Electrochemical sensors

Abstract

To provide a sensor that has a short waiting time before measurement is possible, and after measurement begins, has minimal time lag in the measured value in response to changes in the concentration of the substance being measured. [Solution] An electrochemical sensor for measuring the concentration of a substance to be measured in a solution, comprising an electrode section including an anode electrode having an anode layer and a first liquid-permeable layer, and a cathode electrode having a cathode layer and a second liquid-permeable layer, wherein a substance capable of recognizing the substance to be measured is fixed to the anode layer, and the water absorption rate of the first liquid-permeable layer is greater than the water absorption rate of the second liquid-permeable layer.

Description

Technical Field

[0001] The present disclosure relates to an electrochemical sensor and a method for measuring the concentration of a substance to be measured using the same.

Background Art

[0002] In recent years, with the increasing 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 body's condition. Among them, non-invasive measurements that do not harm the bodies of subjects who do not require blood sampling have attracted attention.

[0003] Patent Document 1 discloses a power generation device including a fuel, an anode, a cathode, and a substrate, wherein the anode contains an enzyme that promotes the oxidation of the fuel, the fuel is contained in the substrate, the anode and the cathode are arranged on the same surface of the substrate so as to contact the substrate respectively, and power is generated by supplying a liquid. Further, when the power generation device has an anode that further contains an enzyme that promotes the oxidation of the measurement target substance, the concentration of the measurement target substance contained in the liquid can be measured, and examples of the measurement target substance include components in biological fluids such as glucose, lactic acid, and uric acid.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] In monitoring the physical state during activity, it is important not only to collect biological fluids and measure the organic substances within them in a batch process, but also to measure the change in the concentration of organic substances in the biological fluids over time due to activity. Furthermore, it is desirable that the method for measuring the concentration of the substance to be measured in the biological fluids be capable of real-time measurement that can respond to changes in concentration. However, the device disclosed in Patent Document 1 had the problem that it took time for the output of the electrical signal to stabilize after contact with the liquid, in other words, the time until measurement was possible ("waiting time") was long. In addition, the device had the problem that the response of the output of the electrical signal to changes in the concentration of the substance to be measured was poor, in other words, there was a large "time delay".

[0006] The present invention provides an electrochemical sensor that has a short waiting time before measurement is possible, and after measurement begins, has little time delay in the measured value in response to changes in the concentration of the substance being measured. [Means for solving the problem]

[0007] A representative embodiment of the present invention is described below. [1] An electrochemical sensor for measuring the concentration of a substance to be measured in a solution, The electrode portion includes an anode electrode comprising an anode layer and a first liquid-permeable layer, and a cathode electrode comprising a cathode layer and a second liquid-permeable layer. The anode layer has a substance fixed to it that has the ability to recognize the substance to be measured. The water absorption rate of the first liquid-conducting layer is greater than the water absorption rate of the second liquid-conducting layer. sensor. [2] The electrochemical sensor according to [1], wherein the substance having the ability to recognize the substance to be measured, which is immobilized on the anode layer, is an enzyme that uses the substance to be measured as a substrate. [3] The electrochemical sensor according to [1], wherein a reversibly redoxable substance is immobilized on at least one of the anode layer or the cathode layer. [4] The electrochemical sensor according to [3], wherein the redox substance is oxidized or reduced by a product produced by the reaction of the substance to be measured with the enzyme. [5] The electrochemical sensor according to [1], wherein a catalyst that promotes the transfer of electrons by the cathode electrode is fixed to the cathode layer. [6] The substance fixed to the anode layer that has the ability to recognize the substance to be measured has the function of oxidizing the substance to be measured, A substance capable of reducing oxygen is immobilized on the cathode electrode. [1] The electrochemical sensor described above. [7] The electrochemical sensor according to [6], wherein the substance having the ability to reduce oxygen, which is fixed to the cathode electrode, is an enzyme having the ability to reduce oxygen. [8] The electrochemical sensor according to [1], wherein the anode layer, the first fluid-permeable layer, the cathode layer, and the second fluid-permeable layer are stacked in this order in the electrode portion. [9] A method for measuring the concentration of a substance to be measured in a solution, Supplying the solution to the anode electrode of the electrochemical sensor described in any one of items (1) to (8); and, To measure the electrical signal corresponding to the concentration of the substance being measured, Methods that include...

[10] The method described in [9], wherein the measurement of the electrical signal includes any of the following: The anode and cathode electrodes of the electrochemical sensor are electrically connected, and the current flowing when a voltage is applied between the anode and cathode electrodes is measured; To measure the potential difference between the anode electrode and the cathode electrode of the electrochemical sensor; or, Connecting a load between the anode electrode and the cathode electrode of the electrochemical sensor, and directly or indirectly measuring at least one of the current flowing through the load, the voltage applied to the load, and the power consumed by the load. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide an electrochemical sensor that has a short waiting time before measurement becomes possible, and after measurement starts, has little time delay in the measured value in response to changes in the concentration of the substance being measured. [Brief explanation of the drawing]

[0009] [Figure 1] Schematic cross-sectional view (A) and top view (B) of the electrode portion of the electrochemical sensor of Example 1. [Figure 2] Schematic cross-sectional view (A) and top view (B) of the electrode portion of the electrochemical sensor of Example 2. [Modes for carrying out the invention]

[0010] The inventors have found that in an electrochemical sensor comprising 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, when the water absorption rate of the first liquid-permeable layer is greater than the water absorption rate of the second liquid-permeable layer, the sensor can measure the change in the concentration of a substance to be measured over time with a short waiting time (hereinafter also simply referred to as "waiting time") and with a short time delay.

