Enzyme sensor

Abstract

To measure the concentrations of multiple substances in a solution with high precision. [Solution] An apparatus for measuring the concentration of a substance to be measured in a solution. The apparatus comprises an electrochemical sensor having a plurality of working electrodes and a tilt output mechanism, wherein the plurality of working electrodes include working electrode A and working electrode B, and working electrode A contains an enzyme that uses the substance to be measured as a substrate, and the output of working electrode A is connected to the tilt output mechanism, which outputs the slope of the potential difference between working electrode A and a reference electrode with respect to time.

Description

Technical Field

[0001] The present invention relates to an apparatus for measuring a substance to be measured in a solution by an electrochemical method.

Background Art

[0002] In recent years, with the increasing health consciousness, there has been a growing need to selectively detect 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 involve procedures that harm the subject's body, such as blood collection, have attracted attention. Biological fluids (such as sweat) that allow non-invasive measurement tend to have a lower concentration of organic substances than blood, and therefore, a more sensitive sensor for non-invasive measurement is required.

[0003] There is an electrochemical sensor that measures a specific substance to be measured, such as a biological substance, by an electrical signal from an electrode in contact with a solution containing the substance to be measured. Furthermore, when a film containing a substance having the ability to selectively recognize the substance to be measured is formed on the electrode of the electrochemical sensor, more selective and sensitive detection of the substance to be measured becomes possible. As such a sensor, an enzyme sensor that enables selective and sensitive detection of substances using an enzyme has attracted attention.

[0004] In monitoring the body's condition, multi-sensing for detecting a plurality of biological substances is a useful technique. For example, by measuring a plurality of biological substances, a more accurate evaluation of the body's condition becomes possible. Also, for example, by arranging a plurality of enzyme electrodes in an array, it is possible to measure the concentration distribution of the substance to be measured. Furthermore, when measuring a biological substance, by measuring values of the measurement environment, such as temperature and pH, together and correcting the measured value of the biological substance using those values, it is possible to obtain a more accurate measured value.

[0005] Non-patent document 1 describes how to simultaneously measure glucose and lactate concentrations in sweat by using a sensor array equipped with a working electrode immobilized with glucose oxidase and a working electrode immobilized with lactate oxidase, and how to improve measurement accuracy by simultaneously measuring skin temperature and using it to correct the sensor response. Patent document 1 discloses an electrochemical measurement electrode characterized in that electrodes are arranged in an array on a substrate, only the electrode portion is selectively modified with a modifier, and the electrodes have multiple groups of electrodes modified with different modifiers.

[0006] Patent Document 2 discloses a method for monitoring the concentration of a target analyte by measuring the time-dependent change in the open-circuit potential between a working electrode on which an oxidoreductase is immobilized and a reference electrode, in the presence of the target analyte. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2002-071620 [Patent Document 2] Special Publication No. 2023-553889 [Non-patent literature]

[0008] [Non-Patent Document 1] W. Gao et.al., Nature, 2016, 529, 509-514 [Overview of the project] [Problems that the invention aims to solve]

[0009] Conventional multi-sensing measurements, such as those described in Patent Document 1, employ a chronoamperometry method to measure the current flowing through the working electrode when the working electrode is set to a predetermined potential. However, when performing multi-sensing measurements using this method, the currents from multiple working electrodes interfere with each other, affecting the measurement. Furthermore, because the total current that can flow through the sensor as a whole is limited, the current that can flow through each individual working electrode is small, resulting in insufficient measurement accuracy and sensitivity. While sequential measurement of multiple working electrodes can avoid problems such as current interference, this makes simultaneous measurement from multiple electrodes impossible. In measurement methods such as those described in Patent Document 2, in order to obtain sufficient measurement accuracy, it is necessary to apply a voltage between the working electrode and the counter electrode to flow a current immediately before measuring the time-dependent change in potential between the working electrode and the counter electrode. This still cannot avoid the problem of current interference, and therefore it has the problem of not being applicable to multi-sensing measurements. For this reason, there is a need for a method for high-precision multi-sensing measurement that enables simultaneous measurement from multiple electrodes. [Means for solving the problem]

[0010] A representative embodiment of the present invention is described below. [1] An apparatus for measuring the concentration of a substance to be measured in a solution, the apparatus being: An electrochemical sensor equipped with multiple working electrodes, and Tilt output mechanism, Equipped with, The plurality of working electrodes include working electrode A and working electrode B, The working electrode A contains an enzyme that uses the substance to be measured as a substrate. The output of the working electrode A is connected to the tilt output mechanism. The slope output mechanism outputs the slope of the potential difference between the working electrode A and the reference electrode with respect to time. Device. [2] The apparatus according to [1], wherein the working electrode A contains a redox substance that can be reversibly oxidized and reduced. [3] The apparatus according to [2], wherein the potential difference between the working electrode A and the reference electrode shifts to the noble side when the redox substance is oxidized by the reaction of the substance to be measured with the enzyme at the working electrode A, and shifts to the detrimental side when the redox substance is reduced by the reaction of the substance to be measured with the enzyme at the working electrode A. [4] The apparatus according to any one of [1] to [3], wherein the working electrode B contains an enzyme that uses the substance to be measured as a substrate. [5] The apparatus according to any one of items [1] to [3], wherein the output of the working electrode B is not connected to the tilt output mechanism. [6] The apparatus according to [5], wherein the output of the working electrode B is an electric current. [7] The apparatus according to any one of [1] to [3], wherein the plurality of working electrodes further include working electrode C. [8] The apparatus according to [7], wherein the working electrode C is connected to a tilt output mechanism. [9] A method for measuring the concentrations of multiple substances to be measured in a solution, Using the apparatus described in any one of items (1) to (3), obtain an electrical signal containing information about the concentration of each of the multiple substances to be measured. Methods that include... [Effects of the Invention]

