Method for electrochemically measuring oxidoreductase using biosensor and biosensor for use in the same

Abstract

To provide a method for electrochemically measuring the quantity of an oxidoreductase.SOLUTION: A biosensor comprises an electrically conductive portion that includes two or more electrodes, and a reagent layer that is disposed in contact with the electrically conductive portion and that includes the ground substance of an oxidoreductase and two or more kinds of mediators. The mediators include a first and a second mediator. The first mediator propagates to the electrodes electrons generated by reaction of the ground substance with the oxidoreductase. The second mediator propagates to the electrodes the electrons propagated from the first mediator. A method for electrochemically measuring oxidoreductase using a bio sensor includes bringing a sample including the oxidoreductase into contact with the reagent layer 15, accumulating electrons in the second mediator by incubating for a prescribed time after contact, applying a voltage between the electrodes after incubation, and measuring the electric signal generated by voltage application, then calculating the amount of the oxidoreductase on the basis of the electric signal.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] This disclosure relates to a method for electrochemically measuring the amount of oxidoreductase using a biosensor, and to a biosensor used in said method. [Background technology]

[0002] A biosensor is a molecular measurement device that uses a detection unit containing biological substances such as enzymes and a transducer (signal conversion device) such as an electrode, and is used to measure various analytes in living organisms, including glucose.

[0003] As a method for measuring glucose concentration using biosensors, electrochemical measurement using enzymes is commonly employed. Various methods applying the Cottrell equation have been reported as one way to accurately measure glucose concentration (Patent Documents 1-6). The Cottrell equation is one of the diffusion equations that depends on the size and shape of the electrode. Since the thickness of the electrode of a biosensor such as a glucose sensor is small compared to the range in which diffusion occurs along with electron transfer (~several hundred μm), the current response when a potential is applied to the electrode is not constant and decays inversely proportional to the square root of time (t) according to the Cottrell equation. -1 / 2 It is known that it decays over time. Patent Document 4 discloses a method for measuring Cottrell current by mixing an analyte and a reagent (oxidizing agent and enzyme, etc.) in a measuring cell having electrodes, allowing them to react, incubating until the enzymatic reaction is complete, and then applying a voltage to the electrodes, in order to prevent differences in measurement values ​​among users.

[0004] On the other hand, for a Cottrell current (decay according to the Cottrell equation) to be established, there must be virtually no change in the concentration of the substance after the voltage is applied, that is, the enzymatic reaction must be substantially completed. For this reason, the method in Patent Document 4 requires a certain reaction time, which has the problem of taking time before measurement. To solve this problem, Patent Document 6 proposes measuring glucose using an electrochemical process that does not involve Cottrell decay. The decay constant of Cottrell decay is -0.5 (constant). Patent Document 6 discloses determining the decay constant during a short reaction (incubation) time in which the enzymatic reaction is not completed, and determining the concentration of the analyte in the sample based on that decay constant and an output signal with transient decay.

[0005] In recent years, methods for electrochemically measuring the concentration of biomolecules other than glucose have been reported (Non-Patent Documents 1-7). Non-Patent Documents 1-4 disclose a method of converting sucrose to glucose using oxidoreductase and measuring it using a glucose meter (BGM), etc. On the other hand, when the concentration of the analyte in the sample is low or very low compared to glucose, there is a problem in that sufficient sensitivity cannot be obtained compared to measuring glucose. For this reason, Non-Patent Document 5 reports methods for improving sensitivity, such as modifying the electrode and localizing multiple electron transport enzymes. Non-Patent Documents 6 and 7 report a method for measuring the concentration of vascular endothelial growth factor (VEGF) in the blood, using glucose dehydrogenase (GDH) as the detection enzyme, measuring the response current of GDH, and converting the concentration of the analyte to GDH. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 3-293556 [Patent Document 2] Patent No. 2651278 [Patent Document 3] Patent No. 2702286 [Patent Document 4] Patent No. 2901678 [Patent Document 5] Patent No. 4018748 [Patent Document 6] Patent No. 5244116 [Non-patent literature]

[0007] [Non-Patent Document 1] Yu Xiang et al., Nature Chemistry, 2012, 3(9): 697-703 [Non-Patent Document 2] Tian Lan et al., Methods Mol Biol. 2015, 1256, 99-109 [Non-Patent Document 3] Naveen K. Singh et al., Biosensors and Bioelectronics, 2021, 180, 113111 [Non-Patent Document 4] Tian Lan et al., Biotechnology Advances, 2016, 34(3) 331-341 [Non-Patent Document 5] Jinhee Lee et al., Electrochemistry, 2015, 83(12) 1085-1090 [Non-Patent Document 6] Yoshihiko Nonaka et al., Electrochemistry, 2012, 80(5) 363-366 [Non-Patent Document 7] Koichi Abe et al., Electrochemistry, 2012, 80(5) 348-352 [Non-Patent Document 8] Nasa Savory et al., Urakami Foundation memoirs, 2014, Vol. 21 136-142 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] In the process of repeated research to provide a method for electrochemically measuring the concentration of a biological substance using a biosensor, the inventors have found a new problem that when analyzing an analyte at a low or extremely low concentration, as described in Non-Patent Documents 6 to 8, the analyte is converted into an oxidoreductase using an aptamer or the like, and when the amount of the oxidoreductase is electrochemically measured, the resulting response current is so small that sufficient sensitivity cannot be obtained. Further, the inventors have found that, in particular, the mediator used in Non-Patent Documents 6 to 8 is of one type (single mediator), and there is a problem that sufficient sensitivity cannot be obtained in measurement using a biosensor.

[0009] In one aspect, the present disclosure provides a method capable of measuring with high sensitivity in the electrochemical measurement of the amount of an oxidoreductase, and a biosensor used therefor.

Means for Solving the Problems

[0010] As one aspect, the present disclosure is a method for electrochemically measuring the amount of an oxidoreductase using a biosensor, wherein the biosensor has a conductive part including two or more electrodes, and a reagent layer disposed in contact with the conductive part and containing a substrate of the oxidoreductase and two or more types of mediators, wherein the mediators include a first mediator and a second mediator, the first mediator is a mediator for transferring electrons generated by the reaction of the substrate and the oxidoreductase to the second mediator, and the second mediator is a mediator for transferring the electrons transferred from the first mediator to the electrode, and the method includes contacting a sample containing an oxidoreductase with the reagent layer, after the contact, incubating for a predetermined time to accumulate the electrons in the second mediator, after the incubation, applying a voltage between the electrodes, measuring an electrical signal generated by the application of the voltage, and The present invention relates to a method that includes calculating the amount of the oxidoreductase based on the aforementioned electrical signal.

[0011] In other aspects, this disclosure relates to a method for electrochemically measuring the amount of oxidoreductase using a biosensor, The aforementioned biosensor is A conductive part including two or more electrodes, The reagent layer comprises the substrate of the oxidoreductase and two or more mediators, and is disposed in contact with the conductive part. The mediator includes a first mediator for transferring electrons generated by the reaction between the substrate and the oxidoreductase to a second mediator, and a second mediator for transferring electrons transferred from the first mediator to the electrode. The method is, The sample containing the oxidoreductase is brought into contact with the reagent layer of the biosensor. After contact, apply a voltage between the electrodes (first application), Measuring the electrical signal generated by the first application (first measurement), Based on the electrical signal obtained by the first measurement, calculate the incubation time. Perform incubation for the time obtained by calculation. After incubation, apply a voltage between the electrodes (second application). Measuring the electrical signal generated by the second application (second measurement), and The present invention relates to a measurement method that includes calculating the amount of the oxidoreductase based on the electrical signal obtained by the second measurement.

[0012] In other aspects, this disclosure relates to a biosensor for electrochemically measuring oxidoreductases, A conductive part including two or more electrodes, The reagent layer comprises the substrate of the oxidoreductase and two or more mediators, and is disposed in contact with the conductive part. The mediator includes a first mediator for transferring electrons generated by the reaction between the substrate and the oxidoreductase to a second mediator, and a second mediator for transferring electrons transferred from the first mediator to the electrode. This disclosure relates to a biosensor for use in the measurement method. [Effects of the Invention]

[0013] According to this disclosure, in one embodiment, a method capable of measuring the amount of oxidoreductase with high sensitivity in electrochemical measurement, and a biosensor for use therein can be provided. [Brief explanation of the drawing]

