oxidase, formate dehydrogenase, pyranose oxidase, pyranose dehydrogenase, Examples of coenzymes for the above enzymes include, but are not limited to, tauropine dehydrogenase. Nicotinamide adenine dinucleotide (NAD), Nucleotide phosphate, flavin adenine dinucleotide (FAD), pyrroloquinoline quinone The above-listed oxidoreductases can be used in the methods described in, for example, Methods in Enzymes Activity measurements using various substrates were performed using the method described in the Journal of Biological Chemistry (vol. 1-602). The oxidoreductases listed above (e.g., glucose dehydrogenase, LOD, The origin of the enzymes (e.g., amadoriase) is not particularly limited, and may be derived from prokaryotes, eukaryotes, or microorganisms. These various oxidoreductases can be derived from fungi, plants, or animals. Any known enzyme can be used, and various commercially available enzyme products can also be used. can.

[0028] In certain embodiments, the oxidoreductase may be FADGDH. ADGDH is flavin adenine dinucleotide-dependent glucose dehydrogenase or refers to flavin adenine dinucleotide-binding glucose dehydrogenase. In one embodiment, commercially available FADGDH may be used. Mucor type FADGDH may be used herein as a variant of DH or its equivalent. means wild-type FADGDH of the genus Mucor and / or its variants, unless otherwise specified. In the present specification, the term "Botryotinia" refers to a plant that is a plant of the same type, and includes both wild-type and modified forms thereof. tryotinia) type FADGDH is the wild-type FADGDH of the genus Botryotinia and / or variants thereof, and unless otherwise specified, refers to both the wild type and variants thereof. As used herein, the term "Aspergillus-type FADGDH" refers to a FADGDH derived from the genus Aspergillus. means wild-type FADGDH of the genus Aspergillus and / or its variants, unless otherwise specified. In the present specification, the term "Penicillium" includes both wild-type and modified forms thereof, unless otherwise specified. Penicillium-type FADGDH is a wild-type FADGDH of the Penicillium genus and and / or variants thereof, and unless otherwise specified, includes both wild-type and variants thereof. As used herein, Circinella-type FADGDH refers to a Circinella-type FADGDH. It refers to wild-type FADGDH of the genus Nella and / or its variants, and unless otherwise specified, wild-type and any of its variants.

[0029] Mucor microorganisms include, but are not limited to, Mucor prainii, Mucor javanicus, Mucor circinelloides f. circinelloides, Mucor guilliermondii, Muco r hiemalis f. silvaticus, Mucor subtiliss imus, and Mucor dimorphosporus. Microorganisms in the genus Botryothinia include, but are not limited to, Botryothinia fucheriana ryotinia fuckeliana. Examples of microorganisms in the genus Aspergillus include, but are not limited to, Aspergillus oocytes. ryzae, Aspergillus sojae, Aspergillus nige r, and Aspergillus terreus. Examples of microorganisms in the genus Penicillium include, but are not limited to, Penicillium scleroides Thiol (Penicillium sclerotiorum), Penicillium jan Penicillium janthinellum and Penicillium patens Examples include Penicillium paneum.

[0030] In the present specification, the modified form of wild-type FADGDH of the genus Mucor is A GDH-specific glycoprotein having a sequence in which amino acid residues are substituted, and which is capable of FAD-dependent glycolysis. In a specific embodiment, the variant is a maltodextrin dehydrogenase. It has low substrate specificity for glucose and galactose, and low substrate specificity for glucose. The number of substituted amino acid residues is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or , 10, for example, 1 to 10, 1 to 5, 1 to 3, for example, 1 to 2.

[0031] As used herein, the variant of wild-type FADGDH of Mucor prainii refers to The amino acid sequence of the wild-type FADGDH is substituted with an amino acid residue, and In certain embodiments, the term "antigen" refers to a gene that has AD-dependent glucose dehydrogenase activity. The modified strain has low reactivity to maltose, xylose and galactose, and The number of substituted amino acid residues is, for example, 1, 2, 3, 4, or , 5, 6, 7, 8, 9, 10, for example 1-10, 1-5, 1-3, for example 1-2 There can be one.

[0032] For example, when the enzyme used is a modified version of the wild-type FADGDH of Botryothinia genus, and a sequence in which an amino acid residue in the amino acid sequence of the wild-type FADGDH is substituted, In certain embodiments, the term "antigen" refers to a substance having FAD-dependent glucose dehydrogenase activity. The modified form has low reactivity to maltose and galactose and high reactivity to glucose. The number of substituted amino acid residues is, for example, 1, 2, 3, 4, 5, 6, It may be 7, 8, 9, 10, for example 1 to 10, 1 to 5, 1 to 3, for example 1 to 2 do.

[0033] For example, the enzyme used is the wild-type FA of Botryotinia fuckeliana. In the case of a variant of FADGDH, the amino acid residues in the amino acid sequence of the wild-type FADGDH are Those with a substituted sequence and FAD-dependent glucose dehydrogenase activity In certain embodiments, the variants have reactivity to maltose and galactose. The number of substituted amino acid residues is, for example, , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, for example, 1-10, 1-5, 1-3 It may be, for example, 1 to 2.

