Enzyme sensor

Abstract

To provide a compact enzyme sensor capable of measuring temporal changes in the concentration of a specific organic substance (target analyte) in a test solution with high precision and minimal time delay.SOLUTION: The enzyme sensor is designed to measure the concentration of a target substance contained in a solution. The enzyme sensor includes an electrode unit 1 that includes an anode electrode 2 including an anode layer 21 and a first liquid-permeable layer 22, a cathode electrode 3 including a cathode layer 31 and a second liquid-permeable layer 32, and a liquid-absorbing layer 4 with higher water absorption capacity than the first and second liquid-permeable layers 22, 32. The enzyme sensor further includes a laminated body 6 in which the anode layer 21, the first liquid-permeable layer 22, the cathode layer 31, the second liquid-permeable layer 32, and the liquid-absorbing layer 4 are stacked in this order.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a sensor for a specific substance (substance to be measured) using an enzyme and a method for measuring the concentration of the specific substance using the same. [Background technology]

[0002] In recent years, with the rise in health consciousness, there has been a growing need for sensors that can selectively detect organic substances in biological fluids such as sweat, saliva, urine, tears, and blood in order to monitor the physical condition. In particular, non-invasive measurements that do not damage the subject's body, such as those that do not require blood sampling, have attracted attention. In particular, when monitoring the physical condition during activity, it is important not only to collect biological fluids and measure the organic substances in them in a batch process, but also to measure the time-dependent changes in the organic substance concentration in the biological fluids due to activity.

[0003] Patent Document 1 discloses a power generation device that generates electricity using glucose as fuel and a method for measuring glucose concentration. Patent Document 2 discloses a system that incorporates an enzyme battery that generates electricity using glucose in urine into a diaper and measures the glucose concentration from the amount of electricity generated by the enzyme battery. Non-Patent Document 1 also discloses a method for measuring the change in ethanol concentration in sweat over time by forming a flow path for sweat, which is the solution to be measured. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO2018 / 062419 publication [Patent Document 2] Japanese Patent Application Publication No. 2020-6129 [Non-patent literature]

[0005] [Non-Patent Document 1] A. Hauke et. al., Lab Chip, 2018, 18, pp.3750 Summary of the Invention [Problem to be solved by the invention]

[0006] Ideally, this type of power generation device would be capable of measuring the concentration of the substance to be measured in real time. However, the power generation device disclosed in Patent Document 1 had a problem in that the electrical signal output corresponding to the time-series change in the concentration of the substance to be measured was not responsive, in other words, there was a problem in that there was a large "time delay." Furthermore, the correlation of the output with the change in the concentration of the substance to be measured was not good, and there was also a problem in that the measurement accuracy of the concentration of the substance to be measured was poor.

[0007] In the system disclosed in Patent Document 2, a large amount of absorbent material is present around the enzyme battery. Therefore, the enzyme battery placed inside the diaper generates electricity using urine (a glucose-containing solution) diffusing from the absorbent material, and measures the concentration of the substance to be measured (glucose) according to the amount of electricity generated. Therefore, there was a problem in that it was not possible to measure the change in glucose concentration over time with a small time delay and with high accuracy.

[0008] Furthermore, the ethanol sensor disclosed in Non-Patent Document 1 uses a solution pump that utilizes an absorbent material, enabling the measurement of ethanol concentration over time. The enzyme electrode that measures the ethanol concentration is placed on the same plane as the absorbent material. As a result, the amount of enzyme contained in the electrode that contributes to the reaction with the solution to be measured is small, resulting in poor output efficiency and poor measurement accuracy.

[0009] In one aspect, the present disclosure aims to provide an enzyme sensor that can measure the change over time in the concentration of a substance to be measured in a solution with a small time delay and high accuracy. [Means for solving the problem]

[0010] In one aspect, the present disclosure provides an enzyme sensor for measuring a concentration of a substance to be measured in a solution containing the substance to be measured, an electrode part including a laminate in which an anode electrode including an anode layer having an enzyme immobilized thereon and a first liquid-permeable layer, a cathode electrode including a cathode layer and a second liquid-permeable layer, and a liquid-absorbing layer are laminated; The present invention relates to an enzyme sensor, wherein the laminate comprises the anode layer, the first liquid-permeable layer, the cathode layer, the second liquid-permeable layer, and the liquid-absorbing layer, laminated in this order.

[0011] The present disclosure relates to one aspect of a method for measuring a substance to be measured using the enzyme sensor of the present invention, supplying a solution containing the substance to be measured to the anode layer; the anode electrode and the cathode electrode are electrically connected, and a value of a current that flows when a predetermined voltage is applied between the anode electrode and the cathode electrode is measured; and calculating an absolute value of the concentration of the substance to be measured based on the current value.

[0012] The present disclosure provides another aspect of a method for measuring a substance to be measured using the enzyme sensor of the present invention, supplying a solution containing the substance to be measured to the anode layer; measuring a potential difference between the anode electrode and the cathode electrode and obtaining a rate of increase of the potential difference; and calculating an absolute value of the concentration of the substance to be measured based on the rate of increase of the potential difference.

[0013] The present disclosure provides yet another aspect of a method for measuring a substance to be measured using the enzyme sensor of the present invention, supplying a solution containing the substance to be measured to the anode layer; connecting a load between the anode electrode and the cathode electrode and directly or indirectly obtaining a measurement value of at least one of a current flowing through the load, a voltage applied to the load, and a power consumed by the load; and calculating an absolute value of the concentration of the substance to be measured based on the measured value. [Effects of the Invention]

