Reagent composition and sensor, and method of producing same

Abstract

To provide a biosensor, and a composition for a sensor using a ruthenium compound. In the production of the above, the occurrence of precipitation should be avoided. The present invention relates to a biosensor comprising an oxidoreductase, a ruthenium compound, and an electron transfer promoter. A method for producing a sensor is also provided, which comprises first adding a powdered oxidoreductase to a solution and dissolving the oxidoreductase in the solution. Then, a powdered ruthenium compound is mixed into the solution in which the oxidoreductase is dissolved. This provides a method for producing a composition for a biosensor.

Description

[Technical field]

[0001] The present disclosure relates to a biosensor comprising an electron transfer promoter, an oxidoreductase, and a metal complex compound. The present invention relates to a method for producing the same. [Background technology]

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

[0003] Patent Document 2 discloses 1-methoxy-5-methylphenazinium ethyl sulfate (mPE We 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 detect the reduction of an acid compound by transferring electrons to an electrode, thereby obtaining a current value. The enzyme layer contains the redox enzyme. In such sensors, mPMS and mPES are The mediator that first accepts electrons from the enzyme is located in the same layer as the oxidoreductase, which is easily accessible. (enzyme layer), whereas ruthenium compounds are present in a different 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 to the electrode. There is a concern that this may complicate the analysis of reaction kinetics. There is concern that the response time and performance of the sensor may be affected by the When multiple types of mediators are used, the first mediator is placed on the enzyme layer. However, it will be common for the second mediator to be located 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 entirety of all documents referenced herein are incorporated by reference. as if each individual document was specifically and individually indicated to be incorporated by reference. The disclosure 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 developed a glucose sensor with an electrode that uses a powder 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. As a result, the ruthenium complex solution The problem we faced was that when the enzyme powder was added, a precipitate formed. The same problem occurred when the product was changed to another FADGDH product. Therefore, precipitation was not used. This issue was not believed to be specific to the FADGDH product.

[0008] The supernatant of the precipitated solution was used to make an electrode, which was then used to measure glucose concentration. In addition, 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 match that of the ruthenium complex solution. The precipitate was thought to be an enzyme, but the absorption spectrum of the precipitate was consistent with that of the enzyme alone. The absorption 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 present invention provides a sensor containing a ruthenium compound and a method for producing the same, which avoids the above 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 precipitation in conventional enzyme sensors that use This is a new challenge. [Means for solving the problem]

[0010] As a result of intensive research, the present inventors have discovered that the respective reagents constituting the reagent composition are mixed in a powder state. Instead of dissolving the oxidoreductase by adding water afterwards, the enzyme solution is dissolved in advance. By adding the liquid to other components, ruthenium compounds do not precipitate and the composition is uniform. Furthermore, the sensor produced using the composition has Compared with sensors fabricated without using such a method, it is possible to quantitatively measure glucose up to a high concentration range. 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] The oxidoreductase is added to a final concentration of 20 to 80 mg / mL. The manufacturing method described in [3] The ruthenium compound is mixed to a final concentration of 180 to 500 mM. 3. The manufacturing method according to claim 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 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 The method of any one of claims 1 to 3, wherein the polymer is selected from the group consisting of: [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, wherein the enzyme is madoriase, lactate dehydrogenase, or sarcosine oxidase. 6. The method for producing a semiconductor device according to any one of claims 1 to 5. Effect of the Invention

[0012] According to the present disclosure, in one embodiment, the ruthenium compound is not precipitated, and the electron transfer is A biosensor combining a promoter, an oxidoreductase, an electrode and a metal complex compound and the biosensor According to the present disclosure, in one embodiment, 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 combining compounds and a method for producing the glucose sensor can be done. [Brief description of the drawings]

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

[0014] In certain embodiments, the present disclosure provides a method for the preparation of ... compound comprising the steps of: an electron transfer facilitator; an oxidoreductase; a metal complex compound; and an electrode. The biosensor includes a 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 includes 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 a working electrode, a counter electrode, and a reference electrode. The electrode may be, for example, a tripolar electrode or a printed electrode. In the 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 a system where the mediator is in the redox state, it receives electrons from the redox enzyme to become reduced and is then attached to the electrode. From this perspective, the compound that functions as a mediator In this specification, these terms are synonymous. do.