[0034] For example, when the enzyme used is a variant of wild-type FADGDH of the genus Aspergillus, The amino acid sequence of the wild-type FADGDH is substituted with an amino acid residue, and In certain embodiments, the term "antigen" refers to a gene that has AD-dependent glucose dehydrogenase activity. The modified form has low reactivity to maltose and galactose and high reactivity to glucose. The number of substituted amino acid residues is, for example, 1, 2, 3, 4, 5, 6, 7, or , 8, 9, 10, for example, 1 to 10, 1 to 5, 1 to 3, for example, 1 to 2 .

[0035] For example, when the enzyme used is a modified version of wild-type FADGDH of the genus Penicillium, The amino acid sequence of the wild-type FADGDH is substituted with an amino acid residue, and In certain embodiments, the term "glucose dehydrogenase" refers to a substance having glucose D-dependent glucose dehydrogenase activity. The modified form has low reactivity to maltose and galactose, and low reactivity to glucose. The number of substituted amino acid residues is, for example, 1, 2, 3, 4, 5, 6, 7, It may be 8, 9, 10, for example 1 to 10, 1 to 5, 1 to 3, for example 1 to 2.

[0036] In some embodiments, the oxidoreductase may be an amadoriase. Madoriase is a flavin adenine dinucleotide-dependent fructosyl peptide oxidoreductase (MADORIASE). flavin adenine dinucleotide-dependent fructosyl amino acid oxidase, or flavin adenine dinucleotide-binding fructosyl peptide oxidase, or Flavin adenine dinucleotide-binding fructosyl amino acid oxidase. In this embodiment, commercially available amadoriase may be used. Modified forms of amadoriase or equivalents thereof may be used. The origin is not particularly limited, and examples thereof include the genus Coniochaeta, Eupenisi Genus Eupenicillium, Pyrenochaeta Genus, Arthrinium, Curvularia ), Neocosmospora, Cryptococcus ptococcus, Phaeosphaeria, Aspergillus Aspergillus genus, Emericella genus, Uro Ulocladium genus, Penicillium genus , Fusarium genus, Achaetomiella Genus, Achaetomium, Thielavia Genus, Chaetomium, Gelasinospora ora genus, Microascus genus, Leptosphaeria osphaeria), Ophiobolus, Pleospora ( Pleospora genus, Coniochaetidium genus, Pi Pichia, Debaryomyces, Corynebacterium Genus Corynebacterium, Agrobacterium from the genus Arthrobacter or Arthrobacter The term "amadoriase" refers to amadoriase, and includes both wild-type and modified forms thereof, unless otherwise specified.

[0037] In some embodiments, the oxidoreductase may be lactate oxidase. Lactate oxidase is a flavin adenine mononucleotide-dependent lactate oxidase, also known as refers to flavin adenine mononucleotide-binding lactate oxidase. In another embodiment, a commercially available lactate oxidase may be used. In this specification, the origin of lactate oxidase is Without being particularly limited, for example, the genus Aerococcus, Streptococcus Streptococcus genus, Pediococcus genus or Enterococcus genus-derived lactate oxidase Unless otherwise specified, the term "wild type" includes both wild-type and modified forms thereof.

[0038] In certain embodiments, the oxidoreductase may be lactate dehydrogenase. Lactate dehydrogenase is a flavin adenine mononucleotide-dependent lactate dehydrogenase. It refers to flavin adenine mononucleotide-binding lactate dehydrogenase. In certain embodiments, lactate dehydrogenase may be commercially available. may use a modified version of a commercially available lactate dehydrogenase or its equivalent. The origin of lactate dehydrogenase is not particularly limited. For example, lactate dehydrogenase can be derived from the genus Pichia, Lactic acid dehyde derived from the genus Ogataea or Candida The term "alginase" refers to an allogenease, and includes both wild-type and modified forms thereof, unless otherwise specified.

[0039] In some embodiments, the oxidoreductase may be sarcosine oxidase. In this context, sarcosine oxidase is a flavin adenine dinucleotide-dependent sarcosine oxidase, or flavin adenine dinucleotide-binding sarcosine oxidase. In certain embodiments, commercially available sarcosine oxidase may be used. In this embodiment, a modified version of a commercially available sarcosine oxidase or an equivalent thereof may be used. The origin of the sarcosine oxidase is not particularly limited, and examples thereof include those derived from the genus Bacillus, Corynebacterium, and the like. Bacterium, Cylindrocarpon, Pseudomonas, and Arthrobacter Unless otherwise specified, the wild-type and its variants are included. This also includes deviations.

[0040] In certain embodiments, FADGDH has an enzyme at the N-terminus or C-terminus of its amino acid sequence. A tag sequence for purification, a peptide sequence, a signal sequence, a cleavage recognition sequence, and / or A recombinant FADGDH to which the cleavage residue of the sequence of It may include those with hydrogenase activity.