[0014] According to the present disclosure, an enzyme sensor can be provided that can measure the change over time in the concentration of a substance to be measured in a solution with a small time delay and high accuracy. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic cross-sectional view of one embodiment of the electrode part of the enzyme sensor of the present disclosure. [Figure 2] Figure 2A is an explanatory diagram illustrating one embodiment of a method for measuring a substance to be measured using the enzyme sensor disclosed herein, and Figure 2B is a graph showing that there is a linear relationship between the concentration of the substance to be measured and the measured current value. [Figure 3] Figure 3A is an explanatory diagram illustrating another embodiment of a method for measuring a substance to be measured using the enzyme sensor disclosed herein, and Figure 3B is a graph showing that there is a linear relationship between the concentration of the substance to be measured and the rate of increase in the measured potential difference. [Figure 4] Figure 4A is a schematic diagram illustrating another embodiment of a method for measuring a substance to be measured using the enzyme sensor disclosed herein, and Figure 4B is a graph showing that there is a linear relationship between the concentration of the substance to be measured and the measured current value, voltage value, or power value. [Figure 5] Figures 5A to 5C are schematic diagrams showing the overlap between the part of the main surface of the first liquid-passing layer constituting the electrode part of the enzyme sensor of Examples 1 to 3 that overlaps with the anode layer and the surface of the liquid-absorbing layer facing the first liquid-passing layer. [Figure 6] FIG. 6A is a schematic cross-sectional view of an electrode portion of the enzyme sensor of Comparative Example 1. FIG. [Figure 7] FIG. 7 is a schematic cross-sectional view of the electrode part of the enzyme sensor of Comparative Example 3. As shown in FIG. DETAILED DESCRIPTION OF THE INVENTION

[0016] In one aspect, the enzyme sensor (hereinafter sometimes abbreviated as "sensor") disclosed herein is an enzyme sensor for measuring the concentration of a substance to be measured (hereinafter sometimes referred to as "substrate") in a solution (hereinafter sometimes referred to as "measured solution") containing the substance to be measured. The inventors of the present invention have discovered that by stacking an anode electrode including an anode layer and a first liquid-permeable layer, and a cathode electrode including a cathode layer and a second liquid-permeable layer above a liquid-absorbing layer, it is possible to measure the change over time in the concentration of a substance to be measured (e.g., an organic substance) in a solution to be measured with high accuracy and with little time delay.

[0017] The present inventors speculate as follows about the details of the mechanism by which the effects of the present disclosure are manifested.

[0018] To measure the concentration of a substance to be measured in a solution to be measured, the anode layer carrying an enzyme must first come into contact with the solution to be measured. When the solution to be measured comes into contact with the anode layer, the solution to be measured is retained in the anode layer. In particular, when a porous layer is used as the anode layer to increase the amount of enzyme carried and enhance electrode activity, the amount of solution retained by the anode electrode increases. However, when the anode layer already holds a large amount of solution, it takes time for the substance to be measured in the newly supplied solution to reach the surface of the anode layer where the enzyme is supported and its vicinity. This results in poor response of the output (electrical signal) corresponding to the concentration change of the substance to be measured in the newly supplied solution to be measured, in other words, a problem of a large "time delay." Furthermore, due to the influence of the solution to be measured already held in the electrode, the concentration change of the substance to be measured on the surface of the anode layer and its vicinity is small, resulting in a decrease in measurement accuracy. As in Non-Patent Document 2, by arranging an anode electrode, a cathode electrode, and a solution-absorbing material such as a water-absorbent polymer in this order parallel to the main surface of the anode electrode, the presence of the absorbent material serves as a driving force to induce a flow of the analyte solution from the anode electrode toward the absorbent material. The analyte solution held by the anode electrode is quickly discharged toward the absorbent material. This reduces the "time delay" in the output (electrical signal) and improves the correlation between the change in the concentration of the analyte at the anode electrode and the output (electrical signal), thereby improving measurement accuracy. However, this method has the drawback of increasing the area of the sensor's electrode, and the proportion of enzymes immobilized on the electrode that come into contact with the solution is small, resulting in poor reaction efficiency. Therefore, in the present disclosure, the electrode section of the sensor is configured to include a laminate in which an anode electrode, a cathode electrode, and a liquid-absorbent layer are stacked in this order. With this configuration, the test solution supplied to the surface of the anode electrode flows in this order in the thickness direction of the anode electrode and the cathode electrode, and the arrangement of the liquid-absorbent layer promotes the flow of the test solution toward the liquid-absorbent layer. As a result, retention of the test solution at the electrode is suppressed, improving the responsiveness of the electrical signal corresponding to changes in the concentration of the test substance; in other words, the degree of "time delay" is reduced. Furthermore, the correlation between the change in the concentration of the substance to be measured and the output (electrical signal) is improved by appropriately discharging the solution to be measured from both electrodes, and the contact efficiency between the solution to be measured and the enzyme is also improved, which improves the accuracy of measuring the concentration of the substance to be measured. Furthermore, the above configuration contributes to reducing the area of the main surface of the electrode part.

[0019] An example of the enzyme sensor of the present disclosure will be described below with reference to the drawings: Figure 1 is a schematic cross-sectional view of the electrode part of an enzyme sensor of one embodiment of the present disclosure.

[0020] The electrode unit 1 shown in FIG. 1 includes a laminate 6 in which an anode electrode 2, a cathode electrode 3, and a liquid-absorbent layer 4 are laminated in this order. The anode electrode 2 includes an anode layer 21 and a first liquid-permeable layer 22, and the cathode electrode 3 includes a cathode layer 31 and a second liquid-permeable layer 32. In the laminate 6, the anode layer 21, the first liquid-permeable layer 22, the cathode layer 31, the second liquid-permeable layer 32, and the liquid-absorbent layer 4 are laminated in this order. The first liquid-permeable layer 22 and the second liquid-permeable layer 32 are also referred to as a first substrate and a second substrate, respectively. In FIG. 1, 7 indicates a solution containing a substance to be measured, and 7 indicates the direction of movement of the solution.

[0021] An enzyme (hereinafter also referred to as "anode enzyme") (not shown) is supported on the anode layer 21, and the enzyme selectively reacts with the analyte to be measured by the sensor of the present disclosure. The anode enzyme is preferably immobilized on or near the surface of the anode layer 21 opposite the surface facing the first liquid-passage layer 22. Furthermore, when the anode electrode 2 contains a redox substance (not shown), it is preferable that both the anode enzyme and the redox substance (not shown) be immobilized directly or indirectly on or near the surface of the anode layer 21 opposite the surface facing the first liquid-passage layer.

[0022] The anode electrode 2 preferably further includes a lead layer (not shown) disposed between the anode layer 21 and the first liquid passage layer 22 for electrically connecting the electrodes.

