Enzyme sensor

Abstract

To provide an enzyme sensor capable of selectively and highly accurately measuring density of measured substance including secular change.SOLUTION: An enzyme sensor 1 for measuring density of measured substance in solution containing the measured substance comprises: a working electrode 2 and a reference electrode 3 which may be in contact with the solution; enzyme and reversibly oxidizable and reducible oxidation-reduction substance respectively fixed to the working electrode 2; and a dV / dt output mechanism 9 capable of outputting, with time, time differential value dV / dt of potential difference between the reference electrode 3 and the working electrode 2 caused by density change of the oxidant and the reductant of the oxidation-reduction substance due to reaction between the measured substance and the enzyme. The potential difference shifts to a noble side when the oxidation-reduction substance is oxidized by reaction between the measured substance and the enzyme while it shifts to a base side when the oxidation-reduction substance is reduced.SELECTED DRAWING: Figure 1

Description

[Technical field]

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

[0002] In recent years, with the rise of health consciousness, there is an increasing need for sensors that selectively detect organic substances in biological fluids such as sweat, saliva, urine, tears, and blood in order to monitor the physical condition, and non-invasive measurements that do not damage the subject's body, such as those that do not require blood sampling, are attracting attention. The concentration of organic substances in biological fluids that can be non-invasively measured tends to be lower than that in blood, and therefore sensors with higher sensitivity are required. As a sensor of this kind, an enzyme sensor that utilizes the high selectivity of enzymes and can selectively detect organic substances with high sensitivity is considered to be a promising candidate.

[0003] In Patent Document 1, the steady-state potential ΔV∞ generated when protons generated by an enzyme reaction are captured by a proton-sensitive membrane on a working electrode is estimated from the rate of change in the initial potential to measure the substrate concentration. Patent Document 2 discloses providing an external resistor between the working electrode and the reference electrode to make the redox reaction of the redox substance reversible. Patent Document 3 discloses a method for measuring the substrate concentration from the change over time in the potential between the working electrode and the reference electrode. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 64-88244 [Patent Document 2] JP 2019-158650 A [Patent Document 3] Special Publication No. 2023-553889 Summary of the Invention [Problem to be solved by the invention]

[0005] The enzyme sensor disclosed in Patent Document 1 includes a working electrode on which an enzyme is immobilized on a pH-sensitive membrane, and utilizes the fact that the potential settles to a stationary potential (constant potential) over time. The enzyme sensor disclosed in Patent Document 1 avoids the problem of it taking time to settle to a stationary potential, which results in a long measurement time, and specifically, it calculates ΔV∞ as an estimated value from a specific linear function to shorten the measurement time. However, this method has a problem in that it is not possible to measure the change over time in the concentration of the substance to be measured in the measurement solution. In addition, in detection using an ion-sensitive field effect transistor (ISFET), there is a problem in that the detection accuracy is low because protons generated in the enzyme reaction are buffered by the measurement solution, which tends to reduce the change in potential.

[0006] In Patent Document 2, an enzyme sensor capable of measuring the change in concentration of a substance to be measured over time is produced by immobilizing a redox substance and an enzyme on a working electrode that can be reversibly oxidized and reduced, and measuring a signal obtained by amplifying the redox potential of the redox substance with an amplifier such as a field effect transistor. The reaction of the redox substance caused by the enzyme reaction is only in one direction, either oxidation or reduction, and as long as the enzyme reaction continues, the potential of the working electrode continues to rise or fall and does not settle at a constant potential. Therefore, in this enzyme sensor, the working electrode and the reference electrode are connected by an external resistor to promote a spontaneous regeneration reaction (oxidation or reduction reaction) of the redox substance, which is an electron transfer mediator, thereby solving the above-mentioned problem of irreversibility. However, there is a problem that the measurement accuracy is reduced because the reference electrode potential is changed by passing a current through the reference electrode, and further, there is a problem that the reference electrode may be damaged by passing a current through the reference electrode.

[0007] In Patent Document 3, a potential is applied between the working electrode and the reference electrode for a short period of time before measuring the potential between the working electrode and the reference electrode serving as a standard electrode. This makes the relationship between the time change in potential and the substrate concentration to be measured complex, making it difficult to incorporate a mechanism for determining the substrate concentration into the sensor.

[0008] In one aspect, the present disclosure aims to provide an enzyme sensor capable of selectively measuring the concentration of a substance to be measured in a solution containing the substance, including changes over time, with high accuracy. [Means for solving the problem]

[0009] The present disclosure provides, in one aspect, a method for producing a method for manufacturing a semiconductor device comprising: An enzyme sensor for measuring a concentration of a substance to be measured in a solution containing the substance, a working electrode and a reference electrode each of which is accessible to the solution; an enzyme and a redox substance that can be reversibly oxidized and reduced, each of which is immobilized on the working electrode; a dV / dt output mechanism capable of outputting a time differential value dV / dt of a potential difference between the reference electrode and the working electrode, which is generated by a change in concentration of an oxidant and a reductant in the redox substance caused by a reaction between the substance to be measured and the enzyme, The potential difference in this enzyme sensor shifts to the noble side when the redox substance is oxidized by the reaction between the analyte and the enzyme, and shifts to the negative side when the redox substance is reduced. In the present disclosure, the "concentration of the substance to be measured" includes not only the absolute value of the concentration of the substance to be measured but also the relative value, and is a concept that also includes, for example, the time derivative value dV / dt that is linearly related to the absolute value of the substrate concentration and can be used to calculate the absolute value of the concentration of the substance to be measured.

[0010] In one aspect, the present disclosure provides a method for measuring a substance to be measured using the enzyme sensor of the present disclosure, bringing a solution containing the substance to be measured into contact with both the working electrode and the reference electrode; obtaining a time differential value dV / dt of a potential difference between the reference electrode and the working electrode over time in a state in which the working electrode and the reference electrode are electrically connected and no voltage is applied between the working electrode and the reference electrode; and calculating the concentration of the substance to be measured using the acquired time differential value dV / dt and the following linear function (i).

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[0011] In one aspect, the present disclosure provides a method for measuring another target substance using the enzyme sensor of the present disclosure, When the electrode system of the enzyme sensor is a two-electrode system, the working electrode and the counter electrode (reference electrode) are brought into contact with a solution containing the substance to be measured, and when the electrode system is a three-electrode system, the working electrode, the counter electrode, and the reference electrode (reference electrode) are brought into contact with a solution containing the substance to be measured. When the electrode system of the enzyme sensor is the two-electrode system, the working electrode and the counter electrode (reference electrode) are electrically connected, and when the electrode system is the three-electrode system, the working electrode, the counter electrode and the reference electrode (reference electrode) are electrically connected, and when the potential of the working electrode reaches a predetermined potential set in advance, in either the two-electrode system or the three-electrode system, a predetermined voltage is applied between the counter electrode and the working electrode to allow a current to flow between the working electrode and the counter electrode, thereby returning the potential of the working electrode to a value before the start of measurement; Obtaining a time differential value dV / dt of the potential difference between the reference electrode and the working electrode over time; and calculating the concentration of the substance to be measured using the acquired time differential value dV / dt and the following linear function (i).

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[0012] In one aspect, the present disclosure provides a measurement system for measuring a concentration of a target substance in a solution containing the target substance, the system comprising: The measurement system includes an enzyme sensor, a signal processing mechanism, and a display unit. The enzyme sensor comprises: a working electrode and a reference electrode each of which is accessible to the solution; an enzyme and a redox substance that can be reversibly oxidized and reduced, each of which is immobilized on the working electrode; a measuring unit capable of measuring a potential difference between the reference electrode and the working electrode over time, the potential difference being generated by a change in concentration of an oxidant and a reductant in the redox substance caused by a reaction between the substance to be measured and the enzyme; The potential difference shifts to the noble side when the redox substance is oxidized by the reaction between the analyte and the enzyme, and shifts to the negative side when the redox substance is reduced. The signal processing mechanism calculates the concentration of the substance to be measured from the potential difference using the following linear function (i): The display unit relates to a measurement system capable of displaying the concentration of the substance to be measured calculated by the signal processing mechanism.