[0041] In one embodiment, the content of FADGDH per glucose sensor is, for example, For example, it can be 0.1 to 50U, 0.5 to 20U, 1 to 10U, for example, 1 to 5U. In this specification, the enzyme unit U of FADGDH is defined as the enzyme unit of 1 μm at 37° C. for 1 minute. is the amount of enzyme required to convert ol of glucose.

[0042] In this specification, the content of FADGDH per glucose sensor is a specific In this embodiment, the present invention is applied to a glucose sensor having one electrode system including a working electrode and a counter electrode. In another embodiment, the content refers to the amount of FADGDH contained in one electrode system. In another embodiment, the amount of FADGDH contained in the reagent layer placed on the The above content is the amount of FADGD that is mixed into the reagent so that it is included in the reaction system when the sample is added. The amount of H.

[0043] In one embodiment, the LOD content per lactate sensor is, for example, 0.1 to It can be, but is not limited to, 50U, 0.5-20U, 1-10U, for example, 1-5U. In this specification, the enzyme unit U of LOD is the enzyme required to oxidize 1 μmol of lactate in 1 minute at 37°C. The amount of enzyme is

[0044] In this specification, the content of LOD per lactate sensor refers to a specific embodiment. 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 is in a reagent layer disposed on one electrode system. In another embodiment, the content refers to the amount of LOD contained in the sample. This refers to the amount of LOD that is mixed into the reagent so that it is included in the reaction system.

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

[0046] Reagent Layer In certain embodiments, the biosensor of the present disclosure includes an electron transfer facilitator, an electrode, an oxidation-reduction enzyme, and a In some embodiments, the biosensor comprises an electrode and a reagent layer. The reagent layer includes an electron transfer promoter, an oxidoreductase, and a metal complex compound. In one embodiment, the electrode includes an electrode portion having a working electrode and a counter electrode. The electrode portion may be disposed on an insulating substrate. In some embodiments, a reagent layer may be disposed on the electrode portion. do.

[0047] In one embodiment, the present disclosure provides a method for treating a catalysis-induced oxidoreductase-containing solution by adding a metal complex compound and The electron transfer accelerator is added in powder form, or a metal complex compound and an electron transfer accelerator are added. After mixing the powders, add the solution containing the oxidoreductase and mix until all the reagents are dissolved. and forming a reagent layer by applying the solution to an electrode while the solution is being applied to the electrode. The present invention provides a method for producing a hydroxybenzoate by adding a metal complex compound and an electron transfer promoter to a solution containing an oxidoreductase. Add the accelerator in powder form, or mix the metal complex compound and electron transfer accelerator in powder form. After mixing, add the solution containing the oxidoreductase and mix until all the reagents are dissolved. The method of forming a reagent layer by applying the solution to the electrode is to Unlike the method of mixing the powder with other reagent components and then dissolving, A well-dissolved reagent composition can be obtained without any problem, and then applied to the electrode. When a biosensor is fabricated, it will be possible to quantify glucose up to high concentrations.

[0048] In one embodiment, the method comprises: A buffer may be added to the solution. A buffer may be added to the solution containing the oxidoreductase. The metal complex compound and the electron transfer promoter may be mixed together in powder form.

[0049] The temperature at which the oxidoreductase, the metal complex compound, and the electron transfer promoter are dissolved is: The temperature is preferably 10 to 40°C. If the mixture is stored at 4°C or below, the metal complex compound will precipitate. If stored at temperatures above 20°C for a long period of time, the oxidoreductase enzymes may be inactivated.

[0050] In one embodiment, the present disclosure provides a biosensor and a method for applying a voltage to an electrode of the biosensor. and a means for measuring the current. The voltage applying means includes a contact portion that can come into contact with the electrode, and a power source (e.g., The disclosed system may include a potentiostat or galvanostat. In some embodiments, the biosensor may be a glucose sensor. In some embodiments, the compound of interest in the sample may be glucose. The present disclosure relates to a method for measuring a glucose concentration using a glucose concentration measurement system. In some embodiments, the biosensor may be a lactate sensor. In this embodiment, the target compound in the sample may be lactic acid. The disclosure provides a method for measuring lactate concentration using a lactate concentration measurement system.

[0051] In one embodiment, the present disclosure provides a method for detecting a compound to be measured by combining a sample that may contain a compound to be measured with an oxidoreductase. contacting the electrodes, applying a voltage to the electrodes, and The present invention provides a method for measuring the concentration of a compound to be measured, which comprises measuring a response current in the presence of The voltage to be applied is not particularly limited, but when a ruthenium compound is used as the metal complex compound, When used, for example, 10 to 1000 mV, 10 to 800 mV, 50 to 500 mV, e.g. For example, the reference electrode may be 0 to 100 mV. Unless otherwise specified in this specification, the reference electrode is a silver electrode. The potential when silver chloride is used is described.

