Transistor type enzyme sensor

Abstract

To provide a transistor type enzyme sensor that can measure the concentration of a measured substance in a solution containing the measured substance selectively and highly accurately including a change in time sequence.SOLUTION: A transistor type enzyme sensor 1 for measuring the concentration of a measured substance in a solution 6 containing the measured substance, includes: a field-effect transistor 10; an action electrode 2' that is electrically connected to a gate electrode 13 of the field-effect transistor 10, and can come into contact with the solution 6; an enzyme and an oxidation-reduction substance capable of reversible oxidation-reduction each of which is fixed to the action electrode 2'; a reference electrode 3 that is electrically connected to a source electrode 11, and can be comes into contact with the solution 6; and a dIds / dt output mechanism 9 that enables time-sequentially acquiring a time differential value dIds / dt of a varying drain current value caused by a change in concentration of an oxidant and reductant in the oxidation-reduction substance produced by the measured substance reacting with the enzyme.SELECTED DRAWING: Figure 1

Description

Technical Field

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

Background Art

[0002] In recent years, with the increasing health consciousness, there has been a growing need for sensors that can selectively detect organic substances in biological fluids such as sweat, saliva, urine, tears, and blood in order to monitor the body's condition. Among them, non-invasive measurements that do not harm the bodies of subjects without taking blood samples have attracted attention. In biological fluids that can be measured non-invasively, the concentration of organic substances tends to be lower than in blood, and therefore, more sensitive sensors are required. As this type of sensor, a transistor-type enzyme sensor that utilizes the amplification action of a transistor and the high selectivity of an enzyme and can selectively detect organic substances with high sensitivity is considered a strong candidate.

[0003] In Patent Document 1, the steady 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 change rate of the initial potential to measure the substrate concentration. Patent Document 2 discloses providing an external resistance between the working electrode and the reference electrode in order to reverse the redox reaction of a redox substance that mediates the transfer of electrons from the enzyme to the working electrode.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] In the enzyme sensor disclosed in Patent Document 1, it includes a working electrode on which an enzyme is immobilized on a pH-sensitive membrane, and utilizes the fact that the potential of the working electrode stabilizes at a steady potential (constant potential) over time. However, the enzyme sensor disclosed in Patent Document 1 had a problem that it took a long time to reach the steady potential. Specifically, ΔV∞ was calculated as an estimated value from a specific linear function to shorten the measurement time. However, this method had a problem that it was impossible to measure the change in the concentration of the analyte in the measurement solution over time. In addition, in detection using an ion-sensitive field-effect transistor (ISFET), since protons generated by the enzyme reaction are buffered in the analyte solution and the potential change tends to be small, there was also a problem of low detection accuracy.

[0006] In Patent Document 2, by immobilizing a redox substance that can be reversibly oxidized and reduced and an enzyme on a working electrode, the reaction of the enzyme is mediated by the redox substance, and by measuring a signal obtained by amplifying the redox potential of the redox substance with an amplifier such as a field-effect transistor, an enzyme sensor capable of measuring the change in the concentration of the analyte over time is created. The reaction of the redox substance caused by the enzyme reaction is only in one direction of 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 stabilize at a constant potential. Therefore, in this enzyme sensor, by connecting the working electrode and the reference electrode with an external resistor, a spontaneous regeneration reaction (oxidation or reduction reaction) of the redox substance, which is an electron transfer mediator, is promoted, and the problem of not stabilizing at the above constant potential is solved. However, there were problems that the measurement accuracy decreased because the potential of the reference electrode was changed by passing a current through the reference electrode, and furthermore, there was a risk that the reference electrode would be damaged by passing a current through the reference electrode.

[0007] An object of the present disclosure is to provide a transistor-type enzyme sensor that can selectively and highly accurately measure the concentration of the analyte in a solution containing the analyte, including changes in the concentration over time, in one aspect.

Means for Solving the Problems

[0008] In one aspect, the present disclosure A transistor-type enzyme sensor for measuring the concentration of the substance to be measured in a solution containing the substance to be measured, a field-effect transistor, and a working electrode electrically connected to the gate electrode of the field-effect transistor and capable of contacting the solution, an enzyme and a redox substance that can be reversibly oxidized and reduced, each fixed to the working electrode, a reference electrode electrically connected to the source electrode of the field-effect transistor and capable of contacting the solution, a time derivative dI of the drain current value that changes due to a change in the concentration of the oxidized form and the reduced form of the redox substance generated by the reaction between the substance to be measured and the enzyme ds / dt that can be output over time, and a dI ds / dt output mechanism, and relates to a transistor-type enzyme sensor including the same.

[0009] In addition, 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. For example, the time derivative dI ds / dt that is linearly related to the absolute value of the concentration of the substance to be measured and can be used for calculating the absolute value of the concentration of the substance to be measured is also included in the concept.

[0010] In one aspect, the present disclosure is a method for measuring a substance to be measured using the transistor-type enzyme sensor of the present invention, contacting a solution containing the substance to be measured with both the working electrode and the reference electrode, and obtaining the time derivative dI ds / dt of the drain current value over time, and relates to a method for measuring a substance to be measured including the same.

[0011] In one aspect, the present disclosure is a method for measuring a substance to be measured using a transistor-type enzyme sensor, wherein the transistor-type enzyme sensor includes a field-effect transistor, and a working electrode electrically connected to the gate electrode of the field-effect transistor and capable of contacting the solution containing the substance to be measured, An enzyme and a redox substance that can be reversibly oxidized and reduced, each fixed to the working electrode, A reference electrode that is electrically connected to the source electrode of the field-effect transistor and can contact the solution, A current measurement unit that can measure over time the drain current value that changes due to the change in the concentration of the oxidized form and the reduced form in the redox substance generated by the reaction between the substance to be measured and the enzyme, A communication interface for transmitting to an external information processing terminal the temporal information of the measured drain current value so that the time differential value dI ds / dt of the drain current value can be calculated, and includes, The measurement method is, Contacting the solution with both the working electrode and the reference electrode, Acquiring the drain current value over time by the current measurement unit, Transmitting the temporal information of the drain current value to the information processing terminal, and obtaining the time differential value dI ds / dt at the information processing terminal, and relates to a measurement method for a substance to be measured including this.