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[0013] According to the present disclosure, it is possible to provide an enzyme sensor capable of selectively measuring the concentration of the substance to be measured, including changes over time, with high accuracy. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 is a schematic diagram of an enzyme sensor according to one embodiment of the present disclosure. [Diagram 2] FIG. 2 is a schematic diagram of an enzyme sensor according to another embodiment of the present disclosure. [Diagram 3]FIG. 3 is a schematic diagram of an enzyme sensor according to another embodiment of the present disclosure. [Figure 4] FIG. 4 is a schematic diagram of an enzyme sensor according to another embodiment of the present disclosure. [Diagram 5] FIG. 5 is a schematic diagram of an enzyme sensor according to another embodiment of the present disclosure. [Figure 6] FIG. 6 is a schematic diagram of an enzyme sensor according to another embodiment of the present disclosure. [Figure 7] FIG. 7 is a plan view of a working electrode formed on a substrate and used in Examples 1 to 7. [Figure 8] FIG. 8 is a schematic diagram illustrating a substrate concentration measurement system used in Examples 1 and 4 to 7. [Figure 9] FIG. 9 is a schematic diagram illustrating the substrate concentration measurement system used in Example 2. [Figure 10] FIG. 10 is a schematic diagram illustrating the substrate concentration measurement system used in Example 3. [Figure 11] FIG. 11 is a graph showing the time differential value of the working electrode potential that changes with time obtained in the experimental procedure of Example 1. [Figure 12] FIG. 12 is a graph showing the relationship between the time differential value of the working electrode potential obtained in FIG. 11 and the glucose concentration. [Figure 13] FIG. 13 is a graph showing the change in potential of the working electrode over time obtained in the experimental procedure of Example 2. [Figure 14] FIG. 14 is a graph showing the relationship between the time derivative of the working electrode potential obtained in the experimental procedure of Example 2 and time. [Figure 15] FIG. 15 is a graph showing the time differential value of the working electrode potential that changes with time obtained in the experimental procedure of Example 3. [Figure 16] FIG. 16 is a graph showing the relationship between the time differential value of the working electrode potential obtained in FIG. 15 and the glucose concentration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] In one embodiment, the enzyme sensor of the present disclosure is an enzyme sensor for measuring the concentration of a substance to be measured (hereinafter also referred to as a "substrate") in a solution containing the substance. The enzyme sensor of the present disclosure includes a working electrode and a reference electrode, each of which can be in contact with the solution, an enzyme and a redox substance, each of which is immobilized on the working electrode, and a dV / dt output mechanism electrically connected to the working electrode and the reference electrode. The dV / dt output mechanism is capable of outputting, over time, a time differential value dV / dt of the potential difference between the reference electrode and the working electrode, which is generated by a change in concentration of the oxidized form and the reduced form of the redox substance due to the reaction between the measured substance and the enzyme. In the enzyme sensor of the present disclosure, when the redox substance is oxidized by the reaction between the measured substance and the enzyme, the potential difference shifts to the noble side, and when the redox substance is reduced, the potential difference shifts to the negative side. With this configuration, the enzyme sensor of the present disclosure can selectively measure the concentration of the measured substance, including changes over time, with high accuracy, for the following reasons.

[0016] As described above, in a measurement system in which an enzyme is immobilized on a pH-sensitive membrane as disclosed in Patent Document 1, it is known that the potential of the enzyme electrode settles to a constant potential over time. However, in a measurement system in which a redox substance and an enzyme are immobilized on an electrode, the redox substance is oxidized or reduced by the reaction between the substrate and the enzyme (enzyme reaction), but the working electrode potential continues to rise or fall and does not settle at a constant potential. Therefore, it is not possible to monitor the change in the concentration of the substrate over time. However, when an enzyme and a redox substance are immobilized on an electrode, the charge generated by the reaction between the substrate and the enzyme can be transferred quickly and efficiently to the electrode via the redox substance. Therefore, even when the substrate concentration is low and the product of the enzyme reaction is small, the enzyme sensor can obtain a sufficient signal (voltage) strength in a short time, and it is believed that the time-dependent change in the concentration of the substrate can be measured with high accuracy by calculating the differential value dV / dt of this signal with respect to time.

[0017] Hereinafter, examples of the enzyme sensor of the present disclosure will be described with reference to the drawings. For the sake of convenience, the measurement target (substrate) is glucose in Fig. 1 to Fig. 6, but the sensing target of the enzyme sensor of the present disclosure is not limited thereto.

[0018] The enzyme sensor 1 of the present disclosure shown in FIG. 1 is a device for measuring the glucose concentration contained in a solution 6. The enzyme sensor 1 of the present disclosure includes a working electrode 2 to which an enzyme and a redox substance are immobilized, a sensing portion 8 including a counter electrode (reference electrode) 3, and a dV / dt output mechanism 9 electrically connected to the working electrode 2 and the counter electrode 3. The working electrode 2 and the counter electrode 3 can each be in contact with a solution 6 containing a substance to be measured, but the counter electrode 3 and the working electrode 2 are arranged so as not to be in electrical contact with each other. The enzyme is not dissolved in the solution 6, and the redox substance is insoluble in the solution 6. The enzyme is "not dissolved" in the solution 6 means that the enzyme is bound to the working electrode by adsorption or chemical bonding, so that even if the working electrode is brought into contact with the solution 6, the enzyme does not substantially migrate into the solution. The redox substance is "insoluble" in the solution 6 means that the solubility of the redox substance in the solution is low, and when the working electrode is brought into contact with the solution, the amount of reduction due to dissolution of the redox substance immobilized on the working electrode into the solution is substantially zero. The reduction in the amount of the immobilized redox substance due to dissolution in the solution is substantially zero means that the solubility of the redox substance in the solution is 100 mg / L or less.

[0019] The measurement of glucose concentration by the enzyme sensor 1 of the present disclosure is performed in a state where the working electrode 2 and the counter electrode 3 are in contact with the solution 6, the working electrode 2 and the counter electrode (reference electrode) 3 are electrically connected, and no voltage is applied to the working electrode 2 and the counter electrode 3, so that no current flows substantially. Here, the state where no current flows substantially means a state where no current flows other than unintended leakage of current such as a leak current. In this state, the dV / dt output mechanism 9 outputs the rate of change of the potential difference generated between the counter electrode 3 and the working electrode 2 with respect to time (hereinafter, the "rate of change of the potential difference with respect to time" may also be referred to as "time differential value dV / dt of the potential difference between the reference electrode and the working electrode" or simply as "time differential value dV / dt"). The "potential difference between the reference electrode and the working electrode" is also called "OCV" (Open Circuit Voltage) or "natural potential", and may also be called "potential of the working electrode" in this application.

[0020] The "immobilization" of the redox substance and the enzyme to the working electrode 2 means that the redox substance and the enzyme are in a state in which they do not move relative to the working electrode 2. The redox substance is preferably directly immobilized to the working electrode 2. The enzyme may be directly immobilized to the working electrode 2, or may be indirectly immobilized via the redox substance. In a preferred embodiment, the redox substance constitutes a redox layer 5 arranged in contact with the working electrode 2, and the enzyme constitutes an enzyme immobilized layer 4 arranged in contact with the redox layer. Alternatively, a mixture layer of the enzyme and the redox substance may be formed on the working electrode 2.

[0021] Next, the operation at the sensing site 8 will be described, taking as an example a case where the substance to be measured (substrate) is glucose. When the enzyme (glucose oxidase) immobilized on the working electrode 2 reacts with glucose in the presence of water and oxygen, glucose becomes gluconic acid and hydrogen peroxide is generated. At the same time, the hydrogen peroxide oxidizes the reductant to the oxidant in the redox substance serving as the electron transfer mediator. This causes a change in the ratio of oxidant to reductant in the electron transfer mediator, and the potential difference between the counter electrode 3 and the working electrode 2 changes accordingly.

[0022] In the enzyme sensor 1 of the present disclosure, since the redox substance and the enzyme are immobilized on the working electrode 2, the charge generated by the reaction between the substrate and the enzyme can be transferred quickly and efficiently to the working electrode 2 via the redox substance. Therefore, even when the substrate concentration is low and the product of the enzyme reaction is small, the enzyme sensor 1 can obtain a sufficient signal (potential of the working electrode) strength in a short time, and by calculating the time differential value dV / dt of this signal, the change in the substrate concentration over time can be measured with high accuracy. For example, the signal can be measured even when the substrate concentration is several tens of μM (mol / L) or less. In addition, since the enzyme sensor 1 of the present disclosure utilizes an enzyme reaction, the substrate concentration can be measured selectively.

[0023] As described later, in the enzyme sensor of the present disclosure, the time differential value dV / dt of the potential difference between the reference electrode and the working electrode and the substrate concentration have a linear relationship (first-order correlation), so that the absolute value of the substrate concentration can be easily calculated from the measurement data of dV / dt. The enzyme sensor 1 of the present disclosure includes a dV / dt output mechanism 9 that can output the dV / dt over time, so that the absolute value of the substrate concentration can be monitored over time.

[0024] In the enzyme sensor 1 of the present disclosure, the enzyme is not dissolved in the solution 6, and the redox substance is insoluble in the solution 6, and each is fixed to the working electrode 2. Therefore, by applying a predetermined voltage between the working electrode 2 and the counter electrode 3 and passing a current between the working electrode 2 and the counter electrode 3, the ratio of the oxidized form and the reduced form can be appropriately controlled to a desired ratio. For example, when the oxidized form of the redox substance increases due to an enzyme reaction, the ratio of the oxidized form and the reduced form can be restored to the ratio at the start of the measurement by passing a current in a direction in which the oxidized form is reduced, thereby reducing the oxidized form. That is, in one embodiment, the enzyme sensor 1 of the present disclosure can be used repeatedly. Therefore, as shown in FIG. 2, in one embodiment, the enzyme sensor 1 of the present disclosure preferably further includes a circuit 10a that allows a predetermined voltage to be applied between the counter electrode 3 and the working electrode 2 to pass a current between the working electrode 2 and the counter electrode 3 as necessary.