[0014] [Figure 1] Figures 1A to 1E are process diagrams showing an example of a method for manufacturing a biosensor in one embodiment of the present disclosure, and show schematic diagrams of the biosensor at each step. [Figure 2] Figure 2 is a cross-sectional view taken along line II of Figure 1E. [Figure 3] Figures 3A to 3E are process diagrams showing an example of a method for manufacturing a biosensor in one embodiment of the present disclosure, and show schematic diagrams of the biosensor at each step. [Figure 4] Figure 4 is a cross-sectional view taken along line II-II in Figure 3E. [Figure 5] Figure 5 is a graph showing an example of the results from Experimental Example 1. It shows the results of evaluating the measurable GDH concentration range by incubating a reaction solution containing a substrate (glucose) and mediators (1-methoxy-5-ethylphenadinium ethyl sulfate (1-mPES) and a ruthenium compound) so that the final concentration of glucose oxidoreductase (GDH) is 0 to 12.5 nM for 5 minutes. [Figure 6]Figure 6 is a graph showing an example of the results from Experimental Example 2. It shows the results of evaluating the relationship between incubation (energy storage) time and response current value by incubating for 5, 10, or 15 minutes using a reaction solution containing a substrate (glucose), mediators (1-mPES and ruthenium compounds), and GDH (final concentrations: 0.56, 5.56, or 55.56 nM). [Figure 7] Figure 7 is a graph showing an example of the results from Experimental Example 3. It shows the results of evaluating the relationship between the substrate concentration in the reaction solution and the response current value by incubating a reaction solution containing a substrate (glucose, final concentration: 2-200 mM), a mediator (1-mPES and a ruthenium compound), and GDH for 5 minutes. [Figure 8] Figure 8 is a graph showing an example of the results from Experimental Example 4. It shows the results of evaluating the relationship between the type of mediator present in the reaction solution and the response current value by incubating a reaction solution containing a substrate (glucose), a mediator (1-mPES and / or a ruthenium compound), and GDH (final concentration: 0-10 nM) for 5 minutes. [Figure 9] Figure 9 is a graph showing an example of the results from Experimental Example 5. It shows the results of evaluating the relationship between the concentration of 1-mPES in the reaction solution and the response current value by incubating a reaction solution containing a substrate (glucose), mediators (1-mPES and a ruthenium compound) and GDH for 5 minutes. [Figure 10] Figure 10 is a graph showing an example of the results from Experimental Example 6. It shows the results confirming that the analyte (adenosine) can be detected by measuring the response current value generated by the oxidoreductase reaction of GDH in the GDH-labeled aptamer, which is released by the reaction of the aptamer with the analyte (adenosine) in the sample, using an adenosine DNA aptamer. [Figure 11] Figure 11 is a graph showing an example of the results from Experimental Example 7. By performing the first application (pre-application), the incubation (charging) time is determined (calculated) for each sample being measured, and the measured response current value obtained after incubation for the calculated time is shown. [Modes for carrying out the invention]

[0015] The inventors have conducted extensive research to provide a method for electrochemically measuring the amount of oxidoreductase in a reaction solution with high sensitivity using a biosensor. In the course of this research, the inventors confirmed that it is difficult to satisfy both the requirements of efficiently receiving electrons generated by the reaction between oxidoreductase and a substrate and continuously storing the received electrons with a single mediator, and that sufficient sensitivity cannot be obtained with a single mediator. Therefore, they discovered that by using a combination of a mediator with high electron storage capacity (second mediator) and a mediator (first mediator) that can rapidly receive electrons generated by the reaction between oxidoreductase and a substrate and transfer them to the second mediator as mediators to be placed in the biosensor, the oxidoreductase reaction between the substrate and oxidoreductase can be advanced, and the electrons generated by the reaction can be stored in the second mediator, thereby enabling electrochemical measurement of the amount of oxidoreductase with high sensitivity. Based on this discovery, they conceived the method described in this disclosure.

[0016] The differences between the measurement method disclosed herein and general biosensors such as glucose measuring devices disclosed in Patent Documents 1 to 6 are as follows. In typical biosensors, sufficient amounts of "oxidoreductase" and "mediator" are provided for the concentration range of the analyte (glucose) in the sample to ensure that the reaction between the analyte (glucose) and the oxidoreductase, and the accompanying electron transfer, proceed smoothly. In a typical glucose sensor, glucose is quantified by measuring the response current value generated when the analyte and oxidoreductase react, and electrons released from glucose are transferred to the electrode via the mediator. That is, when voltage is applied, the enzymatic reaction between the analyte and the oxidoreductase is substantially complete, or has progressed to a degree that allows for the determination of the analyte's concentration, and electron transfer from the analyte (glucose) to the mediator is substantially complete or has progressed sufficiently. In contrast, the measurement method of the present disclosure measures oxidoreductase, and the biosensor is equipped with a "substrate" of the oxidoreductase and a "mediator (second mediator)" for storing electrons generated by the reaction. In the measurement method of the present disclosure, the oxidoreductase to be measured and the substrate are reacted for a predetermined time, the electrons generated by the reaction are stored in the second mediator via the first mediator, and the electrical signal (response current value) generated after storage is measured. That is, the reaction between the oxidoreductase to be measured and the substrate is incubated for a predetermined time within a time range in which the substrate is not depleted. In other words, incubation is terminated at a predetermined time within a time range in which only a portion of the substrate is reacting with the oxidoreductase. The measurement method of the present invention differs from conventional methods using general biosensors in that the amount of oxidoreductase is calculated based on the electrical signal obtained as a result of applying a voltage at a predetermined time. In other words, according to the measurement method of this disclosure, by performing a continuous reaction between oxidoreductase and substrate, and the rapid acceptance of electrons generated by the reaction by a first mediator and their transfer to a second mediator, and the storage of energy by the second mediator, the amount of oxidoreductase in the reaction solution can be measured with high detection sensitivity even when the concentration is extremely low, for example, on the order of pM (e.g., 1-10 pg / L). In addition, since the reaction between oxidoreductase and substrate proceeds in a time-dependent manner, the amount of reaction, i.e., the amount of electron transfer, depends on the amount of oxidoreductase, so the amount of oxidoreductase can be calculated by setting the incubation time to a predetermined time.

[0017] [Measurement method] This disclosure relates, in one embodiment, to a method for electrochemically measuring the amount of oxidoreductase using a biosensor. In one or more embodiments, the measurement method of this disclosure includes using a biosensor comprising at least two mediators, including a first mediator and a second mediator as described below, incubating for a predetermined time to store electrons generated by a continuous reaction between a substrate and oxidoreductase in the second mediator via the first mediator, and then applying a voltage to measure the resulting electrical signal. In this disclosure, "storing electrons in a second mediator" can also be described as the accumulation of a reducing second mediator in one or more embodiments.

[0018] In this disclosure, "electrochemical measurement method" refers to a measurement method using an electrochemical technique, in which, in one or more embodiments, a change in the concentration of a target substance (oxidoreductase) in a reaction solution is converted into an electrical signal on an electrode and output. Examples of electrochemical techniques in one or more embodiments include chronoamperometry, chronocoulometry, and cyclic voltammetry. Examples of electrical signals in this disclosure in one or more embodiments include response current values ​​to applied voltage and response voltage values ​​to applied voltage.

[0019] [Biosensor] A biosensor used in the method of the present disclosure has a conductive portion including two or more electrodes and a reagent layer disposed in contact with the conductive portion. The reagent layer comprises an oxidoreductase substrate and two or more mediators, the mediators comprising at least a first mediator for transferring electrons generated by the reaction of the substrate and the oxidoreductase to a second mediator, and a second mediator for transferring electrons transferred from the first mediator to the electrodes.

[0020] [Oxidoreductase] Examples of oxidoreductases in one or more embodiments include glucose dehydrogenase (GDH), glucose oxidase (GOD), cholesterol oxidase, quinohem ethanol dehydrogenase, sorbitol dehydrogenase, D-fructose dehydrogenase, D-glucoside-3-dehydrogenase, cellobiose dehydrogenase, lactate oxidase (LOD), lactate dehydrogenase (LDH), and uric acid dehydrogenase. In one or more embodiments, the oxidoreductase may have flavin adenine dinucleotide (FAD), pyrroloquinoline quinone (PQQ), nicotinamide adenine dinucleotide (NAD), or nicotinamide adenine dinucleotide lein (NADP) as a coenzyme (also called a catalytic subunit or catalytic domain). Examples of oxidoreductases having a coenzyme include FAD-GDH, PQQ-GDH, NAD-GDH, and NADP-GDH in one or more embodiments. Examples of oxidoreductases include, in one or more embodiments, Aspergillus oryzae-type FAD-GDH (flavin adenine dinucleotide-dependent glucose dehydrogenase or flavin adenine dinucleotide-bound glucose dehydrogenase). In one or more embodiments, the Aspergillus oryzae-type FAD-GDH disclosed in Japanese Patent Application Publication No. 2013-083634 may be used. The contents of said document are incorporated by reference as constituting a part of this disclosure.

[0021] [Substrate] The substrate can be appropriately determined depending on the type of oxidoreductase to be measured. Examples of substrates in one or more embodiments include glucose, cholesterol, sorbitol, D-fructose, cellobiose, lactic acid, and uric acid. In one or more embodiments, a sufficient amount of substrate is placed in the reagent layer of the biosensor.

[0022] Amount of substrate in the reagent layer (1 cm of reagent layer) 3The amount of substrate per cubic centimeter (1 cm³ of the reagent layer) is, in order to suppress the reaction between the substrate and the oxidoreductase from becoming the rate-limiting factor and to increase the measurement sensitivity, 4.4 nmol or more in one or more embodiments, preferably 8.8 nmol or more, 13.2 nmol or more, 17.6 nmol or more, or 22.0 nmol or more, and more preferably 26.4 nmol or more. 3 The upper limit (per unit) is not particularly limited, but in one or more embodiments it is 220 nmol or less, 176 nmol or less, 132 nmol or less, 88 nmol or less, or 44 nmol or less.