[0011] Therefore, the present invention provides an electrochemical sensor for measuring the concentration of a substance to be measured in a solution. The electrochemical sensor of the present invention (which may be abbreviated as "sensor" in this specification below) The electrode portion includes an anode electrode comprising an anode layer and a first liquid-permeable layer, and a cathode electrode comprising a cathode layer and a second liquid-permeable layer. The anode layer has a substance fixed to it that has the ability to recognize the substance to be measured. The water absorption rate of the first liquid-conducting layer is greater than the water absorption rate of the second liquid-conducting layer.

[0012] The aforementioned waiting time and time delay, as well as the mechanism for reducing them, as described above, are presumed to be as follows, although this is not definitive.

[0013] In the measurement using an electrochemical sensor, first, a standard solution (e.g., water) is brought into contact with the anode and the cathode. By contacting the standard solution, an electrochemical reaction occurs and the potentials of the anode and the cathode change. This potential change continues until the diffusion of the standard solution becomes steady or, in the case of measuring the current by applying a predetermined voltage between the anode and the cathode using the chronoamperometry method, until the potential difference between the anode and the cathode becomes the applied voltage. In order to reduce the waiting time until the sensor becomes measurable, it is necessary to quickly bring the potentials of the anode and the cathode after contact with the standard solution to a steady state. Generally, it is considered that if the contact area between the electrode and the solution is increased and the rate of the electrochemical reaction is increased, the potential of the electrode will quickly become steady. However, since oxygen in the air is involved in the reaction at the cathode in the sensor, when the contact area between the cathode and the solution increases, the contact area of that cathode with the air decreases and the reaction rate slows down. In other words, the waiting time until the sensor becomes measurable becomes longer. Also, in the sensor, the reaction between the substance to be measured (substrate) and the enzyme occurs at the anode. The substrate contained in the solution to be measured around the cathode moves to the anode by diffusion and then reacts with the enzyme. Therefore, when the amount of the solution around the cathode increases, the reaction at the anode is delayed. In other words, the time delay of the sensor with respect to the change in the substrate concentration becomes larger. The sensor of the present invention has a structure in which a solution is supplied to the anode layer and the cathode layer of the electrodes through liquid-permeable layers contacting each of them, and the water absorption rate of the liquid-permeable layer (first liquid-permeable layer) contacting the anode layer is greater than the water absorption rate of the liquid-permeable layer (second liquid-permeable layer) contacting the cathode layer. According to this structure, the anode layer can be sufficiently in contact with the solution through the first liquid-permeable layer, and since a part of the cathode layer remains in contact with air, the electrochemical reactions in both the anode layer and the cathode layer proceed smoothly. As a result, the time (waiting time) until the output of the sensor stabilizes is shortened. Also, according to this structure, the amount of the measurement solution held on the cathode electrode side is less than that on the anode electrode, so the time lag with respect to the change in the substrate concentration is reduced. Therefore, according to the present invention, it is possible to provide an electrochemical sensor with a short waiting time until measurement becomes possible and a small time lag with respect to the change in the concentration of the measured substance after the start of measurement.

[0014] <Electrochemical sensor> Hereinafter, the structure of the electrochemical sensor of the present invention will be described.

[0015] (Electrode part) The electrode part of the sensor of the present invention includes 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. Preferably, in the anode electrode, the anode layer and the first liquid-permeable layer are laminated, and in the cathode electrode, the cathode layer and the second liquid-permeable layer are laminated.

[0016] The anode electrode and the cathode electrode in the electrode part may be laminated or juxtaposed so as not to overlap, and there is no limitation on their arrangement as long as the electrical connection is interrupted. In one embodiment, in the electrode part, the anode electrode and the cathode electrode are juxtaposed without overlapping. In a preferred embodiment, in the electrode part, the anode layer, the first liquid-permeable layer, the cathode layer, and the second liquid-permeable layer are laminated in this order.

[0017] The anode electrode preferably further includes a lead layer disposed between the anode layer and the first fluid-permeable layer for electrically connecting the layers. The cathode electrode preferably further includes a lead layer disposed between the cathode layer and the second fluid-permeable layer for electrically connecting the layers.

[0018] (Anode layer, cathode layer) The materials of the anode layer and the cathode layer are not particularly limited, as long as they each contain a conductive material. From the viewpoint of enabling a larger amount of enzyme to be supported, the conductive material is preferably a porous conductive material. Preferred examples of the porous conductive material include porous carbon material and porous nickel, from the viewpoint of enabling a larger amount of enzyme to be supported. From the viewpoint of disposability and biocompatibility, porous carbon material is more preferable. Commercially available porous conductive material can be used. For example, an example of a commercially available porous carbon material is Knobel® (manufactured by Toyo Tanso Co., Ltd.). The anode layer and the cathode layer may contain metal powder from the viewpoint of improving conductivity.