Advantages of the Invention

[0012] According to the present disclosure, it is possible to provide a transistor-type enzyme sensor that can selectively and highly accurately measure the concentration of the substance to be measured including changes over time.

Brief Description of the Drawings

[0013]

Figure 1

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Figure 11

BEST MODE FOR CARRYING OUT THE INVENTION

[0014] The transistor-type enzyme sensor of the present disclosure (hereinafter sometimes abbreviated as "the enzyme sensor of the present disclosure") is, in one aspect, an enzyme sensor for measuring the concentration of a substance to be measured (hereinafter sometimes referred to as "substrate") in a solution containing the substance to be measured. The enzyme sensor of the present disclosure, in one aspect, includes a field-effect transistor, a working electrode, a reference electrode, and dI dsIt includes a / dt output mechanism. The working electrode and the reference electrode can each be in contact with a solution containing the substance to be measured. The working electrode is electrically connected to the gate electrode of a field-effect transistor, and the reference electrode is electrically connected to the source electrode of the field-effect transistor. An enzyme and a redox substance are immobilized on the working electrode. When the working electrode and the reference electrode are in contact with a solution containing the substance to be measured, the substance to be measured reacts with the enzyme, and as a result, the concentrations of the oxidized form and the reduced form of the redox substance change. dI ds The / dt output mechanism can output the time derivative value dI ds / dt of the drain current value that changes due to the concentration change over time.

[0015] The enzyme sensor of the present disclosure can selectively and highly accurately measure the substrate concentration including the change over time for the following reasons.

[0016] In a measurement system in which an enzyme is immobilized on a pH-sensitive film as disclosed in Patent Document 1, it is known that the potential of the enzyme electrode settles to a constant potential over time. In contrast, in a measurement system in which a redox substance and an enzyme are immobilized on an electrode, the redox substance continues to be oxidized or reduced by the reaction between the substrate and the enzyme (enzyme reaction). Therefore, the potential of the working electrode continues to rise or fall with the change in the concentrations of the oxidized form and the reduced form of the redox substance and does not settle to a constant potential. Therefore, the gate voltage also does not settle to a constant voltage, the drain current continues to change, and the drain current and the substrate concentration do not have a one-to-one relationship.

[0017] However, in the region where the drain current and the gate voltage are linearly correlated (hereinafter also referred to as the "linear region"), paying attention to the fact that the potential of the working electrode connected to the gate electrode shows a change depending on the substrate concentration, as will be described in detail later, the derivative value dI of the drain current value with respect to time ds / dt (hereinafter referred to as the "time derivative value dI ds(Also referred to as “ / dt”). It has been found that the concentration of the substrate can be measured over time by determining ( ). In addition, in the present disclosure, by fixing an enzyme and a redox substance to a working electrode, the charge generated by the reaction between the substrate and the enzyme can quickly and efficiently move to the electrode through the redox substance. Then, combined with these and the amplification action by the field effect transistor, even when the substrate concentration is low and the product of the enzyme reaction is small, a sufficient signal (drain current value (I ds )) intensity can be obtained in a short time in the transistor-type enzyme sensor. For example, the signal can be measured even when the substrate concentration is several tens of μM (mol / L) or less. From the above, in the linear operation region, by determining the differential value dI ds of the above signal (drain current value (I ds )) with respect to time, the change in substrate concentration over time can be measured with high precision. In addition, since the enzyme sensor of the present disclosure utilizes an enzyme reaction, the measurement of the substrate concentration can be selectively performed. Note that even when the field effect transistor is used outside the linear operation region, if corrected with mutual conductance, the potential of the working electrode can be obtained, so that the change in substrate concentration over time can be measured with high precision.

[0018] Hereinafter, specific examples of the enzyme sensor of the present disclosure will be described with reference to the drawings. In FIGS. 1 to 4 and FIG. 9, for convenience of explanation, the substrate is glucose, but the sensing target of the enzyme sensor of the present disclosure is not limited thereto.

[0019] The enzyme sensor 1 of the present disclosure is a device for measuring the glucose concentration contained in the solution 6. The enzyme sensor 1 of the present disclosure includes an enzyme-modified electrode 2 in which an enzyme and a redox substance are fixed on a working electrode 2′, a sensing site 8 including a counter electrode (reference electrode) 3, and a dI ds / dt output mechanism 9 and a field effect transistor 10. As shown in FIG. 2, the enzyme-modified electrode 2 includes a working electrode 2′ formed on a substrate 7, and an enzyme 4 and a redox substance 5 fixed on the working electrode 2′.

[0020] In FIG. 1, 18 is a bias voltage power supply, 19 is a drain voltage power supply, and 21 is a current measurement unit. In one aspect of the present disclosure, the bias voltage power supply 18 and the drain voltage power supply 19 may each be the voltage generation function (power supply) of a source meter having a voltage generation function (power supply) and a voltage / current measurement function. The current measurement unit 21 may be the voltage / current measurement function of the source meter. The drain current value output from the current measurement unit 21 may be either a digital signal or an analog signal. However, if the voltage / current measurement function of the source meter is used as the current measurement unit 21, the drain current value can be output as a digital signal. Also, in FIGS. 1, 3, and 4, the current measurement unit 21 is connected to the drain electrode 12 side of the field effect transistor, but it may be connected to the source electrode 11 side.