[0025] The dV / dt output mechanism 9 capable of outputting the time differential value dV / dt may be, for example, one capable of directly outputting dV / dt as a signal by an electric circuit such as a differential circuit (see Figs. 8 to 10), or may be one comprised of a measuring device for measuring a change in potential difference occurring between the reference electrode 3 and the working electrode 2 caused by a change in concentration of oxidized and reducted forms in a redox substance resulting from a reaction between a substrate and an enzyme, an A / D converter for A / D converting the potential difference, and a central processing unit (CPU) including a calculation unit for calculating dV / dt from the obtained digital signal, etc. The dV / dt output mechanism 9 may be any mechanism capable of outputting the time differential value dV / dt.

[0026] Next, the proportional relationship (linear relationship) between the time differential value dV / dt and the absolute value of the substrate concentration will be described. Here, we will mainly explain the case where a redox substance is oxidized by a reaction between a substrate and an enzyme (enzymatic reaction), but the same explanation applies to the case where a redox substance is reduced by a reaction between a substrate and an enzyme (enzymatic reaction).

[0027] The enzyme reaction oxidizes the substrate to generate an oxidizing agent such as hydrogen peroxide, which then oxidizes the redox substance in the electrode. The rate at which the redox substance is oxidized is expressed by the following formula based on chemical reaction kinetics:

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[0028] By solving the above equation (1), the concentrations of the oxidized and reduced forms of the redox substance can be obtained as shown in equation (2).

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[0029] The potential difference between the working electrode 2 and the reference electrode 3 is expressed by the Nernst equation, which gives the potential of an electrode on which a redox substance is immobilized, as shown in equation (3).

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[0030] By substituting equation (2) into equation (3), the potential difference between the working electrode 2 and the reference electrode 3 can be calculated as shown in equation (4).

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[0031] By differentiating both sides of equation (4) with respect to time, we obtain the following equation (5).

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[0032] When the initial oxidant concentration of the redox substance is sufficiently larger than the reductant concentration, the second term on the right side of equation (5) can be ignored and the equation can be approximated as equation (6) below. In equation (6), the time differential value dV / dt of the potential difference between the working electrode 2 and the reference electrode 3 is linearly related to the oxidant concentration. Furthermore, since the oxidant is generated by the enzyme reaction, the oxidant concentration can be considered to be a constant value corresponding to the substrate concentration if the oxidant is in a steady state where it diffuses into the solution and does not remain on the electrode surface. The reaction rate of the enzyme is accurately described by the Michaelis-Menten equation, but in the range where the concentration of the measured substance (substrate) is not high, the reaction rate of the enzyme can be considered to be proportional to the concentration of the measured substance (substrate). Therefore, the concentration of the oxidant generated in the enzyme reaction can be considered to be proportional to the substrate concentration, so that the time differential value dV / dt of the potential difference between the working electrode 2 and the reference electrode 3 can be considered to be proportional to the substrate concentration, as shown in equation (7). Here, C M is the concentration of the substance to be measured (substrate).

[0033]

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[0034] In the above explanation, it is assumed that the enzyme reaction rate is slower than the diffusion rate of the substrate in the solution. However, even if the diffusion rate of the substrate is slower than the enzyme reaction rate, the diffusion rate of the substrate is proportional to the substrate concentration, so the oxidant concentration is also proportional to the substrate concentration, and the rate of change over time of the potential difference generated between the counter electrode (reference electrode) 3 and the working electrode 2 is still proportional to the substrate concentration.

[0035] From the above explanation, it can be seen that the time derivative dV / dt of the potential difference between the working electrode 2 and the reference electrode 3 is proportional to the substrate concentration. When calculating the substrate concentration from dV / dt, it can be expressed as in equation (8) using the proportionality constant A.

[0036]

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[0037]

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[0038] Thus, when an redox substance is reduced by an enzyme reaction, in equations (1) to (9), c is the concentration of the substance that reduces the redox substance, and the enzyme reaction increases the reductant concentration, shifting the working electrode potential 2 to the negative side. When an redox substance is reduced by an enzyme reaction, the rate of change with time (dV / dt) of the potential difference generated between the reference electrode 3 and the working electrode 2 is proportional to the concentration of the substrate, based on the same arguments as when an redox substance is oxidized by an enzyme reaction.

[0039] In the enzyme sensor of the present disclosure, when a redox substance is oxidized or reduced by an enzyme reaction, the time derivative (dV / dt) of the potential difference between the working electrode 2 and the reference electrode 3 is proportional to the substrate concentration. From the viewpoint of highly accurate measurement, however, it is preferable that the redox substance is not oxidized or reduced directly by the enzyme, but is oxidized or reduced by a product generated by the enzyme reaction.

[0040] When a measurement is performed for a long time using the enzyme sensor 1 of the present disclosure, for example, when an oxidized-redox substance is oxidized by an enzyme reaction, the concentration of the reductant becomes very low as the enzyme reaction proceeds. Under such circumstances, the influence of slight oxidation of substances other than the oxidized-redox substance in the electrode cannot be ignored, and the proportional relationship between the time differential value dV / dt of the potential difference and the substrate concentration is gradually impaired. As shown in FIG. 2, it is preferable that the enzyme sensor 1 of the present disclosure includes a circuit 10a as a mechanism for applying a predetermined voltage between the working electrode 2 and the counter electrode (reference electrode) 3 as necessary to pass a current between the working electrode 2 and the counter electrode 3. This is because, by passing the current in the circuit 10a in the direction in which the oxidized substance is reduced, the oxidized substance decreases and the reductant increases, making it possible to repeatedly use the enzyme sensor 1. On the other hand, when an oxidized-redox substance is reduced by an enzyme reaction, a current may be passed in the direction in which the reductant is oxidized.

[0041] Such an embodiment can be achieved, for example, by adding a circuit 10a as a switch, which is adjusted so that when a redox substance is oxidized due to a reaction between a substrate and an enzyme (enzymatic reaction), the working electrode 2 and the counter electrode 3 are brought into a conductive state when the potential of the working electrode 2 exceeds a value slightly smaller than the potential at which the following linear function (ii) holds, and is brought into a non-conductive state when the potential of the working electrode 2 becomes equal to that of the counter electrode 3. When a redox substance is reduced due to a reaction between a substrate and an enzyme (enzymatic reaction), the same can be achieved by using a value for the potential of the working electrode 2 slightly larger than the potential at which the following linear function (ii) holds.

[0042]

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[0043] The "slightly smaller value" is, for example, preferably a value 5 to 10% smaller than the potential at which the linear function (ii) is established, and the "slightly larger value" is, for example, more preferably a value 5 to 10% larger than the potential at which the linear function (ii) is established.

[0044] An electric double layer exists on the electrode surface. Therefore, when a current is passed to reduce or oxidize a redox substance, if the time for passing the current is too short, the electric double layer is merely charged and the redox substance is not oxidized or reduced. Furthermore, the charge stored in the electric double layer adversely affects the subsequent measurement of the potential difference between the working electrode 2 and the reference electrode 3, and the time differential value dV / dt of the potential difference and the substrate concentration no longer show a proportional relationship, as in Patent Document 3. The time for which the current is passed is preferably 1 second or more, more preferably 10 seconds or more, and even more preferably 20 seconds or more, from the viewpoint of sufficiently reducing or oxidizing the redox substance, and is preferably 300 seconds or less, more preferably 200 seconds or less, and even more preferably 100 seconds or less, in order to reduce the influence of interruption of the measurement time due to the current passing.

[0045] The electrode system of the sensing site 8 may be a two-electrode system consisting of a working electrode 2 and a counter electrode (reference electrode) 3 as shown in Figures 1 and 2, but may also be a three-electrode system as shown in Figure 3. That is, the electrode system of the enzyme sensor 1 of the present disclosure may be a three-electrode system further including a reference electrode 11 that can be in contact with a solution containing a substance to be measured, independent of the working electrode 2 and the counter electrode 3. In this case, the reference electrode 11 functions as the reference electrode.

[0046] As shown in Fig. 4, the enzyme sensor 1 of the present disclosure may have the above-mentioned three-electrode system as an electrode system, and may further include, for example, a circuit 10b as a means for applying a predetermined voltage between the working electrode 2 and the counter electrode 3 to pass a current as necessary. In this embodiment, the redox substance fixed to the working electrode 2 can be oxidized or reduced by applying a voltage between the working electrode 2 and the counter electrode 3 to pass a current through the circuit 10b as necessary. Therefore, for example, when an oxidant is generated by the reaction between the substrate and the enzyme (enzyme reaction), the potential of the working electrode 2 is shifted to the negative side before the start of measurement to reduce the ratio of the oxidant at the start of measurement, thereby making it possible to measure the change in the concentration of the substrate over a longer period of time.