[0017] Electron Transfer Enhancer 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 depends on the concentration. In one embodiment, the electron transfer promoter is a mediator. In the present specification, the electron transfer promoter can "modify the function of the mediator." "Modify the function of the oxidoreductase in the absence of an electron transfer promoter" means to modify the function of the oxidoreductase in the absence of an electron transfer promoter. For mediators that are not or hardly ever oxidized, the presence of an electron transfer promoter results in no reduction. This refers to the transfer of electrons from the parent enzyme to the electrode. PMS and mPES can be considered as electron transfer promoters. Delivery promoters are sometimes called mediators.

[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. Can be obtained.

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

[0020] The concentration of the electron transfer promoter in the reagent composition applied to the biosensor is determined based on the measurement objective. There are no limitations as long as the concentration is such that the desired 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 This may be, but is not limited to, ~5mM.

[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 For example, 1 to 4 U. 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 one embodiment, 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] In general, electron transfer promoters and mediators, such as PMS, are unstable in solution. Therefore, it is preferable to apply the compound to the electrode promptly after dissolving it in a solution and store it in a dry state. When preparing a solution containing multiple electron transfer promoters or mediator reagents, The time that the powder remains in solution is longer if it is mixed and then dissolved, rather than preparing a powder and mixing it. Short and preferable.

[0023] The concentration of the oxidoreductase in the reagent composition applied to the biosensor is determined based on the measurement objective. There are no limitations as long as the concentration is such that a certain 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 Examples of 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 in the preparation of a 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 applied to the biosensor is determined based on the measurement objective. There are no limitations as long as the concentration is such that the desired component can be measured, for example, 0.01 to 1000 mM, 0. 0.05-800mM, 0.05-700mM, 1-700mM, 5-600mM, e.g. 1 The concentration may 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 study, 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 as follows: [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 The oxidoreductases include various oxidoreductases classified into EC group 1, such as glycosyltransferases. Soxidase, glucose dehydrogenase, amadoriase (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, Phosphorus oxidase, xanthine oxidase, sarcosine oxidase, D- or L- Lactate oxidase (LOD), ascorbate oxidase, cytochrome oxidase, Alcohol dehydrogenase, cholesterol dehydrogenase, aldehyde dehydrogenase Enzyme, aldehyde oxidase, fructose dehydrogenase (FDH), sorbitol lactate dehydrogenase, D- or L-lactate dehydrogenase, malate dehydrogenase glycerol dehydrogenase, 17B hydroxysteroid dehydrogenase, Stratified 17B dehydrogenase, D- or L-amino acid dehydrogenase, Lysaldehyde 3-phosphate dehydrogenase, 3-hydroxysteroid dehydrogenase Glutathione reductase, 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, for example, Methods in Enzymes The activity of various substrates was measured using the method described in the Journal of The oxidoreductases listed above (e.g., glucose dehydrogenase, LOD, The origin of the enzyme (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 some 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. In another embodiment, commercially available FADGDH may be used. Mucor type FADGDH or its equivalent may be used herein. means wild-type FADGDH of the genus Mucor and / or its modified form, 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 a wild type and a modified form thereof. tryotinia type FADGDH is a wild-type FADGDH of the Botryotinia genus. and / or variants thereof, and unless otherwise specified, the term refers to both the wild type and variants thereof. As used herein, the term "Aspergillus type FADGDH" refers to means wild-type FADGDH of the genus Aspergillus and / or its modified form, unless otherwise specified. In the present specification, the term "Penicillium" includes both wild-type and modified forms thereof. Penicillium type FADGDH is a wild type FADGDH of Penicillium genus and and / or its variants, and unless otherwise specified, includes both the wild type and its variants. As used herein, Circinella genus-type FADGDH refers to Circinella genus-type FADGDH. It means wild-type FADGDH and / or its variants of the genus Nera, and unless otherwise specified, the wild-type and any of its modifications.