[0052] The method for measuring the concentration of the compound to be measured comprises contacting the sample with an oxidoreductase, A voltage may be applied after a certain period of time without applying a potential, or a voltage may be applied simultaneously with contact. The time during which no potential is applied can be greater than 0 seconds and less than 1 minute, for example. The time may be 1 to 30 seconds, for example, 1 to 10 seconds. For example, the oxidoreductase is FADGDH. The target compound can be glucose. Also, an oxidoreductase can be used as the LOD. The compound to be measured can be lactic acid. Similarly, for various oxidoreductases, the substrates recognized by those oxidoreductases are used as measurement targets. The compounds can be used as the target compounds, and a system for measuring the concentration of each of the target compounds is provided. It is served.

[0053] In some embodiments, the reagent layer further comprises a buffer, a surfactant, an inorganic compound, and the like. It may contain other ingredients.

[0054] The buffering agent is not particularly limited, but may be a phosphate buffer, an amine buffer, or Examples of buffers include those containing carboxyl groups. Amine-based buffers include Tris, AC ES, CHES, CAPSO, TAPS, CAPS, Bis-Tris, TAPSO, T ES, Tricine, and ADA are examples of buffers with carboxyl groups. Acetic acid-sodium acetate buffer, malic acid-sodium acetate buffer, malonic acid-sodium acetate buffer, Buffers include sodium citric acid-sodium acetate buffer. Buffers may be used alone or in combination.

[0055] The surfactant is not particularly limited, but may be a nonionic, anionic, cationic or Examples of amphoteric surfactants include carboxybetaine. , sulfobetaines, and phosphobetaines. Examples of suitable anti-inflammatory agents include CHAPS (3-[(3-cholamidopropyl)dimethylammonium chloride) propanesulfonate), CHAPSO(3-[(3-cholamidopropyl)dimethyl methylammonio]-2-hydroxy-1-propanesulfonate), and alkylhydrogen Examples of suitable amines include, but are not limited to, sulfobetaine.

[0056] In some embodiments, the inorganic compound may be a layered inorganic compound, conventionally a glucose The inorganic compounds used in sensors and their equivalents that will be developed in the future can be used. , swelling clay minerals with ion exchange capacity, smectite, bentonite, synthetic fluorine mica , vermiculite, synthetic hectorite, synthetic smectite such as synthetic saponite; synthetic Swellable synthetic mica containing fluorine mica; synthetic mica containing Na-type mica, and combinations thereof These include, but are not limited to:

[0057] In some embodiments, the reagent layer may comprise an enzyme layer. In one embodiment, the enzyme layer containing FADGDH may contain polyacrylamide gel. It may contain additives such as sodium phosphate, trehalose, and glucomannan.

[0058] The reagent layer may be a single layer or a multi-layer structure. Each layer may have one or more components. In this embodiment, the reagent layer is an inorganic gel layer and an enzyme layer containing FADGDH laminated thereon. The reagent layer may be disposed in a dry state on the electrode.

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

[0060] In one embodiment, the glucose sensor comprises a working electrode comprising FADGDH, a counter electrode, and In some cases, a reference electrode is provided. Examples of working electrodes include carbon electrodes, gold electrodes, and platinum electrodes. 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 is an Ag / AgCl electrode. When FADGDH is immobilized, the immobilization method may be a method using a cross-linking reagent, a method of encapsulating in a polymer matrix, a method of covering with a dialysis membrane, Photocrosslinkable polymers, conductive polymers, redox polymers, etc., or ferrocene Or, it is immobilized or electrophoresed in a polymer together with an electron mediator represented by its derivative. The material may be fixed by adsorption onto the electrode, or these may be used in combination. FADGDH was immobilized on a carbon electrode using taraldehyde, and then amine-containing The glutaraldehyde is blocked by treating with a reagent containing glutaraldehyde. It can be immobilized in the same manner.

[0061] Methods for manufacturing glucose sensors are known in the literature, for example, Liu, et. al. Anal. Chem. 2012, 84, 3403-3409 and Tsujimu ra, et. al., J. Am. Chem. Soc. 2014, 136 , 14432-14437 (the entire contents of which are incorporated herein by reference). (Incorporated).

[0062] In one embodiment, the glucose sensor may include printed electrodes, where the electrodes are printed on an insulating substrate. Specifically, the electrodes can be formed by photolithography, screen printing, gravure printing, etc. The insulating substrate can be formed on the substrate by a printing technique such as lithography or flexography. Examples of materials that can be used include silicon, glass, ceramic, polyvinyl chloride, and polyethylene. Examples include polyethylene, polypropylene, polyester, etc. Resistant to various solvents or chemicals Highly resistant materials can be used.

[0063] The glucose concentration can be measured as follows: Put a buffer solution into a thermostatic cell. For electron transfer, a metal complex compound (e.g., a ruthenium compound) is used. etc.) and an electron transfer promoter are used. FADGDH is used as the oxidoreductase. Working electrode A carbon electrode is used as the counter electrode (e.g., a platinum electrode) and a reference electrode (e.g., Ag / AgCl A constant voltage is applied to the carbon electrode, and after the current becomes steady, glucose A sample containing glucose is added and the increase in current is measured. According to the calibration curve, the glucose concentration in the sample can be calculated.