[0047] Even when the electrode system is the three-electrode system, it is preferable to apply a voltage between the working electrode 2 and the counter electrode 3 for a predetermined time, as necessary, and control the current to flow between the working electrode 2 and the counter electrode 3, so that the enzyme sensor 1 can be used repeatedly. This embodiment can be implemented, for example, by adding a circuit 10b as a switch, which is adjusted so that when the redox substance is oxidized due to the reaction between the substrate and the enzyme (enzyme reaction), the circuit becomes conductive when the potential of the working electrode 2 exceeds a value slightly smaller than the potential at which the linear function (ii) is established, and becomes non-conductive when the potential of the working electrode 2 becomes equal to the potential of the working electrode 2 at the start of measurement. When the redox substance is reduced due to the reaction between the substrate and the enzyme (enzyme reaction), the circuit can be implemented similarly by using a value slightly larger than the potential at which the linear function (ii) is established.

[0048] The "slightly smaller value" is, for example, preferably a value 5 to 10% smaller than the potential at which the linear function (ii) is established, and the "slightly larger value" is, for example, preferably a value 5 to 10% larger than the potential at which the linear function (ii) is established.

[0049] As shown in Fig. 5, the enzyme sensor 1 of the present disclosure may include a calculator 13 including a dV / dt output mechanism 9, a signal processing mechanism 16, and a display unit 12. The signal processing mechanism 16 is preferably a mechanism that calculates the absolute value of the concentration of the substrate corresponding to the time differential value dV / dt over time using the following linear function (i), but may also be a mechanism that displays the relative value of the concentration of the substrate. The display unit 12 may be an analog display unit or a digital display unit.

[0050] In one embodiment, the signal processing mechanism 16 includes a storage unit 15 and a central processing unit 14. The storage unit 15 includes a main storage unit (memory) 15a and an auxiliary storage unit (storage) 15b. The main storage unit 15a stores the following linear function (i) for calculating the absolute value of the concentration of the substrate from the time differential value dV / dt. In this embodiment, a program for calculating the absolute value stored in the main storage unit 15a is executed by the central processing unit 14 such as a CPU including a calculation unit and a control unit, and the absolute value of the concentration of the substrate corresponding to the time differential value dV / dt is calculated over time from the following linear function (i). The calculated absolute value of the concentration of the substrate is recorded in the auxiliary storage unit 15b. The calculated absolute value of the concentration of the substrate may be digitally processed so that it can be displayed on the display unit 12 under the control of the control unit. In another embodiment, the signal processing mechanism 16 may be configured to convert the voltage output by the dV / dt output mechanism 9, for example by resistive division, and display it on a voltmeter (not shown) as an absolute value or a relative value of the concentration of the substrate calculated from the following linear function (i): In yet another embodiment, the signal processing mechanism 16 may be configured in an analog manner in which the voltmeter (not shown) is an analog voltmeter whose scale indicates the concentration of the substrate calculated from the linear function (i).

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[0051] Although a measuring unit is not clearly shown in FIGS. 1 to 5, the dV / dt output mechanism 9 may include a measuring unit in the enzyme sensor 1 of the present disclosure shown in FIGS. Furthermore, the enzyme sensor 1 shown in Figures 1 to 5 includes a dV / dt output mechanism 9, but as shown in Figure 6, the enzyme sensor 100 of the present disclosure may be configured to include, instead of the dV / dt output mechanism 9, a measurement unit 101 that can measure the potential difference (OCV) between the reference electrode and the working electrode 2 over time, and a communications interface for transmitting digital information of the obtained potential difference to an external information processing terminal 102.

[0052] The measuring unit 101 is capable of measuring the potential difference (OCV) between the reference electrode 3 and the working electrode 2 over time, which is generated by the change in concentration of the oxidant and the reductant in the redox substance caused by the reaction between the substrate and the enzyme, in a state in which the working electrode 2 and the reference electrode 3 are electrically connected, and in which no voltage is applied to the working electrode 2 and the counter electrode (reference electrode) 3, and substantially no current is flowing. The potential difference (OCV) measured by the measuring unit 101 shifts to the noble side when the redox substance is oxidized by the reaction between the substrate and the enzyme, and shifts to the noble side when the redox substance is reduced. For example, a potentiostat can be used as the measuring unit 101.

[0053] The communication interface may be either wireless or wired. The communication interface means a means for transferring data from the enzyme sensor of the present disclosure to another device or system, and examples of wireless communication interfaces include wi-fi and Bluetooth (registered trademark) technology.

[0054] The information processing terminal 102 is, for example, a digital electronic calculator capable of calculating the time differential value dV / dt of the potential difference, and includes, for example, a signal processing mechanism including a CPU (central processing unit) including a calculation unit and a control unit, a memory unit including memory and storage, an input unit, and a display unit.

[0055] The memory stores the linear function (i) for converting the time derivative dV / dt into the absolute value of the concentration of the substrate, and the CPU executes a program for calculating the absolute value of the substrate concentration stored in the memory to output the change over time in the absolute value of the substrate concentration corresponding to the time derivative dV / dt.

[0056] Thus, the present disclosure provides in one aspect a method for producing a method for treating a cancer cell comprising: An enzyme sensor for measuring a concentration of a substance to be measured in a solution containing the substance, a working electrode and a reference electrode each of which is accessible to the solution; an enzyme and a redox substance that can be reversibly oxidized and reduced, each of which is immobilized on the working electrode; a measuring unit capable of measuring a potential difference between the reference electrode and the working electrode over time, the potential difference being generated by a change in concentration of an oxidant and a reductant in the redox substance caused by a reaction between the substance to be measured and the enzyme; A communication interface for transmitting the time differential value dV / dt of the potential difference to an external information processing terminal capable of calculating the time differential value dV / dt of the potential difference, The potential difference in this enzyme sensor shifts to the noble side when the redox substance is oxidized by the reaction between the analyte and the enzyme, and shifts to the negative side when the redox substance is reduced.

[0057] The present disclosure also provides, in one aspect, A measurement system for measuring a concentration of a substance to be measured in a solution containing the substance, The measurement system includes an enzyme sensor, a signal processing mechanism, and a display unit. The enzyme sensor comprises: a working electrode and a reference electrode each of which is accessible to the solution; an enzyme and a redox substance that can be reversibly oxidized and reduced, each of which is immobilized on the working electrode; a measuring unit capable of measuring a potential difference between the reference electrode and the working electrode over time, the potential difference being generated by a change in concentration of an oxidant and a reductant in the redox substance caused by a reaction between the substance to be measured and the enzyme; The potential difference shifts to the noble side when the redox substance is oxidized by the reaction between the analyte and the enzyme, and shifts to the negative side when the redox substance is reduced. The signal processing mechanism calculates the concentration of the substance to be measured from the potential difference using the following linear function (i): The display unit relates to a measurement system capable of displaying the concentration of the substance to be measured calculated by the signal processing mechanism.

number

[0058] Regardless of how the dV / dt output mechanism 9 and the measurement unit 101 are configured, they have a finite input impedance as long as they measure a potential difference, so that a minute current (leak current) flows through the working electrode 2 connected to them due to the load effect of the dV / dt output mechanism 9 or the measurement unit 101. When a redox substance is oxidized by the reaction between a substrate and an enzyme (enzyme reaction), the oxidized substance is reduced by this minute current, and when a redox substance is reduced by the reaction between a substrate and an enzyme (enzyme reaction), the reduced substance is oxidized. When the reduction of the oxidized substance or the oxidation of the reduced substance by this minute current cannot be ignored compared to the amount of the oxidized substance or the reduced substance generated by the reaction between a substrate and an enzyme (enzyme reaction), the above formula (1) (chemical reaction kinetics formula) and linear function (i) no longer hold for the potential difference between the working electrode 2 and the reference electrode 3 to be measured.

[0059] In order to prevent the reduction of oxidants or oxidation of reductants due to the above-mentioned microcurrent from substantially affecting the enzyme sensor, it is necessary to make the input impedance of the dV / dt output mechanism 9 or the measurement unit 101 sufficiently high in accordance with a conventional method and to make the microcurrent flowing through the working electrode 2 sufficiently small. For example, by using an operational amplifier, the input impedance of the dV / dt output mechanism 9 or the measurement unit 101 can be easily set to a value large enough to ignore the load effect. The input impedance is preferably set, for example, so that the amount of redox substance reduced or oxidized by the current due to the load effect is 50% or less, preferably 10% or less, of the amount of redox substance oxidized or reduced by the enzyme reaction.

[0060] Whether the input impedance of the dV / dt output mechanism 9 or the measuring unit 101 is sufficiently high can be confirmed by observing the situation in which the potential of the working electrode during measurement shifts to the noble side when the redox substance is oxidized due to the reaction between the substrate and the enzyme, and shifts to the negative side when the redox substance is reduced due to the reaction between the substrate and the enzyme.

[0061] Each configuration of the enzyme sensor of the present disclosure will be illustrated in more detail below.

[0062] <Sensing part> [Oxidation-reduction substances] The redox substance constituting the enzyme sensor 1 of the present disclosure is not particularly limited as long as it is a redox substance capable of transferring electrons to an electrode, and any conventionally known substance can be used. Examples of the redox substance include Prussian blue, Meldola blue, tetrathiafulvalene, quinones such as hydroquinone and 1,4-naphthoquinone, ferrocene, ferrocene derivatives, potassium ferrocyanide, ferricyanide, osmium complexes, p-aminophenol, and the like.