[0023] The amount of substrate in the reagent layer (substrate concentration during incubation) is, in order to suppress the reaction between the substrate and oxidoreductase from becoming the rate-limiting factor while promoting the reaction and improving measurement sensitivity, 10 mM or more, preferably 20 mM or more, 30 mM or more, 40 mM or more, or 50 mM or more, and more preferably 60 mM or more, relative to the amount of sample in contact with the reagent layer. The upper limit of the amount of substrate in the reagent layer is not particularly limited, and in 1 or more embodiments, it is 500 mM or less, 400 mM or less, 300 mM or less, 200 mM or less, or 100 mM or less. In one or more embodiments, the substrate concentration during incubation can also be determined according to the amount of oxidoreductase being measured. In one or more embodiments, which are not particularly limited, when the amount of oxidoreductase being measured (the amount expected to be contained in the sample) is 0.01 nM to 60 nM, the substrate concentration during incubation is 10 mM or higher, preferably 20 mM or higher, 30 mM or higher, 40 mM or higher, or 50 mM or higher, and more preferably 60 mM or higher.

[0024] The amount of sample to be brought into contact with the reagent layer can be determined as appropriate, and in one or more embodiments, it is approximately 1 μL to 20 μL.

[0025] [Mediator] In one or more embodiments, the mediator includes ruthenium compounds, 1-Methoxy-PES (1-Methoxy-5-ethylphenazinium ethylsulfate, 1-mPES), 1-Methoxy-PMS (1-Methoxy-5-methylphenazinium methylsulfate, 1-mPMS), phenylenediamine compounds, quinone compounds, ferricyanide compounds, Coenzyme Q0 (2,3-Dimethoxy-5-methyl-p-benzoquinone), AZURE A Chloride (3-amino-7-(dimethylamino)phenothiazin-5-ium chloride), Phenosafranin (3,7-Diamino-5-phenylphenanzinium chloride), 6-Aminoquinoxaline, and Tetrathiafulvalene, etc. As the ruthenium compound, in one or more embodiments, a ruthenium compound that exists in the reaction system as an oxidized ruthenium complex and functions as a mediator (electron transfer agent) can be used. The type of ligand of the ruthenium complex is not particularly limited. As the ruthenium compound, in one or more embodiments, the oxidized ruthenium complex represented by the following chemical formula can be mentioned. [Ru(NH3)5X] n+ Examples of X include NH3, halogen ions, CN, pyridine, nicotinamide, or H2O, and among them, NH3 or halogen ions are preferred. As the halogen ion, in one or more embodiments, Cl - , F - , Br - , and I - can be mentioned. In the formula, n+ represents the valence of the oxidized ruthenium (III) complex determined by the type of X. As the ruthenium complex, in one or more embodiments, those disclosed in JP-A-2018-013400 can be used. The content of this document is incorporated herein by reference as part of the present disclosure. Examples of phenylenediamine compounds in one or more embodiments include N,N-Dimethyl-1,4-phenylenediamine and N,N,N',N'-tetramethyl-1,4-phenylenediaminedihydrochloride. Examples of quinone compounds in one or more embodiments include 1,4-Naphthoquinone, 2-Methyl-1,4-Naphthoquinone (VK3), 9,10-Phenanthrenequinone, 1,2-Naphthoquinone, p-Xyloquinone, Methylbenzoquinone, 2,6-Dimethylbenzoquinone, Sodium 1,2-Naphthoquinone-4-sulfonate, 1,4-Anthraquinone, Tetramethylbenzoquinone, and Thymoquinone. Examples of ferricyanide compounds include calcium ferricyanide in one or more embodiments.

[0026] The combination of mediators used in the measurement method of this disclosure can be appropriately determined in one or more embodiments depending on the oxidoreductase and the material of the electrode of the conductive part. Examples of combinations of a first mediator and a second mediator in one or more embodiments, which are not particularly limited, include combinations of 1-mPES and a ruthenium compound, and combinations of 1-mPMS and a ruthenium compound. Examples of combinations of mediators in one or more embodiments, which are not particularly limited, include combinations in which the first mediator is 1-mPES and the second mediator is a ruthenium compound.

[0027] The amounts of the first mediator and the second mediator in the reagent layer can be appropriately determined in one or more embodiments depending on the type of oxidoreductase to be measured, the types and combinations of the first and second mediators, and the material of the electrodes in the conductive part.

[0028] Amount of the first mediator in the reagent layer (1 cm of the reagent layer) 3 The amount of the first mediator in the reagent layer (per 1 cm of the reagent layer) is, in order to suppress the rate-limiting electron transfer resulting from the reaction with oxidoreductase and to improve measurement sensitivity, 0.22 nmol or more or 0.3 nmol or more in one or more embodiments, preferably 0.35 nmol or more, 0.44 nmol or more or 2.2 nmol or more. 3 The amount per 1 cm of reagent layer may be 44 nmol or more in one or more embodiments, which are not particularly limited. 3 The upper limit (per unit) is not particularly limited, but in one or more embodiments it is 220 nmol or less or 110 nmol or less. Since the first mediator can provide sufficient measurement sensitivity even with a smaller amount in the reagent layer compared to the second mediator, and thus helps to reduce manufacturing costs, the amount of the first mediator in the reagent layer should be 44 nmol or less, 22 nmol or less, 17.6 nmol or less, 13.2 nmol or less, 8.8 nmol or less, or 4.4 nmol or less.

[0029] The amount of the first mediator in the reagent layer (concentration of the first mediator during incubation) is, in one or more embodiments, 0.5 mM or more, or 0.7 mM or more, preferably 0.8 mM or more, 1 mM or more, or 5 mM or more, relative to the amount of sample in contact with the reagent layer, in order to suppress the reaction between the substrate and oxidoreductase from becoming the rate-limiting factor while increasing measurement sensitivity. The amount of the first mediator in the reagent layer (concentration of the first mediator during incubation) may be 100 mM or more in one or more embodiments, which is not particularly limited. The upper limit of the amount of the first mediator in the reagent layer is not particularly limited, and in one or more embodiments, it is 500 mM or less or 250 mM or less. Since the first mediator can provide sufficient measurement sensitivity even with a smaller amount in the reagent layer compared to the second mediator, and in terms of reducing manufacturing costs, the amount of the first mediator in the reagent layer should be 100 mM or less, 50 mM or less, 40 mM or less, 30 mM or less, 20 mM or less, or 10 mM or less.

[0030] Amount of the second mediator in the reagent layer (1 cm of the reagent layer) 3 The amount of the second mediator in the reagent layer (per 1 cm of the reagent layer) is, in order to suppress the rate-limiting electron transfer with the first mediator while enabling electron storage and improving measurement sensitivity, 0.44 nmol or more in one or more embodiments, preferably 4.4 nmol or more, 22 nmol or more, 30.8 nmol or more, or 35.2 nmol or more, and more preferably 39.6 nmol or more or 44 nmol or more. 3 The upper limit (per unit) is not particularly limited, but in one or more embodiments it is 220 nmol or less or 110 nmol or less.

[0031] The amount of the second mediator in the reagent layer (concentration of the second mediator during incubation) is, in one or more embodiments, 1 mM or more, preferably 10 mM or more, 50 mM or more, 70 mM or more, or 80 mM or more, and more preferably 90 mM or more or 100 mM or more, as a percentage of the amount of sample in contact with the reagent layer, in order to suppress electron transfer with the first mediator from becoming the rate-limiting step while enabling electron storage and improving measurement sensitivity. The upper limit of the amount of the second mediator in the reagent layer is not particularly limited, and in one or more embodiments, it is 500 mM or less or 250 mM or less.

[0032] The reagent layer may contain components other than the substrate and mediator of the oxidoreductase. Examples of other components include buffers, surfactants, and binders.

[0033] Examples of buffering agents in one or more embodiments include phosphate buffers, amine buffers, and buffers having carboxyl groups. Examples of amine buffers in one or more embodiments include Tris, ACES, CHES, CAPSO, TAPS, CAPS, Bis-Tris, TAPSO, TES, Tricinine, and ADA. Examples of buffering agents having carboxyl groups in one or more embodiments include citrate buffers, phosphate citrate buffers, acetate-sodium acetate buffers, malic acid-sodium acetate buffers, malonic acid-sodium acetate buffers, and succinic acid-sodium acetate buffers. One type of buffering agent may be used, or two or more types may be used in combination. The pH of the buffering agent is 6.0 to 8.0, preferably 7.3 to 7.4, in one or more embodiments.

[0034] Examples of surfactants in one or more embodiments include TritonX-100 ((p-tert-Octylphenoxy)polyethoxyethanol), Tween20 (Polyoxyethylene Sorbitan Monolaurate), sodium dodecyl sulfate, perfluorooctanesulfonic acid, sodium stearate, alkylaminocarboxylic acid (or its salt), carboxybetaine, sulfobetaine, and phosphobetaine. One surfactant may be used, or two or more may be used in combination.