[0019] The anode layer and the cathode layer may each contain a binder (binder resin). Examples of binders include polyvinylidene fluoride (PVDF), polyvinylidene fluoride-hexafluoropropylene copolymer, styrene-butadiene rubber (SBR), and polyacrylonitrile, which can be used individually or in combination of two or more.

[0020] From the viewpoint of enabling the immobilization of a larger amount of enzyme, the anode layer is preferably 20 μm or more, more preferably 30 μm or more in thickness. On the other hand, from the viewpoint of the passability of the solution to be measured, the thickness of the anode layer is preferably 200 μm or less, more preferably 180 μm or less, even 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.

[0021] From the viewpoint of enabling the immobilization of a larger amount of enzyme, the cathode layer is preferably 20 μm or more, more preferably 30 μm or more in thickness. On the other hand, from the viewpoint of the passability of the solution to be measured, the thickness of the cathode layer is preferably 200 μm or less, more preferably 180 μm or less, even 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.

[0022] The anode layer and the cathode layer can each be formed, for example, by applying a paste containing the porous conductive material (e.g., porous carbon material), the binder, a solvent, and optionally metal powder, onto a lead layer formed on each substrate by screen printing or the like, firing the coating film by heating, and repeating this process until the anode layer and cathode layer reach the desired thickness.

[0023] The lead layers of the anode electrode and the cathode electrode can be formed, for example, by applying a paste containing a conductive material commonly used as a lead layer material to the substrate of each electrode (e.g., the first fluid-permeable layer and the second fluid-permeable layer) and then heating and firing it.

[0024] (1st liquid passing layer, 2nd liquid passing layer) The first and second liquid-permeable layers each absorb the solution to be measured and maintain the electrochemical reaction of the anode and cathode layers in contact with them. Furthermore, the first and second liquid-permeable layers may also serve as substrates if the anode and cathode layers, or the lead layer if necessary, are formed by coating as described above.

[0025] The materials of the first and second permeable layers are not particularly limited, as long as the water absorption rate of the first permeable layer is greater than the water absorption rate of the second permeable layer, and as long as the materials have good permeability to the solution to be measured. For example, the materials of the first and second permeable layers are porous materials such as paper, nonwoven fabric, cloth, and membrane filters. Of these, paper and cloth made from natural materials are preferred from the viewpoint of disposability and biocompatibility. More preferably, from the viewpoint of the range of adjustment of the water absorption rate, the materials of the first and second permeable layers are Japanese paper (washi). Commercially available porous materials can be used.

[0026] From the viewpoint of increasing the contact area between the anode layer and the solution to be measured, the first liquid-permeable layer has a water absorption rate of preferably 5 mm / min or more, more preferably 10 mm / min or more, even more preferably 15 mm / min or more, and even more preferably 20 mm / min or more. On the other hand, from the viewpoint of improving the detection sensitivity of the substance to be measured in the anode layer, the water absorption rate of the first liquid-permeable layer is preferably 90 mm / min or less, more preferably 70 mm / min or less, even more preferably 50 mm / min or less, and even more preferably 30 mm / min or less.

[0027] From the viewpoint of maintaining contact between the cathode layer and the solution to be measured, the water absorption rate of the second liquid-permeable layer should be greater than 0 mm / min, preferably 1 mm / min or more, more preferably 3 mm / min or more, and even more preferably 5 mm / min or more. On the other hand, from the viewpoint of reducing the amount of solution to be measured contained in the cathode layer, the water absorption rate of the second liquid-permeable layer should be preferably 30 mm / min or less, more preferably 25 mm / min or less, even more preferably 20 mm / min or less, and even more preferably 15 mm / min or less.

[0028] However, in the sensor of the present invention, the water absorption rate of the first fluid-permeable layer is greater than that of the second fluid-permeable layer. That is, the ratio of the water absorption rates of the first fluid-permeable layer to the second fluid-permeable layer (first fluid-permeable layer / second fluid-permeable layer) is greater than 1. From the viewpoint of shortening the standby time of the sensor of the present invention, the ratio of the water absorption rates (first fluid-permeable layer / second fluid-permeable layer) is preferably 1.2 or more, more preferably 1.5 or more, even more preferably 2.0 or more, and even more preferably 2.5 or more. On the other hand, from the viewpoint of preventing the accumulation of the solution to be measured inside the sensor, the ratio of the water absorption rates (first fluid-permeable layer / second fluid-permeable layer) is preferably 10 or less, more preferably 8 or less, even more preferably 6 or less, and even more preferably 5 or less. In this specification, the water absorption rate of the fluid-permeable layer is a value measured according to the Klem method (JIS P8141:2004).

[0029] The water absorption rates of the first and second fluid-permeable layers can be made different by manufacturing them from materials with different water absorption rates. Specifically, a material with higher water absorption can be used for the first fluid-permeable layer. Alternatively, the water absorption rate of the first fluid-permeable layer can be made higher than that of the second fluid-permeable layer by applying a treatment to the material of the second fluid-permeable layer to reduce its water absorption (e.g., a water-repellent treatment). Specifically, the water absorption rate of the first fluid-permeable layer can be made higher than that of the second fluid-permeable layer by using the same Japanese paper for the first and second fluid-permeable layers, but applying a water-repellent treatment only to the Japanese paper used for the second fluid-permeable layer.