[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 Botryotinia include, but are not limited to, Botryotinia fucheriana ryotinia fuckeliana. Aspergillus genus microorganisms 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 scleroblasts. Thiol (Penicillium sclerotiorum), Penicillium jansii Penicillium janthinellum and Penicillium patens Examples include Penicillium paneum.

[0030] In the present specification, the modified form of the wild-type FADGDH of the genus Mucor is A gene encoding an FAD-dependent glycoprotein having a sequence in which amino acid residues in the amino acid sequence of GDH have been substituted. In a particular embodiment, the variant is a maltose 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 , 10, for example, 1 to 10, 1 to 5, 1 to 3, for example, 1 to 2.

[0031] In the present specification, the variant of wild-type FADGDH of Mucor prainii means The amino acid sequence of the wild-type FADGDH is substituted with an amino acid residue, and It refers to those having 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 , 5, 6, 7, 8, 9, 10, e.g. 1-10, 1-5, 1-3, e.g. 1-2 There can be one.

[0032] For example, when the enzyme used is a modified version of the wild-type FADGDH of Botryothinia spp. and having 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 "subunit" refers to a subunit 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 DGDH, 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 variant has a 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, e.g. 1-10, 1-5, 1-3 It may be, for example, 1 to 2.

[0034] For example, when the enzyme used is a modified version of the wild-type FADGDH of the Aspergillus genus, The amino acid sequence of the wild-type FADGDH is substituted with an amino acid residue, and It refers to those having 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 , 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 to be used is a modified version of the 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 that has 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 oxytocinase (MADOCA) enzyme that activates fructosyl peptide oxytocin. 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 one embodiment, the amadoriase may be commercially available. Modified forms of amadoriase or their equivalents 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 oryzae Aspergillus genus, Emericella genus, Uro Ulocladium and Penicillium , Fusarium genus, Achaetomiella Genus, Achaetomium, Thielavia Genus, Chaetomium, Gelasinospora ora, Microascus, Leptosphaeria osphaeria), Ophiobolus, Pleospora ( Pleospora genus, Coniochaetidium genus, Pi Pichia, Debaryomyces, Corynebacterium 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 also known as flavin adenine mononucleotide-dependent lactate oxidase. refers to flavin adenine mononucleotide-binding lactate oxidase. In certain embodiments, In another embodiment, a commercially available lactate oxidase may be used. In the present specification, the lactate oxidase is derived from Without being particularly limited, for example, the genus Aerococcus, Streptococcus Streptococcus, Pediococcus or Enterococcus genus-derived lactate oxidase. Unless otherwise specified, the term "protein sequence" includes both the 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. genase, or 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 dehydration derived from the genus Ogataea or Candida The term "algogenase" refers to an enzyme that is an enzyme that is a nucleotide analogue of an agonist, and includes both wild-type and modified forms thereof, unless otherwise specified.

[0039] In some embodiments, the oxidoreductase may be sarcosine oxidase. In this study, sarcosine oxidase is a flavin adenine dinucleotide-dependent sarcosine oxidase, or flavin adenine dinucleotide-binding sarcosine oxidase. In certain embodiments, sarcosine oxidase may be commercially available. In the present 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. For example, the sarcosine oxidase may be derived from the genus Bacillus, Bacterium, Cylindrocarpon, Pseudomonas, and Arthrobacter The sarcosine oxidase may be any of the wild-type and modified forms thereof, unless otherwise specified. This also includes deviations.

[0040] In a particular embodiment, 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 These may include those that have 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. However, this is not limited to this. In this specification, the enzyme unit U of FADGDH is defined as 1 μm at 37° C. for 1 minute. is the amount of enzyme required to convert ol of glucose.

[0042] In the present specification, the content of FADGDH per glucose sensor is a specific In the embodiment, the present invention is used for a glucose sensor having one electrode system having a working electrode and a counter electrode. In another embodiment, the content refers to the amount of FADGDH that can be used 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. This refers to 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 capacity 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 having 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 when the sample is added. This refers to the amount of LOD that is mixed into a reagent so that it is included in the reaction system.