[0021] The working electrode 2' and the counter electrode 3 can each contact the solution 6 containing the 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 4 does not elute into the solution 6, and the redox substance is insoluble in the solution 6. The fact that the enzyme 4 "does not elute" in the solution 6 means that since the enzyme is bound to the working electrode 2' by adsorption or chemical bonding, even when the working electrode 2' is brought into contact with the solution 6, the enzyme 4 does not substantially migrate into the solution. The fact that the redox substance 5 is "insoluble" in the solution 6 means that the solubility of the redox substance 5 in the solution is small, and when the working electrode 2' is brought into contact with the solution 6, the amount of decrease due to the dissolution of the redox substance 5 fixed to the working electrode 2' in the solution 6 is substantially zero. The fact that the amount of decrease due to the dissolution of the fixed redox substance 5 in the solution 6 is substantially zero means that the solubility of the redox substance 5 in the solution 6 is 100 mg / L or less.

[0022] The "immobilization" of the redox substance 5 and the enzyme 4 on the working electrode 2' means that the redox substance 5 and the enzyme 4 are in a state where they do not move relative to the working electrode 2'. The redox substance 5 is preferably directly immobilized on the working electrode 2'. The enzyme 4 may be directly immobilized on the working electrode 2' or may be indirectly immobilized via the redox substance 5. In a preferred embodiment, the redox substance 5 constitutes a redox layer disposed in contact with the working electrode 2', and the enzyme 4 constitutes an enzyme immobilization layer disposed in contact with the redox layer. Also, a mixture layer of the enzyme 4 and the redox substance 5 may be formed on the working electrode 2'.

[0023] The measurement of the substrate 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. When the working electrode 2' and the counter electrode 3 are in contact with the solution 6, the ratio of the oxidized form to the reduced form in the redox substance 5 changes due to the reaction between the substrate in the solution 6 and the enzyme 4. In response to this change in ratio, the potential difference between the reference electrode 3 and the working electrode 2' changes, and the voltage applied to the gate electrode 13 changes. Also, the measurement of the substrate concentration by the enzyme sensor 1 of the present disclosure is performed, for example, in a state where a bias voltage is applied to the counter electrode (reference electrode) 3 and a drain voltage is applied to the drain electrode 12 so that the field effect transistor 10 operates in the linear operation region. Since substantially no current flows through the gate electrode 13 of the field effect transistor, even a minute change in the working electrode potential due to a minute change in the ratio of the oxidized form to the reduced form of the redox substance generated by the reaction of a small amount of substrate and enzyme becomes a signal (drain current value (I ds )) of sufficient intensity due to the amplification effect of the field effect transistor 10. Then, the time derivative value dI ds / dt of the drain current is output over time by the dI ds / dt output mechanism 9.

[0024] Next, for example, taking the case where the substrate is glucose as an example, the operation at the sensing site 8 will be described.

[0025] The enzyme (glucose oxidase) fixed to the working electrode 2' reacts with glucose in the presence of water and oxygen, converting glucose into gluconic acid and generating hydrogen peroxide. At the same time, the hydrogen peroxide oxidizes the reduced form of the redox substance, which serves as an electron transfer mediator, to its oxidized form. This causes a change in the ratio of the oxidized form to the reduced form in the electron transfer mediator. Then, in response to this change in ratio, the potential difference between the reference electrode 3 and the working electrode 2' changes, and the voltage applied to the gate electrode 13 also changes.

[0026] In the enzyme sensor 1 of the present disclosure, since the redox substance 5 and the enzyme 4 are fixed on the working electrode 2', the charge generated by the reaction between the substrate and the enzyme 4 can quickly and efficiently move to the working electrode 2' through the redox substance 5. Therefore, even when the substrate concentration is low and the product of the enzyme reaction is small, combined with the amplification effect of the field-effect transistor 10, a sufficient signal (drain current value) intensity can be obtained in a short time in the enzyme sensor 1. Also, the enzyme sensor 1 of the present disclosure has a dI ds / dt output mechanism 9 that can output the time differential value (dI ds / dt) over time. By obtaining the time differential value (dI ds / dt), the change in substrate concentration over time can be measured with high precision. And, for example, by using a plot (calibration curve data) in which the glucose concentration and the time differential value dI ds / dt are in a proportional relationship, the change in the absolute value of the substrate concentration over time can be measured with high precision.

[0027] Regarding the output characteristics of the field-effect transistor 10, when the gate voltage (V gs ) is greater than the drain voltage (V ds ), it becomes a linear operation region where the drain current (I ds ) increases linearly with respect to the gate voltage (V gs ). On the other hand, when the gate voltage (V gs ) is smaller than the drain voltage (V ds ), the gate voltage (V gs ) and the drain current (I ds) does not have a linear relationship. If the field effect transistor 10 is operated in the linear operation region, as described later, the drain current (I ds ) with respect to time (time derivative: dI ds / dt) and the substrate concentration have a correlation (linear relationship), which is preferable because it facilitates the processing of measurement data for obtaining the substrate concentration. However, as described above, even when the field effect transistor 10 is used outside the linear operation region, the substrate concentration can be obtained by correcting with the mutual conductance.

[0028] The "rate of change of the drain current value (I ds ) with respect to time" (time derivative: dI ds / dt) can be output over time. The dI ds / dt output mechanism 9 can be any mechanism as long as it can output the time derivative value dI ds / dt. For example, the voltage output of the differential circuit 22 in FIG. 9 may be converted to dI ds / dt. Alternatively, the dI ds / dt output mechanism 9 may include a central processing unit (CPU) 14 including an arithmetic unit that calculates the time derivative value dI ds / dt from the drain current value I ds measured by the current measurement unit 21. The central processing unit (CPU) 14 may be built into the enzyme sensor of the present disclosure as shown in FIGS. 1 and 3, or may be a component that constitutes an external information processing terminal 102 as in another aspect of the present disclosure shown in FIG. 4.