[0030] The first and second fluid-permeable layers may be provided with a function to prevent electrical connection between the anode electrode and the cathode electrode. Alternatively, a separator may be placed between the anode electrode and the cathode electrode to prevent electrical connection between them. As the separator, a separator commonly used as a battery material can be used. Preferably, the separator is a porous polymer sheet, paper, etc., and more preferably a porous polyethylene sheet, a porous polypropylene sheet, etc.

[0031] (enzyme) A substance having the ability to recognize the substance to be measured by the sensor of the present invention (hereinafter also referred to as the "recognition substance") is immobilized on the anode layer. The recognition substance is not particularly limited as long as it has the ability to selectively recognize the substance to be measured. From the viewpoint of availability and ease of handling, the recognition substance is preferably a biologically derived substance (such as a ligand). Examples of the recognition substance include enzymes, nucleic acids, antibodies, receptors, organelles, microorganisms, cells, tissues, etc., of which enzymes are preferred from the viewpoint of ease of handling. Preferably, the recognition substance immobilized on the anode layer is an enzyme that uses the substance to be measured as a substrate.

[0032] The enzyme immobilized on the anode layer (anodic enzyme) can be either an enzyme that can directly exchange electrons with the electrode through a reaction with the substance to be measured, or an enzyme that can exchange electrons with the working electrode via a product of a reaction with the substance to be measured. Examples of such anodic enzymes include glucose oxidase (GOD), glucose dehydrogenase (GDH), L-lactate oxidase, L-lactate dehydrogenase, fructose dehydrogenase, invertase, amylase, urease, uricase, amino acid oxidase, bilirubin oxidase, cholesterol oxidase, alcohol oxidase, alcohol dehydrogenase, and creatininase. However, there are no particular restrictions on the type of enzyme used, and it can be appropriately selected depending on the substance to be measured. For example, if the substance to be measured is glucose, examples of enzymes that can be used include glucose oxidase (GOD) and glucose dehydrogenase (GDH), and if the substance to be measured is lactate, examples of L-lactate oxidase and L-lactate dehydrogenase.

[0033] A catalyst that promotes the transfer of electrons by the cathode electrode may be immobilized on the cathode layer. For example, if the enzyme immobilized on the anode layer has the function of oxidizing the substance to be measured, a catalyst having the ability to reduce oxygen is immobilized on the cathode layer. The catalyst immobilized on the cathode layer is preferably an organic substance, and more preferably an enzyme, from the viewpoint of disposability and biocompatibility. An example of an enzyme immobilized on the cathode layer (cathode enzyme) is an enzyme having the ability to reduce oxygen, and examples of such enzymes include bilirubin oxidase and laccase.

[0034] (Redox substances) To improve the sensitivity and accuracy of measurements by the sensor, a mediator (substance that mediates the exchange of electrons with the electrodes) may be immobilized on the anode layer and the cathode layer. The mediator may be any substance that can be reversibly oxidized or reduced (hereinafter also referred to as "oxidation-reduction substance"). Preferably, the oxidation-reduction substance is an aromatic substance that can be reversibly oxidized or reduced. Examples of mediators immobilized on the anode layer (anodic mediators) include benzoquinone, 1,2-naphthoquinone, 1,4-naphthoquinone, thionine acetate, azure A, azure B, azure C, phenothiazine, methylene blue, and Prussian blue, of which thionine acetate, azure A, azure B, and azure C are preferred. Examples of mediators (cathode mediators) fixed to the cathode electrode include ABTS(2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)).

[0035] In this specification, "immobilization" of a substance such as a recognition substance or mediator to the anode or cathode layer means that the substance is in a state where it does not move relative to the anode or cathode layer. For example, the recognition substance may be directly immobilized on the anode layer, or it may be indirectly immobilized via another substance such as a mediator, or a layer of a mixture of the recognition substance and the mediator may be formed on the anode layer. The same applies to the cathode layer. Since the recognition substance or mediator is immobilized on the anode or cathode layer by chemical or physical bonding, it remains substantially immobilized on the anode or cathode layer even after the electrode portion has been brought into contact with the solution to be measured.

[0036] There are no particular restrictions on the fixing position of the recognition substance or mediator on the anode or cathode layer, but from the viewpoint of more effectively obtaining the effect of promoting oxidation or reduction reactions, it is preferable that the substance be fixed on the surface of the anode or cathode layer where contact with the solution to be measured begins and in the vicinity thereof.

[0037] (covering material) To prevent misalignment of the anode and cathode electrodes, the electrode portion may be covered and fixed with a covering material. For example, the electrode portion can be fixed with the covering material by preparing a pair of covering materials, sandwiching the electrode portion between the pair of covering materials, and sealing their periphery. The covering material is not particularly limited as long as it allows the solution to be measured to pass through, and mesh material is one example. When measuring substances in sweat, a preferred example of the covering material from the viewpoint of smooth supply of sweat to the anode layer is a sweat-absorbing sports mesh.