[0011] According to the present invention, in multi-sensing measurement using multiple working electrodes, the output from at least one working electrode is used as the slope of the open-circuit potential with respect to time, thereby improving adverse effects on measurement accuracy caused by current interference between the multiple electrodes. The present invention makes it possible to simultaneously and accurately measure the concentrations of multiple substances to be measured. Furthermore, the apparatus of the present invention can also be applied to measuring the concentration of the same substance to be measured at different locations in a solution. [Brief explanation of the drawing]

[0012] [Figure 1] A schematic diagram of one embodiment of the apparatus of the present invention. [Figure 2] Plan view (A) and side view (B) of the working electrode used in the example. [Figure 3] Schematic diagram explaining the measurement system for glucose concentration used in Example 1. [Figure 4] Measurement results in Example 1. A: Change in the slope of the potential difference at working electrode A according to glucose concentration (top), change in the current at working electrode B according to lactate concentration (bottom). B: Relationship between the slope of the potential difference at working electrode A and the glucose concentration in the solution (top), relationship between the current at working electrode B and the lactate concentration in the solution (bottom). [Figure 5] Temporal change in the potential difference at working electrode A output from Circuit 2. [Figure 6] Measurement results in Example 2. A: Change in the slope of the potential difference at working electrode A according to glucose concentration. B: Relationship between the slope of the potential difference at working electrode A and the glucose concentration in the solution.

Mode for Carrying Out the Invention

[0013] The present invention provides an apparatus that enables more accurate multi-sensing measurement.

[0014] The present inventor has previously found that in an enzyme sensor using a working electrode containing an enzyme that recognizes a substance to be measured, the differential value (rate of change with respect to time) of the potential difference (open circuit potential; OCP) between the working electrode and the reference electrode when no current flows through the working electrode is proportional to the concentration of the substance to be measured. The concentration of the substance to be measured can be determined from the slope of the OCP of the working electrode with respect to time.

[0015] Furthermore, the present inventor has found that even in multi-sensing measurement using a plurality of working electrodes, by providing a working electrode for determining the concentration of the substance to be measured from the slope of the OCP with respect to time, it becomes possible to reduce the interference of current between the plurality of working electrodes.

[0016] The present invention provides an apparatus for measuring the concentration of a substance to be measured in a solution. The apparatus of the present invention comprises an electrochemical sensor having a plurality of working electrodes, the plurality of working electrodes including at least working electrode A and working electrode B, each designed to measure the concentration of the substance to be measured. Of the plurality of working electrodes, at least the output of working electrode A is connected to a slope output mechanism, the slope output mechanism outputs the slope of the potential difference (OCP) between working electrode A and a reference electrode with respect to time.

[0017] The apparatus of the present invention enables the simultaneous and highly accurate measurement of the concentrations of multiple substances in a solution. Furthermore, the apparatus of the present invention can also be applied to measuring the concentration (concentration distribution) of the same substance at different locations within a solution.

[0018] The following describes exemplary embodiments of the apparatus of the present invention.

[0019] Figure 1 is a schematic diagram representing one embodiment of the apparatus of the present invention. The apparatus shown in Figure 1 includes an electrochemical sensor comprising a plurality of working electrodes, including working electrode A and working electrode B. A tilt output mechanism is connected to the output of working electrode A. The output of working electrode B is connected to an electrical signal output mechanism.

[0020] Each of the plurality of working electrodes may contain a substance (also referred to herein as "recognition substance") that has the ability to recognize a predetermined substance to be measured. When the electrochemical sensor is brought into contact with a solution containing the substance to be measured, the recognition substance contained in each working electrode reacts with the corresponding substance to be measured, or the electrochemical reaction of the substance produced by the reaction changes the electrical properties of the working electrode, thereby causing each working electrode to output an electrical signal containing information regarding the concentration of the corresponding substance to be measured.

[0021] The following provides more detailed examples of each component of the apparatus of the present invention.

[0022] <Electrochemical Sensor> The electrochemical sensor in the apparatus of the present invention comprises a plurality of working electrodes and a counter electrode (reference electrode). The working electrodes and the counter electrode are arranged so that each can come into contact with a solution containing the substance to be measured, but they do not come into electrical contact with each other.

[0023] In one embodiment, the electrode system of the electrochemical sensor is a two-electrode system consisting of a working electrode and a counter electrode (reference electrode), as shown in Figure 1. In another embodiment, the electrode system of the electrochemical sensor may be a three-electrode system, as shown in Figure 3, which further includes a reference electrode that can come into contact with a solution containing the substance to be measured, independently of the working electrode and the counter electrode. In such a three-electrode system, the reference electrode functions as the reference electrode. The working electrode, the counter electrode, and the reference electrode, if necessary, may be independent electrodes, or they may be electrodes formed on the same substrate.