[0063] The redox layer 5 can be formed by dropping or coating a solution containing a redox substance on the working electrode 2 and then drying it. The solution is, for example, a dispersion liquid in which the redox substance is dispersed in a dispersion medium. The unfixed redox substance is preferably removed by washing, and the washing liquid can be, for example, pure water or a buffer solution. The total amount of the redox substance fixed on the working electrode 2 is appropriately determined, for example, so that the above linear function (ii) holds during a desired measurement time, depending on the application (type of substrate) of the enzyme sensor 1 of the present disclosure. In addition, mixing a conductive material into the solution is preferable because it increases the conductivity of the redox layer 5. Examples of the conductive material contained in the redox layer 5 include conductive carbon materials, and among them, carbon nanotubes (CNTs) are preferable because they have good conductivity, a large specific surface area, and can be used to easily fix the enzyme and redox substance on the working electrode 2. The carbon nanotubes may be either single-walled carbon nanotubes or multi-walled carbon nanotubes.

[0064] [Reducing agent] When the redox substance is oxidized by an enzyme reaction, it is preferable that a reducing agent is contained in advance in addition to the redox substance in the redox substance layer 5. If a reducing agent is contained in advance in the redox substance layer, the potential of the working electrode at the start of measurement can be set to a more negative side, so that the time derivative dV / dt of the potential at which the above linear function (i) holds can be measured for a longer period of time.

[0065] The type of reducing agent is not particularly limited as long as it can reduce the redox substance, and any known reducing agent can be used. Examples of reducing agents include thiol compounds such as mercaptopropanediol, mercaptoethanol, mercaptomethanol, and thiophenol, thiourea, ascorbic acid, oxalic acid, formic acid, gallic acid, uric acid, lithium aluminum hydride, sodium borohydride, and hydrazine.

[0066] The molar ratio of the reducing agent to the redox substance contained in the redox substance layer (reducing agent / redox substance) is preferably 0.1 or more, more preferably 0.5 or more, and even more preferably 0.8 or more, from the viewpoint of enabling long-term measurement of the time derivative dV / dt of the potential at which the above linear function (i) holds, and is preferably 30 or less, more preferably 20 or less, and even more preferably 10 or less, from the viewpoint of making the concentration of the oxidized form of the redox substance larger than the concentration of the reduced form to maintain the proportional relationship between the time derivative dV / dt of the potential difference and the substrate concentration.

[0067] [Oxidizing agent] When the redox substance is reduced by an enzyme reaction, it is preferable that an oxidizing agent is already contained in addition to the redox substance in the redox substance layer 5. If an oxidizing agent is already contained in the redox substance layer, the potential of the working electrode at the start of measurement can be set to a more noble side, so that the time derivative dV / dt of the potential at which the above linear function (i) holds can be measured for a longer period of time.

[0068] The type of oxidizing agent is not particularly limited as long as it can oxidize the redox substance, and any known oxidizing agent can be used. Examples of the oxidizing agent include peroxides such as hydrogen peroxide, potassium nitrate, and chromic acid.

[0069] The molar ratio of the oxidant to the redox substance contained in the redox substance layer (oxidant / redox substance) is preferably 0.1 or more, more preferably 0.5 or more, and even more preferably 0.8 or more, from the viewpoint of enabling long-term measurement of the time derivative dV / dt of the potential at which the above linear function (i) holds, and is preferably 30 or less, more preferably 20 or less, and even more preferably 10 or less, from the viewpoint of making the reductant concentration of the redox substance higher than the oxidant concentration to maintain the proportional relationship between the time derivative dV / dt of the potential difference and the substrate concentration.

[0070] [enzyme] The enzyme constituting the sensing portion 8 is not particularly limited as long as it is an enzyme that can donate and receive electrons through a reaction, and is appropriately applied depending on the sensing target (substrate), but an enzyme that selectively reacts with the substrate is preferable. For example, when the sensing target is glucose contained in blood, urine, etc., examples of the enzyme include glucose oxidase (GOD) and glucose dehydrogenase (GDH). Other examples of enzymes that can be used depending on the sensing target contained in the biological fluid include lactate oxidase, lactate dehydrogenase, urease, uricase, amino acid oxidase, bilirubin oxidase, cholesterol oxidase, alcohol oxidase, alcohol dehydrogenase, etc.

[0071] The enzyme immobilization layer 4 can be formed, for example, by dropping or coating a solution containing an enzyme and a binder such as chitosan onto the redox layer 5, and then drying. The above solution is, for example, a mixture of a solution in which an enzyme is dissolved and a solution in which a binder is dissolved. Unimmobilized enzymes are preferably removed by washing, and the washing solution can be, for example, pure water or a buffer solution. In addition to chitosan, polyvinyl butyral, cellulose acetate, polyvinyl alcohol, poly-L-lysine, 2-methacryloyloxyethylphosphocholine polymer, fibroin, etc. can be used as the binder. In addition, the enzyme immobilization layer can be formed by covalently crosslinking enzymes with each other using a crosslinking agent such as glutaraldehyde.

[0072] In this manner, an enzyme-modified electrode is obtained in which the enzyme and the redox substance are immobilized on the working electrode 2. The total amount of the redox substance and the total amount of the enzyme immobilized on the working electrode 2 are appropriately determined depending on the application (type of substrate) of the enzyme sensor of the present disclosure, for example, so that the above linear function (ii) holds true during a desired measurement time.

[0073] [Working electrode, reference electrode, counter electrode] The working electrode 2, the counter electrode 3, and the reference electrode 11, which is included as necessary, constituting the enzyme sensor 1 of the present disclosure each consist of, for example, a conductive layer formed on a substrate 7. The material of these conductive layers is selected taking into consideration the reaction products with the enzyme. Typically, metals such as gold, palladium, platinum, rhodium, indium, or iridium, carbon materials, etc. are mentioned. Gold or platinum is preferable, but a conductive material that does not react with a solution containing a substance to be measured may also be used. Preferably, the counter electrode 3 is platinum, the working electrode 2 is gold, and the reference electrode 11 is a gold-Ag / AgCl electrode. The conductive layer is formed, for example, by evaporating these metals at any position on the substrate 7. Alternatively, a separately prepared metal thin film may be attached to the substrate 7 to form various electrodes. In addition, the conductive layer (electrode) may be formed by a conventionally known method such as vacuum deposition, electron beam, sputtering, plating, CVD, ion plating coating, inkjet, or printing, depending on the material. The counter electrode 3 may be formed using a metal wire or a metal plate without using the substrate and conductive layer. The reference electrode may also be formed by sealing a reference electrode material such as Ag / AgCl in a glass tube without using the substrate and conductive layer. Furthermore, in a two-electrode system that does not include a reference electrode 11 in addition to the counter electrode 3, an electrode that serves as a reference potential such as Ag / AgCl may be used as the counter electrode 3 in order to clarify the reference for the potential of the working electrode 2.

[0074] [Base material] The material of the substrate may be, for example, a sheet made of polyimide (PI) resin, polyester resin, polyamide resin, epoxy resin, polysulfone resin, or the like, or a film-like flexible material made of these.

[0075] [Method for measuring the concentration of the substance to be measured (substrate)] Next, a method for measuring the concentration of a substance to be measured (sensing target) of the present disclosure using the enzyme sensor of the present disclosure (hereinafter sometimes abbreviated as the "measurement method of the present disclosure") will be described.

[0076] The measurement method of the present disclosure is a method for measuring the concentration of a substance to be measured (eg, a biological substance) over time, and in one embodiment, comprises at least the following steps (A) and (B). The measurement method of the present disclosure may further include step (C) in addition to steps (A) and (B). In one embodiment of the measurement method of the present disclosure, since the time differential value dV / dt is acquired over time, the following steps (A) to (C) proceed almost simultaneously. (A) A solution 6 containing the substance to be measured is brought into contact with both the working electrode and the reference electrode. (B) obtaining a time differential value dV / dt of the potential difference between the reference electrode and the working electrode over time while the working electrode and the reference electrode are electrically connected and no voltage is applied to the working electrode and the reference electrode and substantially no current flows. (C) Using the time differential value dV / dt, obtaining an absolute value of the concentration of the substrate corresponding to the time differential value dV / dt from the following linear function (i):

[0077]

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[0078] In the step (C), the time differential values ​​dV / dt obtained in the step (B) are converted into absolute values ​​of the substrate concentration. Therefore, according to the measurement method of the present disclosure including the steps (A) to (C), it is possible to measure the change over time in the absolute value of the substrate concentration.

[0079] When it is desired to measure only the relative change in the substrate concentration, the above step (C) is not performed, and the time differential value dV / dt obtained in the above step (B) may be used as it is.

[0080] The above (C) may be performed by the enzyme sensor of the present disclosure or by an external information processing terminal. When the above (C) is performed by an external information processing terminal, the above (B) includes obtaining the time differential value dV / dt and then transmitting the time differential value dV / dt to the external information processing terminal.