[0023] The cathode electrode 3 preferably further includes a lead layer (not shown) disposed between the cathode layer 31 and the second liquid passage layer 32 for electrically connecting the electrodes. When the cathode electrode 3 includes a cathode enzyme (not shown), the cathode enzyme is preferably immobilized on or in the vicinity of the surface of the cathode layer 31 opposite the surface facing the second liquid passage layer 32. When the cathode electrode 3 includes a redox substance (not shown), both the cathode enzyme and the redox substance are preferably immobilized directly or indirectly on the surface of the cathode layer 31 opposite the surface facing the second liquid passage layer 32.

[0024] The lead layers of the anode electrode 2 and the cathode electrode 3 can be formed, for example, by applying a paste containing a conductive material generally used as a material for lead layers to the first substrate and the second substrate (first liquid-permeable layer, second liquid-permeable layer), and then firing the paste by heating.

[0025] The term "immobilization" of the enzyme and the redox substance means that the enzyme and the redox substance are in a state where they do not move relative to the anode layer 21 or the cathode layer 31. There are no particular restrictions on the immobilization position of the enzyme as long as they are immobilized on the anode layer and the cathode layer, respectively. However, from the viewpoint of effectively obtaining the enzyme's promoting effect of oxidation or reduction, it is preferable that the enzyme be immobilized on or near the surface of the anode layer and the cathode layer where the contact with the test solution begins. The enzyme may be immobilized directly to the anode layer 21 or the cathode layer 31, or may be immobilized indirectly via a redox substance. Alternatively, a mixture layer of the enzyme and the redox substance may be formed on the anode layer 21 or the cathode layer 31. Preferably, from the viewpoint of effectively obtaining the enzyme's effect of promoting oxidation or reduction, the enzyme is immobilized to the anode layer 21 or the cathode layer 31 via a redox substance. Specifically, a solution containing a redox substance may be applied to one main surface of each of the anode layer 21 and the cathode layer 31 and dried, and then a solution containing the enzyme may be applied thereon and dried.

[0026] (anode layer, cathode layer) The materials for the anode layer 21 and the cathode layer 31 are not particularly limited as long as they each contain a conductive material. However, the conductive material is preferably a porous conductive material from the viewpoint of being able to support a larger amount of enzyme. As the porous conductive material, porous carbon materials and porous nickel are preferred from the viewpoint of being able to support a larger amount of enzyme, and porous carbon materials are more preferred from the viewpoint of disposability and biocompatibility. Preferred commercially available porous carbon materials include, for example, Knobel (registered trademark) (manufactured by Toyo Tanso Co., Ltd.). Furthermore, the anode layer 21 and the cathode layer 31 may contain metal powder from the viewpoint of improving conductivity.

[0027] A binder (binder resin) may be contained in each of the anode layer 21 and the cathode layer 31. Examples of binders that can be used include polyvinylidene fluoride (PVDF), vinylidene fluoride-hexafluoropropylene copolymer, styrene butadiene rubber (SBR), and polyacrylonitrile, either alone or in combination.

[0028] The anode layer 21 and the cathode layer 31 can be formed by, for example, applying a paste containing a porous carbon material, a binder, a solvent, and optionally metal powder onto lead layers formed on a first substrate and a second substrate by screen printing or the like, sintering the coating by heating, and repeating this process until the anode layer 21 and the cathode layer 31 reach the desired thickness.

[0029] The thickness of the anode layer 21 is preferably large from the viewpoint of immobilizing a larger amount of enzyme, and specifically, is preferably 5 μm or more, more preferably 8 μm or more. On the other hand, from the viewpoint of permeability of the test solution, the thickness of the anode layer 21 is preferably 200 μm or less, more preferably 180 μm or less, more preferably 150 μm or less, and even more preferably 100 μm or less.

[0030] The thickness of the cathode layer 31 is preferably large from the viewpoint of immobilizing a larger amount of enzyme, and specifically, is preferably 20 μm or more, more preferably 30 μm or more. On the other hand, from the viewpoint of the permeability of the test solution, the thickness of the cathode layer 31 is preferably 200 μm or less, more preferably 180 μm or less, more preferably 150 μm or less, even more preferably 100 μm or less, even more preferably 80 μm or less, and even more preferably 50 μm or less.

[0031] (1st liquid passing layer, 2nd liquid passing layer) The first and second liquid-permeable layers serve as substrates for forming lead layers and the anode layer 21 and cathode layer 31 by coating or other methods. The materials for the first and second liquid-permeable layers are not particularly limited as long as they are suitable as substrates and have good permeability for the test solution. However, from the viewpoints of disposability and biocompatibility, natural materials such as paper and cloth are preferred, and Japanese paper is more preferred. In the present disclosure, the first and second liquid-permeable layers may serve to prevent short-circuiting between the anode electrode 2 and the cathode electrode 3. The laminate 6 may further include a separator disposed between the anode electrode 2 and the cathode electrode 3 to prevent short-circuiting. Separators commonly used in battery materials can be used as the separator. Specifically, porous polymer sheets or paper are preferred, and porous polyethylene sheets, porous polypropylene sheets, etc. are more preferred.

[0032] (enzyme) The anode enzyme promotes the oxidation of the analyte. The anode enzyme can be selected depending on the type of analyte to be oxidized. The anode electrode may contain only one or more types of anode enzyme. When the analyte is lactic acid, the anode enzyme is preferably at least one selected from oxidases and dehydrogenases, more preferably at least one selected from glucose oxidase, glucose dehydrogenase, fructose dehydrogenase, invertase, glucose dehydrogenase, amylase, glucose dehydrogenase, L-lactate oxidase, L-lactate dehydrogenase, and alcohol dehydrogenase, and from the viewpoint of easy availability, L-lactate oxidase is even more preferable.

[0033] When the substance to be measured is glucose, the anode enzyme is preferably at least one selected from oxidases and dehydrogenases, more preferably at least one selected from glucose oxidase, glucose dehydrogenase, fructose dehydrogenase, invertase, glucose dehydrogenase, amylase, glucose dehydrogenase, L-lactate oxidase, L-lactate dehydrogenase, and alcohol dehydrogenase, and from the viewpoint of easy availability, glucose oxidase is even more preferable.