[0038] <Measurement of the substance to be measured> The sensor of the present invention can be used to measure the concentration of a substance to be measured in a solution. The substance to be measured can be any substance that can be selectively recognized by the recognition substance of the anode electrode, and its type is not particularly limited. Preferably, the substance to be measured is an organic substance, such as biologically derived substances such as glucose, lactic acid, fructose, uric acid, amino acids, bilirubin, cholesterol, alcohol, creatinine, and ascorbic acid, as well as viruses and antibodies in environmental samples. Applications of the sensor of the present invention include, for example, a biosensor that measures organic substances and bodily fluids in biological samples, and a biosensor that senses viruses and antibodies in environmental samples. More specifically, examples include a blood glucose sensor that measures the sugar concentration in the blood, a urine glucose sensor that measures the sugar concentration in the urine, a lactic acid sensor that measures the lactic acid concentration in sweat, and a virus sensor that measures viruses present in food or sewage.

[0039] The following describes one embodiment of the operation of the sensor of the present invention in the process of measuring the concentration of a substance to be measured. In this embodiment, lactic acid is used as the substance to be measured, and an enzyme (lactate oxidase) that uses the substance to be measured as a substrate is used as the recognition substance contained in the anode electrode of the sensor. However, the substance to be measured and the recognition substance that can be used in the present invention are not limited to these.

[0040] In this embodiment, an enzyme is immobilized as a recognition substance, and a redox substance is immobilized as a mediator on the anode electrode of the sensor. When the enzyme on the anode electrode reacts with lactic acid in the presence of the mediator, the lactic acid is converted to pyruvate, and a reduced mediator is produced. This causes a change in the ratio of oxidized to reduced forms in the redox substance, and the potential difference between the anode electrode and the cathode electrode changes in accordance with this change. The electrical signal produced by this reaction between the substance to be measured and the enzyme should be measured.

[0041] As for the method of measuring the electrical signal, conventional methods for measuring electrical signals can be used, such as chronoamperometry, cyclic voltammetry, pulse voltammetry, a method for measuring the slope of the open-circuit potential (OCP) with respect to time, and a method for measuring impedance.

[0042] In one embodiment, the anode electrode and cathode electrode of the sensor of the present invention are electrically connected, and the current flowing when a voltage is applied between the anode electrode and the cathode electrode is measured. For example, a linear function representing the relationship between the concentration of the substance to be measured and the magnitude of the potential difference between the anode electrode and the cathode electrode can be determined, and the concentration of the substance to be measured corresponding to the magnitude of the measured potential difference can be calculated based on this linear function. More preferably, the rate of change of the potential difference between the anode electrode and the cathode electrode with respect to time is determined. Based on the linear function representing the relationship between the concentration of the substance to be measured and the rate of change of the potential difference with respect to time, the concentration of the substance to be measured can be calculated.

[0043] In another embodiment, the magnitude of the current flowing between the anode electrode and the cathode electrode of the sensor of the present invention is measured. For example, a linear function representing the relationship between the concentration of the substance to be measured and the magnitude of the current can be determined, and based on this linear function, the concentration of the substance to be measured corresponding to the measured magnitude of the current can be calculated.

[0044] In yet another embodiment, a load is connected between the anode and cathode electrodes of the sensor of the present invention, and at least one of the current flowing through the load, the voltage applied to the load, or the power consumed by the load is measured directly or indirectly. A linear function representing the relationship between the measured value and the concentration of the substance to be measured can be obtained, and the concentration of the substance to be measured corresponding to the measured value can be calculated based on the linear function.

[0045] <Measurement method> Next, a method for measuring a substance to be measured in a solution using the sensor of the present invention (hereinafter sometimes abbreviated as "the measurement method of the present invention") will be described.

[0046] The measurement method of the present invention includes measuring the concentration of a substance to be measured in a solution using the sensor of the present invention. More specifically, the measurement of the concentration of the substance to be measured includes the following steps: Supplying the solution (solution to be measured) to the anode electrode of the sensor; and, To measure the electrical signal corresponding to the concentration of the substance being measured.

[0047] The following are exemplary embodiments of the measurement of the electrical signal in the measurement method of the present invention: The anode electrode and cathode electrode of the aforementioned sensor are electrically connected, and the current flowing when a voltage is applied between the anode electrode and the cathode electrode is measured; Measuring the potential difference between the anode electrode and the cathode electrode of the aforementioned sensor; or, Connecting a load between the anode electrode and cathode electrode of the sensor, and directly or indirectly measuring at least one of the following: the current flowing through the load, the voltage applied to the load, or the power consumed by the load.

[0048] The signals, such as current or voltage, obtained at the electrode may be subjected to processing such as amplification, filtering, or differentiation as necessary. Circuits for these processes can be provided within the sensor of the present invention. Alternatively, the electrical signals measured by the sensor may be transmitted externally and processed by external equipment.

[0049] Therefore, the sensor of the present invention may further include a circuit section that includes a circuit for performing processing such as amplification, filtering, and differentiation on the signal from the electrode section. The sensor of the present invention may further include an external transmission means for transmitting the electrical signal measured by the sensor to an external source. The sensor of the present invention may further include a housing for protecting the electrode section and the circuit section, and fixing means for detachably fixing the sensor to a substrate such as skin.

[0050] The calculation of the concentration of the substance to be measured based on the electrical signal measured by the sensor is performed by a device such as a computer connected to the sensor. Therefore, the present invention can further provide a device for measuring the concentration of a substance to be measured, comprising the sensor of the present invention and a device for calculating the concentration of the substance to be measured from the electrical signal measured by the sensor. An example of such a device is a computer equipped with a central processing unit (CPU) that includes a calculation unit for calculating the concentration of the substance to be measured. Furthermore, the device may include a memory storing the linear function used to calculate the concentration of the substance to be measured, an auxiliary storage unit (storage) for storing the electrical signal from the sensor, and an output unit for displaying the calculation result of the concentration of the substance to be measured.