[0035] Examples of binders in one or more embodiments include resin binders and layered inorganic compounds. Examples of resin binders in one or more embodiments include butyral resin binders and polyester resin binders. Examples of layered inorganic compounds include those described in WO2005 / 043146. Examples of layered inorganic compounds in one or more embodiments include swelling clay minerals having ion exchange capacity. Examples of layered inorganic compounds in one or more embodiments include bentonite, smectite, vermiculite, and synthetic fluorinated mica. The binder may be of one type or two or more types may be used in combination.

[0036] In one or more embodiments, the reagent layer is substantially free of oxidoreductase. In one or more embodiments, the reagent layer is substantially free of oxidoreductase that reacts with the substrate contained in the reagent layer. In this disclosure, "substantially free" means the amount of oxidoreductase in the reagent layer (1 cm of the reagent layer). 3 In one or more embodiments, the amount per unit is less than 0.1 U, less than 0.05 U, or less than 0.01 U. In this disclosure, "U" is a unit of enzyme activity, meaning the amount of enzyme that acts on 1 μmol of substrate per minute under optimal conditions.

[0037] [Conductive parts] The conductive part includes two or more electrodes. In one or more embodiments, the electrodes may be an electrode pair including a working electrode and a counter electrode, or a three-electrode system electrode pair including a working electrode, a counter electrode, and a reference electrode. In one or more embodiments, the conductive part may further have a sensing electrode. In one or more embodiments, conductive materials can be used as the electrode material. Examples of conductive materials in one or more embodiments include metallic materials and carbon materials. Examples of metallic materials in one or more embodiments include gold (Au), platinum (Pt), silver (Ag), ruthenium (Ru), palladium (Pd), and nickel (Ni) alloys. Examples of nickel alloys in one or more embodiments include nickel-vanadium alloys, nickel-tungsten alloys, and nickel-ruthenium alloys. Examples of carbon materials in one or more embodiments include graphite, carbon nanotubes, graphene, and mesoporous carbon.

[0038] In one or more embodiments, electrodes include thin-film electrodes formed by depositing a metallic material onto a substrate. In one or more embodiments, film deposition methods include screen printing, physical vapor deposition (PVD, e.g., sputtering), or chemical vapor deposition (CVD).

[0039] In one or more embodiments, the conductive portion is formed on a substrate. In one or more embodiments, an insulating substrate can be used as the substrate. In one or more embodiments, examples of insulating substrate materials include thermoplastic resins, thermosetting resins, glass, ceramics, and paper. In one or more embodiments, examples of thermoplastic resins include polyetherimide (PEI), polyethylene terephthalate (PET), and polyethylene (PE). Examples of thermosetting resins include polyimide resins and epoxy resins.

[0040] The measurement method of the present disclosure includes contacting a sample containing oxidoreductase with a reagent layer of a biosensor, incubating for a predetermined time after contact to accumulate electrons generated by the reaction between oxidoreductase and a substrate in a second mediator in the reagent layer via a first mediator, applying a voltage between electrodes of the conductive part of the biosensor after incubation, measuring the electrical signal generated by the application of the voltage, and calculating the amount of oxidoreductase based on the measured electrical signal.

[0041] In this disclosure, "incubating" means carrying out a redox reaction between an oxidoreductase in a sample and a substrate in a reagent layer, and may include transferring electrons generated by the redox reaction to a second mediator via a first mediator, and accumulating (storing) the transferred (received) electrons in the second mediator. In this disclosure, "incubating" and "storing energy" may be used synonymously.

[0042] In the measurement method of this disclosure, the incubation time after contact with the sample is, in one or more embodiments, 1 minute or more, and in order to accumulate a larger amount of electrons in the second mediator and increase the resulting electrical signal, 2 minutes or more, 3 minutes or more, 4 minutes or more, 5 minutes or more, 6 minutes or more, 7 minutes or more, or 10 minutes or more. The upper limit of the incubation time is not particularly limited, and in one or more embodiments, examples include the time until all of the substrate contained in the reagent layer is transferred by oxidation-reduction reactions. In one or more embodiments where the upper limit of the incubation time is not particularly limited, it is 30 minutes or less, 20 minutes or less, or 15 minutes or less.

[0043] In the measurement method of the present disclosure, the incubation time is preferably, in one or more embodiments, a time range in which the substrate contained in the reagent layer is not depleted by reaction with the oxidoreductase to be measured. Therefore, in one or more embodiments of the measurement method of the present disclosure, the incubation time may be set to a time range in which the substrate contained in the reagent layer is not depleted by reaction with the oxidoreductase.

[0044] In one or more embodiments of the measurement method of the present disclosure, incubation after contact is preferably performed in a non-voltage state. In one or more embodiments of the measurement method of the present disclosure, it is preferable not to apply voltage until incubation for a predetermined time after contact is completed.

[0045] In one or more embodiments, a voltage can be applied between electrodes by applying a constant voltage between the electrodes (electrode pair). In one or more embodiments, which are not particularly limited, a voltage can be applied to the electrodes by applying a positive voltage to the working electrode relative to the counter electrode. In one or more embodiments, the voltage applied to the working electrode is +50mV to +500mV relative to the counter electrode, preferably +100mV to +200mV, and more preferably +200mV.

[0046] In one or more embodiments, the measurement of the electrical signal can be determined according to the method used for measurement, depending on the amount of oxidoreductase. When calculating the amount of oxidoreductase using chronoamperometry, the electrical signal is measured in one or more embodiments by measuring the response current value after a certain period of time following voltage application. The constant period of voltage application can be 20 seconds or less, 15 seconds or less, 10 seconds or less, 9 seconds or less, 8 seconds or less, 7 seconds or less, 6 seconds or less, 5 seconds or less, 4 seconds or less, 3 seconds or less, or 2 seconds or less, in one or more embodiments. When calculating the amount of oxidoreductase using chronocoulometry, in one or more embodiments, the electrical signal is measured by continuously measuring the response current value over time in order to obtain the integrated value of the response current. When calculating the amount of oxidoreductase using cyclic voltammetry, in one or more embodiments, the electrical signal is measured by continuously measuring the current value corresponding to the voltage during sweep in order to obtain the cyclic voltammetry waveform.

[0047] In one or more embodiments, the amount of oxidoreductase based on electrical signals can be calculated using methods such as chronoamperometry, chronocoulometry, and cyclic voltammetry. When using chronoamperometry, in one or more embodiments, the relationship between the integrated value of the response current and the amount (concentration) of the substance to be measured (oxidoreductase) can be determined in advance using a calibration curve, and the amount of the substance to be measured can be calculated by applying the measured integrated value of the response current to the calibration curve.

[0048] In one or more embodiments, the measurement method of this disclosure may include applying a short-term solution (pre-application) immediately after contact between the sample and the reagent layer, and measuring the electrical signal generated by such application. By pre-application and measuring the resulting electrical signal, in one or more embodiments, the incubation (energy storage) time can be varied according to the amount of oxidoreductase (the substance to be measured) in the sample, thereby shortening the measurement time, for example. Furthermore, the electrical signal generated after incubation (energy storage) can be kept within a predetermined range, which can reduce the load on the measuring device.

[0049] Therefore, in other embodiments, the measurement method of the present disclosure includes contacting a sample containing oxidoreductase with the reagent layer of a biosensor; applying a voltage between electrodes after contact (first application); measuring the electrical signal generated by the first application (first measurement); calculating an incubation time based on the electrical signal obtained by the first measurement; incubating for the calculated time; applying a voltage between electrodes after incubation (second application); measuring the electrical signal generated by the second application (second measurement); and calculating the amount of oxidoreductase based on the electrical signal obtained by the second measurement.

[0050] In the measurement method of this disclosure, the first application (pre-application) can be performed immediately after the sample is brought into contact with the reagent layer in one or more embodiments. In one or more embodiments, the first application (pre-application) is preferably performed 0.01 seconds to 10 seconds or 0.01 seconds to 5 seconds after contact.

[0051] In the measurement method of this disclosure, the application time in the first measurement (pre-measurement) is preferably 0.01 seconds to 10 seconds or 0.01 seconds to 5 seconds in one or more embodiments.

[0052] The measurement method of this disclosure includes calculating an incubation time based on an electrical signal obtained by a first measurement. In one or more embodiments, the incubation time can be calculated (determined) based on a predetermined threshold (value of the electrical signal). In one or more embodiments, there may be one threshold or two or more thresholds. In one or more embodiments, the thresholds can be appropriately determined depending on the material of the electrodes and the type of mediator. In one or more embodiments, which are not particularly limited, if the threshold is set to three levels (high concentration, medium concentration, and low concentration) according to the concentration of oxidoreductase, if the electrical signal (response current value) obtained by the first measurement exceeds 2000 nA, the incubation time is determined to be 1 minute or less because the concentration of oxidoreductase is high; if the electrical signal (response current value) is 1000 nA or more and 2000 nA or less, the incubation time is determined to be more than 1 minute but less than 5 minutes (or, for example, 3 minutes) because the concentration of oxidoreductase is medium; and if the electrical signal (response current value) is less than 1000 nA, the incubation time can be determined to be 5 minutes or more because the concentration of oxidoreductase is low.