[0081] In the measurement method of the present disclosure, after carrying out the above steps (A) to (C) for a predetermined period of time, the following step (D) may be further carried out. (D) When the potential of the working electrode reaches a predetermined potential, a predetermined voltage is applied between the working electrode and the counter electrode to cause a current to flow between the working electrode and the counter electrode, thereby returning the potential of the working electrode to the value before the start of measurement. The above (D) can be performed whether the electrode system of the enzyme sensor is a two-electrode system consisting of a working electrode and a counter electrode, or a three-electrode system consisting of a working electrode, a counter electrode, and a reference electrode. The above (D) can be performed by the enzyme sensor including, as a switch, circuits 10a and 10b adjusted so that the working electrode and the counter electrode are in a conductive or non-conductive state depending on the potential of the working electrode.

[0082] As described above, when the amount of the reductant or oxidant in the measurement system becomes too small due to the enzyme reaction, the correlation between the time derivative dV / dt and the substrate concentration becomes poor. The continuous measurement time of the concentration of the analyte by the enzyme sensor of the present disclosure is, for example, the time at which the linear function (i) is established at most. The "predetermined potential" is a value slightly smaller than the potential of the working electrode 2 at which the linear function (i) is satisfied when the redox substance is oxidized by the enzymatic reaction between the substrate and the enzyme, and a value slightly larger than the potential of the working electrode 2 at which the linear function (i) is satisfied when the redox substance is reduced by the enzymatic reaction between the substrate and the enzyme.

[0083] The "slightly smaller value" is, for example, a value that is preferably 5 to 10% smaller than the potential of the working electrode 2 at which the above linear function (i) is established, and the "slightly larger value" is, for example, a value that is preferably 5 to 10% larger than the potential of the working electrode 2 at which the above linear function (i) is established.

[0084] In one embodiment of the enzyme sensor of the present disclosure used in the measurement method of the present disclosure, the reference electrode is a reference electrode constituting a three-electrode system. That is, in this embodiment, the enzyme sensor includes a working electrode, a counter electrode, and a reference electrode (reference electrode). By adjusting the voltage applied between the working electrode and the counter electrode so that the potential of the working electrode relative to the reference electrode (reference electrode) corresponds to the desired ratio of oxidized form to reduced form, the ratio of oxidized form to reduced form of the redox substance can be set to a desired value before the start of measurement. As a result, for example, when an oxidized form is generated by the reaction of the substrate with the enzyme (enzyme reaction), the potential of the working electrode is shifted to the negative side before the start of measurement to lower the ratio of oxidized form at the start of measurement, and the change in the concentration of the substrate over time can be measured for a longer period of time.

[0085] The use of the enzyme sensor of the present disclosure is not particularly limited as long as it can utilize the substrate specificity of oxidoreductase, and examples of such uses include biosensors that sense organic substances or body fluids in biological samples, and biosensors that sense viruses, antibodies, etc. in environmental samples. More specifically, examples of such uses include blood glucose level sensors that measure the sugar concentration in blood, urine glucose level sensors that measure the sugar concentration in urine, lactate sensors that measure the lactate concentration in sweat, and virus sensors that exist in food, sewage, etc. EXAMPLES

[0086] The present disclosure will be described in more detail below with reference to examples, but these are merely illustrative and the present disclosure is not limited to these examples.

[0087] Example 1 [Working electrode] Au was vacuum-deposited through a metal mask onto a substrate (polyimide film, 125 μm thick) 70 to a thickness of 50 nm, forming a working electrode 21 having the pattern shown in Fig. 7. Next, 5 μl of a mixed solution obtained by mixing a 0.2 wt% carbon nanotube aqueous dispersion solution (manufactured by TUBALL) and an aqueous solution containing 1 wt% Prussian blue (a redox material, manufactured by Aldrich) at a volume ratio of 10:1 was dropped onto a circular portion 21a of the working electrode 21 having a diameter of 3 mm, and the mixture was dried in a thermostatic chamber at 80°C for 10 minutes, forming a carbon nanotube layer containing Prussian blue as a redox layer.

[0088] Next, 10 μl of a mixed solution of PBS solution (phosphate buffered saline, pH=7.2, Life Technologies) containing 1 wt% glucose oxidase (enzyme, Aldrich) and 2 wt% acetic acid (Fujifilm Wako Pure Chemical Industries) containing 1 wt% chitosan (Aldrich) in a volume ratio of 1:2 was dropped onto the Prussian blue-containing carbon nanotube layer (redox layer) and dried at 25° C. for 20 hours. After that, the unimmobilized enzyme was washed and removed with PBS solution, and dried at room temperature to form an enzyme immobilization layer, and an enzyme-modified electrode in which the enzyme and redox substance were immobilized on the working electrode 21 was obtained.

[0089] When the fabricated working electrode is immersed in a glucose solution, the redox substance (Prussian blue) is oxidized by hydrogen peroxide produced by the enzyme reaction.

[0090] [Counter electrode (reference electrode)] A silver / silver chloride electrode was used as the counter electrode.

[0091] [Method of measuring substrate concentration] Using the measurement system of FIG. 8, the concentration of the substance to be measured (substrate) was measured as follows. As shown in FIG. 8, an enzyme-modified electrode in which an enzyme and an oxidizing / reducing substance were immobilized on a working electrode 21 was connected to the non-inverting input terminal of a non-inverting amplifier circuit 27. The counter electrode 22 was connected to a ground potential. The enzyme-modified electrode and the counter electrode 22 were immersed in 10 mL of a PBS solution (a solution to be measured), and the solution to be measured was stirred at 500 rpm using a stirrer 24. First, measurement of the time differential value dV / dt of the potential of the working electrode 21 output by the differentiation circuit 28 was started. After 20 seconds, 1 μL of a 100 mM D-glucose solution was added to the stirred PBS solution to adjust the glucose concentration in the measurement solution to 10 μM. Thereafter, the glucose concentration was successively changed to 20 μM, 40 μM, 60 μM, and 100 μM, and a graph was obtained in which the time differential value dV / dt of the potential of the working electrode 21 for each glucose concentration was continuously measured, as shown in FIG.

[0092] [Quantitative determination of the measured substance] From the time differential value dV / dt for each glucose concentration shown in Fig. 11, a plot (calibration curve data) was obtained in which the glucose concentration and the time differential value dV / dt were in a proportional relationship, as shown in Fig. 12. This plot was consistent with the following linear function (i). The following linear function (i) was stored in the memory of the external information processing terminal (digital electronic computer) mentioned above, and a program for calculating the absolute value of the substrate concentration using the linear function (i), which was also stored in the memory, was executed, whereby the glucose concentration corresponding to the time differential value dV / dt was output.

[0093]

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[0094] Example 2 [Working electrode] The working electrode 21 (see FIG. 9) and the enzyme-modified electrode including it were prepared in the same manner as in Example 1. [Counter electrode (reference electrode)] A copper electrode was used as the counter electrode 22.

[0095] [Method of measuring substrate concentration] Using the measurement system of FIG. 9, the concentration of the substance to be measured (substrate) was measured as follows. 9, an enzyme-modified electrode in which an enzyme and an oxidizing / reducing substance were immobilized on a working electrode 21 was connected to a counter electrode 22 by a switch 25 for controlling current flow and a power source 26 for applying a predetermined potential, and the enzyme-modified electrode was then connected to a non-inverting input terminal of a non-inverting amplifier circuit 27. The enzyme-modified electrode and the counter electrode 22 were immersed in 10 mL of a PBS solution of 30 μM glucose (measurement target solution), and the measurement target solution was stirred at 500 rpm using a stirrer 24. Thereafter, the following steps (1) to (3) were carried out.

[0096] (1) With the switch 25 open so that no current flows between the working electrode 21 and the counter electrode 22, the change in potential of the working electrode 21 was measured by the non-inverting amplifier circuit 27, and the result was that the potential increased to the noble side over time. (2) When the potential of the working electrode 21 measured by the non-inverting amplifier circuit 27 reached 0.180 V, the switch 25 was closed to apply a voltage of 0.115 V between the working electrode 21 and the counter electrode 22 for 10 seconds, thereby energizing the working electrode 21 and the counter electrode 22 and returning the potential of the working electrode 21 to 0.115 V. (3) When the potential of the working electrode 21 returned to the predetermined value (0.115 V), the switch 25 was immediately opened so that no current was flowing between the working electrode 21 and the counter electrode 22, and the change in the potential of the working electrode 21 was again measured by the non-inverting amplifier circuit 27.

[0097] After that, the above steps (2) and (3) were repeated six times to obtain a graph showing the change in the potential of the working electrode 21 over time, as shown in Fig. 13. Furthermore, the time differential value of the potential of the working electrode 21 was measured by the differentiation circuit 28 connected to the non-inverting amplifier circuit 27, to obtain a graph showing a constant time differential value dV / dt for the potential of the working electrode 21 that changes over time, as shown in Fig. 14. While a current is flowing between the working electrode 21 and the counter electrode 22, the measurement of the time differential value dV / dt of the potential of the working electrode 21 in Fig. 14 is stopped. The input impedance of the non-inverting input terminal of the non-inverting amplifier circuit 27 was 1 MΩ or more.