[0034] When the substance to be measured is fructose, the anode enzyme is preferably at least one selected from oxidases and dehydrogenases, more preferably at least one selected from glucose oxidase, glucose dehydrogenase, fructose dehydrogenase, invertase, glucose dehydrogenase, amylase, glucose dehydrogenase, L-lactate oxidase, L-lactate dehydrogenase, and alcohol dehydrogenase, and even more preferably fructose dehydrogenase.

[0035] When the substance to be measured is sucrose, the anode enzyme is preferably at least one selected from oxidases and dehydrogenases, more preferably at least one selected from glucose oxidase, glucose dehydrogenase, fructose dehydrogenase, invertase, glucose dehydrogenase, amylase, glucose dehydrogenase, L-lactate oxidase, L-lactate dehydrogenase, and alcohol dehydrogenase, and even more preferably invertase.

[0036] When the substance to be measured is alcohol, the anode enzyme is preferably at least one selected from oxidases and dehydrogenases, more preferably at least one selected from glucose oxidase, glucose dehydrogenase, fructose dehydrogenase, invertase, glucose dehydrogenase, amylase, glucose dehydrogenase, L-lactate oxidase, L-lactate dehydrogenase, and alcohol dehydrogenase, and even more preferably alcohol dehydrogenase.

[0037] The cathode enzyme is not particularly limited as long as it promotes the reduction of oxygen in the air, but from the viewpoints of disposability and biocompatibility, organic substances are preferred, examples of which include bilirubin oxidase, laccase, etc. The cathode enzyme may be of one type or of two or more types.

[0038] (oxidation-reduction substances) The redox substance (anode mediator) contained in the anode electrode 2 is preferably an aromatic substance that can be reversibly oxidized and reduced, more preferably at least one selected from benzoquinone, 1,2-naphthoquinone, 1,4-naphthoquinone, thionine, azure A, azure B, azure C, phenothiazine, methylene blue, and Prussian blue, and even more preferably at least one selected from benzoquinone, 1,2-naphthoquinone, and 1,4-naphthoquinone, and even more preferably 1,2-naphthoquinone, because it does not dissolve into the solution to be measured.

[0039] An example of the redox substance (cathode mediator) contained in the cathode electrode is ABTS (2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid).

[0040] (Liquid absorption layer) The liquid-absorbent layer 4 has the function of inducing a flow of the test solution in the direction from the anode electrode 2 toward the liquid-absorbent layer 4. In order to prevent the test solution from being absorbed by the absorbent layer 4 before the test substance in the test solution newly supplied to the anode layer 2 reacts with the enzyme to generate a current, the absorbent layer 4 is disposed in the laminate 6 on the side opposite to the side to which the test solution is supplied with respect to the anode layer 21.

[0041] The material of the liquid-absorbent layer 4 is not particularly limited as long as it has the function of inducing a flow of the test solution in the direction from the anode electrode 2 toward the liquid-absorbent layer 4. Preferably, the material contains a material with higher absorbency than the materials constituting the anode electrode 2 and the cathode electrode 3. High absorbency refers to the ability to hold a large amount of solution. Examples include absorbent materials such as sponges and water-absorbent polymers, and preferably water-absorbent polymers. Examples of water-absorbent polymers include hydrogel materials, which are used in diapers and are capable of absorbing and retaining water. For example, polymers or copolymers of acrylic acid or alkali metal acrylates can be used. Specific examples include polyacrylic acid and its salts, and polymethacrylic acid and its salts, specifically partial sodium salts of acrylic acid polymers.

[0042] The shape of the water-absorbent polymer is not particularly limited, and may be, for example, spherical, tufted, blocky, bale-shaped, fibrous, irregularly shaped, or particles of a combination thereof. From the viewpoint of increasing the uniformity of the water-absorbent polymer dispersion during preparation of the liquid-absorbent layer 4 and improving the absorption performance of the liquid-absorbent layer 4, it is preferable to use water-absorbent polymer particles of the same shape, and spherical particles are preferred.

[0043] The liquid-absorbent layer 4 preferably comprises at least one absorbent sheet material selected from Japanese paper, nonwoven fabric, and tissue paper. From the viewpoints of shape retention and handleability, it preferably comprises Japanese paper, and from the viewpoint of liquid absorption, it comprises at least one absorbent sheet material selected from nonwoven fabric and tissue. The liquid-absorbent layer is preferably a composite material of an absorbent sheet material and a water-absorbent polymer, more preferably a composite material of a nonwoven fabric and a water-absorbent polymer. These composite materials can be formed by spraying a water-absorbent polymer on one main surface of an absorbent sheet material.

[0044] The weight per unit area (basis weight) of the liquid-absorbent sheet material is preferably 4 g / m from the viewpoint of maintaining the shape of the liquid-absorbent layer. 2 More than 6g / m 2 From the viewpoint of preventing backflow due to the osmotic pressure of the test solution held inside the liquid-absorbent sheet, the liquid-absorbent sheet preferably has a density of 100 g / m 2 Less than 50 g / m 2 More preferably 30 g / m or less 2 The following is the result.

[0045] The weight per unit area of the water-absorbent polymer in the liquid-absorbent layer is preferably 60 g / m from the viewpoint of reducing the "time delay" and improving the measurement accuracy. 2 More preferably, 100 g / m 2 or more, and preferably 500 g / m 2 Less than 400 g / m 2 The following is the result.

[0046] The area of the surface of the liquid-absorbent layer 4 facing the first liquid-permeable layer 22, when viewed in the thickness direction of the laminate 6, is at least 0.2 times, more preferably at least 0.4 times, and even more preferably at least 0.5 times the area of the portion of the main surface of the first liquid-permeable layer 22 that overlaps with the anode layer 21, and is preferably at most 2.5 times, more preferably at most 2.0 times, and even more preferably at most 1.0 times. If the area exceeds 2.5 times, the proportion of the solution to be measured that is absorbed directly into the absorbent layer 4 without passing through the anode layer 21 becomes too large, which is disadvantageous from the perspective of reducing the "time lag" and increasing the maximum output of the sensor.