[0053] [Samples containing oxidoreductase] In the measurement method of this disclosure, the sample containing oxidoreductase may, in one or more embodiments, be the sample itself containing oxidoreductase, or it may be a sample in which a component in the sample has been converted to oxidoreductase. Examples of samples in one or more embodiments include biological samples. Examples of biological samples in one or more embodiments include blood and urine and samples derived therefrom, cell extracts, and cell culture media. In one or more embodiments, the sample containing oxidoreductase is a sample for measuring the amount of oxidoreductase and includes samples that may contain oxidoreductase, and samples in which a component in the sample may contain oxidoreductase (or is suspected of containing oxidoreductase) through conversion.

[0054] In one or more embodiments of the measurement method of the present disclosure, an analyte such as a hormone or a low molecular weight compound can be measured with high sensitivity by converting the analyte into an oxidoreductase and preparing a "sample containing oxidoreductase." According to the measurement method of the present disclosure, in one or more embodiments, an analyte at an extremely low concentration can be measured with high sensitivity by converting it into an oxidoreductase.

[0055] One or more embodiments of a method for converting an analyte into an oxidoreductase include a method using a molecular recognition element, such as an aptamer modified (labeled) with an oxidoreductase. For example, by using a substance in which an oxidoreductase is released, separated, or activated when the aptamer (molecular recognition element) binds to the analyte, it may be possible to perform measurements by converting the analyte into an oxidoreductase. For example, by using an oxidoreductase-modified molecular recognition element immobilized on magnetic beads so as to be released from the magnetic beads when the analyte binds to them, it is possible to release an oxidoreductase appropriate to the analyte.

[0056] Therefore, in one or more embodiments, the measurement method of this disclosure may include converting the analyte in the sample into an oxidoreductase to prepare a sample containing the oxidoreductase.

[0057] In one or more embodiments, the measurement method of the present disclosure may include, in a biosensor in which a second reagent layer containing a substance capable of converting an analyte in the sample into an oxidoreductase is positioned upstream of a first reagent layer, introducing the sample into the second reagent layer to prepare a sample containing an oxidoreductase, and bringing the prepared sample into contact with a reagent layer (first reagent layer) containing a substrate and a mediator.

[0058] In one or more embodiments, the analytes include low molecular weight compounds, peptides, proteins, antigens, hormones, immune cells, rare cells, sugars, toxins, viral particles, and metals. Therefore, in one or more embodiments, the measurement method of this disclosure can be used for the quantitative determination of low molecular weight compounds, peptides, proteins, antigens, hormones, immune cells, rare cells, sugars, toxins, viral particles, and metals. In one or more embodiments, the measurement method of this disclosure is not used for the measurement (e.g., quantitative determination) of substrates of oxidoreductases contained in the reagent layer of the biosensor used for measurement (e.g., glucose, cholesterol, ethanol, sorbitol, fructose, cellobiose, lactic acid, and uric acid).

[0059] Therefore, this disclosure relates, in other embodiments, to a method for electrochemically measuring an analyte in a sample using a biosensor. The biosensor used in the method of this embodiment has a conductive part including two or more electrodes, a second reagent layer containing a substance capable of converting the analyte into an oxidoreductase, and a first reagent layer disposed in contact with the conductive part, the first reagent layer and the second reagent layer being arranged in a flow channel, and the second reagent layer being located upstream of the first reagent layer. The first reagent layer includes the substrate of the oxidoreductase and two or more mediators, the mediator including a first mediator for transferring electrons generated by the reaction between the substrate and the oxidoreductase to the second mediator, and a second mediator for transferring electrons transferred from the first mediator to the electrode. The electrodes, substrate and mediator in the biosensor of this embodiment are as described above.

[0060] In one or more embodiments, the second reagent layer and the first reagent layer may be arranged in contact with each other or spaced apart.

[0061] The measurement method of the present disclosure can be carried out in the same manner as the measurement method described above, except that in one or more embodiments, a sample containing the analyte is used as the sample, and the biosensor comprises a second reagent layer including a molecular recognition element.

[0062] [Method for fabricating a biosensor] In one or more embodiments, the biosensor of this disclosure can be prepared by preparing a substrate on which a conductive portion including two or more electrodes is formed, and placing a reagent layer containing an oxidoreductase substrate and two or more types of mediators thereon. The reagent layer is placed on at least a portion of the working electrode of two or more electrodes. The method of placing the reagent is not particularly limited, but for example, it can be done by spotting the reagent solution onto a portion of the electrode and letting it dry. More specifically, the reagent layer can be formed by preparing a dispersion containing, for example, an oxidoreductase, two or more mediators, and optionally a buffer and binder, dispensing this dispersion onto the electrode, and letting it dry.

[0063] The biosensors of this disclosure can be fabricated in one or more embodiments by following the procedure shown in Figures 1A to E. However, the method for fabricating the biosensors in this disclosure is not limited to the procedure shown below.

[0064] As shown in Figure 1A, an electrode system including a working electrode 12, a counter electrode 11, a reference electrode and a detection electrode (1) 13, and a detection electrode (2) 14 is formed on the substrate 10 by sputtering. As shown in Figures 1A to E, by providing the detection electrode (1) 13 and the detection electrode (2) 14, the detection electrode (1) 13 and the counter electrode 11 can detect when the sample has reached the detection electrode (1) 13. Furthermore, the detection electrode (2) 14 and the working electrode 12 can detect when the sample has reached the detection electrode (2) 14. This allows the incubation time to be calculated from the time the sample is detected by the detection electrode (1) 13 and the time the sample is detected by the detection electrode (2) 14. Next, a reagent layer is formed. The reagent layer 15 only needs to be placed on a portion of the working electrode 12, and as shown in Figure 1B, the reagent layer 15 may be placed so as to be in contact with the detection electrode (1) 13, the counter electrode 11, and a portion of the working electrode 12. The reagent layer 15 can be formed by preparing a reagent solution (dispersion) containing at least an oxidoreductase substrate and two or more types of mediators, and optionally a buffer and binder, and then placing (dispensing) this solution onto the detection electrode (1) 13, the counter electrode 11, and the working electrode 12 and drying it. In addition, a water-stopping section 17 may be formed at the end of the flow channel that is formed later. In one or more embodiments, the water-stopping section 17 can be formed by applying a water-stopping agent such as a water-stopping polymer and drying it. This prevents the sample that comes into contact with the water-stopping section 17 from moving further downstream in the flow channel by causing it to gel. Next, as shown in Figure 1C, a spacer 18 is placed on the substrate 10 on which the reagent layer 15 is placed. The spacer 18 may be placed via an insulating layer. In one or more embodiments, the spacer 18 can be formed using a resin film and tape or the like. The spacer 18 is positioned so that the electrode formed at one end on the side where the water-stopping portion 17 is formed is exposed. Next, as shown in Figure 1D, a cover 19 is placed on top of the spacer 18. The space around the opening of the spacer 18, surrounded by the substrate 10 and the cover 19, becomes a flow path. The cover 19 is provided with a through-hole 20 that serves as a sample supply section and a through-hole 21 that serves as a vent. The through-hole 20 and the vent are formed to be located above the reagent layer 15 and the watertight section 17, respectively. In one or more embodiments, the material of the cover 19 can be various plastics such as PET. As shown in Figure 2, a space (flow path) 23 is formed surrounded by the substrate 10 and the cover 19, and the sample supplied to the through-hole 20 that serves as the sample supply section can move through the reagent layer 15 to the through-hole 21 that serves as the vent. Finally, as shown in Figure 1E, an O-ring type water-repellent tape 22 is placed around the through-hole 20, which serves as the sample supply section of the cover 19. This prevents the sample from flowing out of the through-hole 20 and the flow path.

[0065] Figures 3A-E and 4 show other examples of the method for fabricating the biosensor of this disclosure. Figure 4 is a cross-sectional view taken along line II-II of Figure 3E. In one or more embodiments, the biosensor of the present disclosure may include a first reagent layer 15 and a second reagent layer 16 as reagent layers, as shown in Figures 3A-E and Figure 4. The first reagent layer 15 contains at least an oxidoreductase substrate and two or more mediators, and the second reagent layer 16 contains a molecular recognition element modified with oxidoreductase. As in the biosensor of this embodiment, by having a first reagent layer 15 and a second reagent layer 16, in one or more embodiments, the time it takes for the sample to pass through the second reagent layer can be adjusted depending on the material of the second reagent layer, and the reaction time between the sample and the molecular recognition element can be appropriately changed. Furthermore, the detection electrode (1) 13 and the counter electrode 11 can detect when the sample has reached the detection electrode (1) 13. In addition, the detection electrode (2) 14 and the working electrode 12 can detect when the sample has reached the detection electrode (2) 14. The incubation time can also be calculated from the time when the sample is detected by the detection electrode (1) 13 and the time when the sample is detected by the detection electrode (2) 14.