[0051] As exemplary embodiments of the present invention, the following substances, manufacturing methods, uses, or methods are further disclosed herein. However, the present invention is not limited to these embodiments.

[0052] [1] An electrochemical sensor for measuring the concentration of a substance to be measured in a solution, The electrode portion includes an anode electrode comprising an anode layer and a first liquid-permeable layer, and a cathode electrode comprising a cathode layer and a second liquid-permeable layer. The anode layer has a substance fixed to it that has the ability to recognize the substance to be measured. The water absorption rate of the first liquid-conducting layer is greater than the water absorption rate of the second liquid-conducting layer. sensor. [2] Preferably, the electrochemical sensor according to [1], wherein the substance having the ability to recognize the substance to be measured, which is immobilized on the anode layer, is an enzyme that uses the substance to be measured as a substrate. [3] Preferably, an electrochemical sensor according to [1] or [2], wherein a reversibly redoxable substance is immobilized on at least one of the anode layer or the cathode layer. [4] Preferably, the redox substance is oxidized or reduced by a product produced by the reaction of the substance to be measured with the enzyme, according to [3]. [5] Preferably, an electrochemical sensor according to any one of [1] to [4], wherein a catalyst that promotes the transfer of electrons by the cathode electrode is fixed to the cathode layer. [6] Preferably, the substance fixed in the anode layer that has the ability to recognize the substance to be measured has the function of oxidizing the substance to be measured, Preferably, a substance having the ability to reduce oxygen is immobilized on the cathode electrode. An electrochemical sensor as described in any one of items [1] to [5]. [7] Preferably, the substance having the ability to reduce oxygen fixed to the cathode electrode is an enzyme having the ability to reduce oxygen, according to [6], the electrochemical sensor. [8] Preferably, in the electrode portion, the anode layer, the first permeable layer, the cathode layer, and the second permeable layer are stacked in this order, the electrochemical sensor according to any one of [1] to [7]. [9] The electrochemical sensor according to any one of [1] to [8], wherein the thickness of the anode layer is preferably 20 μm or more, more preferably 30 μm or more, on the other hand preferably 200 μm or less, more preferably 180 μm or less, even 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.

[10] The electrochemical sensor according to [9], wherein the thickness of the cathode layer is preferably 20 μm or more, more preferably 30 μm or more, on the other hand preferably 200 μm or less, more preferably 180 μm or less, even 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.

[11] The electrochemical sensor according to any one of [1] to

[10] , wherein the water absorption rate of the first liquid-permeable layer is preferably 5 mm / min or more, more preferably 10 mm / min or more, even more preferably 15 mm / min or more, even more preferably 20 mm / min or more, as measured by the Klem method (JIS P8141:2004), on the other hand, preferably 90 mm / min or less, more preferably 70 mm / min or less, even more preferably 50 mm / min or less, and even more preferably 30 mm / min or less.

[12] The electrochemical sensor according to

[11] , wherein the water absorption rate of the second liquid-permeable layer is preferably 1 mm / min or more, more preferably 3 mm / min or more, and even more preferably 5 mm / min or more, as measured by the Klem method (JIS P8141:2004), on the other hand, preferably 30 mm / min or less, more preferably 25 mm / min or less, even more preferably 20 mm / min or less, and even more preferably 15 mm / min or less.

[13] The electrochemical sensor according to any one of [1] to

[12] , wherein the ratio of the water absorption rates of the first fluid-permeable layer to the second fluid-permeable layer (water absorption rate of the first fluid-permeable layer / water absorption rate of the second fluid-permeable layer) is preferably 1.2 or more, more preferably 1.5 or more, even more preferably 2.0 or more, and even more preferably 2.5 or more, while on the other hand, preferably 10 or less, more preferably 8 or less, even more preferably 6 or less, and even more preferably 5 or less.

[14] A method for measuring the concentration of a substance to be measured in a solution, Supplying the solution to the anode electrode of the electrochemical sensor described in any one of items [1] to

[13] ; and, To measure the electrical signal corresponding to the concentration of the substance being measured, Methods that include...

[15] The method described in

[14] , wherein the measurement of the electrical signal includes any of the following: The anode and cathode electrodes of the electrochemical sensor are electrically connected, and the current flowing when a voltage is applied between the anode and cathode electrodes is measured; To measure the potential difference between the anode electrode and the cathode electrode of the electrochemical sensor; or, Connecting a load between the anode electrode and the cathode electrode of the electrochemical sensor, and directly or indirectly measuring at least one of the current flowing through the load, the voltage applied to the load, and the power consumed by the load. [Examples]

[0053] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0054] 1. Water-repellent treatment of Japanese paper Washi paper (Shoun, a type of Japanese paper made by Hanshiya) was soaked in a water-repellent agent (NY-50, manufactured by Nikka Chemical Co., Ltd.), dried at room temperature for 24 hours, and the surface was made water-repellent.