[0024] The three-electrode system described above may be equipped with a potential control mechanism that controls the potential of the counter electrode so that the potential of the working electrode relative to the reference electrode is a predetermined value. The potential control mechanism is not particularly limited as long as it is a mechanism that can set the potential of the working electrode to a predetermined value relative to the reference electrode, and conventionally known mechanisms can be used. Examples of the potential control mechanism include a mechanism using an analog circuit with an operational amplifier, a mechanism using a microprocessor with a potentiostat function (for example, the ADuCM355 from Analog Devices), a mechanism that digitally converts the working electrode potential and inputs it to a microprocessor, the microprocessor calculates the counter electrode potential necessary to make the working electrode potential a predetermined value, and applies the calculated potential to the counter electrode, and a mechanism that receives working electrode potential data from an external device and applies the potential corresponding to the potential data to the counter electrode. Of these, from the viewpoint of miniaturizing the device of the present invention, a mechanism using an analog circuit with an operational amplifier is preferred.

[0025] When the plurality of working electrodes are brought into contact with a solution containing the substance to be measured, the corresponding substance undergoes an electrochemical reaction at each of the plurality of working electrodes, causing changes in the electrical state of each working electrode, such as the potential of the working electrode relative to the counter electrode, the current flowing when the working electrode and the counter electrode are electrically connected, and the impedance between the working electrode and the counter electrode. An electrical signal reflecting this change in electrical state is output from each working electrode. The output electrical signal may be any signal, such as voltage, current, power, or frequency and phase as an AC signal, but voltage or current is preferred. In one embodiment, the working electrode A outputs a signal of the potential difference between the working electrode A and the reference electrode.

[0026] When the electrochemical sensor converts the current flowing when the working electrode and the counter electrode are electrically connected into an electrical signal and outputs it, the electrochemical sensor may include a mechanism to set the potential of the working electrode relative to the counter electrode to a predetermined value. Furthermore, when the electrochemical sensor converts the impedance between the working electrode and the counter electrode into an electrical signal and outputs it, the electrochemical sensor may include a mechanism to apply a predetermined DC voltage, or an AC voltage with a predetermined amplitude and frequency, between the working electrode and the counter electrode.

[0027] [Working electrode] The number of working electrodes included in the electrochemical sensor is not particularly limited, as long as it includes at least working electrode A connected to the tilt output mechanism and working electrode B. In one embodiment, the plurality of working electrodes included in the electrochemical sensor consist of working electrode A and working electrode B. In another embodiment, the plurality of working electrodes include working electrode A and working electrode B, and one or more further working electrodes (hereinafter also referred to as working electrode C).

[0028] The output of the working electrode A is the potential difference generated between the working electrode A and the reference electrode, and the slope output mechanism connected to the working electrode A outputs the slope of this potential difference. More specifically, when the working electrode A, which contains a recognition substance, is brought into contact with a solution containing the substance to be measured on the working electrode A, the recognition substance and the corresponding substance to be measured react, or an electrochemical reaction occurs between the substance produced by this reaction, resulting in the transfer of electrons to and from the working electrode A. A signal of the potential difference between the working electrode A and the reference electrode is output and input to the slope output mechanism. The slope output mechanism outputs the slope of the output signal from the working electrode A. That is, it outputs the slope of the potential difference between the working electrode A and the reference electrode with respect to time.

[0029] Signals from working electrodes other than working electrode A are converted as necessary depending on the type of electrical signal output mechanism and output in an appropriate format. For example, the output of working electrode B may be a current, voltage, or potential difference between working electrode B and a reference electrode. The output of working electrode B may be connected to a slope output mechanism, or it may be connected to an electrical signal output mechanism different from the slope output mechanism. In one embodiment of working electrode B, when working electrode B containing a recognition substance is brought into contact with a solution containing the substance to be measured, a signal of the potential difference between working electrode B and a reference electrode is output. In another embodiment of working electrode B, when a voltage is applied between working electrode B and a counter electrode, and working electrode B is brought into contact with a solution containing the substance to be measured, a signal of the current flowing between working electrode B and the counter electrode is output. When working electrode B is connected to a slope output mechanism, it is preferable that the slope output mechanism outputs the slope of the potential difference between working electrode B and a reference electrode.

[0030] On the other hand, if the working electrode C is connected to an electrical signal output mechanism that allows current to flow through the working electrode B, it is preferable from the viewpoint of high-precision multi-sensing that it is connected to a tilt output mechanism or an electrical signal output mechanism that prevents current from flowing through the working electrode C. Furthermore, if the working electrode B is connected to a tilt output mechanism or an electrical signal output mechanism that prevents current from flowing through the working electrode B, the working electrode C may be connected to a tilt output mechanism, but may also be connected to an electrical signal output mechanism different from the tilt output mechanism. Furthermore, if there are multiple working electrodes C, and the working electrode B is connected to an electrical signal output mechanism that allows current to flow through it, it is preferable that each one is separately connected to a tilt output mechanism or an electrical signal output mechanism that prevents current from flowing through the working electrode C. Furthermore, if there are multiple working electrodes C, and the working electrode B is connected to a tilt output mechanism or an electrical signal output mechanism that prevents current from flowing through the working electrode B, it is preferable that all but at least one of the multiple working electrodes C are connected to a tilt output mechanism or an electrical signal output mechanism that prevents current from flowing through the working electrode C. In one embodiment, when the output of the working electrode B is a current, the output of the working electrode C is a voltage or a potential difference between the working electrode C and the reference electrode, and this potential difference is preferably sent to a slope output mechanism and converted into a slope. In another embodiment, when the output of the working electrode B is a voltage or a potential difference between the working electrode B and the reference electrode, and this potential difference is sent to a slope output mechanism and converted into a slope, the output of the working electrode C is a current or a voltage or a potential difference between the working electrode C and the reference electrode, and this potential difference is preferably sent to a slope output mechanism and converted into a slope.