[0066] As shown in Figure 3B, the second reagent layer 16 is positioned to be in contact with the detection electrode (1) 13 and the counter electrode 11, and the first reagent layer 15 is positioned to be in contact with the counter electrode 11 and a portion of the working electrode 12. The second reagent layer 16 can be formed by placing a substrate impregnated with a molecular recognition element modified with oxidoreductase on top of the detection electrode (1) 13 and the counter electrode 11. Examples of substrates in one or more embodiments include glass filters and bead separation membranes. The first reagent layer 15 can be formed by preparing a reagent solution (dispersion) containing at least a substrate for oxidoreductase and two or more types of mediators, with buffers and binders dispersed as needed, placing (dispensing) this solution on top of the detection electrode (1) 13, the counter electrode 11 and the working electrode 12, and drying it. The biosensor of this embodiment can be manufactured in the same manner as the biosensors shown in Figures 1A to E, except that it forms two reagent layers: a first reagent layer 15 and a second reagent layer 16.

[0067] This disclosure may relate to one or more embodiments, not limited to the following. [A1] A method for electrochemically measuring the amount of oxidoreductase using a biosensor, The aforementioned biosensor is A conductive part including two or more electrodes, Distended in contact with the conductive portion, it has a reagent layer containing the substrate of the oxidoreductase and two or more types of mediators, The mediator comprises a first mediator and a second mediator, wherein the first mediator is a mediator for transferring electrons generated by the reaction between the substrate and the oxidoreductase to the second mediator, and the second mediator is a mediator for transferring electrons transferred from the first mediator to the electrode. The method is, A sample containing an oxidoreductase is brought into contact with the reagent layer. After contact, the electrons are accumulated in the second mediator by incubation for a predetermined time. After incubation, apply a voltage between the electrodes. To measure the electrical signal generated by the application of the aforementioned voltage, and A method comprising calculating the amount of the oxidoreductase based on the electrical signal. [A2] The method according to [A1], comprising setting the incubation time to a time range in which the substrate contained in the reagent layer is not depleted by reaction with the oxidoreductase. [A3] Amount of the substrate in the reagent layer (1 cm of the reagent layer) 3 The method according to [A1] or [A2], wherein the amount per unit is 4.4 nmol or more. [A4] The incubation time is 1 minute or more, as described in any of [A1] to [A3]. [A5] The method according to any one of [A1] to [A4], wherein the oxidoreductase is glucose dehydrogenase and the substrate is glucose. [A6] The method according to any one of [A1] to [A5], wherein the combination of the first mediator and the second mediator is a combination of 1-methoxy-5-ethylphenadinium ethyl sulfate (1-mPES) and a ruthenium compound, or a combination of 1-methoxy-5-methylphenadinium methyl sulfate (1-mPMS) and a ruthenium compound. [B1] A method for electrochemically measuring the amount of oxidoreductase using a biosensor, The aforementioned biosensor is A conductive part including two or more electrodes, The reagent layer comprises the substrate of the oxidoreductase and two or more mediators, and is disposed in contact with the conductive part. The mediator comprises a first mediator and a second mediator, wherein the first mediator is a mediator for transferring electrons generated by the reaction between the substrate and the oxidoreductase to the second mediator, and the second mediator is a mediator for transferring electrons transferred from the first mediator to the electrode. The method is, The sample containing the oxidoreductase is brought into contact with the reagent layer of the biosensor. After contact, apply a voltage between the electrodes (first application), Measuring the electrical signal generated by the first application (first measurement), Based on the electrical signal obtained by the first measurement, calculate the incubation time. Perform incubation for the calculated time. After incubation, apply a voltage between the electrodes (second application). Measuring the electrical signal generated by the second application (second measurement), and A measurement method comprising calculating the amount of the oxidoreductase based on an electrical signal obtained by a second measurement. [C1] A biosensor for electrochemically measuring oxidoreductase, A conductive part including two or more electrodes, The reagent layer comprises the substrate of the oxidoreductase and two or more mediators, and is disposed in contact with the conductive part. The mediator comprises a first mediator and a second mediator, wherein the first mediator is a mediator for transferring electrons generated by the reaction between the substrate and the oxidoreductase to the second mediator, and the second mediator is a mediator for transferring electrons transferred from the first mediator to the electrode. A biosensor for use in any of the measurement methods described in [A1] to [A6] and [B1]. [C2] A biosensor for electrochemically measuring oxidoreductase, A conductive part including two or more electrodes, The reagent layer comprises the substrate of the oxidoreductase and two or more mediators, and is disposed in contact with the conductive part. The mediator is a biosensor comprising a first mediator for transferring electrons generated by the reaction between the substrate and the oxidoreductase to a second mediator, and a second mediator for transferring electrons transferred from the first mediator to the electrode. [C3] The sensor according to [C1] or [C2], wherein the oxidoreductase is glucose dehydrogenase and the substrate is glucose. [C4] The sensor according to any one of [C1] to [C3], wherein the combination of the first mediator and the second mediator is a combination of 1-methoxy-5-ethylphenadinium ethyl sulfate (1-mPES) and a ruthenium compound, or a combination of 1-methoxy-5-methylphenadinium methyl sulfate (1-mPMS) and a ruthenium compound. [D1] A method for electrochemically measuring an analyte in a sample using a biosensor, The aforementioned biosensor is A conductive part including two or more electrodes, A second reagent layer containing a molecular recognition element modified with oxidoreductase, The device comprises the substrate of the oxidoreductase and two or more mediators, and has a first reagent layer positioned in contact with the conductive portion. The mediator includes a first mediator for transferring electrons generated by the reaction between the substrate and the oxidoreductase to a second mediator, and a second mediator for transferring electrons transferred from the first mediator to the electrode. The method is, Introducing the aforementioned sample into the biosensor, By incubating for a predetermined time, electrons are accumulated in the second mediator. After incubation, apply a voltage between the electrodes. To measure the electrical signal generated by the aforementioned application, and A measurement method comprising calculating the amount of the oxidoreductase based on the measured values. [D2] The method according to [D1], comprising setting the incubation time to a time range in which the substrate contained in the first reagent layer is not depleted by reaction with the oxidoreductase. [D3] Amount of the substrate in the first reagent layer (1 cm of the reagent layer) 3 The method according to [D1] or [D2], wherein the amount per unit is 4.4 nmol. [D4] The incubation time is 1 minute or more, the method according to any of [D1] to [D3]. [D5] The method according to any one of [D1] to [D4], wherein the oxidoreductase is glucose dehydrogenase and the substrate is glucose. [D6] The method according to any one of [D1] to [D5], wherein the combination of the first mediator and the second mediator is a combination of 1-methoxy-5-ethylphenadinium ethyl sulfate (1-mPES) and a ruthenium compound, or a combination of 1-methoxy-5-methylphenadinium methyl sulfate (1-mPMS) and a ruthenium compound.

[0068] The present disclosure will be further described below with reference to examples, but these are illustrative and the disclosure is not limited to these examples. [Examples]

[0069] The following reagents and electrodes were used in the experimental example. [buffer] 0.5M phosphate buffer (pH 7.3-7.4, 0.5M NaCl, 0.25% Tween20) [Mediator] Ruthenium compound (Ru(NH3)3Cl6, manufactured by Dojin Chemical Research Institute Co., Ltd.) 1-mPES (1-methoxy-5-ethylphenadinium ethyl sulfate, manufactured by Dojin Chemical Laboratories Co., Ltd.) [GDH] FAD-dependent Glucose Dehydrogenase (Product name: Glucose Dehydrogenase “Amano 8”, MW: 180,000, manufactured by Amano Enzyme Co., Ltd.) [Substrate] glucose [electrode] DEP-CHIP (model name: DEP-ER-N, round gold electrode, printed electrode for immobilization (3-electrode system), external dimensions: 12.5 mm x 4 mm x 0.3 mm, working electrode area: 3.67 mm 2 (Manufactured by BioDevice Technology Co., Ltd.)

[0070] [Experimental Example 1] GDH was measured electrochemically using the following procedure. <Instructions> 1. Dispense 50 μl of buffer into a PCR tube. Dispense 50 μl of 1M glucose into tube 2.1. Dispense 50 μl of 5 mM 1-mPES into tube 3.2. Dispense 50 μl of 500 mM Ru compound into tube 4.3. Dispense 50 μl of GDH solution into tube 5.4 so that the final concentration is 0-12.5 nM, and immerse the electrode connected to the measuring instrument in the reaction solution. 6. Start measuring the incubation time when the last GDH solution is dispensed. 7. Five minutes after the start of measurement, apply voltage to the electrodes and perform electrochemical measurement. Applied voltage: 200mV Measurement time: 15 seconds Sampling interval: 0.1 seconds 8. Measurement complete

[0071] The composition of the reaction solution (tube 6) is as follows: <Composition of the reaction solution> 0.1M phosphate buffer (pH 7.3-7.4) 0.1M NaCl 0.05% Tween20 200mM Glucose 1mM 1-mPES 100mM Ru(NH3)3Cl6 0-12.5 nM GDH

[0072] The results obtained are shown in Figures 5A and 5B. Figures 5A and 5B are graphs of the response values ​​2 seconds after the start of application, and Figure 5B is an enlarged view of the low concentration range (0 to 0.8 nM). As shown in Figures 5A and 5B, GDH concentrations in the range of 0.024 nM (24 pM) to 12.5 nM could be detected by incubating (storing) the sample for 5 minutes. In other words, GDH in the pM order could be measured by incubating (storing) the sample for 5 minutes. By obtaining response values ​​at a short time after application (for example, 2 seconds after application), highly linear quantitative analysis of GDH was possible.