[0029] Next, it will be explained that the rate of change of the drain current value with respect to time (time derivative: dI ds / dt) and the substrate concentration are in a proportional relationship in the linear operation region. Here, the case where the redox substance is oxidized by the enzyme reaction will be described, but the same explanation applies when the redox substance is reduced by the enzyme reaction. The following formula (1) is a modified form of the Nernst equation.

[0030]

Equation

[0031] In formula (1), [OX]0 is the concentration of the initial oxidized form, [OX] is the change in the concentration of the oxidized form (when the redox substance is oxidized by the enzyme reaction, this change is an increase). [RED]0 is the concentration of the initial reduced form, and [RED] is the change in the concentration of the reduced form (when the redox substance is oxidized by the enzyme reaction, this change is a decrease). R is the gas constant, T is the temperature (K), F is the Faraday constant, z is the number of electrons transferred in the redox reaction of one molecule of the oxidized form, and V0 is the potential of the initial working electrode. V is the potential of the working electrode that changes with the change in the concentrations of the oxidized and reduced forms of the redox substance, that is, the potential of the gate electrode. The meanings of these symbols are the same for the following formulas (2) to (6) and the following linear functions (i) to (ii). As can be seen from the above formula (1), when the concentration of the oxidized form increases due to the enzyme reaction, the potential of the working electrode shifts to the noble side.

[0032] When the changes in the concentrations of the oxidized and reduced forms are small compared to the total amount of the redox substance, formula (1) can be approximated as the following formula (2). Furthermore, since the increase in the amount of the oxidized form and the decrease in the amount of the reduced form are usually equal, it can be approximated as the following formula (3). In formula (3), the potential V of the working electrode and the increase in the concentration of the oxidized form [OX] are in a linear relationship.

[0033]

Number

[0034] In the enzyme sensor of the present disclosure, the reaction rate of the enzyme can be regarded as the rate of increase in the concentration of the oxidized form of the redox substance. Although the reaction rate of the enzyme is accurately described by the Michaelis-Menten equation, in the range where the concentration of the sensing target (substrate) is not high, the reaction rate of the enzyme can be regarded as proportional to the concentration of the sensing target (substrate). Therefore, the rate of change of the potential difference generated between the reference electrode and the working electrode with respect to time (dV / dt) can be regarded as proportional to the concentration of the sensing target (substrate) as shown in the following equation (4). Here, k is the reaction rate constant of the enzyme, and D is the thickness of the electrode. The same applies to the following linear function (i).

[0035] [Number]

[0036] The time derivative dI of the drain current value of the field-effect transistor ds / dt is expressed by the following equation (5) when the mutual conductance of the field-effect transistor is G. In view of equation (4), the final equation (6) is obtained. In the enzyme sensor 1 of the present disclosure, when the field-effect transistor is operated in the linear region, the mutual conductance of the field-effect transistor 10 becomes a constant value, and the rate of increase in the oxidized form d[OX] / dt and the derivative of the drain current value with respect to time (dI ds / dt) are proportional. Since the substrate concentration [M] is proportional to the reaction rate of the enzyme, that is, the rate of increase in the oxidized form d[OX] / dt, the substrate concentration [M] and the derivative of the drain current value with respect to time (dI ds / dt) are also proportional. When the concentration of the oxidized form increases due to the enzyme reaction, the potential of the working electrode shifts to the noble side. At this time, when an n-type field-effect transistor is used as the field-effect transistor, the drain current increases on the positive side.

[0037] [Number]

[0038] In the above description, it is assumed that the enzymatic reaction rate is slower than the diffusion rate of the substrate in the solution. However, even when the diffusion rate of the substrate is slower than the enzymatic reaction rate, since the diffusion rate of the substrate is proportional to the substrate concentration, the rate of increase in the concentration of the oxidized form is also proportional to the substrate concentration. Therefore, even when the diffusion rate of the substrate is slower than the enzymatic reaction rate, the derivative value of the drain current value with respect to time is proportional to the concentration of the substrate.

[0039] When a long-term measurement is performed using the enzyme sensor 1 of the present disclosure, for example, when a redox substance is oxidized by an enzymatic reaction, due to the increase in the concentration of the oxidized form, the proportional relationship between the substrate concentration [M] and the derivative value of the drain current value with respect to time (dI ds / dt) is gradually impaired. Therefore, the enzyme sensor 1 of the present disclosure may be provided with a circuit (not shown) as a mechanism for applying a predetermined voltage between the working electrode 2' and the counter electrode (reference electrode) 3 as needed to allow a current to flow between the working electrode 2' and the counter electrode 3. By flowing the current in the direction in which the oxidized form is reduced in the circuit, the oxidized form is reduced, and the proportional relationship between the substrate concentration [M] and the derivative value of the drain current value with respect to time (dI ds / dt) is restored, enabling repeated use of the enzyme sensor 1, which is preferable. On the other hand, when a redox substance is reduced by an enzymatic reaction, a current may be flowed in the direction in which the reduced form is oxidized.

[0040] As shown in FIGS. 1, 3, 4, and 9, the electrode system of the sensing site 8 may be a two-electrode system including the working electrode 2' and the counter electrode (reference electrode) 3, or may be a three-electrode system (not shown). 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 that can be in contact with the solution 6 containing the substance to be measured, independently of the working electrode 2' and the counter electrode 3. In this case, the reference electrode functions as a reference electrode.