[0047] The anode electrode 2, cathode electrode 3, and liquid-absorbent layer 4 constituting the laminate 6 may be bonded to adjacent layers, or may simply be stacked without being bonded. To prevent misalignment of the anode electrode 2, cathode electrode 3, and liquid-absorbent layer 4, the laminate 6 may be sandwiched between a pair of covering materials 5 such as mesh materials, each having an area larger than the main surfaces of the anode electrode 2, cathode electrode 3, and liquid-absorbent layer 4, and the edges of the covering materials 5 may be sealed. The sealing method is not particularly limited. While the mesh material is not particularly limited, in the case of measuring substances in sweat, sweat-absorbent sports mesh is preferred for smooth supply of sweat to the anode layer 21. A preferred commercially available product is Aquadry® (registered trademark) manufactured by Teijin Limited, which quickly absorbs sweat by capillary action.

[0048] Next, a method for measuring the concentration of a substance to be measured (sensing target) of the present disclosure using the enzyme sensor of the present disclosure (hereinafter sometimes abbreviated as "the measurement method of the present disclosure") will be described.

[0049] In one embodiment, the measurement method of the present disclosure comprises the following steps A to C. Step A: Supplying a solution 7 containing a substance to be measured to the anode layer 21 (FIG. 1) Step B: The anode electrode 2 and the cathode electrode 3 are electrically connected, and a predetermined voltage is applied between the anode electrode and the cathode electrode, and the current value (measured value) that flows is measured (FIG. 2A). Step C: Calculating the absolute value of the concentration of the substance to be measured based on the current value (FIG. 2B); In this embodiment, the above step B is a method for measuring an electrical signal using an electrochemical technique called chronoamperometry (CA measurement). In step C, the current value is used to calculate the absolute value of the concentration of the substance to be measured corresponding to the current value from a linear function of the concentration of the substance to be measured and the current value.

[0050] In another embodiment, the measurement method of the present disclosure includes the following steps A to C. Step A: Supplying a solution 7 containing a substance to be measured to the anode layer 21 (FIG. 1) Step B: Measure the potential difference (measured value) between the anode layer and the cathode layer and obtain the rate of increase of the potential difference (FIG. 3A). Step C: Calculating the absolute value of the concentration of the substance to be measured based on the rate of increase of the voltage difference (FIG. 3B). In this embodiment, the potential difference measured in the above step B is also called the natural potential (OCV), and is the voltage in the state where no voltage or current is applied. In step C, the absolute value of the concentration of the substance to be measured corresponding to the rate of increase in the potential difference is calculated from a linear function of the concentration of the substance to be measured and the rate of increase in the potential difference.

[0051] In yet another embodiment, the measurement method of the present disclosure includes the following steps A to C. Step A: Supplying a solution 7 containing a substance to be measured to the anode layer 21 (FIG. 1). Step B: Connect a load between the anode electrode 2 and the cathode electrode 2 and directly or indirectly obtain measurements of at least one of the current flowing through the load (measurement I), the voltage applied to the load (measurement II), and the power consumed by the load (measurement III) (Figure 4A). Step C: Calculating the absolute concentration of the substance to be measured based on the measured value (FIG. 4B). In step C, the absolute values of the concentrations of the measured substance corresponding to the measurements I to III are obtained from a linear function between the concentration of the measured substance and measurement value I, a linear function between the concentration of the measured substance and measurement value II, or a linear function between the concentration of the measured substance and measurement value III.

[0052] In the measurement method of the present disclosure, the measured values are acquired over time, so steps A to C proceed almost simultaneously. When the potential difference between the anode electrode 2 and the cathode electrode 3 before voltage application is smaller than the applied voltage, the current flows in the direction of the applied voltage, but when it is larger than the applied voltage, the current flows in the opposite direction to the applied voltage. Either direction of current may be used to measure the concentration of the substance to be measured.

[0053] In the sensor of the present disclosure, an electrical signal is generated by the following mechanism. The above mechanism will be explained below using an example in which the solution containing the substance to be measured is sweat containing lactic acid, lactate oxidase as an enzyme and 1,2-naphthoquinone as a redox substance are immobilized on the anode layer 21, and bilirubin oxidase (BOD) as an enzyme and ABTS (2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) as a redox substance are immobilized on the cathode layer 31.

[0054] When sweat containing lactic acid is supplied to the anode layer 21, the lactic acid is oxidized by lactate oxidase (an enzyme), and at that time, 1,2-naphthoquinone (a redox substance) releases electrons (e - ) is directly obtained and reduced. Meanwhile, at the cathode electrode 3, oxygen (O2) in the air is reduced by bilirubin oxidase (BOD). At this time, the electrons (e - ) is transferred to oxygen (O2) via the redox substance ABTS at the cathode electrode 3. In this way, a current flows between the two electrodes.

[0055] The current density between the two electrodes increases or decreases depending on the concentration of lactic acid supplied to the anode layer 21. There is a linear relationship (first-order correlation) between the concentration of lactic acid and the current density. Therefore, by using the time-dependent current density data obtained by CA measurement, the absolute value of the lactic acid concentration can be calculated from a linear function between the concentration of the substance to be measured and the current value, which is prepared in advance. The calculated concentration information can be transmitted to the outside by an external transmission means provided in the sensor.

[0056] The enzyme sensor of the present disclosure may further include, in addition to the electrode unit 1 and a circuit unit (not shown) that converts the redox reaction in the electrode unit 1 into an electrical signal, a fixing means for detachably fixing the enzyme sensor to an adherend such as skin, and a housing for protecting the electrode unit 1 and the circuit unit. Furthermore, as a means for obtaining the absolute value of the concentration of the substance to be measured using the electrical signal, the enzyme sensor may further include, for example, a memory in which the linear function is stored, or a central processing unit (CPU) including a calculation unit that calculates the absolute value of the concentration of the substance to be measured. The enzyme sensor may also include an auxiliary memory unit (storage) that stores the acquired electrical signal.

[0057] The enzyme sensor of the present disclosure may be used in any application that can utilize the substrate specificity of oxidoreductases, including, for example, a biosensor for sensing organic substances or body fluids in biological samples, or a biosensor for sensing viruses or antibodies in environmental samples. More specifically, examples include a blood glucose sensor for measuring the glucose concentration in blood, a urine glucose sensor for measuring the glucose concentration in urine, a lactate sensor for measuring the lactate concentration in sweat, and a virus sensor for detecting viruses present in food or sewage.

[0058] The present application further discloses the following enzyme sensor and a method for measuring a substance to be measured using the enzyme sensor.