[0045] In one embodiment, the biosensor is a size that is typically used for measuring samples. For example, the glucose sensor may include a sensor that detects glucose, such as blood, in a particular embodiment. 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 oxidoreductase, In one embodiment, 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 comprising the steps of: The electron transfer promoter is added in powder form, or a metal complex compound and an electron transfer promoter are added. After mixing the powders, add the solution containing the oxidoreductase and mix until all the reagents are dissolved. and applying the solution to an electrode while the solution is in a liquid state to form a reagent layer. The present invention provides a method for producing a catalysis enzyme comprising 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 the 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 trouble, and then it can be applied to the electrode. When the biosensor is fabricated, it becomes possible to quantitatively measure glucose up to a high concentration range.

[0048] In one embodiment, the method comprises the steps of: 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 in powder form.

[0049] The temperature at which the oxidoreductase, the metal complex compound, and the electron transfer promoter are dissolved is The temperature range is preferably 10 to 40°C. If the mixture is stored at 4°C or lower, 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 method for detecting a voltage applied to an electrode of a biosensor. and a means for measuring the current. The voltage applying means includes a contact portion that can contact the electrode, and a power source (e.g. The system of the present disclosure 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 can be glucose. The present disclosure relates to a method for measuring glucose concentration using a glucose concentration measuring system. In some embodiments, the biosensor may be a lactate sensor. In one embodiment, the target compound in the sample may be lactate. 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 a method comprising: 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, comprising 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 target compound includes 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 and the measurement is performed. 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 will be offered.

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

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

[0055] The surfactant is not particularly limited, but may be a nonionic, anionic, cationic, or Amphoteric surfactants include carboxybetaine, amphoteric surfactant, and carboxyl group surfactant. , sulfobetaines, and phosphobetaines. Examples of the acetylcholinesterase inhibitor include CHAPS (3-[(3-cholamidopropyl)dimethylammonium chloride). propanesulfonate), CHAPSO(3-[(3-cholamidopropyl)dimethyl ethylammonio]-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, and is conventionally a glucose The inorganic compounds may be those used in sensors or equivalents developed in the future. , swelling clay minerals with ion exchange capacity, smectite, bentonite, synthetic fluorine mica , synthetic smectites such as vermiculite, synthetic hectorite, and synthetic saponite; synthetic Swellable synthetic mica including fluorine mica; synthetic mica including sodium mica, and combinations of these. These include, but are not limited to:

[0057] In some embodiments, the reagent layer may include an enzyme layer. In some embodiments, the enzyme layer may include an F In one embodiment, the enzyme layer containing FADGDH may have a polyacrylamide gel. Additives such as sodium phosphate, trehalose, and glucomannan may be included.

[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 fluids, 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 included. The working electrode is a carbon electrode, a gold electrode, a platinum electrode, etc. 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. In the case of immobilizing FADGDH, the immobilization method may be: A method using a crosslinking agent, 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 electropositive in a polymer together with an electron mediator such as its derivative. The material may be fixed on the electrode by adsorption or by a combination of these methods. FADGDH was immobilized on a carbon electrode using aldehyde and then amine-containing The glutaraldehyde reaction is blocked by treating with a reagent that blocks the glutaraldehyde reaction. It can be immobilized in the same manner.

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

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

[0063] The glucose concentration can be measured as follows. A buffer solution is placed in 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 is 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 includes a means for applying a predetermined voltage for a certain period of time, A measuring device equipped with a means for measuring the electrical signal generated by the measurement and a means for converting the electrical signal into the concentration of the substance to be measured. The same is true for biosensors, including lactate sensors.

[0065] The electrochemical measurements of the present disclosure can be amperometric, potentiometric, or coulometric. In one embodiment, in an electrochemical assay, the reduced electron transfer substance is reacted with the application of a potential. The current value when the oxidation state is reached is measured.