[0064] The glucose sensor is a device that applies a predetermined voltage for a certain period of time. A measuring instrument equipped with a means for measuring the electrical signal generated by the instrument, 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 some embodiments, in electrochemical assays, the reduced electron carrier is reacted with the application of a potential. The current value when the material is oxidized is measured.

[0066] In some embodiments, the methods of the present disclosure do not involve medical procedures. In certain embodiments, the disclosed methods involve diagnosing diabetes. In certain embodiments, the methods of the present disclosure can be used for blood glucose monitoring. In some embodiments, the methods of the present disclosure may be used in combination with conventional methods. In one embodiment, the method of the present disclosure includes the method disclosed in Japanese Patent Application Laid-Open No. 2013-083634 In one embodiment, the method disclosed herein is not limited to the method disclosed in Japanese Patent Application Laid-Open No. 2018-014666. The method described in US Pat. No. 5,455,555 is excluded.

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

[0068] [Comparative Example 1] Glucose dehydrogenase (Kikkoman Biochemifa Corporation, Derived from the Cole genus, product name: FADGDH-AA) 120 mg, hexaammineruthenium chloride Ru(III) (Tokyo Chemical Industry Co., Ltd., hereafter referred to as Ru) 186 mg, mPMS 0.68 mg (Dojindo Laboratories), dipotassium phosphate 33.6 mg (Fujifilm) 100mg (Fujifilm Wako Pure Chemical Industries, Ltd.), 22.6mg monopotassium dihydrogen phosphate (Fujifilm Wako Pure Chemical Industries, Ltd.) (manufactured by the company) were mixed in powder form, and 2.0 mL of ultrapure water was added to the mixture and left at 25°C. When mixed with water, it did not dissolve well and significant precipitates were observed (Fig. 3). The weight was measured and found to be 56.2 mg.

[0069] Next, the mixing process was slightly changed and a test was carried out. First, 93 mg of powdered Ru was weighed out. 1.0 mL of ultrapure water was added to this and mixed at 25°C or 37°C. All of the powder dissolved well (final concentration of Ru was 300 mM). , mPMS 0.34mg, dipotassium phosphate monobasic 16.8mg, dipotassium phosphate monobasic 11.3 mg of sodium was added in powder form and mixed. All of the components were dissolved well. Finally, the mixed solution in which these components were dissolved was When 60 mg of FADGDH-AA was added in powder form, precipitates were immediately observed. Furthermore, when the weight of FADGDH-AA was changed to 20 mg, a similar precipitate was observed. .

[0070] The resulting precipitate was collected, washed with ultrapure water, and then redissolved, and the absorption spectrum was measured. As a result, peaks were observed around 210 nm and 270 nm, and the precipitates were ruthenium. It was found to be a Zn complex.

[0071] Instead of FADGDH-AA, FADGDH derived from Aspergillus sp. (BBI Co., Ltd.) ) and the same test was carried out, and the occurrence of similar precipitates was observed. In a 2 mL system, the precipitate was collected and weighed, and it was 69.6 mg. This indicates that the components in powder form are mixed and dissolved. The precipitation that occurs by this method is not a phenomenon specific to a particular enzyme, FADGDH-AA. It was also confirmed that the enzyme alone was dissolved in the solution and the absorption spectrum was measured. The absorption spectrum of the precipitate showed a waveform different from that of the enzyme. I confirmed that there was no.

[0072] Next, the order of dissolution was changed and further tests were carried out. 93 mg of powdered Ru was weighed out. 1.0 mL of ultrapure water was added to dissolve the solution, and FADGDH-AA When 60 mg of the enzyme powder was added, a precipitate was immediately observed. Precipitation occurs simply by adding the powder to the Ru solution, and the precursors to this are mPMS and diphosphate monobasic. No difference was observed in this phenomenon whether sodium or monopotassium dihydrogen phosphate was added or not. Ta.

[0073] Next, tests were conducted by changing the Ru concentration. First, 155 mg of powdered Ru was weighed. 1.0 mL of ultrapure water was added to this and mixed at 25°C or 37°C. All of the Ru dissolved well (final concentration of Ru was 500 mM). PMS 0.34mg, dipotassium phosphate monobasic 16.8mg, potassium phosphate monobasic 11.3 mg of powder was added to each of the samples in order and mixed. All of the components were dissolved well. Finally, FA When 60 mg of DGDH-AA was added in powder form, a precipitate was observed.

[0074] Furthermore, tests were conducted by changing the Ru concentration. First, 56 mg of powdered Ru was weighed. 1.0 mL of ultrapure water was added to this and mixed at 25°C or 37°C. All of the Ru dissolved well (final concentration of Ru was 180 mM). PMS 0.34mg, dipotassium phosphate monobasic 16.8mg, potassium phosphate monobasic 11.3 mg of powder was added to each of the samples in order and mixed. All of the components were dissolved well. Finally, FA When 60 mg of DGDH-AA was added in powder form, a precipitate was observed.