[0098] Example 3 [Working electrode] The working electrode 29 (see FIG. 10) and the enzyme-modified electrode including it were prepared in the same manner as in Example 1. [Counter electrode] A platinum electrode was used as the counter electrode 30. [Reference electrode (standard electrode)] A silver / silver chloride electrode was used as the reference electrode 31.

[0099] [Method of measuring substrate concentration] FIG. 10 shows another embodiment of a system for measuring the concentration of a substance to be measured (substrate). As shown in FIG. 10, a circuit 34 was used to control the potential of the counter electrode 30 so that the reference electrode 31 was at ground potential. An enzyme-modified electrode in which an enzyme and an oxidizing / reducing substance were immobilized on the working electrode 29 was connected to a switch 35 that controls the current flow between the working electrode 29 and the counter electrode 30 by applying a predetermined voltage and a non-inverting input terminal of a non-inverting amplifier circuit 37. The enzyme-modified electrode, the counter electrode 30, and the reference electrode 31 were immersed in 10 mL of PBS solution. The PBS solution was stirred at 500 rpm using a stirrer 33, and then the switch 35 was closed for 100 seconds to first energize the working electrode 29 and the counter electrode 30. The potential of the working electrode 29 relative to the reference electrode 31 at this time was set to 0.015 V. Next, the switch 35 was opened so that the working electrode 29 and the counter electrode 30 were not energized, and then the measurement of the time differential value dV / dt of the potential of the working electrode 29 output by the differentiation circuit 38 was started. After 40 seconds, 1 μL of 100 mM D-glucose solution was added to the stirred PBS solution to adjust the glucose concentration in the measurement solution to 10 μM, and then the glucose concentration was continuously changed to 30 μM, 50 μM, 75 μM, 100 μM, and 200 μM. Since the working electrode potential reached 0.08 V during measurement of the PBS solution with a glucose concentration of 100 μM, the measurement of the time differential of the working electrode potential by the differentiation circuit 38 was stopped, and the switch 35 was closed for 80 seconds so that the working electrode 29 and the counter electrode 30 were energized, and the potential of the working electrode 29 was returned to 0.015 V. When the potential of the working electrode 29 returned to 0.015 V, the switch 35 was immediately opened so that the working electrode 29 and the counter electrode 30 were not energized, and the measurement of the time differential value dV / dt of the potential of the working electrode 29 by the differentiation circuit 38 was resumed. 15, a graph was obtained in which the time differential value dV / dt of the potential of the working electrode 29 for each glucose concentration was continuously measured. It was further confirmed that the glucose concentration could be measured in real time.

[0100] [Quantitative determination of the measured substance] From the time differential value dV / dt for each glucose concentration shown in Fig. 15, a plot (calibration curve data) was obtained in which the glucose concentration and the time differential value dV / dt were in a proportional relationship, as shown in Fig. 16. This plot was consistent with the linear function (i) above. The linear function (i) was stored in the memory of the external information processing terminal (digital electronic computer) above, and a program for calculating the absolute value of the substrate concentration using the linear function (i), which was also stored in the memory, was executed, whereby the glucose concentration corresponding to the time differential value dV / dt was output.

[0101] Example 4 [Working electrode] The working electrode 21 (see FIG. 8) and the enzyme-modified electrode including it were prepared in the same manner as in Example 1. [Counter electrode (reference electrode)] The counter electrode 22 was a silver / silver chloride electrode. [Measurement of electrode potential and continuous measurement time] Using a measurement system as shown in FIG. 8, an enzyme-modified electrode in which an enzyme and an oxidizing / reducing substance were immobilized on a working electrode 21 was connected to a non-inverting input terminal of a non-inverting amplifier circuit 27. The counter electrode 22 was connected to a ground potential. The enzyme-modified electrode and the counter electrode 22 were immersed in 10 mL of a PBS solution (measurement solution), and the measurement solution was stirred at 500 rpm using a stirrer 24. First, the potential of the working electrode 21 output by the non-inverting amplifier circuit 27 was measured and found to be 0.015 V. Next, the glucose concentration in the measurement solution was adjusted to 10 μM by adding 10 μL of a 100 mM D-glucose solution to the stirred PBS solution. After that, the time during which the time differential value dV / dt of the potential at which the linear function (i) holds could be continuously measured was measured and found to be 70 seconds.

[0102] Example 5 [Working electrode] The working electrode 21 (see FIG. 8) was prepared in the same manner as in Example 4. An enzyme-modified electrode was prepared in the same manner as in Example 1, except that 5 μl of a mixed solution obtained by mixing a 0.2 wt % carbon nanotube aqueous dispersion (manufactured by TUBALL) and an aqueous solution (Prussian blue content: 1 mass %) obtained by adding Prussian blue (redox material, manufactured by Aldrich) to an aqueous solution of ascorbic acid (reducing agent, manufactured by Tokyo Chemical Industry Co., Ltd.) adjusted to 100 mM was dropped onto a circular portion 21a having a diameter of 3 mm of the working electrode 21 (see FIG. 7) at a volume ratio of 10:1. The molar ratio (reducing agent / redox material) of ascorbic acid to Prussian blue was 8.6. [Counter electrode (reference electrode)] The counter electrode 22 was a silver / silver chloride electrode. [Measurement of electrode potential and continuous measurement time] Using a measurement system as shown in FIG. 8, an enzyme-modified electrode in which an enzyme and an oxidizing / reducing substance were immobilized on a working electrode 21 was connected to a non-inverting input terminal of a non-inverting amplifier circuit 27. The counter electrode 22 was connected to a ground potential. The enzyme-modified electrode and the counter electrode 22 were immersed in 10 mL of a PBS solution (measurement solution), and the measurement solution was stirred at 500 rpm using a stirrer 24. First, the potential of the working electrode 21 output by the non-inverting amplifier circuit 27 was measured, and found to be −0.05 V. Next, the glucose concentration in the measurement solution was adjusted to 100 μM by adding 10 μL of a 100 mM D-glucose solution to the stirred PBS solution. After that, the time during which the time differential value dV / dt of the potential at which the linear function (i) holds could be continuously measured was measured, and found to be 200 seconds.

[0103] Example 6 [Working electrode] The working electrode 21 (see FIG. 8) was prepared in the same manner as in Example 4. An enzyme-modified electrode including the working electrode 21 was prepared in the same manner as in Example 4, except that 5 μl of a mixed solution obtained by mixing a 0.2 wt % carbon nanotube aqueous dispersion (manufactured by TUBALL) and an aqueous solution (Prussian blue content: 1 mass %) obtained by adding Prussian blue (redox material, manufactured by Aldrich) to an aqueous solution of 3-mercapto-1,2-propanediol (reducing agent, manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) adjusted to 50 mM was dropped onto the circular portion 21a having a diameter of 3 mm of the working electrode 21. The molar ratio (reducing agent / redox material) of 3-mercapto-1,2-propanediol to Prussian blue was set to 5.0. [Counter electrode (reference electrode)] The counter electrode 22 was a silver / silver chloride electrode. [Measurement of electrode potential and continuous measurement time] Using a measurement system as shown in FIG. 8, an enzyme-modified electrode in which an enzyme and an oxidizing / reducing substance were immobilized on a working electrode 21 was connected to a non-inverting input terminal of a non-inverting amplifier circuit 27. The counter electrode 22 was connected to a ground potential. The enzyme-modified electrode and the counter electrode 22 were immersed in 10 mL of a PBS solution (measurement solution), and the measurement solution was stirred at 500 rpm using a stirrer 24. First, the potential of the working electrode 21 output by the circuit 27 was measured, and it was −0.04 V. Next, 10 μL of a 100 mM D-glucose solution was added to the stirred PBS solution to adjust the glucose concentration in the measurement solution to 100 μM. After that, the time during which the time differential value dV / dt of the potential at which the linear function (i) holds could be continuously measured was measured, and it was 150 seconds.

[0104] Example 7 [Working electrode] The working electrode 21 (see FIG. 8) was prepared in the same manner as in Example 4. An enzyme-modified electrode including the working electrode 21 was prepared in the same manner as in Example 4, except that 5 μl of a mixed solution obtained by mixing a 0.2 wt % carbon nanotube aqueous dispersion solution (manufactured by TUBALL) and an aqueous solution (Prussian blue content: 1 mass %) obtained by adding Prussian blue (redox material, manufactured by Aldrich) to an aqueous solution of thiourea (reducing agent, manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) adjusted to 10 mM in a volume ratio of 10:1 was dropped onto the circular portion 21a having a diameter of 3 mm of the working electrode 21. The molar ratio of thiourea to Prussian blue (reducing agent / redox material) was 3.0. [Counter electrode (reference electrode)] The counter electrode 22 was a silver / silver chloride electrode. [Measurement of electrode potential and continuous measurement time] Using a measurement system as shown in FIG. 8, an enzyme-modified electrode in which an enzyme and an oxidizing / reducing substance were immobilized on a working electrode 21 was connected to a non-inverting input terminal of a non-inverting amplifier circuit 27. The counter electrode 22 was connected to a ground potential. The enzyme-modified electrode and the counter electrode 22 were immersed in 10 mL of a PBS solution (measurement solution), and the measurement solution was stirred at 500 rpm using a stirrer 24. First, the potential of the working electrode 21 output by the circuit 27 was measured, and found to be −0.02 V. Next, the glucose concentration in the measurement solution was adjusted to 100 μM by adding 10 μL of a 100 mM D-glucose solution to the stirred PBS solution. After that, the time during which the time differential value dV / dt of the potential at which the linear function (i) holds could be continuously measured was measured, and found to be 100 seconds.