[0066] In some embodiments, the methods of the present disclosure do not involve a medical procedure. In certain embodiments, the disclosed methods include the step of diagnosing diabetes. In certain embodiments, the methods of the present disclosure can be used for blood glucose monitoring. These do not require medical judgment. In some embodiments, the methods of the present disclosure In one embodiment, the method of the present disclosure includes the method disclosed in Japanese Patent Publication No. 2013-083634. In one embodiment, the method disclosed herein is not limited to the method disclosed in Japanese Patent Application Laid-Open No. 2018-013666. The method described in US Pat. No. 5,4555 is excluded.

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

[0068] [Comparative Example 1] Glucose Dehydrogenase (Kikkoman Biochemipha Corporation, Cole genus derived, product name: FADGDH-AA) 120mg, 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 22.6 mg of monopotassium dihydrogen phosphate (FUJIFILM Wako Pure Chemical Industries, Ltd.) (manufactured by the company) were mixed in powder form, 2.0 mL of ultrapure water was added to the mixture, and the mixture was left at 25°C. When mixed with water, it did not dissolve well and significant precipitation was observed (Figure 3). The weight of the recovered product was 56.2 mg.

[0069] Next, a test was carried out with some changes to the mixing process. First, 93 mg of powdered Ru was weighed out. 1.0 mL of ultrapure water was added to each solution and mixed at 25°C or 37°C. All of the powder dissolved well (final concentration of Ru was 300 mM). , mPMS 0.34 mg, dipotassium phosphate 16.8 mg, dihydrogen phosphate 1 calcium When 11.3 mg of sodium was added in powder form to each of the samples and mixed, no precipitate was observed. All 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, a precipitate was immediately observed. Even when the weight of FADGDH-AA was changed to 20 mg, the same 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] In place of FADGDH-AA, FADGDH derived from Aspergillus sp. (BBI) ) 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, yielding 69.6 mg. The result showed that the powders of each component were mixed and dissolved, and 37% of Ru was precipitated. The precipitation that occurs by this method is not specific to a particular enzyme, FADGDH-AA. It was also confirmed that the enzyme was not dissolved in water. The absorption spectrum of the precipitate showed a waveform different from that of the enzyme. I confirmed that there was no.

[0072] Next, further tests were carried out by changing the order of dissolution. 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 powder was added, a precipitate was immediately observed. Precipitation occurs simply by adding the powder to a Ru solution. 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 out. When 1.0 mL of ultrapure water was added to this and mixed at 25°C or 37°C, a powder All of the Ru dissolved well (final concentration of Ru was 500 mM). PMS 0.34mg, dipotassium phosphate 16.8mg, potassium phosphate dihydrogen 1 When 11.3 mg of powder was added to each of the samples and mixed, no precipitate was observed. All of the components were dissolved well. Finally, the mixture was diluted with 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 out. When 1.0 mL of ultrapure water was added to this and mixed at 25°C or 37°C, a powder All of the Ru dissolved well (final concentration of Ru was 180 mM). PMS 0.34mg, dipotassium phosphate 16.8mg, potassium phosphate dihydrogen 1 When 11.3 mg of powder was added to each of the samples and mixed, no precipitate was observed. All of the components were dissolved well. Finally, the mixture was diluted with 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 has been reported that the oxidoreductase in powder form is dissolved in Ru. By adding the enzyme to the liquid, or by mixing the oxidoreductase and Ru in powder form, As a hypothesis, the reason why precipitation occurs significantly after dissolution is that FADGDH does not show any Due to this, electrons flow to Ru, which converts Ru into hexaammineruthenium chloride ( It is believed that ruthenium chloride (III) was converted to hexaammineruthenium chloride (II) and 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 Biochemipha Co., Ltd., product The test was carried out using a FPOX-CE (product name). 93 mg of powdered 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 Next, 0.34 mg of mPMS and 1 aqueous solution of phosphoric acid were added to the Ru solution. potassium phosphate dibasic 16.8mg, potassium dihydrogen phosphate monobasic 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 the mixed solution containing the above ingredients. Upon addition, a precipitate was observed.