[0075] Without wishing to be bound by any particular theory, it is believed that the oxidoreductase in powder form is dissolved in Ru. By adding the enzyme to the liquid, or by mixing the powdered oxidoreductase and Ru, As a hypothesis, the reason why precipitation occurs significantly after dissolution is that FADGDH does not show any This causes electrons to flow to Ru, which then converts Ru into hexaammineruthenium chloride ( III) to hexaammineruthenium chloride(II), which is thought to have 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. Since the reagents used do not contain any glucose, an enzyme reaction may have occurred during dissolution. The possibility is extremely low.

[0076] Comparative Example 2 Instead of FADGDH-AA, amadoriase (Kikkoman Biochemifa Co., Ltd.) The test was carried out using a powdered Ru (product name: FPOX-CE). 93 mg of Ru was weighed out. When 1.0 mL of ultrapure water was added and mixed at 25°C or 37°C, the powder was completely dissolved. Next, 0.34 mg of mPMS and 1 aqueous solution of phosphoric acid were added to the Ru solution. Dipotassium phosphate 16.8mg, monopotassium dihydrogen phosphate 11.3mg, When the powders were added and mixed, all the ingredients dissolved well without any precipitates. Finally, 60 mg of FPOX-CE in powder form was added to this mixed solution. Upon addition, a precipitate was observed.

[0077] Comparative Example 3 Instead of FADGDH-AA, sarcosine oxidase (Kikkoman Biochemifa The test was carried out using a ruthenium dioxide detector (manufactured by SOD-EP Co., Ltd.). 93 mg of powdered ruthenium was weighed out. Then, 1.0 mL of ultrapure water was added to the mixture and mixed at 25°C or 37°C. Next, 0.34 mg of mPMS and phosphorus were added to the Ru solution. Dipotassium dihydrogen phosphate 16.8 mg, and monopotassium dihydrogen phosphate 11.3 mg, in that order. All of the components were added in powder form and mixed, and all of the components dissolved well without forming any precipitates. Finally, 60 mg of powdered SOD-EP was added to the mixed solution containing these ingredients. When added in this state, precipitates were observed.

[0078] Comparative Example 4 Instead of FADGDH-AA, lactate dehydrogenase (Kikkoman Biochemifa) The test was carried out using a ruthenium dioxide detector (manufactured by SANYO Chemical Industries, Ltd., product name: LDH-E). 93 mg of powdered Ru was weighed out. When 1.0 mL of ultrapure water was added to this and mixed at 25°C or 37°C, the powder All of the Ru dissolved well. Next, 0.34 mg of mPMS and 1 mg of phosphoric acid were added to the Ru solution. Potassium dihydrogen 16.8 mg, potassium dihydrogen phosphate 11.3 mg, When the powder was added and mixed, all the ingredients dissolved well without any precipitates. Finally, 60 mg of LDH-E in powder form was added to this mixed solution in which these ingredients had been dissolved. When added, precipitates were observed.

[0079] [Example 1] On the other hand, instead of mixing the oxidoreductase in powder form, it is first dissolved and then mixed. The following test was carried out using the method described above. First, 60 mg of powdered FADGDH-AA was After weighing, 1.0 mL of ultrapure water was added and dissolved, and the mixture was dissolved well. When 93 mg of powdered Ru was added to the solution, it dissolved well without forming any precipitates. Next, 0.34 mg of powdered mPMS and powdered dipotassium phosphate 16.8 mg of phosphate buffer, 11.3 mg of potassium dihydrogen phosphate in powder form were added in that order and mixed. Upon examination, the solution dissolved without depositing any material.

[0080] We also performed a test in which the order of adding the enzyme solutions was changed. First, 93 mg of Ru, mPMS 0.34mg, dipotassium phosphate monobasic 16.8mg, monopotassium phosphate dibasic 11 0.3mg of powder was mixed with this, and 60mg of FADGDH-AA was added in advance to ultrapure water. 1.0 mL of the solution dissolved in step 1 was added and mixed. Even in this case, a precipitate was generated. It dissolved well without any problem.

[0081] From the above results, it was found that the solution containing the oxidoreductase, the metal complex compound, and the electron transfer promoter When adjusting the product, the amount of ruthenium varies depending on the process of dissolving these ingredients. There is a phenomenon in which the compound precipitates and precipitates, which is a technical problem in the production of biosensors. It was confirmed that this was a problem. In addition, as a means to solve this phenomenon, It has been newly discovered that it is effective to dissolve the enzyme powder before mixing. It was.

[0082] As will be shown in the following examples, the "prevention of precipitation of ruthenium compounds" disclosed in the present invention The industrial importance of solving this problem lies in providing a biosensor that can perform measurements with high accuracy. This phenomenon is a major issue for the fabrication of biosensors. In addition, when mixing and preparing the reagent composition as a pre-process before applying the reagent composition to the electrode, There is a process for preparing a solution in which the seed components are dissolved. If precipitation occurs during this process, It is expected that the ruthenium concentration will be very uneven in the composition. In addition, many ruthenium particles are filtered to remove solids before application. This can lead to the compound being removed from the reagent composition as a precipitate.