[0055] 2. Measurement of water absorption rate The water absorption rate of the permeable layer (Japanese paper) was measured using the Klem method (JIS P8141:2004). A piece of Japanese paper cut to a width of 15 mm and a length of 300 m was prepared. A marking line was drawn 15 mm from one of the shorter sides, and a weight clip was attached between the marking line and the shorter side. The Japanese paper was then suspended so that the marking line was at the water surface, and the height of the water surface rise from the marking line was measured. The height was recorded every 30 seconds for 10 minutes, the water absorption rate was calculated from the value during the first 30 seconds, and the value after 10 minutes was defined as the degree of water absorption (Klem absorption height).

[0056] 3. Fabrication of the anode electrode Untreated Japanese paper (Shoun, a type of calligraphy paper made by Hanshiya) was used as the base material (first permeable layer). Carbon paste (JELCON, CH-8, manufactured by Jujo Chemical) was applied to one main surface of the Japanese paper and baked at 120°C for 15 minutes. This application and baking of carbon paste was repeated six times, and a total of six carbon layers were laminated to create the lead layer. Next, 0.75 g of porous carbon Knobel (manufactured by Toyo Tanso), 2.6 mL of N-methyl-2-pyrrolidone (special grade WAKO Pure Chemical), and 7.6 mL of PVDF resin (manufactured by Kureha, KF Polymer; L#9305) were added and mixed to prepare a paste containing porous carbon Knobel (Toyo Tanso Knobel). The prepared paste was applied onto the lead layer described above and baked at 60°C for 24 hours. The application and baking of the paste was repeated three times to laminate a total of three porous carbon layers. In this way, an anode layer and a first permeable layer consisting of three porous carbon layers were laminated. Next, thionine acetate (manufactured by Tokyo Chemical Industry Co., Ltd.) was dissolved in methanol to prepare a 50 mM thionine acetate methanol solution. 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 fix the anode mediator to the anode layer. Then, L-lactate oxidase (LCO301, manufactured by Toyobo Co., Ltd.) was dissolved in 0.1 M PBS solution (pH 7.4) to prepare an L-lactate oxidase solution. 20 μL (enzyme amount 40 U) of this L-lactate oxidase solution was applied to the anode layer to fix the anode enzyme to the anode layer. In this way, an anode electrode was prepared containing an anode layer with the anode mediator and anode enzyme fixed, and a first liquid-permeable layer.

[0057] 4. Fabrication of the cathode electrode Water-repellent treated Japanese paper (Hanshiya Seigansenshi Shoun) was used as the base material (second permeable layer). On one main surface of the Japanese paper, a lead layer consisting of six carbon layers was formed in the same manner as in "1. Fabrication of the anode electrode," and then three porous carbon layers were formed. In this way, a cathode layer consisting of three porous carbon layers and the second permeable layer were laminated. Next, bilirubin oxidase (WAK-AMA-BO3, manufactured by Amano Enzyme) was dissolved in 0.1 M PBS solution (pH 7.4) to obtain a bilirubin oxidase solution. 20 μL (2.5 U of enzyme) of the bilirubin oxidase solution was applied to the cathode layer to immobilize the cathode enzyme. In this way, a cathode electrode was prepared containing the enzyme-immobilized cathode layer and a second permeable layer.

[0058] 5. Fabrication of the electrode portion of the electrochemical sensor (Example 1) Half of a 12mm x 25mm square piece of Japanese paper (6mm x 25mm) was treated to be water-repellent according to 1. above. A 5mm x 25mm square anode electrode (anode layer thickness 40μm) was fabricated on the untreated portion of the Japanese paper (first permeable layer) according to 3. above, and a 5mm x 25mm square cathode electrode (cathode layer thickness 40μm) was fabricated on the water-repellent treated portion of the Japanese paper (second permeable layer) according to 4. above. The resulting electrodes were covered and sealed with a sports mesh (Teijin, Sorel Up sweat-absorbing material) to create the electrode portion of an electrochemical sensor.

[0059] Figure 1 shows a schematic cross-sectional view (A) and a top view (B) of the electrode portion of the sensor fabricated in Example 1. The electrode portion shown in Figure 1 includes an anode electrode comprising an anode layer and a first fluid-permeable layer, and a cathode electrode comprising a cathode layer and a second fluid-permeable layer. The anode layer and the first fluid-permeable layer are laminated, and the cathode layer and the second fluid-permeable layer are laminated. Furthermore, the anode electrode and the cathode electrode are placed side by side and sandwiched between covering materials (sports mesh) above and below. The arrows in Figure 1A indicate the direction of movement of the solution to be measured. Japanese paper is used for the first and second liquid-permeable layers. The Japanese paper in the second liquid-permeable layer is treated to be water-repellent, and as a result, the water absorption rate of the first liquid-permeable layer is greater than that of the second liquid-permeable layer. On the conductive layer of the anode layer, an oxidation-reduction substance and an enzyme (not shown) are immobilized on the side opposite to the surface in contact with the first fluid-permeable layer. The enzyme immobilized on the anode layer (hereinafter also referred to as "anodic enzyme") selectively reacts with the substance to be measured by the sensor of the present invention and oxidizes it. On the cathode layer, an enzyme (not shown) having the ability to reduce oxygen is immobilized on the side opposite to the surface in contact with the second fluid-permeable layer.