[0077] [Comparative Example 3] Instead of FADGDH-AA, sarcosine oxidase (Kikkoman Biochemi The test was carried out using a ruthenium oxide (SOD-EP, product name: SOD-EP) manufactured by the company. 93 mg of powdered Ru was weighed out. Then, 1.0 mL of ultrapure water was added to the mixture and mixed at 25°C or 37°C. The Ru solution was then added with 0.34 mg of mPMS and 0.2 mg of phosphorus. 16.8 mg of dipotassium dihydrogen phosphate, 11.3 mg of monopotassium dihydrogen phosphate, All of the components were added in powder form and when mixed, all of the components dissolved well without forming any precipitates. Finally, 60 mg of SOD-EP in powder form was added to the mixed solution containing these ingredients. When it was added in this state, precipitates were observed.

[0078] [Comparative Example 4] Lactate dehydrogenase (Kikkoman Biochemifa Co., Ltd.) instead of FADGDH-AA The test was carried out using a 93 mg powder of Ru. 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 components were dissolved well without any precipitates. Finally, 60 mg of LDH-E in powder form was added to the 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 by the method described below. First, 60 mg of powdered FADGDH-AA was The enzyme was weighed and dissolved in 1.0 mL of ultrapure water. When 93 mg of powdered Ru was added to the solution, it dissolved well without forming any precipitate. Next, 0.34 mg of powdered mPMS and 0.34 mg of powdered dipotassium phosphate 16.8 mg of the mixture, 11.3 mg of powdered potassium dihydrogen phosphate, and 10.5 mg of the powdered potassium phosphate were added in that order and mixed. The solution dissolved without depositing any precipitate.

[0080] We also performed a test in which the order of addition of the enzyme solutions was changed. First, 93 mg of Ru, mPMS 0.34mg, dipotassium phosphate monobasic 16.8mg, dipotassium phosphate monobasic 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, precipitation occurred. 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 In adjusting the amount of ruthenium, the process of dissolving these ingredients varies slightly. There is a phenomenon in which a compound precipitates and forms a precipitate, which is a technical problem in the production of biosensors. It was confirmed that this was a problem. In addition, as one method to solve this phenomenon, It has been newly discovered that it is effective to dissolve the enzyme powder before mixing. It was.

[0082] As shown in the following example, the present invention discloses "inhibition of precipitation of ruthenium compounds." 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. In addition, when mixing and preparing the reagent composition as a pre-process for applying the reagent composition to the electrodes, There is a process to prepare 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 non-uniform in the composition. In addition, most of the ruthenium is removed by filtering the material before application. This can lead to the compound being removed from the reagent composition as a precipitate.

[0083] Thus, the amount of ruthenium compounds in the electrode preparation reagent composition is greater than expected. If the concentration is reduced, the biosensor will not have the desired performance. This resulted in only being able to load a significantly reduced amount of the material 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. In 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 two 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 this is the case, some of the issues can be addressed by adjusting the initial mix amounts. However, if a similar method is used in 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 the sensor performance to deteriorate. There is a risk of this happening.

[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 preparation is also divided into each layer, and the reagent dissolution and application are repeated. It is technically possible to return the oxidoreductase to its original state by intentionally placing the oxidoreductase and Ru in separate layers. It is conceivable that the placement of the filter can suppress the occurrence of precipitation, and in fact, this has not been the case in the prior art. In the field of nanotechnology, such a method is commonly used, and there are few disclosures of a method for producing a sensor in a single layer. Little is known about this, and the issue of precipitation of ruthenium compounds as described above has not yet come to light. It had not been done.

[0086] The present inventors intend to fabricate sensors 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 nium compounds was brought to light for the first time, 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 to the solution.