[0059] <1> An enzyme sensor for measuring the concentration of a substance to be measured in a solution containing the substance to be measured, an electrode part including a laminate in which an anode electrode including an anode layer and a first liquid-permeable layer, a cathode electrode including a cathode layer and a second liquid-permeable layer, and a liquid-absorbing layer are laminated; an anode enzyme is immobilized on the anode layer; In the laminate, the anode layer, the first liquid-permeable layer, the cathode layer, the second liquid-permeable layer, and the liquid-absorbing layer are laminated in this order. <2> The liquid-absorbing layer contains a water-absorbing polymer. <1> The enzyme sensor according to claim 1. <3> The liquid-absorbent layer contains a composite of a water-absorbent polymer and a nonwoven fabric. <1> The enzyme sensor according to claim 1. <4> The cathode layer further comprises a cathode enzyme immobilized on the cathode layer. <1> from <3> 10. The enzyme sensor according to any one of the preceding items. <5> the anode enzyme has the ability to oxidize the analyte; <1> from <4> 10. The enzyme sensor according to any one of the preceding items. <6> The anode electrode further includes a reversibly oxidizable and reducible substance fixed to the anode layer. <5> The enzyme sensor according to claim 1. <7> the cathode enzyme has the ability to reduce the analyte; <4> The enzyme sensor according to claim 1. <8> The cathode electrode further comprises a reversibly oxidizable and reducible substance fixed to the cathode layer. <7> The enzyme sensor according to claim 1. <9> The aforementioned <1> from <8> A method for measuring a substance to be measured using the oxygen sensor according to any one of the above items, supplying a solution containing the substance to be measured to the anode layer; the anode electrode and the cathode electrode are electrically connected, and a value of a current that flows when a predetermined voltage is applied between the anode electrode and the cathode electrode is measured; calculating an absolute value of the concentration of the substance to be measured based on the current value. <10> The aforementioned <1> from <8> A method for measuring a substance to be measured using the oxygen sensor according to any one of the above items, supplying a solution containing the substance to be measured to the anode layer; measuring a potential difference between the anode electrode and the cathode electrode and obtaining a rate of increase of the potential difference; calculating an absolute value of the concentration of the substance to be measured based on the rate of increase of the potential difference. <11> The aforementioned <1> from <8> A method for measuring a substance to be measured using the oxygen sensor according to any one of the above items, supplying a solution containing the substance to be measured to the anode layer; connecting a load between the anode electrode and the cathode electrode and directly or indirectly obtaining a measurement value of at least one of a current flowing through the load, a voltage applied to the load, and a power consumed by the load; and calculating an absolute value of the concentration of the substance to be measured based on the measured value. [Example]

[0060] Hereinafter, the present disclosure will be described in more detail with reference to examples, but these are merely illustrative examples and the present disclosure is not limited to these examples.

[0061] 1. Preparation of Anode Electrode Japanese paper (Hanshiya's Gasenshi Shoun) was prepared as the substrate (first liquid-permeable layer), and carbon paste (Jujo Chemical's JELCON, CH-8) was applied to one main surface of the Japanese paper and baked for 15 minutes at 120°C. This carbon paste application and baking process was repeated six times, and a total of six carbon layers were laminated to create the lead part. Next, 2.6 mL of N-methyl-2-pyrrolidone (special grade WAKO Pure Chemicals) and 7.6 mL of PVDF resin (Kureha, KF Polymer-L #9305) were added to 0.75 g of porous carbon Knobel (manufactured by Toyo Tanso), and these were mixed to prepare a paste containing porous carbon Knobel (manufactured by Toyo Tanso). Next, a paste containing porous carbon Knobel (Knobel manufactured by Toyo Tanso) was applied onto the lead portion and fired at 60°C for 24 hours. The application and firing of the paste was repeated three times to stack a total of three porous carbon layers. In this way, an anode electrode was produced that included an anode layer made of three porous carbon layers and a first liquid passage layer. Next, 1,2-naphthoquinone (Tokyo Chemical Industry Co., Ltd.) was dissolved in acetonitrile to obtain a 100 mM acetonitrile solution of 1,2-naphthoquinone (Tokyo Chemical Industry Co., Ltd.). 20 μL of this solution was applied to one main surface of the porous carbon layer (anode layer) and dried under reduced pressure at room temperature for 90 minutes to immobilize the anode mediator on the anode layer. Next, L-lactate oxidase (Toyobo Co., Ltd. LCO301) was dissolved in 0.1 M PBS solution (pH 7.4) to obtain an L-lactate oxidase solution. 20 μL of the L-lactate oxidase solution (enzyme amount: 40 U) was applied to immobilize the anode enzyme on the anode layer. In this way, the anode mediator and anode enzyme were immobilized on the anode layer.

[0062] 2. Fabrication of the Cathode Electrode Japanese paper (Hanshiya-made Gasenshi Shoun) was prepared as the substrate (second liquid-permeable layer), and a lead portion consisting of six carbon layers was formed on one main surface of the Japanese paper in the same manner as in "1. Preparation of anode electrode," followed by the formation of three porous carbon layers. In this way, a cathode electrode was prepared, including a cathode layer consisting of three porous carbon layers and a second liquid-permeable layer. Next, ABTS (Tokyo Chemical Industry Co., Ltd.) was dissolved in a 0.1 M PBS solution (pH 7.4) to obtain a 50 mM ABTS (Tokyo Chemical Industry Co., Ltd.) solution. 20 μL of this solution was applied to one main surface of the porous carbon layer (cathode layer) and dried under reduced pressure at room temperature for 90 minutes to immobilize the anode mediator on the cathode layer. Next, bilirubin oxidase (WAK-AMA-BO3, Amano Enzyme Co., Ltd.) was dissolved in a 0.1 M PBS solution (pH 7.4) to obtain a bilirubin oxidase solution. 20 μL of the bilirubin oxidase solution (enzyme amount: 40 U) was applied to immobilize the cathode enzyme on the cathode layer. In this way, the anode mediator and cathode enzyme were immobilized on the cathode layer.