[0041] As shown in FIG. 3, in one aspect, the enzyme sensor 1 of the present disclosure may further include a storage unit 15 and a display unit 16. The storage unit 15 includes a main storage unit (memory) 15a and an auxiliary storage unit (storage) 15b. In the main storage unit 15a, the following linear function (i) for calculating the absolute value of the substrate concentration from the time differential value dI ds / dt is stored. In such an aspect, a program for calculating the absolute value stored in the main storage unit 15a by a central processing unit 14 such as a CPU including an arithmetic unit and a control unit is executed, and the time differential value dI ds / dt corresponding to the absolute value of the substrate concentration is calculated over time from the following linear function (i). The calculated absolute value of the substrate concentration is recorded in the auxiliary storage unit 15b. Further, the calculated absolute value of the substrate concentration is displayed on the display unit 16 under the control of the control unit.

[0042]

Equation

[0043] The enzyme sensor 1 shown in FIGS. 1, 3, and 9 includes a dI ds / dt output mechanism 9 in the enzyme sensor 1 itself. However, as shown in FIG. 4, the enzyme sensor 100 of the present disclosure may be configured to include a communication interface for transmitting the drain current value obtained by the current measurement unit 21 to an external information processing terminal 102.

[0044] 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. For example, examples of the wireless communication interface include technologies such as wi-fi and Bluetooth (registered trademark).

[0045] The information processing terminal 102 is, for example, a digital electronic computer capable of calculating the time differential value dI ds / dt, and includes, for example, a CPU including an arithmetic unit and a control unit, a storage unit including a memory and a storage, an input unit, and a display unit.

[0046] In the memory, the linear function (i) for converting the time differential value dI ds / dt into the absolute value of the concentration of the substrate is stored, and a program for calculating the absolute value of the substrate concentration stored in the memory is executed by the CPU, and the time differential value dI ds The absolute value of the concentration of the substrate corresponding to / dt over time is output and displayed on the display unit.

[0047] Each component of the enzyme sensor of the present disclosure will be illustrated in more detail below.

[0048] <Sensing site> [Redox substance] The redox substance constituting the enzyme sensor of the present disclosure is not particularly limited as long as it is a redox substance capable of transferring electrons to the electrode, and conventionally known ones 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 ferricyanide, ferricyanide, osmium complexes, p-aminophenol, and the like.

[0049] The redox layer can be formed by dropping or coating a solution containing the redox substance 5 on the working electrode 2' and then drying. The solution is, for example, a dispersion in which the redox substance is dispersed in a dispersion medium. It is preferable that the unfixed redox substance is removed by washing, and as the washing liquid, for example, pure water or a buffer solution can be used. The total amount of the redox substance 5 fixed on the working electrode 2' is appropriately determined according to the use (type of substrate) of the enzyme sensor 1 of the present disclosure, etc., so that the linear function (ii) holds during the desired measurement time. In addition, it is preferable to mix a conductive material into the solution to increase the conductivity of the redox layer.

[0050]

Number

[0051] [Conductive material] Examples of the conductive material contained in the redox layer include conductive carbon materials. Among them, carbon nanotubes (CNT) are preferable because they have good conductivity, a large specific surface area, and can fix enzymes and redox substances well on the working electrode 2'. The carbon nanotubes may be either single-walled carbon nanotubes or multi-walled carbon nanotubes.

[0052] [Enzyme] The enzyme constituting the sensing site 8 is not particularly limited as long as it can transfer electrons by a reaction, and is appropriately applied according to the sensing target (substrate), but an enzyme that selectively reacts with the substrate is preferable. For example, when glucose contained in blood, urine, etc. is the sensing target, examples of the enzyme include glucose oxidase (GOD) and glucose dehydrogenase (GDH).

[0053] The enzyme immobilization layer can be formed, for example, by dropping or coating a solution containing an enzyme and a binder such as chitosan on the redox layer and then drying. The above solution is, for example, a mixture of a solution in which the enzyme is dissolved and a solution in which the binder is dissolved. It is preferable that the unfixed enzyme is removed by washing, and as the washing solution, for example, pure water or a buffer solution can be used. As the binder, in addition to chitosan, polyvinyl butyral, cellulose acetate, polyvinyl alcohol, poly-L-lysine, 2-methacryloyloxyethyl phosphorylcholine polymer, fibroin, etc. can be used. Also, an enzyme immobilization layer can be formed by cross-linking enzymes with a covalent bond using a cross-linking agent such as glutaraldehyde.

[0054] In this way, an enzyme-modified electrode on which the enzyme 4 and the redox substance 5 are fixed on the working electrode 2' is obtained. The total amount of the redox substance 5 and the total amount of the enzyme 4 fixed on the working electrode 2' are appropriately determined according to the use (type of substrate) of the enzyme sensor of the present disclosure, etc., for example, so that the above linear function (ii) holds during the desired measurement time.

[0055] [Working electrode, reference electrode, counter electrode] The working electrode 2′, counter electrode (reference electrode) 3 that constitute the enzyme sensor 1 of the present disclosure each consist of, for example, a conductive layer formed on a substrate 7. The materials of these conductive layers are selected in consideration of the reaction products with the enzyme. Typically, metals such as gold, palladium, platinum, rhodium, indium, or iridium, carbon materials, etc. may be mentioned. Preferably, it is gold or platinum, but a conductive material that does not react with the solution containing the substance to be measured may also be used. Preferably, the working electrode 2′ is gold, and the counter electrode (reference electrode) 3 is an electrode having a reference potential such as Ag / AgCl. The conductive layer is formed, for example, by vapor-depositing these metals at an arbitrary position on the substrate 7. Alternatively, a separately prepared metal thin film may be attached to the substrate 7 to be used as various electrodes. Also, the conductive layer (electrode) may be formed by a conventionally known method such as vacuum vapor deposition, electron beam, sputtering, plating, CVD, ion plating coating, inkjet, printing, etc., depending on the material. The counter electrode (reference electrode) 3 may be formed using a metal wire or metal plate without using the substrate and the conductive layer.

