WO2005093400A1

WO2005093400A1 - Fuel cell type wireless enzyme sensor

Info

Publication number: WO2005093400A1
Application number: PCT/JP2005/003108
Authority: WO
Inventors: Koji Sode
Original assignee: Ultizyme International Ltd.
Priority date: 2004-03-25
Filing date: 2005-02-25
Publication date: 2005-10-06
Also published as: JPWO2005093400A1

Abstract

[PROBLEMS] The new principle of an enzyme sensor capable of continuously measuring the concentration of a substrate wirelessly, and not including a power supply. [MEANS FOR SOLVING PROBLEMS] An enzyme sensor system comprising a disposable sensor chip composed of a circuit that resonates on receiving a radio wave supplied from the outside wirelessly, an enzyme fuel cell that applies potential to the circuit, the fuel cell being an enzyme fuel cell using enzyme as an anode that changes potential depending on the substrate concentration to be measured, and a resonance circuit that converts a change in the power of the enzyme fuel cell into a change in resonance frequency, and an external controller that performs control/display/radio wave reception and transmission on the sensor chip from the outside. A target substrate can be measured wirelessly by an enzyme sensor characterized by detecting the concentration of the substrate depending on the concentration of a detection item and using an electromotive force change as an index, in an electromotive force produced by transferring electrons produced by an anode enzyme reaction to an external electron receptor at a cathode.

Description

Specification

Fuel cell type wireless enzyme sensor

Technical field

The present invention relates to an enzyme sensor.

Background art

[0002] An enzyme sensor is a sensor in which an enzyme is immobilized on an electrode surface such as an oxygen electrode or a hydrogen peroxide electrode, and the enzyme reaction detects the concentration of a compound used as a substrate by the enzyme as a signal of the electrode. . For example, a glucose sensor that can easily and quickly measure a blood glucose level has been developed. On the other hand, the number of diabetic patients tends to increase year by year, and diagnosis of diabetes and home management of patients are very important. Therefore, glucose sensors that can easily and quickly measure blood glucose levels have been developed. Darcos oxidase (GOD) is most often used as a glucose sensor element. As the principle of GOD glucose detection, an oxygen electrode type that detects oxygen consumed during the oxidation reaction of GOD glucose or a hydrogen peroxide electrode type that detects generated hydrogen peroxide has been developed. Was.

[0003] However, in this method, since a high applied potential is affected by other redox substances in the blood, since the 1980s, a mediator type that reduces the applied potential using various electron mediators has been used. Sensors have been developed. However, GOD cannot transfer accurate electrons to oxygen instead of mediator when the dissolved oxygen concentration is high, so accurate measurement cannot be performed.

[0004] Therefore, glucose dehydrogenase (GDH) has been attracting attention as an ideal mediator-type sensor element that is not affected by the concentration of dissolved oxygen. Among the GDHs, the enzyme-linked PQQ glucose dehydrogenase (PQQGDH) has a high catalytic activity and a high turnover number, so that when a mediator such as phenazine methosulfate is used, the response current value is high. High response time no longer. That is, accurate and quick measurement is possible. Also, since it is a coenzyme-bound type, it is not necessary to add an expensive coenzyme to the reaction solution. Furthermore, if the enzyme is water-soluble, a surfactant is not required in the buffer solution, and there is an advantage that the enzyme is easy to handle. QQGDH (PQQGDH-B) is very ideal as an element of a glucose sensor. In any case, continuous and reliable development of a blood glucose measurement device is desired.

[0005] However, when these oxidoreductases are applied to an enzyme electrode, it is necessary to apply a constant potential in order to reoxidize the reduced electron acceptor as a result of the enzymatic reaction. External power supply is indispensable. Furthermore, in order to apply a potential, there is a problem that, in addition to the reducing substance generated as a result of the enzymatic reaction, for example, various oxidized compounds present in a living body are oxidized on the electrode, and a contaminant signal is exhibited. In addition, a disposable glucose sensor used in a normal autoglycemic diagnostic apparatus collects blood by itself, attaches the blood sample to a sensor chip, and uses the sensor chip with a power supply capable of applying a potential. It is not suitable for constant monitoring of blood glucose because it is plugged into the main unit. Furthermore, in the recently developed continuous glucose monitoring system, so-called Continuous Glucose Monitoring System, and CGMS, a conventional enzyme sensor chip is attached to the body surface, and the sensor body including the power supply is fixed to the body, so that It is intended to measure blood sugar. However, miniaturization is difficult in principle because a power supply is required. Disclosure of the invention