[0060] (Example 2) Two sheets of 6mm x 25mm square Japanese paper were prepared, and one sheet was treated with a water-repellent coating according to step 1 above. Using the untreated Japanese paper (first permeable layer), a 5mm x 25mm square anode electrode (anode layer thickness 40μm) was fabricated according to step 3 above, and using the water-repellent treated Japanese paper (second permeable layer), a 5mm x 25mm square cathode electrode (cathode layer thickness 40μm) was fabricated according to step 4 above. Next, the cathode electrode and anode electrode were laminated in this order. The resulting laminate was covered with a sports mesh (Teijin, Sorel Up sweat-absorbing material) to create the electrode portion of the electrochemical sensor.

[0061] Figure 2 shows a schematic cross-sectional view (A) and a top view (B) of the electrode portion of the sensor fabricated in Example 2. The electrode portion shown in Figure 2 has a structure in which the anode electrode and the cathode electrode are stacked and sandwiched between covering materials (sports mesh) on the top and bottom. The arrow in Figure 2A indicates the direction of movement of the solution to be measured. The Japanese paper of the second liquid-permeable layer is treated with a water-repellent coating, and as a result, the water absorption rate of the first liquid-permeable layer is greater than that of the second liquid-permeable layer.

[0062] (Comparative Example 1) The electrode portion of the electrochemical sensor was fabricated using the same procedure as in Example 1, except that the entire Japanese paper was treated with a water-repellent coating. Both the first and second liquid-permeable layers of Japanese paper were treated with a water-repellent coating, and therefore, the water absorption rates of the first and second liquid-permeable layers were equivalent.

[0063] 6. Evaluation of electrochemical sensors The working electrode was connected to the cathode electrode of the electrode section prepared in step 5 above, and the counter electrode and reference electrode were connected to the anode electrode. Chronoamperometry (CA) measurements were performed while maintaining the potential of the cathode electrode relative to the anode electrode at +0.1 (V). Specifically, the cathode electrode was immersed in pure water for 30 seconds. After the current decrease over 10 seconds fell to less than 10 nA, 1 μL / min of phosphate buffer (pH 7.4) containing dissolved sodium L-lactate was added every minute, and the current density one minute after each addition was plotted. The time it took for the decrease in current to fall below 10 nA within 10 seconds after the start of CA measurement was evaluated as the "waiting time". In addition, the concentration of sodium L-lactate in the phosphate buffer in which the added sodium L-lactate was dissolved was changed from 0 → 1 → 3 → 5 → 10 → 15 → 25 → 50 → 50 → 25 → 15 → 10 → 5 → 3 → 1 → 0 mM, and the "time delay" for this concentration change was evaluated. Table 1 shows the "waiting time" and the time from when 50 mM sodium L-lactate phosphate buffer was added until the sensor output reached its maximum, which is defined as the "time delay".

[0064] [Table 1]

Claims

1. An electrochemical sensor for measuring the concentration of a substance to be measured in a solution, The electrode portion includes an anode electrode comprising an anode layer and a first liquid-permeable layer, and a cathode electrode comprising a cathode layer and a second liquid-permeable layer. The anode layer has a substance fixed to it that has the ability to recognize the substance to be measured. The water absorption rate of the first liquid-conducting layer is greater than the water absorption rate of the second liquid-conducting layer. sensor.

2. The electrochemical sensor according to claim 1, wherein the substance having the ability to recognize the substance to be measured, which is immobilized on the anode layer, is an enzyme that uses the substance to be measured as a substrate.

3. The electrochemical sensor according to claim 1, wherein a reversibly redoxable substance is immobilized on at least one of the anode layer or the cathode layer.

4. The electrochemical sensor according to claim 3, wherein the redox substance is oxidized or reduced by a product produced by the reaction of the substance to be measured with the enzyme.

5. The electrochemical sensor according to claim 1, wherein a catalyst that promotes the transfer of electrons by the cathode electrode is fixed to the cathode layer.

6. A substance fixed to the anode layer that has the ability to recognize the substance to be measured has the function of oxidizing the substance to be measured, A substance capable of reducing oxygen is immobilized on the cathode electrode. The electrochemical sensor according to claim 1.

7. The electrochemical sensor according to claim 6, wherein the substance having the ability to reduce oxygen, which is fixed to the cathode electrode, is an enzyme having the ability to reduce oxygen.

8. The electrochemical sensor according to claim 1, wherein in the electrode portion, the anode layer, the first fluid-permeable layer, the cathode layer, and the second fluid-permeable layer are stacked in this order.

9. A method for measuring the concentration of a substance to be measured in a solution, Supplying the solution to the anode electrode of the electrochemical sensor according to any one of claims 1 to 8; and, To measure the electrical signal corresponding to the concentration of the substance being measured, Methods that include...

10. The method according to claim 9, wherein the measurement of the electrical signal includes any of the following: The anode electrode and cathode electrode of the electrochemical sensor are electrically connected, and the current flowing when a voltage is applied between the anode electrode and the cathode electrode is measured; Measuring the potential difference between the anode electrode and the cathode electrode of the electrochemical sensor; or, Connecting a load between the anode electrode and the cathode electrode of the electrochemical sensor, and directly or indirectly measuring at least one of the current flowing through the load, the voltage applied to the load, and the power consumed by the load.

Images