[0031] The tilt output mechanism to which the working electrodes B and C can be connected may be the same tilt output mechanism to which the working electrode A is connected, or it may be a different mechanism to which the working electrode A is connected, depending on the type of tilt output mechanism. For example, when a computer is used as the tilt output mechanism, working electrode A and working electrode B or C can be connected to the same tilt output mechanism (i.e., the same computer).

[0032] The substances to be measured by the plurality of working electrodes may be different for each electrode, or they may be the same for some or all of the electrodes. In one embodiment, the plurality of working electrodes consist of several groups of electrodes for measuring different substances to be measured, each of which has at least one working electrode, and a common substance that reacts with a substance to be measured, but different from that of the other groups, is fixed to the electrodes of each electrode group. In another embodiment, the plurality of working electrodes measure different substances to be measured from each other. Preferably, the substances to be measured by at least working electrode A and working electrode B are different.

[0033] The working electrodes A and B are immobilized with a substance (recognition substance in this specification) that has the ability to recognize the respective substance to be measured. The substance to be measured by the working electrodes A and B can be any substance that can be selectively recognized by the recognition substance that may be contained in each electrode, and the type is not particularly limited. Preferably, the substance to be measured is an organic substance, such as bio-derived substances such as glucose, lactic acid, fructose, urea, uric acid, amino acids, bilirubin, cholesterol, alcohol, creatinine, and ascorbic acid. The recognition substances immobilized on the working electrodes A and B may be the same, but preferably, different recognition substances that recognize different substances to be measured are immobilized on the working electrodes A and B, respectively.

[0034] A recognition substance may be fixed to an operating electrode other than operating electrodes A and B (operating electrode C). Preferably, the recognition substance fixed to operating electrode C is different from that of operating electrodes A and B. If there are multiple operating electrodes C, the recognition substances fixed to each may be the same or different.

[0035] Alternatively, the working electrode C may be configured as, for example, a proton-sensitive membrane for measuring the pH of a solution, or as an ion-sensitive membrane that selectively adsorbs specific ions for detecting specific ions. Therefore, the working electrode C may measure the concentration of organic substances in the same way as the working electrodes A and B, or it may measure temperature, pH, ion concentration, etc.

[0036] In this specification, "fixation" of a substance, such as a recognition substance or a redox substance, to an electrode means that the substance is in a state where it does not move relative to the electrode. For example, the recognition substance may be directly fixed to the surface of the conductive layer of the working electrode, or it may be indirectly fixed via another substance, such as a redox substance. The redox substance is preferably fixed directly to the surface of the conductive layer. In one embodiment, the redox substance constitutes a redox layer arranged in direct contact with the surface of the conductive layer of the working electrode, and the recognition substance constitutes a recognition substance fixed layer arranged in direct contact with the redox layer. In another embodiment, a layer of a mixture of the recognition substance and the redox substance is formed on the surface of the conductive layer of the working electrode. For example, the redox layer can be formed by dropping or coating a solution containing the redox substance onto the surface of the conductive layer of the working electrode, and then drying it. The solution containing the redox substance is, for example, a dispersion in which the redox substance is dispersed in a dispersion medium. After the formation of the redox layer, any redox substance not fixed to the electrode is preferably removed by washing. As the washing solution, for example, pure water or a buffer solution can be used. Since the recognition substance is fixed to the electrode by chemical or physical bonding, it remains substantially fixed to the electrode even after the electrode is brought into contact with a solution containing the substance to be measured.

[0037] [Recognition substance] The recognition substance has the ability to selectively recognize the substance to be measured, and from the viewpoint of availability and ease of handling, biologically derived substances (such as ligands) are preferred. Examples of the recognition substance include enzymes, nucleic acids, antibodies, receptors, organelles, microorganisms, cells, and tissues, and among these, enzymes are preferred from the viewpoint of ease of handling. Preferably, at least the recognition substance of the working electrode A is an enzyme. More preferably, the recognition substances of both the working electrode A and the working electrode B are enzymes. Preferably, different enzymes are immobilized as recognition substances on the working electrode A and the working electrode B, respectively.

[0038] [enzyme] The enzyme used as the recognition substance can be either an enzyme that uses the substance to be measured as a substrate and can directly exchange electrons with the electrode through a reaction with the substrate, or an enzyme that can exchange electrons with the working electrode via the product of a reaction with the substrate. Examples of such enzymes include glucose oxidase (GOD), glucose dehydrogenase (GDH), lactate oxidase, 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 glucose contained in blood, sweat, urine, etc., is the substance to be measured, examples of enzymes that can be used include glucose oxidase (GOD) and glucose dehydrogenase (GDH).