[0056] Also, as shown in FIGS. 1, 3, 4, and 9, 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, or may be a three-electrode system. That is, the electrode system of the enzyme sensor of the present disclosure may be a three-electrode system that further includes a reference electrode (not shown) that can be in contact with the solution containing the substance to be measured, independently of the working electrode 2′ and the counter electrode 3. In this case, the reference electrode functions as a reference electrode. In this case, the counter electrode is preferably platinum, the working electrode is preferably gold, and the reference electrode is preferably an electrode having a reference potential such as Ag / AgCl.

[0057] When the electrode system of the sensing site 8 is a three - electrode system, before the measurement starts, the ratio of the oxidized form to the reduced form of the redox substance can be set to a desired value by adjusting the voltage applied between the working electrode and the counter electrode so that the potential of the working electrode with respect to the reference electrode (reference electrode) becomes the potential corresponding to the desired ratio of the oxidized form to the reduced form. Thus, for example, when an oxidized form is generated by a reaction between a substrate and an enzyme (enzyme reaction), by shifting the potential of the working electrode to the negative side before the measurement starts to lower the ratio of the oxidized form at the start of the measurement, etc., the change over time in the concentration of the substrate can be measured for a longer period of time.

[0058] [Base material] As the material of the above - mentioned base material, for example, it may be a sheet composed of a polyimide (PI) resin, a polyester resin, a polyamide resin, an epoxy resin, a polysulfone resin, etc., or it may be a film - like flexible material made of these.

[0059] [Method for measuring the concentration of the substance to be measured (substrate)] Next, a method for measuring the concentration of the 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.

[0060] The measurement method of the present disclosure is a method for measuring the concentration of a substance to be measured (for example, a biological substance) over time. In one aspect, it includes 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 the measurement method of the present disclosure, in one aspect, since the time - differential value dI ds / dt is obtained over time, the following (A) to (C) proceed almost simultaneously. (A) Contacting a solution 6 containing the substance to be measured with both the working electrode and the reference electrode. (B) Obtaining the time - differential value dI ds / dt over time while the field - effect transistor is operating in the linear operation region. (C) The time - differential value dI dsUsing / dt, from the following linear function (i), obtain the absolute value of the concentration of the substrate corresponding to the time derivative dI ds / dt.

[0061]

Equation

[0062] The time derivative dI ds / dt obtained in the above step (B) is converted into the absolute value of the substrate concentration in each of the above step (C). Therefore, according to the measurement method of the present disclosure including the above steps (A) to (C), the change over time of the absolute value of the substrate concentration can be measured.

[0063] When it is desired to measure only the relative change in the substrate concentration, the above step (C) is not performed, and the time derivative dI ds / dt obtained in the above step (B) may be used as it is.

[0064] 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 transmitting the time derivative dI ds / dt over time to the external information processing terminal.

[0065] In addition, in one aspect, the measurement method of the present disclosure measures the drain current value over time in the current measurement unit constituting the enzyme sensor of the present disclosure, and uses the obtained drain current value to calculate the time derivative dI ds / dt at an external information processing terminal. That is, the above (B) may include the following (b1) and (b2). In this aspect, the above (C) is performed by an external information processing terminal.

[0066] (b1) Acquiring the value of the drain current (analog information or digital information) over time in a state where the field effect transistor operates in the linear operation region. (b2) Transmit the time-dependent information of the drain current value obtained in (b1) to an external information processing terminal, and obtain the time differential value dI ds / dt at the information processing terminal.

[0067] The measurement method of the present disclosure may further perform the following (D) after executing the above (A) to (C) for a predetermined time.

[0068] (D) When the potential of the working electrode (the potential of the gate electrode) reaches a predetermined preset potential, apply a predetermined voltage between the working electrode and the counter electrode to allow a current to flow between the working electrode and the counter electrode, and return the potential of the working electrode to the value before the start of measurement.

[0069] The above (D) can be performed regardless of whether the electrode system of the enzyme sensor is a two-electrode system composed of a working electrode and a counter electrode or a three-electrode system composed of a working electrode, a counter electrode, and a reference electrode. In the case of the three-electrode system, the above (D) can be performed by applying a voltage between the working electrode and the counter electrode to allow a current to flow between the working electrode and the counter electrode so that the potential difference between the working electrode and the reference electrode becomes a predetermined value, and returning the potential of the working electrode to the value before the start of measurement. The above (D) can be executed by including, as a switch, a circuit (not shown) in which the enzyme sensor adjusts so that the working electrode and the counter electrode become in a conductive state or a non-conductive state according to the potential of the working electrode.

[0070] As the use of the enzyme sensor of the present disclosure, there is no particular limitation as long as it is a use that can utilize the property of the substrate specificity of the redox enzyme. For example, a biosensor for sensing organic substances or body fluids in a biological sample, or a biosensor for sensing viruses, antibodies, etc. in an environmental sample can be mentioned. More specifically, a blood glucose sensor for measuring the sugar concentration in blood, a urine sugar sensor for measuring the sugar concentration in urine, a lactate sensor for measuring the lactate concentration in sweat, a virus sensor existing in foods, sewage, etc. can be mentioned.

Example

[0071] Hereinafter, the present disclosure will be described in more detail with reference to examples, which are illustrative only and the present disclosure is not limited to these examples.

[0072] (Example 1) [Working electrode] Through a metal mask, Au was vacuum-deposited on a substrate (polyimide film, thickness 125 μm) 7 to a thickness of 50 nm to form a working electrode 2' in the pattern shown in Fig. 2. Next, on the circular portion with a diameter of 3 mm in the working electrode 2', a mixed solution of a 0.2 wt% carbon nanotube aqueous dispersion solution (manufactured by TUBALL) and an aqueous solution containing 1 wt% Prussian blue (redox substance, manufactured by Aldrich) in a volume ratio of 10:1 was dropped in an amount of 5 μl and dried in a constant temperature bath at 80°C for 10 minutes to form a Prussian blue-containing carbon nanotube layer as a redox layer.