[0073] [Experimental Example 2] GDH was measured electrochemically using the following procedure. <Instructions> 1. Dispense 50 μl of buffer into a PCR tube. Dispense 50 μl of 1M glucose into tube 2.1. Dispense 50 μl of 5 mM 1-mPES into tube 3.2. Dispense 50 μl of 500 mM Ru compound into tube 4.3. Dispense 50 μl of GDH solution into tube 5.4 so that the final concentrations are 0.56, 5.56, and 55.56 nM, and immerse the electrode connected to the measuring instrument in the reaction solution. 6. Start measuring the energy storage time when the last GDH solution is dispensed. 7. After a predetermined time (5, 10, or 15 minutes), apply voltage to the electrodes and perform electrochemical measurements. Applied voltage: 200mV Measurement time: 30 seconds Sampling interval: 0.1 seconds 8. Measurement complete

[0074] The composition of the reaction solution (tube 6) is as follows: <Composition of the reaction solution> 0.1M phosphate buffer (pH 7.3-7.4) 0.1M NaCl 0.05% Tween20 200mM Glucose 1mM 1-mPES 100mM Ru(NH3)3Cl6 0.56nM, 5.56nM, 55.56nM GDH

[0075] The results obtained are shown in Figures 6A-C. Figures 6A-C show the relative values ​​(sensitivity ratios) when the response value after an incubation time of 5 minutes is set to 100. The response value used was the response value at 2 seconds after the start of application. As shown in Figures 6A-C, an increase in the response value dependent on the incubation time was observed at all GDH concentrations.

[0076] [Experimental Example 3] GDH was measured electrochemically using the following procedure. <Instructions> 1. Dispense 50 μl of buffer into a PCR tube. Dispense 50 μl of glucose into tube 2.1 so that the final concentration is 2-200 mM. Dispense 50 μl of 5 mM 1-mPES into tube 3.2. Dispense 50 μl of 500 mM Ru compound into tube 4.3. Dispense 50 μl of 5 nM GDH solution into tube 5.4, and immerse the electrode connected to the measuring instrument in the reaction solution. 6. Start measuring the energy storage time when the last GDH solution is dispensed. 7. After the predetermined time (5 minutes) has elapsed, apply voltage to the electrodes and perform electrochemical measurements. Applied voltage: 200mV Measurement time: 10 seconds Sampling interval: 0.1 seconds 8. Measurement complete

[0077] The composition of the reaction solution (tube 6) is as follows: <Composition of the reaction solution> 0.1M phosphate buffer (pH 7.3-7.4) 0.1M NaCl 0.05% Tween20 2-200mM Glucose 1mM 1-mPES 100mM Ru(NH3)3Cl6 1nM GDH

[0078] The results obtained are shown in Figure 7. The response value used was the response value at 2 seconds after the start of application. As shown in Figure 7, under the conditions of this experimental example, a high response current exceeding 200 nA was obtained when the glucose concentration (substrate concentration) was 10 mM or higher. When the glucose concentration (substrate concentration) was 60 mM or higher (60 to 200 mM), the response value did not change (no decrease in the response value was observed). In other words, it was confirmed that even when the substrate (glucose) concentration in the reaction solution is high (for example, when the biological sample contains a high concentration of the substance that serves as the substrate for the oxidoreductase reaction), the enzyme (GDH) can be measured without being inhibited by the glucose substrate.

[0079] [Experimental Example 4] GDH was measured electrochemically using the following procedure. Dispensing was performed so that the concentration of the mediator in the reaction solution was as shown in the table below. <Instructions> 1. Dispense 50 μl of buffer into a PCR tube. Dispense 50 μl of 1M glucose into tube 2.1. Dispense 50 μl of 5 mM 1-mPES into tube 3.2 (dispense buffer if 1-mPES is not included). Dispense 50 μl of 500 mM Ru compound into tube 4.3 (dispense buffer if the solution does not contain Ru compound). Dispense 50 μl of GDH solution into tube 5.4 so that the final concentration is 0-10 nM, and immerse the electrode connected to the measuring instrument in the reaction solution. 6. Start measuring the energy storage time when the last GDH solution is dispensed. 7. After the predetermined time (5 minutes) has elapsed, apply voltage to the electrodes and perform electrochemical measurements. Applied voltage: 200mV Measurement time: 10 seconds Sampling interval: 0.01 seconds 8. Measurement complete

[0080] The composition of the reaction solution (tube 6) is as follows: <Composition of the reaction solution> 0.1M phosphate buffer (pH 7.3-7.4) 0.1M NaCl 0.05% Tween20 200mM Glucose 1-mPES and Ru(NH3)3Cl6 are listed in the table below. 0-10 nM GDH [Table 1]

[0081] The results are shown in Figure 8. The response values ​​used were the average of the response values ​​from 2.0 to 2.1 seconds after the start of application. As shown in Figure 8, sufficient response values ​​could not be obtained with only one type of mediator. In particular, when only the ruthenium compound was used, no response values ​​could be obtained at any GDH concentration. This is thought to be because the ruthenium compound was unable to receive many electrons from the GDH or was unable to store the electrons it received. In contrast, by using two types of mediators (ruthenium compound and 1-mPES), high response values ​​were obtained at all GDH concentrations.

[0082] [Experimental Example 5] GDH was measured electrochemically using the following procedure. <Instructions> 1. Dispense 50 μl of buffer into a PCR tube. Dispense 50 μl of 1M glucose into tube 2.1. Dispense 50 μl of 5 mM or 500 mM 1-mPES into tube 3.2. Dispense 50 μl of 500 mM Ru compound into tube 4.3. Dispense 50 μl of 5 nM GDH solution into tube 5.4, and immerse the electrode connected to the measuring instrument in the reaction solution. 6. Start measuring the energy storage time when the last GDH solution is dispensed. 7. After the predetermined time (5 minutes) has elapsed, apply voltage to the electrodes and perform electrochemical measurements. Applied voltage: 200mV Measurement time: 10 seconds Sampling interval: 0.01 seconds 8. Measurement complete

[0083] The composition of the reaction solution (tube 6) is as follows: <Composition of the reaction solution> 0.1M phosphate buffer (pH 7.3-7.4) 0.1M NaCl 0.05% Tween20 200mM Glucose 1 mM or 100 mM 1-mPES 100mM Ru(NH3)3Cl6 1nM GDH

[0084] The results are shown in Figure 9. The response value used was the average of the response values ​​from 2.0 to 2.1 seconds after the start of application. As shown in Figure 9, by increasing the amount of 1-mPES (first mediator) to 100 mM, the sensitivity (response value) could be more than doubled.

[0085] [Experimental Example 6] Using an adenosine DNA aptamer modified (labeled) with GDH as a molecular recognition element, the amount of GDH in the reaction solution was measured electrochemically according to the following procedure. The adenosine DNA aptamer is an aptamer that recognizes and binds to adenosine as a target molecule recognition site, is labeled with GDH, and is immobilized on a magnetic bead. When adenosine binds to the aptamer, the GDH-labeled aptamer is released from the bead. <Instructions> 1. Dispense 100 μl of adenosine DNA aptamer-immobilized beads into a PCR tube (final concentration: 6 pmol / 100 μl). 2. Collect the magnetization and discard the liquid. Dispense 120 μl of adenosine solution (adenosine dissolved in buffer) into tube 3.2 (final adenosine concentration: 0, 7.5, 12.5, 37.5, 75, 125, 375, or 750 μM). After reacting for 4.5 minutes, magnetize for 2 minutes and collect the supernatant. 5. Dispense 50 μl of buffer into the PCR tube. Dispense 50 μl of 1 M glucose into a 6.5 tube. Dispense 50 μl of 5 mM 1-mPES into a 7.6 tube. Dispense 50 μl of 500 mM Ru compound into tube 8.7. Dispense 50 μl of each of the four samples (including the GDH-labeled aptamer) into a tube 9.8 and connect it to the measuring instrument. 10. Start measuring the energy storage time when the last 4 samples are dispensed. 11. After the predetermined time (5 minutes) has elapsed, apply voltage to the electrodes and perform electrochemical measurements. Applied voltage: 200mV Measurement time: 10 seconds Sampling interval: 0.01 seconds 12. Measurement complete

[0086] The composition of the reaction solution (10 tubes) is as follows: <Composition of the reaction solution> 0.1M phosphate buffer (pH 7.3-7.4) 0.1M NaCl 0.05% Tween20 200mM Glucose 1mM 1-mPES 100mM Ru(NH3)3Cl6 GDH-labeled aptamers released from magnetic beads according to adenosine concentration

[0087] The measurement was performed in the same manner as described above, except that an adenosine solution dissolved in plasma was used instead of a buffer.

[0088] The results are shown in Figure 10. The response value used was the average of the response values ​​at 2.0 to 2.1 seconds after the start of application. As shown in Figure 10, although there was a difference in sensitivity between plasma and buffer, an increase in response current dependent on the concentration of the substance being measured (adenosine) was confirmed. In other words, we were able to detect the concentration of the target substance using the GDH-modified aptamer released by the reaction of the aptamer with the analyte (adenosine).