[0039] [Redox substances] If it is difficult for the recognition material to directly exchange electrons with the working electrode, a reversibly redoxable substance (also referred to herein as "redox substance") may be further immobilized on the working electrode, and electron exchange with the working electrode may occur via the redox substance. Alternatively, even if the recognition material can directly exchange electrons with the working electrode, the redox substance may be immobilized on the working electrode to improve the sensitivity and accuracy of the measurement. Preferably, the redox substance immobilized on the working electrode is insoluble in the solution containing the substance to be measured. Here, "insoluble" in the solution for the redox substance means that when the working electrode on which the redox substance is immobilized is brought into contact with the solution, the amount of the redox substance lost from the working electrode due to dissolution into the solution is substantially zero, meaning that the solubility of the redox substance in the solution is 100 mg / L or less.

[0040] The redox substance immobilized on the working electrode is not particularly limited as long as it is a redox substance that can be oxidized or reduced by a recognition substance or a substance produced by a reaction between the recognition substance and the substance to be measured, and can transfer electrons to the electrode; conventionally known substances can be used. Examples of such redox substances include Prussian blue, Meldora blue, thionine, azur A, azur B, azur C, phenothiazine, tetrathiafulvalene, quinones such as hydroquinone and 1,4-naphthoquinone, ferrocene, ferrocene derivatives, potassium ferrocyanide, ferricyanide, osmium complex, p-aminophenol, ABTS(2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)), and others.

[0041] [Conductive layer] The conductive layer of the working electrode can be formed on a substrate. The material of the conductive layer can be selected considering its reactivity with the solution and the reaction products between the substance to be measured and the recognition substance. For example, the material of the conductive layer is a material that does not react with the solution containing the substance to be measured. Typically, the material of the conductive layer can be a metal such as gold, palladium, platinum, rhodium, indium, iridium, titanium, or copper, or a carbon material, and is preferably gold or platinum.

[0042] The conductive layer can be formed, for example, by depositing the metal at any position on the substrate. Alternatively, an electrode may be constructed by attaching a separately prepared thin metal film to the substrate. Alternatively, the conductive layer (or electrode) may be formed by conventionally known methods such as vacuum deposition, electron beam deposition, sputtering, plating, CVD, ion plating coating, inkjet printing, etc., depending on the material.

[0043] Examples of the materials for the base material include flexible materials in the form of sheets or films, composed of materials such as polyimide (PI) resin, polyester resin, polyamide resin, epoxy resin, polysulfone resin, paper made from natural materials, and cloth.

[0044] Preferably, the plurality of working electrodes (i.e., working electrodes A, B, and C) are formed from the same material, except for the recognition substance and, optionally, the oxidation-reduction substance.

[0045] [Reference electrode, counter electrode] The counter electrode and, if necessary, the reference electrode included in the electrochemical sensor can each be composed of a conductive layer formed on a substrate, similar to the working electrode. Alternatively, the counter electrode or reference electrode can be formed without using a substrate and a conductive layer. For example, the counter electrode may be formed using a metal wire, a metal plate, etc., or the reference electrode may be a glass tube with a reference electrode material such as Ag / AgCl sealed inside, or a silver / silver chloride electrode may be formed using silver / silver chloride paste on a conductive layer of the substrate. In one preferred embodiment of a two-electrode system, the conductive layer of the working electrode is gold, and the counter electrode (reference electrode) is a silver / silver chloride electrode. In one preferred embodiment of a three-electrode system, the conductive layer of the working electrode is gold, the counter electrode is platinum, and the reference electrode is a silver / silver chloride electrode.

[0046] A redox substance may also be immobilized on the counter electrode to facilitate the transfer of electrons between it and the solution. Examples of redox substances immobilized on the counter electrode include the redox substances that can be used for the working electrode as described above.

[0047] [Other components] To selectively measure the substance to be measured, a film that selectively permeates the substance to be measured may be formed on the surface of the working electrode on which the recognition substance is immobilized, in contact with the solution containing the substance to be measured. Examples of materials for the film include polymers that form hydrogels, and more specific examples of usable polymers include polycaprolactone, gelatin, gelatin methacrylate, alginate, alginate methacrylate, chitosan, chitosan methacrylate, glycol chitosan, glycol chitosan methacrylate, hyaluronic acid (HA), HA methacrylate, and other non-crosslinked natural or synthetic polymer chains. Another example of a usable polymer is a water-soluble photosensitive resin in which a photosensitive group is directly bonded to a water-soluble polymer.