Problems to be solved by the invention

An object of the present invention is to provide a new principle of an enzyme sensor that can continuously measure the concentration of a substrate wirelessly and does not include a power source. Means for solving the problem

[0007] The present invention includes a circuit that resonates by radio waves supplied wirelessly from the outside, and an enzyme fuel cell that applies a potential to the circuit. Furthermore, a fuel cell is an enzyme fuel cell that uses an enzyme for the anode, where the potential (electromotive force) changes depending on the concentration of the substrate to be measured. In addition, a disposable sensor chip consisting of a resonance circuit that converts the change in electromotive force of the enzyme fuel cell into a change in resonance frequency is used, and this is used for external force control, display, and radio wave transmission. It is an enzyme sensor system that also comprises the power of an external controller for receiving.

[0008] Further, in response to the electromotive force of the fuel cell, a change in the potential is used as a change in the capacitance of the capacitor to change the resonance frequency of the circuit. Particularly, the capacitor is constituted by a resonance circuit using a varicap diode.

[0009] Preferably, the enzyme used for the anode is acid oxidase, particularly when glucose is to be measured, glucose oxidase or glucose dehydrogenase. In addition, glucose dehydrogenase is an enzyme containing pyro-mouth quinolinine quinone (PQQ) or flavin adenine dinucleotide (FAD) as a coenzyme.

[0010] More specifically, the present invention relates to an enzyme sensor characterized in that the concentration of a substrate is measured by an electromotive force generated by passing electrons generated by an enzyme reaction at an anode to an electron acceptor at a cathode. It is.

[0011] Further, the present invention is the above-described enzyme sensor, further comprising an oscillation circuit, wherein a resonance frequency in the oscillation circuit changes based on the electromotive force, and the resonance frequency is detected to detect a substrate. An enzyme sensor characterized by measuring a concentration.

[0012] The present invention is the enzyme sensor described above, wherein the resonance frequency is detected by a radio wave supplied from the outside.

[0013] Further, the present invention is the enzyme sensor described above, wherein the change in electromotive force depending on the substrate concentration at the anode changes the resonance frequency in the oscillation circuit.

[0014] Further, the present invention is the enzyme sensor described above, characterized by using a resonance circuit that converts a change in electromotive force into a change in capacitor capacitance.

[0015] Further, the present invention is the above-mentioned enzyme sensor, characterized by using a barrier diode to convert a change in electromotive force into a change in capacitor capacitance.

[0016] Further, the present invention relates to an enzyme sensor including a circuit that resonates with a radio wave supplied wirelessly by an external force,

[0017] The resonance frequency of the circuit changes depending on the potential applied to the circuit,

[0018] The change in the potential is caused by a change in electromotive force of an enzyme fuel cell using an enzyme as an anode,

[0019] The change in the electromotive force depends on the concentration of the substrate to be measured, It is an enzyme sensor.

[0020] The present invention is the above-mentioned enzyme sensor, characterized by using an enzyme using glucose as a substrate as an anode enzyme.

[0021] The present invention also provides the enzyme sensor described above, wherein the enzyme is a glucose oxidase.

[0022] Further, the present invention is the above enzyme sensor, wherein the enzyme is glucose dehydrogenase.

[0023] Further, the present invention is the above enzyme sensor, wherein the glucose dehydrogenase has pyroquinoline quinone as a coenzyme.

[0024] Further, the present invention is the above-mentioned enzyme sensor, wherein the glucose dehydrogenase has flavin adenyldinucleotide (FAD) as a coenzyme. Brief Description of Drawings

FIG. 1 shows a configuration diagram of an enzyme fuel cell of the present invention.

FIG. 2 shows a circuit diagram of a fuel cell type wireless enzyme sensor of the present invention.

FIG. 3 shows the glucose concentration dependence of the output current of an enzyme fuel cell using the catalytic subunit of glucose dehydrogenase using FAD as a coenzyme of the present invention as an anode enzyme.

FIG. 4 shows the glucose concentration dependence of the output voltage of an enzyme fuel cell using the catalytic subunit of glucose dehydrogenase using FAD of the present invention as a coenzyme as an anode enzyme.

FIG. 5 shows the glucose concentration dependence of the output of an enzyme fuel cell using the catalytic subunit of glucose dehydrogenase using FAD of the present invention as a coenzyme as an anode enzyme.