[0073] Next, 10 μl of a mixed solution obtained by mixing a PBS solution (phosphate buffered saline, pH = 7.2, manufactured by Life Technologies) containing 1 wt% glucose oxidase (enzyme, manufactured by Aldrich) and a 2 wt% acetic acid (manufactured by Fujifilm Wako Pure Chemical Corporation) solution containing 1 wt% chitosan (manufactured by 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. Then, the unfixed enzyme was washed and removed with a PBS solution and dried at room temperature to form an enzyme immobilization layer, and an enzyme-modified electrode 2 in which the enzyme and the redox substance were immobilized on the working electrode 2' was obtained.

[0074] [Reference electrode] An Ag / AgCl electrode was used as the reference electrode.

[0075] [Method for measuring substrate concentration] The concentration of the substance to be measured (substrate concentration) was measured using a measurement system as shown in Fig. 4. Specifically, after connecting the enzyme-modified electrode 2 to the gate electrode 13 of a MOSFET (2N7000TA, manufactured by Fairchild Semiconductor), the enzyme-modified electrode 2 and the Ag / AgCl reference electrode 3 (reference electrode) were immersed in 10 ml of PBS solution. The PBS solution was stirred at 500 rpm using a stirrer 20. Next, the source electrode 11 and the drain electrode 12 of the MOSFET were connected to a source meter (manufactured by Keithley) as the drain voltage power supply 19 and the current measurement unit 21, and the Ag / AgCl reference electrode 3 was connected to a source meter (manufactured by Keithley) as the bias voltage power supply 18. A bias voltage of 0.5 V was applied to the Ag / AgCl reference electrode 3, and a drain voltage of 0.1 V was applied to the drain electrode 12, and the time change of the drain current value measured by the current measurement unit 21 was monitored. At the applied bias voltage and drain voltage, the field-effect transistor 10 is in the linear operating region. After confirming that a steady current was flowing through the drain electrode 12, the glucose concentration in the measurement solution was adjusted to 100 μM by adding 10 μl of 100 mM D-glucose PBS solution to the stirred PBS solution.

[0076] As a result, as shown in FIG. 5, a change in the drain current value that increased linearly with time was observed. This drain current value (digital information) was transmitted to an external information processing terminal and time-differentiated to obtain a time-differentiated value dI ds / dt as shown in FIG. 6. Thereafter, measurements were similarly performed using the newly fabricated working electrode in PBS solutions with a glucose concentration of 25 μM and 200 μM, respectively, to obtain a time-differentiated value dI ds / dt of the drain current value that was approximately constant according to the glucose concentration as shown in FIG. 7.

[0077] [Quantification of the Substance to be Measured] From the time-differentiated values dI ds / dt for each glucose concentration shown in FIG. 7, as shown in FIG. 8, the glucose concentration and the time-differentiated value dI dsA plot (calibration curve data) in which / dt is in a proportional relationship was obtained. For example, using this calibration curve data or the linear function (i), the time derivative value dI shown in FIG. 4 ds can be output by obtaining / dt over time in the measurement system shown in FIG. 4, thereby obtaining the change over time in the absolute value of the glucose concentration in the solution to be measured.

[0078] (Example 2) [Working electrode] The working electrode and the enzyme-modified electrode containing the same were prepared in the same manner as in Example 1.

[0079] [Reference electrode (reference electrode)] An Ag / AgCl electrode was used as the reference electrode.

[0080] [Method for measuring substrate concentration] Using the measurement system shown in FIG. 9, the concentration of the substance to be measured (substrate) was measured as follows. After connecting the enzyme-modified electrode 2 to the gate electrode 13 of the MOSFET (2N7000TA, manufactured by Fairchild Semiconductor), the enzyme-modified electrode 2 and the Ag / AgCl reference electrode 3 (reference electrode) were immersed in 10 ml of PBS solution. The PBS solution was stirred at 500 rpm using a stirrer 20. Next, the source electrode 11 and the drain electrode 12 of the MOSFET were connected to a source meter (manufactured by Keithley) as a drain voltage power supply 19 and a current measurement unit 21, and the Ag / AgCl reference electrode 3 was connected to a source meter (manufactured by Keithley) as a bias voltage power supply 18. A bias voltage of 2 V was applied to the Ag / AgCl reference electrode 3, and a drain voltage of 0.1 V was applied to the drain electrode 12. The time change of the drain current value was monitored as a differential output. At the applied bias voltage and drain voltage, the field-effect transistor 10 was in the linear operating region. After confirming that a constant differential output was obtained, 20 μl of 100 mM D-glucose PBS solution was added to the stirred PBS solution one by one, so that the glucose concentration in the measurement solution was continuously changed to 200 μM, 400 μM, and 600 μM. The results of the continuously measured differential output are shown in FIG. 10. As shown in FIG. 11, the differential output of the drain current showed a value proportional to the glucose concentration, and it was confirmed that the glucose concentration could be measured in real time. In FIGS. 10 and 11, the voltage output of the current differential circuit 22 is converted to dI ds / dt and plotted.

Industrial Applicability

[0081] The enzyme sensor of the present disclosure measures the substrate concentration using the time differential value dI ds / dt of the drain current value, and is useful as, for example, a concentration measuring device when the substrate concentration is low (for example, several tens of μM).