[0063] 3. Preparation of water-absorbent sheet (liquid-absorbent layer) SMS nonwoven fabric (weight per unit area 10g / m 2 ) and tissue (weight per unit area 16g / m 2) was prepared, and an adhesive was applied in a predetermined pattern to one main surface of the SMS nonwoven fabric. Further, an acrylic acid polymer partial sodium salt (weight per unit area: 110 g / m) was applied to a part of the adhesive-applied area. 2 ) was sprayed evenly. Next, the tissue paper was placed on the surface of the SMS nonwoven fabric on which the acrylic acid polymer partial sodium salt had been sprayed, with the adhesive interposed therebetween, to integrate the SMS nonwoven fabric, the acrylic acid polymer partial sodium salt, and the tissue paper, thereby producing a water-absorbent sheet (liquid-absorbent layer) in which the acrylic acid polymer partial sodium salt, the SMS nonwoven fabric, and the tissue were integrated.

[0064] 4. Preparation of the electrode part of the enzyme sensor In Examples 1 to 5, the electrode part of the sensor shown in FIG. 1 was produced. Example 1 As shown in Figure 1, a 5 mm x 25 mm square cathode electrode (cathode layer thickness: 40 μm) 3 and a 5 mm x 25 mm square anode electrode (anode layer thickness: 40 μm) 2 were laminated in this order on a 5 mm x 20 mm square water-absorbent sheet (liquid-absorbent layer 4). The cathode electrode 3 and the anode electrode 2 were positioned so that their substrates (second liquid-permeable layer 32 and first liquid-permeable layer 22), respectively, faced the liquid-absorbent layer 4. The resulting laminate was covered and sealed with sports mesh (Teijin, sweat-absorbent Sorel Up) to prepare the electrode part of the enzyme sensor. As shown in FIG. 5A, the area of the surface of the liquid-absorbing layer 4 on the first liquid-permeable layer 22 side was set to be 1.0 times the area (5 mm × 20 mm) of the portion 9 (region surrounded by a dotted line) of the main surface of the first liquid-permeable layer 22 that overlapped with the anode layer 21, when viewed in the thickness direction of the laminate.

[0065] Example 2 As shown in Figure 1, a 5 mm x 25 mm square cathode electrode (cathode layer thickness: 40 μm) 3 and a 5 mm x 25 mm square anode electrode (anode layer thickness: 40 μm) 2 were laminated in this order on a water-absorbent sheet (liquid-absorbent layer 4) cut into a 2.5 mm x 20 mm square. The cathode electrode 3 and anode electrode 2 were positioned so that their base materials (second liquid-permeable layer 32 and first liquid-permeable layer 22) faced the liquid-absorbent layer 4. The resulting laminate was covered with sports mesh (Teijin, sweat-absorbent Sorel Up) to prepare the electrode part of the enzyme sensor. As shown in FIG. 5B, the area of the surface of the liquid-absorbing layer 4 facing the first liquid-permeable layer 22 was set to be 0.5 times the area (5 mm × 20 mm) of the portion 9 (region surrounded by a dotted line) of the main surface of the first liquid-permeable layer 22 that overlapped with the anode layer 21, when viewed in the thickness direction of the laminate.

[0066] Example 3 As shown in Figure 1, a 5 mm x 25 mm square cathode electrode (cathode layer thickness: 40 μm) 3 and a 5 mm x 25 mm square anode electrode (anode layer thickness: 40 μm) 2 were laminated in this order on a water-absorbent sheet (liquid-absorbent layer 4) cut into a 10 mm x 20 mm square. The cathode electrode 3 and anode electrode 2 were positioned so that their base materials (second liquid-permeable layer 32 and first liquid-permeable layer 22) faced the liquid-absorbent layer 4. The resulting laminate was covered with sports mesh (Teijin, sweat-absorbent Sorel Up) to prepare the electrode part of the enzyme sensor. As shown in FIG. 5C, the area of the surface of the liquid-absorbing layer 4 facing the first liquid-permeable layer 22 was set to 2.0 times the area (5 mm × 20 mm) of the portion 9 (area surrounded by a dotted line) of the main surface of the first liquid-permeable layer 22 that overlapped with the anode layer 21 when viewed in the thickness direction of the laminate.

[0067] Example 4 An electrode part of an enzyme sensor was produced in the same manner as in Example 1, except that the thickness of the anode layer in Example 1 was set to 10 μm.

[0068] Example 5 An electrode part of an enzyme sensor was produced in the same manner as in Example 1, except that the thickness of the anode layer in Example 1 was set to 95 μm.

[0069] (Comparative Example 1) As shown in Fig. 6, a 5mm x 25mm square cathode electrode (cathode layer 310, 40µm thick) 30 and a 5mm x 25mm square anode electrode (anode layer 210, 40µm thick) 20 were laminated in this order on a water-absorbent sheet (liquid-absorbent layer 40) cut into a 5mm x 20mm square. The cathode electrode 30 and the anode electrode 20 were positioned so that their substrates (second liquid-permeable layer 320 and first liquid-permeable layer 220) were opposite the liquid-absorbent layer 40. The resulting laminate was covered with a sports mesh (Teijin, sweat-absorbent Sorel Up) 50 to prepare an electrode section for the enzyme sensor. The area of the surface of the liquid absorbing layer 40 on the first liquid passage layer 220 side was set to be equal to the area of the portion of the main surface of the first liquid passage layer 220 that overlaps with the anode layer 210 when viewed in the thickness direction of the laminate.

[0070] (Comparative Example 2) The electrode part of Comparative Example 2 was produced in the same manner as the electrode part of Example 1, except that no water-absorbent sheet (liquid-absorbent layer) was used.

[0071] (Comparative Example 3) 7, an anode lead (not shown), a cathode lead (not shown), an anode electrode 20, and a cathode electrode 30 were prepared on one main surface of the Japanese paper (Hanshiya-made Gasenshi Shoun) 90 used in [1. Preparation of the Anode Electrode] above using the same materials and in the same manner as in [1. Preparation of the Anode Electrode] and [2. Preparation of the Cathode Electrode] above, and a mediator and an enzyme were immobilized on the anode layer 210 and the cathode layer 310, respectively. Next, the anode electrode 20, the cathode electrode 30, and the absorbent sheet 40 prepared in [3. Preparation of the Absorbent Sheet (Liquid-Absorbent Layer)] were arranged in this order on one main surface of the Japanese paper 90, and the resulting laminate was covered with sports mesh 50 (Teijin, sweat-absorbent Sorel Up) to prepare the electrode part of the enzyme sensor. The surface of the anode layer 210 facing the Japanese paper 90, the surface of the cathode layer 310 facing the Japanese paper 90, and the surface of the liquid-absorbent layer 40 facing the Japanese paper 90 each measure 5 mm × 20 mm. The surface of the first liquid-permeable layer 220 facing the anode layer 210 and the surface of the second liquid-permeable layer 320 facing the cathode layer 310 all measure 5 mm × 25 mm. In Figure 7, 70 denotes a solution containing the substance to be measured, and 80 denotes the direction of movement of the solution.