[0083] Thus, the amount of ruthenium compound in the reagent composition for electrode preparation exceeds the expected amount. If the concentration is reduced, the biosensor will not have the desired performance. This meant that only a significantly reduced amount could be loaded compared to the initial concentration. This may result in a deterioration in the quantitative measurement of glucose, which is the original purpose of the biosensor. At the laboratory level, when preparing a prototype of a reagent composition, a person skilled in the art usually prepares each reagent individually. The process involves weighing out the components, dissolving them in each solution, mixing them, and applying them to the sensor. This is because the powders are mixed directly and dissolved in one go in the manufacturing process. It is believed that the problem has not gone unnoticed by those skilled in the art.

[0084] Such precipitation occurs on a very small scale and during short preparation operations. If so, some of the issues can be addressed by adjusting the initial formulation amount, etc. However, if a similar method is used in actual industrial production on a certain scale or larger, PM In the case of a mediator or electron transfer promoter that is unstable in solution, such as S, The mediator deteriorates over time from application to drying, which causes a deterioration in sensor performance. There is a risk of injury.

[0085] As disclosed in Patent Documents 1 and 2, instead of mixing all the components, multiple layers are used. A separate mediator is placed in each layer, and the mixture is prepared separately for each layer, with the reagent dissolved and applied repeatedly. It is technically possible to return the oxidoreductase to its original state, and in this case, the oxidoreductase and Ru are intentionally placed in separate layers. It is conceivable that the placement of the precipitation can be suppressed, and in fact, in the prior art, In the field of semiconductors, such a method is commonly used, and there are few disclosed examples of fabricating a sensor with a single layer. The issue of precipitation of ruthenium compounds as described above has not yet been brought to light. It had not been done.

[0086] The present inventors intend to fabricate a sensor in a single layer, in which case, Considering the need to shorten the time that the mediator and electron transfer promoter remain in liquid state as much as possible, It was thought that it would be preferable to mix each reagent in powder form and then make a solution, but this would result in the The problem of precipitation of ammonium compounds was first brought to light, and the solution to this problem was Add a ruthenium compound to the enzyme solution or dissolve the ruthenium compound in the solution. It has now been discovered that this problem can be solved by adding an aqueous solution containing an enzyme.

[0087] In this example, the amount of liquid was varied in the range of 0.5 to 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. can be obtained.

[0088] [Example 2] Based on the findings of Example 1, a monolayer containing FADGDH-AA, Ru, and mPMS was prepared. We will investigate whether glucose can be quantified using this sensor and whether chronoamperometry can be performed using printed electrodes. Specifically, FADGD was added to PBS at a final concentration of 17.8 U / mL. 100 μL of a solution containing H-AA, 300 mM Ru, and 1 mM mPMS was applied to the printed electrode. The printed electrode was a carbon working electrode (12.6 mm 2 ), and a silver reference electrode was printed on Screen-printed electrons (Drop Sense DRP-110 manufactured by Drop Sense) and a dedicated connector (DRP- CAC) was connected to an ALS Electrochemical Analyzer 814D (manufactured by BAS). The applied voltage was then set to +100 mV (vs. Ag / AgCl). The final concentration was 10-3 Add glucose to make the glucose concentration 0 mM and start chronoamperometry measurement. The results of measuring the current value 30 seconds after the start of the treatment are shown in Figure 4. As shown in Figure 4, The current value increased in a concentration-dependent manner up to the base, showing high linearity.

[0089] Subsequently, in the above test, the concentration of Ru was changed to a final concentration of 30 mM, 150 mM, or 180 mM. As a result, compared to when the final concentration of Ru was 300 mM, In both cases, the linearity of the current value deteriorated when glucose was added (Fig. 5, Fig. 6) The current value increased when 20 mM glucose was added and when 30 mM glucose was added. The results of comparing the current values ​​are shown in Table 1. The closer (b) / (a) is to 100%, the better the linearity.

[0090] [Table 1]

[0091] From Table 1, the lower the concentration of the ruthenium compound in the reagent composition (20 mM glucose), the Compared with the change in (3) (current value when 10 mM glucose was added) - (current value when 10 mM glucose was added), The change in the current value when 0 mM glucose was added minus the current value when 20 mM glucose was added is In other words, the linearity of the current value when it increases depending on the glucose concentration deteriorated. Therefore, the precipitation and removal of ruthenium compounds from the composition may affect the sensor performance. was found to worsen the

[0092] In Example 1 of Patent Document 1, the hexaammineruthenium chloride per sensor The amount of amide (III) was 20 μg. This was measured using the GLUCO self-testing glucose kit. It is equivalent to the CARD™ X-SENSOR and requires approximately 0.6 μL of blood. If the amount of liquid that reaches the sensor is 0.3 to 0.4 μL, then the amount of hexaammineruthenium chloride The concentration of helide (III) is thought to be 162-215 mM. The concentration of hexaammineruthenium chloride(III) was 30% lower than expected. The range was 3 to 151 mM, and it was thought that there was a high possibility that linearity would deteriorate.