FIG. 6 shows the glucose concentration dependence of the output current of an enzyme fuel cell using the glucose dehydrogenase complex using FAD of the present invention as a coenzyme as an anodic enzyme.

FIG. 7 shows the glucose concentration dependency of the output voltage of an enzyme fuel cell using the glucose dehydrogenase complex using FAD of the present invention as a coenzyme as an anode enzyme.

[Fig. 8] Fig. 8 shows the dependence of the output of an enzyme fuel cell on the dulcose concentration when the glucose dehydrogenase complex of the present invention using FAD as a coenzyme was used as an anodic enzyme.

FIG. 9 shows a block diagram of a fuel cell type wireless enzyme sensor of the present invention. FIG. 10 shows a block diagram of a fuel cell type wireless enzyme sensor (including a signal amplifier (amplifier)) of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

[0026] As the enzyme used for the anode of the present invention, various acid reductases can be used. For example, alcohol, glucose, cholesterol, fructosylamine, glycerin, uric acid oxidase with FAD as coenzyme, alcohol, glucose, glycerin dehydrogenase with FAD as coenzyme, alcohol, glucose, glycerin with PQQ as coenzyme Dehydrogenase. In particular, when glucose is to be measured, glucose dehydrogenase using glucose oxidase or FAD or PQQ as a coenzyme is desired. This may be a microorganism producing the enzyme, an enzyme isolated and purified from cells, or an enzyme recombinantly produced in E. coli or the like.

[0027] The fuel cell used in the present invention is an enzyme fuel cell characterized in that an oxidase or a dehydrogenase is fixed to an anode. The force source may be an electrode using an enzyme that reduces oxygen, such as pyrilvin oxidase, or an electrode combining an appropriate electron acceptor. The anode can be configured to include an electron acceptor together with the enzyme. That is, an electron obtained by the enzymatic reaction may be passed to an artificial electron acceptor, and this may be oxidized on an electrode.

[0028] Alternatively, a dehydrogenase that can transfer electrons directly to an electrode, such as an enzyme having cytochrome in the electron transfer subunit, can form an anode without adding an artificial electron acceptor. A carbon electrode, a gold electrode, a platinum electrode, or the like can be used as an electrode material for the anode and the force source. The artificial electron acceptor of the anode is not particularly limited, and examples thereof include an osmium complex, a ruthenium complex, phenazine methosulfate, and derivatives thereof.

[0029] The enzyme of force sword is not particularly limited, but pyrylvin oxidase and laccase can be applied. The artificial electron acceptor of the force sword is not particularly limited, but potassium ferricyanide, ABTS and the like can be used.

[0030] In the present invention, various oxidases can be used as the anode enzyme! /, And dehydrogenase can be used. In particular, when measuring glucose, glucose oxidizing enzyme, P Glucose dehydrogenase using QQ or FAD as a coenzyme can be used.

In the present invention, as a method for attaching an enzyme to an electrode, a method in which an enzyme is directly mixed with an electrode material such as a carbon paste is used. Alternatively, the immobilized enzyme may be prepared and then mounted on an electrode using a general enzyme immobilization method. For example, after mixing the two, cross-linking treatment is performed with a di-crosslinking reagent such as daltaraldehyde, and the mixture is immobilized in a synthetic polymer such as a photo-crosslinkable polymer, a conductive polymer, or an acid-reducing polymer, or a natural polymer matrix. There is a method. The mixed protein thus prepared is mixed with the carbon paste or mixed with the carbon paste and then subjected to a crosslinking treatment, and the mixture prepared is mounted on an electrode made of carbon, gold, platinum, or the like. .

[0032] Further, when the enzyme is mounted on the electrode in this way, the artificial electron acceptor can be immobilized at the same time. Typically, glucose dehydrogenase using FAD as a coenzyme, FADGDH, and methoxyphenazine methosulfate (mPMS) are mixed, further mixed with a carbon paste, and then freeze-dried. This is mounted on a carbon electrode, and immersed in an aqueous solution of daltaraldehyde in that state to crosslink the protein and create an enzyme electrode.

[0033] In the enzyme fuel cell used in the present invention, oxidation or dehydrogenation appeal using the measurement object as a substrate is fixed to the anode electrode. An oxygen reductase is immobilized on the force sword. The electrode thus prepared is used as an anode and an electrode for a force source. For the anode, for example, m-PMS can be used as a human electron acceptor, and for the force source, for example, ABTS can be used as an artificial electron acceptor. A battery is constructed by connecting a variable resistor between the two electrodes, and the resulting current or voltage value is measured by adding a sample containing the substrate to be measured.