Explanation of Signs

[0082] 1,100 Enzyme sensor 2 Enzyme-modified electrode 2' Working electrode 3 Counter electrode (reference electrode) 4 Enzyme 5 Redox substances 6 Solution containing the substance to be measured 7 Substrate 8 Sensing site 9 dI ds / dt output mechanism 11 Source electrode 12 Drain electrode 13 Gate electrode 10 Field-effect transistor 14 Central processing unit 15 Recording unit 15a Main recording unit 15b Auxiliary storage unit 16 Display unit 18 Bias voltage power supply 19 Drain voltage power supply 20 Stirrer 21 Current measurement unit 22 Differentiating circuit 102 Information processing terminal

Claims

1. A transistor-type enzyme sensor for measuring the concentration of a substance to be measured in a solution containing the substance to be measured, a field-effect transistor, a working electrode electrically connected to the gate electrode of the field-effect transistor and capable of contacting the solution, an enzyme and a redox substance that can be reversibly oxidized and reduced, each immobilized on the working electrode, a reference electrode electrically connected to the source electrode of the field-effect transistor and capable of contacting the solution, The time differential value dI of the drain current value that changes due to the change in concentration of the oxidized form and the reduced form in the redox substance that occurs as a result of the reaction between the substance to be measured and the enzyme ds / dt can be obtained over time. ds / dt output mechanism.

2. The transistor-type 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-immobilized layer disposed in contact with the redox layer.

3. The transistor-type enzyme sensor according to claim 1 or 2, wherein the redox layer contains a conductive carbon material.

4. The transistor-type enzyme sensor according to claim 3, wherein the conductive carbon material contains carbon nanotubes.

5. The transistor-type enzyme sensor, The time differential value dI ds a storage unit storing the following linear function (i) for calculating the absolute value of the concentration of the substrate from / dt; From the linear function (i), a central processing unit that calculates over time the absolute value of the concentration of the substrate corresponding to the time derivative value dI ds / dt, further comprising a transistor-type enzyme sensor according to any one of claims 1 to 4. 【Number 1】 In the above linear function (i), Ids is the drain current value, [M] is the concentration of the substance to be measured, [OX] 0 is the concentration of the initial oxidized form, [RED] 0 is the concentration of the initial reduced form, R is the gas constant, T is the temperature (K), F is the Faraday constant, z is the number of electrons transferred in the redox reaction of one molecule of the oxidized form, D is the thickness of the working electrode, and k is the reaction rate constant.

6. A method for measuring a substance to be measured using the transistor-type enzyme sensor according to any one of claims 1 to 5, contacting a solution containing the substance to be measured with both the working electrode and the reference electrode, The time derivative value dI of the drain current value ds obtaining the value dI / dt over time, and a method for measuring a substance to be measured including this.

7. The acquisition of the time differential value dI ds / dt is performed in a state where the field effect transistor operates in a linear operation region, and the method for measuring a substance to be measured according to claim 6.

8. The transistor-type enzyme sensor, The time differential value dI ds a storage unit storing the following linear function (i) for calculating the absolute value of the concentration of the substrate from / dt; From the linear function (i), a central processing unit that calculates over time the absolute value of the concentration of the substrate corresponding to the time derivative value dI ds / dt, further comprising: the time differential value dI ds calculating an absolute value of the concentration of the substance to be measured by using the following linear function (i) and the time differential value dI / dt, the method for measuring a substance to be measured according to claim 7 or 8, further comprising this step. 【Number 2】 In the above linear function (i), Ids is the drain current value, [M] is the concentration of the substance to be measured, [OX] 0 is the concentration of the initial oxidized form, [RED] 0 is the concentration of the initial reduced form, R is the gas constant, T is the temperature (K), F is the Faraday constant, z is the number of electrons transferred in the redox reaction of one molecule of the oxidized form, D is the thickness of the working electrode, and k is the reaction rate constant.

9. A method for measuring a substance to be measured using a transistor-type enzyme sensor, The transistor-type enzyme sensor, a field-effect transistor, a working electrode electrically connected to the gate electrode of the field-effect transistor and capable of contacting a solution containing the substance to be measured, an enzyme and a redox substance that can be reversibly oxidized and reduced, each immobilized on the working electrode, a reference electrode electrically connected to the source electrode of the field-effect transistor and capable of contacting the solution, a current measurement unit capable of measuring over time a drain current value that changes due to a change in the concentration of an oxidized form and a reduced form in the redox substance generated by the reaction between the substance to be measured and the enzyme, A communication interface for transmitting information over time of the measured drain current value to an external information processing terminal capable of calculating a time derivative value dI ds / dt of the drain current value, and The measurement method, contacting the solution with both the working electrode and the reference electrode, acquiring the drain current value over time by the current measurement unit, Transmit the information on the drain current value over time to the information processing terminal, and obtain, at the information processing terminal, the time differential value dI ds / dt of the drain current value, the method for measuring a substance to be measured including this step.

10. The method for measuring a substance to be measured according to claim 9, wherein the acquisition of the drain current value is performed in a state where the field-effect transistor operates in a linear operation region.

11. The information processing terminal, The time differential value dI ds a storage unit storing the following linear function (i) for calculating the absolute value of the concentration of the substrate from / dt; From the linear function (i), a central processing unit that calculates over time the absolute value of the concentration of the substrate corresponding to the time derivative value dI ds / dt, further comprising: The time differential value dI ds Calculating the absolute value of the concentration of the substance to be measured by using dt and the following linear function (i), which is a method for measuring the substance to be measured. [Number 3] In the above linear function (i), Ids is the drain current value, [M] is the concentration of the substance to be measured, [OX] 0 is the concentration of the initial oxidized form, [RED] 0 is the concentration of the initial reduced form, R is the gas constant, T is the temperature (K), F is the Faraday constant, z is the number of electrons transferred in the redox reaction of one molecule of the oxidized form, D is the thickness of the working electrode, and k is the reaction rate constant.

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