[0089] [Experimental Example 7] GDH was measured electrochemically using the following procedure. [device making] Electrodes comprising a counter electrode, a working electrode, and a reference electrode were fabricated by sputtering and trimming metal onto a PET substrate. A device having a capillary structure was then fabricated using the obtained electrodes. <Instructions> 1. Dispense 50 μl of buffer into a PCR tube. Dispense 50 μl of 1M glucose into tube 2.1. Dispense 50 μl of 5 mM 1-mPES into tube 3.2. Dispense 50 μl of 500 mM Ru compound into tube 4.3. Dispense 50 μl of GDH into tube 5.4 so that the final concentrations are 0.56 nM, 5.56 nM, and 55.56 nM. 6. Immediately after dispensing (approximately 10 seconds after dispensing), draw a portion of the sample from tube 5 into one of the above devices. 7. Immediately after aspiration, apply voltage to the electrode and perform electrochemical measurement (first application and first measurement). Applied potential: 200mV Measurement time: 5 seconds Sampling interval: 0.1 seconds After dispensing in 8.5, a predetermined incubation time (1, 3, or 5 minutes) is elapsed, and a voltage is applied to the electrode for electrochemical measurement (second application and second measurement). Applied voltage: 200mV Measurement time: 15 seconds Sampling interval: 0.1 seconds 9. Measurement complete

[0090] The composition of the reaction solution (tubes 7 and 8) is as follows: <Composition of the reaction solution> 0.1M phosphate buffer (pH 7.3-7.4) 0.1M NaCl 0.05% Tween20 200mM Glucose 1mM 1-mPES 100mM Ru(NH3)3Cl6 0.56, 5.56, and 55.56 nM GDH

[0091] Some of the results are shown in Figures 11A and 11B. Figure 11A shows the measurement results of the response current value during the first measurement (pre-application), and Figure 11B shows the measurement results of the response current value during the second measurement. The response value in Figure 11A is the response value at 0.5 seconds after the start of application, and the response value in Figure 11B is the response value at 2.0 seconds after the start of application. As shown in Figure 11A, in the first measurement (pre-application), samples with high GDH concentration showed high current values, while samples with low GDH concentration showed low current values. The incubation time in the second measurement was varied according to the current value in the first measurement. The results are shown in Figure 11B. Samples with high current values ​​in the first measurement (current value of 2000 nA or more at 0.5 seconds after the start of measurement) could be sufficiently measured with 1 minute of incubation (energy storage). When the pre-application current value was between 1000 nA and 2000 nA, measurement was possible with 3 minutes of incubation (energy storage). When the current value in the first measurement was less than 1000 nA, a response value could be detected after 5 minutes of incubation (energy storage). In other words, by pre-setting threshold values ​​and incubation (energy storage) times for each threshold value for the current value of the first measurement, and then comparing the measured value of the first measurement with the threshold and varying the incubation (energy storage) time, it is possible to perform faster measurements, reduce the load on the device, and set a smaller counter electrode area in the electrode structure.

[0092] [Fabrication of biosensors] The biosensor was fabricated using the following procedure, as shown in Figures 3A to 3E. As shown in Figure 3A, a PET substrate 10 (length: 50 mm, width: 6 mm, thickness: 250 μm) was prepared, and a metal was sputtered onto one surface of the substrate, and then trimmed to form a sensing electrode (1) 13, a counter electrode 11, an working electrode 12, and a reference electrode and sensing electrode (2) 14. Next, as shown in Figure 3B, a first reagent layer 15, a second reagent layer 16, and a water-sealing section 17 were provided on the substrate 10 on which the electrodes were formed. The second reagent layer 16 was provided on top of the detection electrode (1) 13 and the counter electrode 11 so as to be in contact with them. The first reagent layer 15 was provided on top of the counter electrode 11 and a portion of the working electrode 12 so as to be in contact with them. The water-sealing section 17 was provided on top of the reference electrode and the detection electrode (2) 14. The second reagent layer 16 was formed by placing a glass filter containing a molecular recognition element modified with oxidoreductase. The filter of the second reagent layer 16 had a diameter of 3 mm. The first reagent layer 15 was formed by placing a reagent solution containing the substrate of oxidoreductase, the first and second mediators, and a buffer and drying it. The water-sealing section 17 was formed by placing a water-sealing polymer. Next, as shown in Figure 3C, a spacer 18 having an opening that exposes the second reagent layer 16, the first reagent layer 15, and the water-sealing portion 17 was placed on the substrate 10 on which the second reagent layer 16, the first reagent layer 15, and the water-sealing portion 17 were formed. The spacer 18 was also positioned so that the electrode at one end (the right side in the figure) was exposed. Next, as shown in Figure 3D, a cover 19 equipped with a through-hole 20 that serves as a sample supply section and a through-hole 21 that serves as a ventilation opening was placed on the spacer 18. The space of the opening of the spacer 18, surrounded by the substrate 10 and the cover 19, becomes the flow path. The height of the flow path was set to approximately 100 μm. The through-hole 20 that serves as the sample supply section and the through-hole 21 that serves as the ventilation opening were formed to be located above the second reagent layer 16 and the water-stopping section 17, respectively. The through-hole 20 that serves as the sample supply section had a diameter of 4 mm, and the through-hole 21 that serves as the ventilation opening had a diameter of 0.5 mm. Finally, as shown in Figure 3E, an O-ring type water-repellent tape 22 (inner diameter: 2 mm and outer diameter: 6 mm) was placed on top of the cover 19. The water-repellent tape 22 was positioned to cover the area around the through-hole 20, which serves as the sample supply section, thereby preventing the sample from flowing out of the second reagent layer 16 (filter). Figure 4 is a cross-sectional view taken along line II-II in Figure 3E. As shown in Figure 4, a space (channel) 23 is formed surrounded by the substrate 10 and the cover 19, and the sample supplied to the through-hole 20, which serves as the sample supply section, can move through this space (channel) 23, passing through the second reagent layer 16 and the first reagent layer 15 to the through-hole 21, which serves as the ventilation opening.

Claims

1. A method for electrochemically measuring the amount of an oxidoreductase using a biosensor, comprising: The biosensor comprises: a conductive portion including two or more electrodes; a reagent layer disposed in contact with the conductive portion and containing a substrate for the oxidoreductase and two or more types of mediators; the mediator includes a first mediator and a second mediator, the first mediator being a mediator for transferring electrons generated by a reaction between the substrate and the oxidoreductase to the second mediator, and the second mediator being a mediator for transferring the electrons transferred from the first mediator to the electrode; The method comprises: contacting a sample containing an oxidoreductase with the reagent layer; After the contact, incubation is carried out for a predetermined time, thereby allowing the electrons to accumulate in the second mediator; After incubation, applying a voltage between the electrodes; measuring an electrical signal generated by the application of the voltage; and calculating the amount of the oxidoreductase based on the electrical signal.

2. The method according to claim 1 , further comprising setting the incubation time within a range in which the substrate contained in the reagent layer is not depleted by the reaction with the oxidoreductase.

3. The amount of the substrate in the reagent layer (1 cm of the reagent layer) 3 3. The method of claim 1 or 2, wherein the amount of hydroxybenzoates per 1000 kJ / kg is 4.4 nmol or more.

4. The method according to any one of claims 1 to 3, wherein the incubation time is 1 minute or more.

5. The method of claim 1 , wherein the oxidoreductase is glucose dehydrogenase and the substrate is glucose.

6. 6. The method according to claim 1, wherein the combination of the first mediator and the second mediator is a combination of 1-methoxy-5-ethylphenazinium ethyl sulfate (1-mPES) and a ruthenium compound, or a combination of 1-methoxy-5-methylphenazinium methyl sulfate (1-mPMS) and a ruthenium compound.

7. A biosensor for electrochemically measuring an oxidoreductase, comprising: a conductive portion including two or more electrodes; a reagent layer that contains a substrate for the oxidoreductase and two or more types of mediators and is disposed in contact with the conductive part; the mediator includes a first mediator and a second mediator, the first mediator being a mediator for transferring electrons generated by a reaction between the substrate and the oxidoreductase to the second mediator, and the second mediator being a mediator for transferring the electrons transferred from the first mediator to the electrode; A biosensor for use in the measurement method according to any one of claims 1 to 6.

8. A method for electrochemically measuring the amount of an oxidoreductase using a biosensor, comprising: The biosensor comprises: a conductive portion including two or more electrodes; a reagent layer that contains a substrate for the oxidoreductase and two or more types of mediators and is disposed in contact with the conductive part; the mediator includes a first mediator and a second mediator, the first mediator being a mediator for transferring electrons generated by a reaction between the substrate and the oxidoreductase to the second mediator, and the second mediator being a mediator for transferring the electrons transferred from the first mediator to the electrode; The method comprises: contacting a sample containing the oxidoreductase with the reagent layer of the biosensor; After contacting, applying a voltage between the electrodes (first application); measuring an electrical signal generated by the first application (first measurement); calculating an incubation time based on the electrical signal obtained by the first measurement; Incubating for the calculated time; After incubation, applying a voltage between the electrodes (second application); measuring the electrical signal generated by the second application (second measurement); and and calculating the amount of the oxidoreductase based on the electrical signal obtained by the second measurement.

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