[0048] <Tilt Output Mechanism> The slope output mechanism in the apparatus of the present invention is connected to the output of the working electrode A and outputs the slope (rate of change) of the potential difference between the working electrode A and the reference electrode with respect to time. Examples of the slope output mechanism include a method of outputting the derivative of the OCP of the working electrode A using a differentiating circuit, a method of digitally converting the OCP and calculating the slope of the OCP over a predetermined time period from the digital data using a computer, and a method of displaying a graph of the open-circuit voltage over time on a display device such as a liquid crystal or CRT and reading the slope from the graph. Conventional circuits can be used for the differentiating circuit. The differentiating circuit may be either a passive differentiating circuit or an active differentiating circuit. Examples of the passive differentiating circuit include a CR differentiating circuit using a capacitor and a resistor, and an RL differentiating circuit combining an inductor and a resistor. Examples of the active differentiating circuit include a differentiating circuit using an operational amplifier, a capacitor, and a resistor, and a differentiating circuit using an operational amplifier, an inductor, and a resistor. Of these, an active differentiating circuit is preferred from the viewpoint of the accuracy of the calculated differential value, and a differentiating circuit using an operational amplifier, a capacitor, and a resistor is even more preferred from the viewpoint of miniaturizing the circuit. When calculating using a computer, the predetermined time for determining the slope of the OCP is the time during which the graph of the OCP can be considered as a straight line. That is, it is preferable that the predetermined time be sufficiently smaller than the radius of curvature when the graph of the OCP is a curve, and as large as possible when the graph is a straight line.

[0049] The measurement of the substance to be measured using the working electrode A is performed without applying a voltage between the working electrode A and the reference electrode, in a state where virtually no current is flowing. Here, a state where virtually no current is flowing means a state in which no current is flowing other than unintended current leakage such as leakage current. In order to avoid the influence of such leakage current on the measurement, the input impedance of the slope output mechanism must be sufficiently high. Whether the input impedance of the slope output mechanism is sufficiently high can be confirmed using the working electrode A on which the enzyme and the redox substance are immobilized. If the redox substance is oxidized due to the reaction between the substance to be measured and the enzyme, the OCP of the working electrode A is observed to continuously shift to the noble side. If the redox substance is reduced due to the reaction between the substance to be measured and the enzyme, the OCP is observed to continuously shift to the despicable side. In such cases, it can be confirmed that the input impedance of the slope output mechanism is sufficiently high.

[0050] If the electrical signal output by the working electrode A is small, an amplification circuit may be inserted between the working electrode A and the tilt output mechanism so that the amplified electrical signal is sent to the tilt output mechanism.

[0051] <Electrical signal output mechanism> The method for measuring the substance to be measured at working electrodes other than working electrode A (working electrodes B, C, etc.) is not particularly limited as long as it is capable of measuring electrical signals. Conventional methods for measuring electrical signals can be used as such measurement methods, and examples include chronoamperometry, cyclic voltammetry, pulse voltammetry, methods for measuring the slope of OCP with respect to time, and methods for measuring impedance. From the viewpoint of reducing the effects of current interference and improving measurement accuracy and sensitivity, it is preferable to measure the slope of OCP with respect to time at all working electrodes, including working electrodes A, B, and C, or at working electrodes other than working electrode B. When using a method that passes current through the working electrode, such as chronoamperometry, a counter electrode in contact with the solution containing the substance to be measured may be used, and current may be passed between the working electrode and the counter electrode.

[0052] The outputs of working electrodes other than working electrode A (working electrodes B, C, etc.) are connected to an electrical signal output mechanism corresponding to the measurement method of the substance being measured, which is adopted for each electrode. Therefore, examples of such electrical signal output mechanisms include not only the slope output mechanism described above, but also mechanisms that output current, voltage, or impedance. Depending on the type of mechanism, the electrical signal output mechanisms to which working electrodes B and C can be connected may be the same mechanism or different mechanisms. For example, if a computer is used as the electrical signal output mechanism, working electrodes B and C can be connected to the same electrical signal output mechanism (i.e., the same computer).

[0053] If the electrical signal output by the working electrode B or C is minute, an amplification circuit may be inserted between the working electrode and the electrical signal output mechanism so that the amplified electrical signal is sent to the electrical signal output mechanism.

[0054] <Measurement of the substance to be measured> The following describes the process for measuring the concentration of a substance to be measured using the apparatus of the present invention, based on one embodiment of the apparatus of the present invention.

[0055] In one embodiment, the circuit for working electrode A in the electrochemical sensor of the present invention consists of working electrode A and a reference electrode (reference electrode). The circuit for working electrode B in the sensor consists of working electrode B, a counter electrode, and a reference electrode (reference electrode). When working electrodes A and B of the sensor and the counter electrode are brought into contact with the solution to be measured, the working electrode and the counter electrode or reference electrode are electrically connected, and measurement begins. Here, the solution to be measured may be a solution containing the substance to be measured for working electrodes A and B, but this is not necessarily required; it is sufficient if it may contain either of the substances to be measured for working electrodes A and B. In this embodiment, the substance to be measured for working electrode A is glucose, and the substance to be measured for working electrode B is lactic acid, but the substances to be measured in the present invention are not limited to these.

[0056] The glucose concentration is measured using the working electrode A without applying a voltage between the working electrode A and the counter electrode, in a state where virtually no current is flowing. The working electrode A has an oxidation-reduction substance and an enzyme (glucose oxidase) immobilized on it as a recognition substance. When the enzyme on the working electrode A reacts with glucose in the solution to be measured in the presence of water and oxygen, glucose is converted to gluconic acid and hydrogen peroxide is produced. At the same time, the hydrogen peroxide converts the oxidation-reduction substance from its reduced form to its oxidized form. This causes a change in the ratio of oxidized to reduced forms of the oxidation-reduction substance, and the potential difference between the counter electrode and the working electrode A changes in accordance with this change. In this embodiment, the potential difference between the counter electrode and the working electrode A shifts to the noble side when the oxidation-reduction substance is oxidized, and to the detrimental side when the oxidation-reduction substance is reduced.