[0072] 5. Evaluation of Enzyme Sensors The working electrode was connected to the cathode electrode of the electrode part of the obtained enzyme sensor, and the counter electrode and reference electrode were connected to the anode electrode. CA (chronoamperometry) measurement was performed while maintaining the potential of the cathode electrode relative to the anode electrode at +0.3 (V). Specifically, 100 μL of 0.1 mol / L phosphate buffer solution (pH 7.4) was dropped onto the anode layer side of the electrode part, and then phosphate buffer solution (pH 7.4) containing dissolved L-lactate sodium was dropped at a rate of 1 μL / min every minute, and the current density after 1 minute of dropping was plotted. The concentration of sodium L-lactate in the phosphate buffer solution into which the sodium L-lactate was dissolved was changed from 0 to 1 to 3 to 5 to 10 to 25 to 50 to 75 to 100 to 75 to 50 to 25 to 10 to 5 to 3 to 1 to 0 mM, and the "time delay" and maximum output in response to this concentration change were evaluated. The time from when 100 mM L-lactate sodium phosphate buffer was added until the sensor output reached its maximum is shown as "time delay" in Table 1 below. The maximum outputs of the sensors in Examples 2, 3, 4, and 5 and Comparative Examples 1, 2, and 3 are also shown in Table 1 below as relative values, with the maximum output of the sensor in Example 1 set at 100. The maximum outputs were calculated by fitting data from several points near the maximum output with a quadratic function.

[0073] [Table 1]

[0074] As can be seen from a comparison of Examples 1, 4, and 5, the thicker the anode layer, the higher the maximum output of the enzyme sensor. This is presumably because the thicker the anode layer, the better the amount of anode enzyme retained. From the results of the Examples, it was confirmed that the thickness of the anode layer is preferably 8 μm or more and 200 μm or less. Furthermore, a comparison between Example 4 and Example 5 shows that increasing the thickness of the anode layer does not significantly affect the "time delay." Furthermore, the "time delay" is significantly larger in Comparative Examples 1 and 2. This confirms that the magnitude of the "time delay" is significantly affected by the arrangement and stacking order of the anode layer, first liquid passage layer, cathode layer, second liquid passage layer, and liquid absorbing layer. Furthermore, the evaluation results for Examples 1 to 3 show that if the area ratio (area of the surface of the liquid-absorbent layer facing the first liquid-permeable layer / area of the portion of the main surface of the first liquid-permeable layer overlapping with the anode layer) is too large, the time lag increases, and if it is too small, the maximum output of the sensor decreases. It was confirmed that an area ratio of 0.5 to 2.0 is preferable. [Explanation of symbols]

[0075] 1. Electrode part of enzyme sensor 2 anode electrode 21 Anode layer 22 First liquid-permeable layer (or first substrate) 3. Cathode electrode 31 Cathode layer 32 Second liquid-permeable layer (or second substrate) 4 Liquid absorption layer 5 Covering material 6 Laminate 7 Solution to be measured 8. Direction of movement of the solution to be measured

Claims

1. An enzyme sensor for measuring the concentration of a substance to be measured in a solution containing the substance to be measured, an electrode part including a laminate in which an anode electrode including an anode layer and a first liquid-permeable layer, a cathode electrode including a cathode layer and a second liquid-permeable layer, and a liquid-absorbing layer are laminated; an anode enzyme is immobilized on the anode layer; In the laminate, the anode layer, the first liquid-permeable layer, the cathode layer, the second liquid-permeable layer, and the liquid-absorbing layer are laminated in this order.

2. The enzyme sensor according to claim 1 , wherein the liquid-absorbing layer comprises a water-absorbing polymer.

3. The enzyme sensor according to claim 1 , wherein the liquid-absorbing layer comprises a composite of a water-absorbing polymer and a nonwoven fabric.

4. The enzyme sensor according to claim 1 , further comprising a cathode enzyme immobilized on the cathode layer.

5. 4. The enzyme sensor according to claim 1, wherein the anode enzyme has the ability to oxidize the substance to be measured.

6. The enzyme sensor according to claim 5 , wherein the anode electrode further comprises a reversibly oxidizable and reducible substance immobilized on the anode layer.

7. The enzyme sensor according to claim 4 , wherein the cathode enzyme has the ability to reduce the substance to be measured.

8. The enzyme sensor according to claim 7 , wherein the cathode electrode further comprises a reversibly oxidizable and reducible substance immobilized on the cathode layer.

9. A method for measuring a substance to be measured using the oxygen sensor according to any one of claims 1 to 3, supplying a solution containing the substance to be measured to the anode layer; the anode electrode and the cathode electrode are electrically connected, and a value of a current that flows when a predetermined voltage is applied between the anode electrode and the cathode electrode is measured; calculating an absolute value of the concentration of the substance to be measured based on the current value.

10. A method for measuring a substance to be measured using the oxygen sensor according to any one of claims 1 to 3, supplying a solution containing the substance to be measured to the anode layer; measuring a potential difference between the anode electrode and the cathode electrode and obtaining a rate of increase of the potential difference; calculating an absolute value of the concentration of the substance to be measured based on the rate of increase of the potential difference.

11. A method for measuring a substance to be measured using the oxygen sensor according to any one of claims 1 to 3, supplying a solution containing the substance to be measured to the anode layer; connecting a load between the anode electrode and the cathode electrode and directly or indirectly obtaining a measurement value of at least one of a current flowing through the load, a voltage applied to the load, and a power consumed by the load; and calculating an absolute value of the concentration of the substance to be measured based on the measured value.

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