In particular, the addition of a sample changes the voltage value in a substrate concentration-dependent manner, and the voltage value changes the capacitance of the varicap diode constituting the resonance circuit, resulting in a change in the resonance frequency. I do. By supplying an appropriate radio wave from the outside and searching for the resonating frequency, the concentration of the substrate can be measured. That is, the correlation between the resonance frequency and the substrate concentration is recorded in advance, a calibration curve is created based on the correlation, and the substrate concentration of the unknown sample can be measured from the observed resonance frequency. [0035] For example, when measuring glucose as a target substrate, FADGDH, Myrothecium sp.-derived pyrilrubin oxidase (BOD) (provided by Amano Enzym) is mixed with a carbon paste, lyophilized, and then lyophilized. The surface is then filled and cross-linked with 1% dataraldehyde to prepare the enzyme-immobilized anode and force sword, respectively. The electrode thus produced is used as an anode and an electrode for a force sword. The anode is an m-PMS as an electronic mediator, and the force sword is an ABTS as an electronic mediator. By constructing a battery by connecting both electrodes with a variable resistor and adding a sample containing glucose, the current value and voltage value change in a concentration-dependent manner. By measuring this with an external radio wave using a resonance circuit, the glucose concentration of the unknown sample can be measured.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples. Example 1

[0038] A heat-resistant glucose dehydrogenase catalytic subunit using FAD as a coenzyme was prepared according to a previous report, and the anode electrode was fixed. The enzyme used was recombinantly produced using Escherichia coli. Anode reaction solution lOOmM ppb (pH 7.0), 200 mM m— PMS 50 ^ Approx. 100 1 (final concentration; ImM or 2 mM), 2M glucose 100 1 (final concentration; 20 mM) To 10 ml.

The force source electrode was prepared by mixing Myrothecium sp.-derived bilirubin oxidase (Bilirubin Oxidase; BOD) (provided by Amano Enzym) with 20 mg of carbon paste and freeze-drying. The amount of enzyme used was 50 U according to a previous report. After mixing this well, it was filled only on the surface of the carbon paste electrode already filled with about 40 mg of carbon paste, and polished on filter paper. These electrodes, and stirred at 10mM MOPS buf fer (pH7. 0 ) 30 min in room temperature with 1% glutaraldehyde, further ^{lOmM Tris buffer (pH 7. 0} ) was stirred at 2 0 min at room temperature in. This electrode is kept in lOmM MOPS buffer (pH 7.0) for 1 hour or less. Stir at room temperature to equilibrate. Except during measurement, the cells were stored at 4 ° C in 10 mM MOPS buffer (pH 7.0). The thus prepared BOD electrode and force sword reaction solution were mixed with 100 mM ppb (pH 7.0) 98001 and 25 mM ABTS 2001 (final concentration; 0.5 mM), and the total amount was used as 10 mM. The electrodes and the reaction solution were set in separate thermostat cells for the anode and the power source, and the cells were connected by a salt bridge (a 2.17M KC1 solution solidified with 30% agarose) to construct a battery. A variable resistor and digital multimeter were connected between each electrode (Fig. 1). The measurement was performed at 25 ° C. The load was changed stepwise from 1 Ω to 1 MΩ with a variable resistor, and the resulting current and voltage values were measured with a digital multimeter. The anode, force sword and digital multimeter were connected in series for current measurement and connected in parallel for voltage measurement. The power was determined by the product of the current value and the voltage value. Example 2

A cell was constructed with an m-PMS concentration of 2 mM in an enzyme fuel cell using an anode electrode on which a 20 U catalytic subunit was fixed. A load of resistance (40 kΩ) was applied, the glucose concentration of the anode was gradually increased by the OmM force, and the current and voltage values obtained at each glucose concentration were measured with a digital multimeter to calculate the power. . The current density and the power density were determined by the quotient of the obtained current value and power value with respect to the electrode surface area (7.1 × 10 ″ ² cm ² ).