[0093] [Example 3] A dissolution test was carried out with varying concentrations of oxidoreductase. First, powdered FADGDH-A 80 mg of A was weighed out and dissolved in 1.0 mL of ultrapure water, which resulted in good dissolution. After that, 93 mg of powdered Ru was added to this enzyme solution, and the reaction proceeded smoothly without any precipitates. Subsequently, 0.34 mg of powdered mPMS and 1 mg of powdered phosphate were added. Dipotassium hydrogen 16.8mg, powdered monopotassium dihydrogen phosphate 11.3mg When the mixture was added to the above and mixed, it dissolved without forming any precipitate.

[0094] We also conducted tests with different Ru concentrations. First, we investigated the effect of powdered FADGDH-AA 6 1.0 mg of the compound was weighed out and dissolved in 1.0 mL of ultrapure water, which resulted in a good dissolution. When 155 mg of powdered Ru was added to this enzyme solution, the enzyme was successfully purified without any precipitates. Subsequently, 0.34 mg of powdered mPMS and powdered monohydrogen phosphate were added. Add 16.8 mg of potassium phosphate dibasic and 11.3 mg of potassium phosphate dibasic in powder form in that order. When the solution was added and mixed, it dissolved without forming any precipitate.

[0095] [Example 4] Instead of FADGDH-AA, amadoriase (Kikkoman Biochemifa Co., Ltd.) The test was carried out using FPOX-CE (product name: FPOX-CE). First, the powdered FPOX-CE 60 mg was weighed out and dissolved in 1.0 mL of ultrapure water, which resulted in good dissolution. When 93 mg of powdered Ru was added to this enzyme solution, it dissolved well without any precipitates. Next, 0.34 mg of powdered mPMS and 0.34 mg of powdered phosphate monobasic were added. 16.8 mg of sodium and 11.3 mg of powdered potassium dihydrogen phosphate were added in that order. When mixed, the solution dissolved without any precipitates. Even after 16 hours at 28°C, no precipitates derived from Ru were observed. did not come out.

[0096] [Example 5] Instead of FADGDH-AA, sarcosine oxidase (Kikkoman Biochemifa The test was carried out using powdered SOD-EP. 60 mg of the compound was weighed out and dissolved in 1.0 mL of ultrapure water, which showed good dissolution. After that, 93 mg of powdered Ru was added to this enzyme solution, and the mixture was successfully purified without any precipitates. Subsequently, 0.34 mg of powdered mPMS and powdered monohydrogen phosphate were added. Add 16.8 mg of potassium phosphate dibasic and 11.3 mg of potassium phosphate dibasic in powder form in that order. When the solution was added and mixed, it dissolved without any precipitates. There was no product.

[0097] [Example 6] Instead of FADGDH-AA, lactate dehydrogenase (Kikkoman Biochemifa) The test was carried out using a powdered LDH-E (manufactured by the company LDH-E). g was weighed out and dissolved in 1.0 mL of ultrapure water, which was found to be well dissolved. When 93 mg of powdered Ru was added to the enzyme solution, it dissolved well without forming any precipitates. Next, 0.34 mg of powdered mPMS and 0.34 mg of powdered dipotassium phosphate were added. 16.8 mg of sodium hydroxide and 11.3 mg of powdered potassium dihydrogen phosphate were added in that order. When mixed, the mixture dissolved without depositing any precipitate.

[0098] When oxidoreductase (powder) was added to Ru solution, precipitates were observed. This phenomenon is caused by FADG. This is true not only for DH, 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, In this case, no precipitate was observed. For both hydroxylase and lactate dehydrogenase, the enzyme must be dissolved first. Therefore, those skilled in the art can easily understand the effect of other types of oxidation reduction. Similarly, the appearance of precipitates can be avoided by dissolving the parent enzyme first. , and understand that. [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. This 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. Process A method for producing a composition for an oxidoreductase sensor, comprising:

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

3. The ruthenium compound according to claim 1 is mixed to a final concentration of 180 to 500 mM. The manufacturing method described above.

4. (iii) After step (i), the solution in which the oxidoreductase is dissolved and the second electron transfer promoter powder are mixed. or a second electron transfer promoter in a state where the second electron transfer promoter is dissolved in the solution. further comprising the step of mixing an accelerator solution; The second electron transfer promoter is phenazine methosulfate (PMS), 1-methoxy PM S (mPMS), and 1-methoxy-5-ethylphenazinium ethyl sulfate (m 2. The method of claim 1, wherein the polyisoprene is selected from the group consisting of PEG (polyethylene glycol stearate).

5. The second electron transfer promoter is mixed to a final concentration of 0.1 to 100 mM.

4. The manufacturing method according to claim 4.

6. The oxidoreductase is flavin-dependent glucose dehydrogenase (FADGDH), lyase, lactate dehydrogenase, or sarcosine oxidase according to claim 1 . Manufacturing method.

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