[0057] The signal of the potential difference between the counter electrode and the working electrode A is fed into a slope output mechanism. This slope output mechanism outputs the slope (rate of change) of the potential difference with respect to time. Since the slope of the potential difference is proportional to the concentration of the substance being measured, the output from the slope output mechanism can be obtained as information about the concentration of the substance being measured (glucose).

[0058] The working electrode B has an oxidation-reduction substance and an enzyme (lactate oxidase) immobilized on it as a recognition substance. When the working electrode B and the counter electrode come into contact with the solution to be measured, the lactic acid in the solution reacts with the enzyme on the working electrode B, converting the lactic acid to pyruvate and generating hydrogen peroxide. This hydrogen peroxide converts the oxidation-reduction substance from its reduced state to its oxidized state. The current flowing between the working electrode B and the counter electrode at this time is the output signal of the working electrode B. The magnitude of the current at the working electrode B and the output of the working electrode B can be obtained as information about the concentration of the substance to be measured (lactic acid).

[0059] 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.

[0060] [1] An apparatus for measuring the concentration of a substance to be measured in a solution, the apparatus being: An electrochemical sensor equipped with multiple working electrodes, and Tilt output mechanism, Equipped with, The plurality of working electrodes include working electrode A and working electrode B, The working electrode A contains an enzyme that uses the substance to be measured as a substrate. The output of the working electrode A is connected to the tilt output mechanism. The slope output mechanism outputs the slope of the potential difference between the working electrode A and the reference electrode with respect to time. Device. [2] Preferably, the apparatus according to [1], wherein the working electrode A contains a redox substance that can be reversibly oxidized and reduced. [3] Preferably, the apparatus according to [2], wherein the potential difference between the working electrode A and the reference electrode shifts to the noble side when the redox substance is oxidized by the reaction of the substance to be measured with the enzyme at the working electrode A, and shifts to the detrimental side when the redox substance is reduced by the reaction of the substance to be measured with the enzyme at the working electrode A. [4] Preferably, the apparatus according to any one of [1] to [3], wherein the working electrode B contains an enzyme that uses the substance to be measured as a substrate. [5] Preferably, the apparatus according to [4], wherein the working electrode B contains a redox substance that can be reversibly oxidized and reduced. [6] Preferably, the substance to be measured by the working electrode B is a different substance from the substance to be measured by the working electrode A, the apparatus according to any one of [1] to [5]. [7] Preferably, the apparatus according to any one of [1] to [6], wherein the output of the working electrode B is not connected to the tilt output mechanism. [8] Preferably, the apparatus according to [7], wherein the output of the working electrode B is an electric current. [9] Preferably, the apparatus according to any one of [1] to [8], wherein the plurality of working electrodes further include working electrode C.

[10] Preferably, the apparatus according to [9], wherein the working electrode C contains an enzyme that uses the substance to be measured on the working electrode C as a substrate.

[11] Preferably, the apparatus according to

[10] , wherein the working electrode C contains a redox substance that can be reversibly oxidized and reduced.

[12] Preferably, the substance to be measured of the working electrode C is a different substance from the substance to be measured of the working electrode A and the substance to be measured of the working electrode B, the apparatus according to any one of [9] to

[11] .

[13] Preferably, the apparatus according to any one of [9] to

[12] , wherein the output of the working electrode B is an electric current, and the output of the working electrode C is a voltage or a potential difference generated between the working electrode C and a reference electrode.

[14] Preferably, the apparatus according to

[13] , wherein the working electrode C is connected to a tilt output mechanism.

[15] A method for measuring the concentrations of multiple substances to be measured in a solution, Using the apparatus described in any one of items (1) to (14), obtain an electrical signal containing information about the concentration of each of the multiple substances to be measured. Methods that include... [Examples]

[0061] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0062] (Fabrication of electrochemical sensors) The electrochemical sensor was fabricated using the following method. [Working electrode A] A working electrode with the pattern shown in Figure 2 was formed. Gold was vacuum deposited onto a substrate (polyimide film, 125 μm thick) through a metal mask to a thickness of 50 nm. Next, a mixed solution was prepared by mixing a 0.2 mass% aqueous dispersion of carbon nanotubes (CNTs) (manufactured by TUBALL) and an aqueous solution containing 1 mass% Prussian blue (PB) (redox substance, manufactured by Aldrich) in a volume ratio of 10:1. 5 μL of this mixed solution was dropped onto a 3 mm diameter circular portion of the working electrode and dried in an 80°C constant temperature bath for 10 minutes to form a Prussian blue-containing carbon nanotube layer as a redox layer.

[0063] Next, a mixed solution was prepared by mixing a PBS solution (phosphate-buffered saline, pH 7.2, Life Technology) containing 1% by mass of enzyme (glucose oxidase, Aldrich) with a 2% by mass acetic acid solution (Fujifilm Wako Pure Chemical Industries) containing 1% by mass of chitosan (Aldrich) in a volume ratio of 1:2. 10 μL of this mixed solution was dropped onto the redox layer and dried at 25°C for 20 hours. After that, the unfixed enzyme was washed off with PBS solution, and the mixture was dried at room temperature to form an enzyme-immobilized layer, thereby obtaining an enzyme-modified electrode in which the enzyme and redox substances were immobilized on the working electrode.