[0041] Figures 3, 4 and 5 show the glucose concentration dependence of the current, voltage and power of the battery in which the glucose dehydrogenase catalytic subunit 20U using FAD as a coenzyme is immobilized. Electric power was obtained by the addition of glucose, and the electric power increased in a glucose concentration-dependent manner. Thus, the glucose concentration of the unknown sample can be measured from the output of the present enzyme fuel cell. Example 3

[0043] A fuel cell was constructed in which an electrode on which a glucose dehydrogenase complex having FAD as a coenzyme was immobilized was used as an anode. 20 U (290 μg) of glucose dehydrogenase complex was mixed with 20 mg of carbon paste and freeze-dried. After mixing well, the surface of the carbon paste electrode previously filled with about 40 mg of carbon paste was filled and polished on filter paper. These electrodes were stirred in lOOmM p.p.b. (pH 7.0) containing 1% glutaraldehyde for 30 minutes at room temperature, and further stirred in lOmM Tris buffer (pH 7.0) for 20 minutes at room temperature. These electrodes were stirred at room temperature in lOOmM p.p.b. (pH 7.0) for 1 hour or more. This electrode was stored at 100C in lOOmM p.p.b. (pH 7.0) except during measurement. Anode reaction solution PuOmM ppb (pH 7.0) 9700 μ 200 mM m—PMS 1001 (final concentration; 2 mM), 2M glucose (final concentration; 40 mM) were mixed to make a total volume of 10 ml. The prepared BOD electrode and force sword reaction solution were mixed with 100 mM pp b. (PH 7.0) 9800 μ25 mM ABTS 200 / z 1 (final concentration; 0.5 mM) to make the total amount 10 mM. Each electrode and reaction solution were set in a separate thermostat cell for the anode and the power source, and the cells were connected by a salt bridge (a 2.17M KC1 solution solidified with 30% agarose). A variable resistor and digital multimeter were connected between each electrode. The measurement was performed at 25 ° C. The load was varied stepwise from 1 Ω to 1ΜΩ with a variable resistor, and the resulting current and voltage values were measured with a digital multimeter. The anode, force sword, and digital multimeter were connected in series for current measurement, and connected in parallel for voltage measurement. Power was obtained by multiplying current and voltage

Example 4

[0045] A resistance load of 40kΩ is applied, the glucose concentration at the anode is increased stepwise from OmM, and the current and voltage values obtained at each glucose concentration are measured with a digital multimeter to calculate the power. did. Immobilize glucose dehydrogenase complex 20U with FAD as coenzyme Figures 6, 7, and 8 show the glucose concentration dependence of the battery current, voltage and power. Electric power was obtained by the addition of glucose, and the electric power increased in a glucose concentration-dependent manner. Thus, the glucose concentration of the unknown sample can be measured from the output of the present enzyme fuel cell.

Industrial applicability

The present invention provides a new principle of an enzyme sensor that can continuously measure the concentration of a substrate wirelessly and does not include a power source.

Claims

The scope of the claims

[1] An enzyme sensor that measures the concentration of a substrate by the electromotive force generated by passing electrons generated by an enzymatic reaction at the anode to an electron acceptor in a force sword

[2] The enzyme sensor according to [1], further comprising an oscillation circuit, wherein the resonance frequency in the oscillation circuit changes based on the electromotive force, and the resonance frequency is detected to detect the substrate. An enzyme sensor for measuring a concentration.

[3] The enzyme sensor according to item 2, wherein the resonance frequency is detected by a radio wave supplied from the outside.

[4] The enzyme sensor according to item 2, wherein the change in electromotive force depending on the substrate concentration at the anode changes the resonance frequency in the oscillation circuit.

[5] The enzyme sensor according to item 4, characterized by using a resonance circuit that converts a change in electromotive force into a change in capacitance of a capacitor.

[6] The enzyme sensor according to item 5, wherein a norcap diode is used to convert a change in electromotive force into a change in capacitor capacity.

[7] An enzyme sensor comprising a circuit that resonates with radio waves supplied wirelessly from the outside,

The resonance frequency of the circuit changes depending on the potential applied to the circuit, and the change in the potential is caused by a change in the electromotive force of an enzyme fuel cell using an enzyme as an anode;

The change in the electromotive force depends on the concentration of the substrate to be measured,

Enzyme sensor.

[8] The enzyme sensor according to any one of Items 1 to 7, wherein an enzyme using glucose as a substrate is used as an anode enzyme.

[9] The enzyme sensor according to item 8, wherein the enzyme is a glucose oxidase.

[10] The enzyme sensor according to item 8, wherein the enzyme is glucose dehydrogenase.

[11] Glucose dehydrogenase is characterized in that it has a pyro-mouth quinoline quinone as a coenzyme Item 11. The enzyme sensor according to item 10, wherein

Item 11. The enzyme sensor according to Item 10, wherein the glucose dehydrogenase has flavin adenine dinucleotide (FAD) as a coenzyme.