[0087] In addition, when the amount of liquid in this example was varied in the range of 0.5 to 2 mL, The same phenomenon was observed regardless of the amount of liquid, so it is believed that the amount of liquid 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 the sensor and whether chronoamperometry can be performed using printed electrodes. Specifically, FADGD was diluted with 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 onto the printed electrode. The printed electrode was a carbon working electrode (12.6 mm 2 ) and a silver reference electrode was printed on the SCREEN-PRINTED ELECTRODES (Drop Sense DRP-110 by Drop Sense) and a dedicated connector (DRP- CAC) was connected to an ALS Electrochemical Analyzer 814D (manufactured by BAS). The applied voltage was +100mV (vs. Ag / AgCl). The final concentration was 10-3 Add glucose to make the glucose concentration 0 mM and start chronoamperometry measurement. The current value measured 30 seconds after the injection is 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] Next, in the above test, the concentration of Ru was changed to a final concentration of 30 mM, 150 mM, or 180 mM. The results were as follows: Compared with the final Ru concentration of 300 mM, In both cases, the linearity of the current value deteriorated when glucose was added (Figure 5, 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 The change in current when 10 mM glucose was added was compared to the change in current when 10 mM glucose was added. The change in current when 0 mM glucose was added minus the current when 20 mM glucose was added is In other words, the linearity of the current value when it increases depending on the glucose concentration becomes poor. 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 done using the GLUCOSE self-test kit. It is equivalent to the CARD(TM) 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 hexaammineruthenium chloride The concentration of helide (III) is considered to be 162 to 215 mM. The concentration of ruthenium 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 the linearity would deteriorate.

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

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

[0095] [Example 4] Instead of FADGDH-AA, amadoriase (Kikkoman Biochemipha Co., Ltd., product First, the powdered FPOX-CE 60 mg was weighed out, and 1.0 mL of ultrapure water was added to dissolve it, which was found to dissolve well. When 93 mg of powdered Ru was added to this enzyme solution, it dissolved well without any precipitate. Next, 0.34 mg of powdered mPMS and 0.2 mg of powdered phosphate monobasic were added. 16.8 mg of sodium, 11.3 mg of powdered potassium dihydrogen phosphate were added in that order. When mixed, it dissolved without any precipitate. Even after 16 hours at 28°C, no precipitate derived from Ru was observed. did not come out.

[0096] [Example 5] Instead of FADGDH-AA, sarcosine oxidase (Kikkoman Biochemi The test was carried out using powdered SOD-EP. 60 mg of the compound was weighed out, and 1.0 mL of ultrapure water was added to dissolve the compound. After that, 93 mg of powdered Ru was added to the enzyme solution, and the enzyme was successfully isolated without any precipitates. Then, 0.34 mg of powdered mPMS and 0.34 mg of powdered monohydrogen phosphate were added. Add 16.8 mg of potassium dihydrogen phosphate and 11.3 mg of potassium dihydrogen phosphate in powder form in that order. When the solution was added and mixed, it dissolved without any precipitate. There was no discharge.

[0097] [Example 6] Lactate dehydrogenase (Kikkoman Biochemifa Co., Ltd.) instead of FADGDH-AA The test was carried out using 60 ml of powdered LDH-E. g was weighed out and dissolved in 1.0 mL of ultrapure water, which showed good dissolution. When 93 mg of powdered Ru was added to the enzyme solution, it dissolved well without any precipitate. Next, 0.34 mg of powdered mPMS and 0.2 mg of powdered potassium phosphate were Add 16.8 mg of sodium hydroxide and 11.3 mg of powdered potassium dihydrogen phosphate in that order. When mixed, the mixture dissolved without depositing any precipitate.

[0098] When oxidoreductase (powder) was added to the Ru solution, a precipitate was 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 into the solution, In this case, no precipitate was observed. For both synthase 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, for the parent enzyme, the appearance of precipitates can be avoided by dissolving the enzyme first. , I understand. [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. 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 is mixed to a final concentration of 180 to 500 mM according to claim 1. The manufacturing method described above.

4. (iii) After step (i), the solution in which the oxidoreductase is dissolved and a powder of a second electron transfer promoter are mixed. Mix and dissolve the second electron transfer promoter in the solution, or dissolve the second electron transfer promoter 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 ethylenediaminetetraacetate (PES).

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

5. The manufacturing method according to claim 4.

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

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