[0105] [Table 1]

[0106] As can be seen from a comparison between Example 4 and Examples 5 to 7, by previously containing a reducing agent in the redox layer, the time during which the time derivative dV / dt of the potential for which the linear function (i) is valid can be continuously measured can be extended. [Industrial Applicability]

[0107] Measurement of substrate concentration using the enzyme sensor disclosed herein utilizes the potential of the working electrode, and does not require passing a current through the working electrode or a solution containing the substance to be measured. Therefore, this is useful, for example, when the substrate concentration is low (e.g., several tens of μM) or when measuring the concentrations of multiple substrates simultaneously. [Explanation of symbols]

[0108] 1. Enzyme Sensor 2, 21, 29 Working electrode 3, 17, 22, 30 Counter electrode 4 Enzyme immobilization layer 5. Redox layer 6, 19, 23, 32 Measurement solution 7 Base material 8 Sensing Part 9 dV / dt output mechanism 101 Measurement section 10a, 10b circuits 11, 31 Reference electrode 12 Display section 13 Arithmetic unit 14 Central Processing Unit 15 Recording section 15a Main Record Section 15b Auxiliary storage 16 Signal Processing Mechanism 20, 24, 33 Stirrer 25, 35 Working electrode-counter electrode current switch 26, 36 power supply 27, 37 Non-inverting amplifier circuit 28, 38 Differential circuit 34 Circuit

Claims

1. An enzyme sensor for measuring the concentration of a substance to be measured in a solution containing the substance to be measured, a working electrode and a reference electrode each of which is in contact with the solution; an enzyme and a redox substance that can be reversibly oxidized and reduced, each immobilized on the working electrode; a dV / dt output mechanism that can output, over time, a time differential value dV / dt of the potential difference between the reference electrode and the working electrode that is generated by a change in concentration of an oxidant and a reductant in the redox substance resulting from a reaction between the substance to be measured and the enzyme; Including, the dV / dt output mechanism is a differentiator circuit; or the dV / dt output mechanism includes an A / D converter that performs A / D conversion of the potential difference, and a central processing unit that includes a calculation unit that calculates dV / dt from the digital signal obtained from the A / D converter; The potential difference shifts to the noble side when the redox substance is oxidized by the reaction between the analyte and the enzyme, and shifts to the negative side when the redox substance is reduced. Enzyme sensor.

2. An enzyme sensor for measuring the concentration of a substance to be measured in a solution containing the substance, a working electrode and a reference electrode each of which is in contact with the solution; an enzyme and a redox substance that can be reversibly oxidized and reduced, each immobilized on the working electrode; a dV / dt output mechanism that can output, over time, a time differential value dV / dt of the potential difference between the reference electrode and the working electrode that is generated by a change in concentration of an oxidant and a reductant in the redox substance resulting from a reaction between the substance to be measured and the enzyme; From the time differential value dV / dt, the following linear function (i): [Equation 1] (In the linear function (i) above, V is the potential difference between the reference electrode and the working electrode, t is time, C M is the concentration of the substance to be measured, and A is a proportionality constant). a mechanism for calculating, over time, an absolute value of the concentration of the substance to be measured corresponding to the time differential value dV / dt by Including, The potential difference shifts to the noble side when the redox substance is oxidized by the reaction between the analyte and the enzyme, and shifts to the negative side when the redox substance is reduced. Enzyme sensor.

3. 3. The enzyme sensor according to claim 1, wherein the redox substance is oxidized or reduced by a product produced by the reaction between the substance to be measured and the enzyme.

4. 3. The enzyme sensor according to claim 1, wherein the enzyme is an enzyme capable of donating and receiving electrons through the reaction.

5. an enzyme-modified electrode on which the enzyme and the redox substance are immobilized; The enzyme-modified electrode comprises: When the redox substance is oxidized by the reaction between the substance to be measured and the enzyme, the sample further contains a reducing agent. When the redox substance is reduced by the reaction between the substance to be measured and the enzyme, the measurement method further contains an oxidizing agent. The enzyme sensor according to claim 1 or 2.

6. 3. The enzyme sensor according to claim 1, wherein the potential difference shifts to the noble side as the redox substance is oxidized by the reaction between the analyte and the enzyme.

7. The electrode system of the enzyme sensor is a two-electrode system consisting of the working electrode and a counter electrode, or a three-electrode system consisting of the working electrode, a counter electrode, and a reference electrode, The reference electrode is the counter electrode constituting the two-electrode system or the reference electrode constituting the three-electrode system. The enzyme sensor according to claim 1 or 2.

8. 3. The enzyme sensor according to claim 1, wherein the redox substance constitutes a redox layer disposed in contact with the working electrode, and the enzyme constitutes an enzyme immobilization layer disposed in contact with the redox layer.

9. The enzyme sensor of claim 8 , wherein the redox layer comprises a conductive carbon material.

10. The enzyme sensor of claim 9 , wherein the conductive carbon material comprises carbon nanotubes.

11. The enzyme sensor according to claim 7, further comprising a circuit that applies a predetermined voltage between the counter electrode and the working electrode in either the two-electrode system or the three-electrode system, thereby enabling a current to flow between the working electrode and the counter electrode.

12. 3. The enzyme sensor according to claim 2, further comprising a display unit that displays the calculated absolute value of the concentration of the substance to be measured.

13. A method for measuring a substance to be measured using the enzyme sensor according to claim 1 or 2, bringing a solution containing the substance to be measured into contact with both the working electrode and the reference electrode; obtaining a time differential value dV / dt of a potential difference between the reference electrode and the working electrode over time in a state where the working electrode and the reference electrode are electrically connected and no voltage is applied between the working electrode and the reference electrode; The obtained time differential value dV / dt and the following linear function (i): [Equation 2] (In the linear function (i) above, V is the potential difference between the reference electrode and the working electrode, t is time, C M is the concentration of the substance to be measured, and A is a proportionality constant.) Calculating the concentration of the substance to be measured using A method comprising:

14. A method for measuring a substance to be measured using the enzyme sensor according to claim 11, When the electrode system of the enzyme sensor is the two-electrode system, the working electrode and the counter electrode are brought into contact with a solution containing the substance to be measured, and when the electrode system of the enzyme sensor is the three-electrode system, the working electrode, the counter electrode, and the reference electrode are brought into contact with a solution containing the substance to be measured; When the electrode system of the enzyme sensor is the two-electrode system, the working electrode and the counter electrode are electrically connected, and when the electrode system of the enzyme sensor is the three-electrode system, the working electrode, the counter electrode, and the reference electrode are electrically connected; When the potential of the working electrode reaches a predetermined potential, in either the two-electrode system or the three-electrode system, a predetermined voltage is applied between the counter electrode and the working electrode to cause a current to flow between the working electrode and the counter electrode, thereby returning the potential of the working electrode to a value before the start of measurement. Obtaining a time differential value dV / dt of the potential difference between the reference electrode and the working electrode over time; The obtained time differential value dV / dt and the following linear function (i): [Equation 3] (In the linear function (i) above, V is the potential difference between the reference electrode and the working electrode, t is time, C M is the concentration of the substance to be measured, and A is a proportionality constant.) Calculating the concentration of the substance to be measured using A method comprising:

15. A measurement system for measuring a concentration of a substance to be measured in a solution containing the substance to be measured, the measurement system includes an enzyme sensor, a signal processing mechanism, and a display unit; The enzyme sensor comprises: a working electrode and a reference electrode each of which is in contact with the solution; an enzyme and a redox substance that can be reversibly oxidized and reduced, each immobilized on the working electrode; a measuring unit that can measure, over time, a potential difference between the reference electrode and the working electrode that occurs due to a change in concentration of an oxidant and a reductant in the redox substance resulting from a reaction between the substance to be measured and the enzyme; Including, The potential difference shifts to the noble side when the redox substance is oxidized by the reaction between the analyte and the enzyme, and shifts to the negative side when the redox substance is reduced. The signal processing mechanism calculates the following linear function (i) from the potential difference: [Equation 4] (In the linear function (i) above, V is the potential difference between the reference electrode and the working electrode, t is time, C M is the concentration of the substance to be measured, and A is a proportionality constant.) Calculating the concentration of the substance to be measured using The display unit can display the concentration of the substance to be measured calculated by the signal processing mechanism. Measurement system.

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