[0064] [Working electrode B] Except for using L-lactate oxidase (manufactured by Toyobo) instead of glucose oxidase as the enzyme, working electrode B was obtained using the same procedure as for working electrode A.

[0065] [Reference electrode (standard electrode)] A silver / silver chloride electrode was used as the reference electrode.

[0066] [Counter electrode] A platinum electrode was used as the counter electrode.

[0067] (Example 1) [Measurement System] Using the electrodes described above, the measurement system shown in Figure 3 was constructed. Circuit 1 was used to control the potential of the counter electrode so that the reference electrode was at ground potential. Working electrode A was connected to the non-inverting amplifier circuit of circuit 2, and further connected to the differentiating circuit of circuit 3. Working electrode B was connected to the current-to-voltage conversion circuit of circuit 4. Working electrode A, working electrode B, the reference electrode, and the counter electrode were immersed in 10 mL of PBS solution (solution to be measured), and the solution to be measured was stirred at 500 rpm using a stirring bar.

[0068] [Measurement of the concentration of the substance being measured] Using the aforementioned measurement system, the concentration of the substance to be measured in the solution was measured as follows. Measurement of the slope (derivative value) of the potential difference of working electrode A relative to the reference electrode with respect to time, output from circuit 3, was started. Simultaneously, the voltage at the non-inverting input terminal of the current-voltage conversion circuit of circuit 4 was adjusted so that the potential difference of working electrode B relative to the reference electrode was -0.1V, and measurement of the current of working electrode B output from circuit 4 was started. Ten seconds after the start of measurement, lactic acid was added to the solution to adjust the lactic acid concentration in the solution to 0.1 mM. Furthermore, eighty seconds after the start of measurement, glucose was added to the solution to adjust the glucose concentration in the solution to 0.1 mM. Thereafter, every 40 seconds, the glucose concentration of the solution was increased sequentially to 0.2 mM, 0.5 mM, and 1.0 mM, and every 70 seconds, the lactic acid concentration of the solution was increased sequentially to 0.2 mM and 0.4 mM. As shown in Figure 4A, a change in the slope of the potential difference across working electrode A, dependent on glucose concentration (upper figure), and a change in the current across working electrode B, dependent on lactate concentration (lower figure), were observed.

[0069] [Quantitative determination of the substance being measured] Under each glucose concentration, when the slope of the potential difference across working electrode A with respect to time became constant, the slope at that point was plotted against the glucose concentration in the solution. Similarly, when the current across working electrode B became constant under each lactate concentration, the current at that point was plotted against the lactate concentration in the solution. As shown in Figure 4B, the slope of the potential difference across working electrode A was proportional to the glucose concentration, and the current across working electrode B was proportional to the lactate concentration.

[0070] (Example 2) The slope of the potential difference of working electrode A relative to the reference electrode with respect to time was calculated using a computer. During the measurement in Example 1, the signal of the potential difference of working electrode A output from circuit 2 was A / D converted and transmitted to the computer without going through circuit 3. Figure 5 shows the temporal change in the potential difference of working electrode A output from circuit 2. The slope of the potential difference of working electrode A was calculated on the computer. Similar to Example 1, a change in the slope of the potential difference of working electrode A dependent on glucose concentration was confirmed (Figure 6A), and the slope of the potential difference was proportional to the glucose concentration (Figure 6B).

Claims

1. An apparatus for measuring the concentration of a substance to be measured in a solution, the apparatus is An electrochemical sensor equipped with multiple working electrodes, and Tilt output mechanism, Equipped with, The plurality of working electrodes include working electrode A and working electrode B, The working electrode A contains an enzyme that uses the substance to be measured as a substrate. The output of the working electrode A is connected to the tilt output mechanism. The slope output mechanism outputs the slope of the potential difference between the working electrode A and the reference electrode with respect to time. Device.

2. The apparatus according to claim 1, wherein the working electrode A contains a redox substance that can be reversibly oxidized and reduced.

3. The apparatus according to claim 2, wherein the potential difference between the working electrode A and the reference electrode shifts to the noble side when the redox substance is oxidized by the reaction of the substance to be measured with the enzyme at the working electrode A, and shifts to the detrimental side when the redox substance is reduced by the reaction of the substance to be measured with the enzyme at the working electrode A.

4. The apparatus according to any one of claims 1 to 3, wherein the working electrode B contains an enzyme that uses the substance to be measured on the working electrode B as a substrate.

5. The apparatus according to any one of claims 1 to 3, wherein the output of the working electrode B is not connected to the tilt output mechanism.

6. The apparatus according to claim 5, wherein the output of the working electrode B is an electric current.

7. The apparatus according to any one of claims 1 to 3, wherein the plurality of working electrodes further include a working electrode C.

8. The apparatus according to claim 7, wherein the working electrode C is connected to a tilt output mechanism.

9. A method for measuring the concentrations of multiple substances to be measured in a solution, Using the apparatus according to any one of claims 1 to 3, obtain an electrical signal containing information about the concentration of each of a plurality of substances to be measured. Methods that include...

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