JP3259010B2 - Enzyme electrode and measuring device using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the concentration of a specific component in a liquid, particularly a body fluid (eg, blood, urine, etc.) by the action of an enzyme.
Specific components (eg glucose, cholesterol, etc.)
The present invention relates to an enzyme electrode for measuring the concentration of an enzyme. More specifically, an electrode system having a working electrode and a counter electrode made of different types of materials is applied to apply a voltage necessary for the potentiostatic electrolysis of an oxidizing or reducing compound converted by an enzymatic reaction. The present invention relates to an enzyme electrode which can be performed by electromotive force and a method for producing such an electrode. Further, the present invention relates to a measuring instrument using such an electrode and a method for measuring concentration.

[0002]

2. Description of the Related Art An enzyme electrode in which an enzyme-immobilized phase and an electron mediator (mediator) are coated, for example, in a layer on an electrode sensitive portion is widely used as a biosensor in the clinical field. For example, taking a glucose sensor as an example, a simple blood glucose meter using a disposable glucose sensor has already been put into practical use for the purpose of managing a blood glucose level of a diabetic patient. Japanese Patent Publication Nos. 2-59424, 4-40658 and U.S. Pat. No. 5,108,564 describe an enzyme electrode as a glucose sensor, and such an electrode has been put to practical use.

The principle of measurement using an enzyme electrode using an electron carrier as described above is as follows: a substrate S (a component whose concentration is to be measured, for example, glucose) is produced by an enzyme E (for example, glucose oxidase). When oxidized to product P (gluconolactone), the active center of enzyme E changes from oxidized Eox to reduced Ered. The reduced Ered returns to Eox via the oxidized compound Mox that acts as an electron carrier of the enzyme, and the electron transfer medium becomes Mred. Simultaneously, Mred is electrolyzed by an appropriate applied voltage on the working electrode, and the substrate concentration can be measured by measuring the oxidation current obtained at this time.

[0004] The principle of this measurement can be expressed by the following equation: When measuring glucose concentration using glucose oxidase (GOD), glucose + GOD (FAD) → gluconolactone + GOD (FADH ₂ ) (1) GOD (FADH) ₂ ) + 2Mox → GOD (FAD) + 2Mred + 2H ⁺ (2) 2Mred → 2Mox + 2e ⁻ (3) (FAD is flavin adenine dinucleotide (flav
in adenine dinucleotide)). The measuring method using such an enzyme electrode is described in the above-mentioned patent publication, and can be referred to in the present invention.

[0005] Among such enzyme electrodes, the shape of a disposable enzyme electrode for measuring glucose concentration is shown in FIG.
Are used as biosensors. In this sensor, the working electrode 1 and the counter electrode 2 were prepared by dispersing the same type of graphite in a binder such as polyvinyl chloride to obtain a paste-like material, which was printed by screen printing to form both layered electrodes. Then, it is formed by firing. Next, a paste-like mixture containing glucose oxidase (glucose oxidase, GOD) and an electron carrier M is applied on the electrode formed as described above, dried, and solidified. Both electrodes are at least partially covered with a reagent layer 3, and a spacer 4 having a sample inlet 6 and a cover 5 having an air outlet 7 are adhered thereon by a bonding means such as a double-sided tape to measure glucose concentration. Complete the enzyme electrode for use.

[0006] Liquid samples such as whole blood, plasma, urine and saliva come into contact with the sample inlet 6 and automatically begin to be sucked into the reagent layer 3 by capillary action, whereby air is discharged from the outlet 7. Then, the entire reagent layer 3 is filled with the sample. Immediately after the suction of the sample is completed, the dissolution of the reagent layer 3 starts, and the enzyme reaction proceeds according to the formula (1). As the electron carrier M, ferrocene, potassium ferricyanide, benzoquinone, or the like is used.
The reduced Mred generated in step (1) is subjected to electrolytic oxidation as shown in equation (3) by applying a constant voltage in the range of 0.3 to 0.6 V to the electrode system from an application circuit using an external power supply. Since the current value (2e ⁻ ) obtained at this time is directly proportional to the glucose concentration, the glucose concentration in the sample can be obtained by measuring this current value.

[0007]

The biosensor of FIG. 1 as described above is used for a measuring instrument such as a blood glucose meter for self-management. In the present specification, a measuring device is a device capable of measuring the concentration of a target component using an enzyme electrode, that is, calculating the concentration of the target component based on a current value obtained from the enzyme electrode. Means that the user can be informed of the concentration.
Such a measuring instrument is required to be small, thin and / or light, so-called pocket size, so as to be easy to carry, and also required to be able to measure quickly, easily, with high accuracy, and the like. .

However, when the above-described conventional biosensor is used for a blood glucose meter, an external power supply is required as a voltage applying means required for the expression (3), and an alkaline dry battery or a lithium battery is not used. It is built into the blood glucose meter as a means. For this reason, a blood glucose meter having a structure thinner than the thickness of the battery cannot be provided, and therefore, there is a limit to miniaturization and weight reduction. Further, since the battery used in the blood glucose meter has a service life, it is necessary to periodically replace the battery.
Furthermore, there is a problem of complication such as preparing a spare battery or always carrying a spare battery. Of course, there is also a demand that the current value consumed by the enzyme electrode should be small. Therefore, as long as a conventional enzyme electrode is used, the above requirements have not been sufficiently satisfied.

Such a request is not limited to the enzyme electrode for measuring blood glucose level described above as an example, but various measuring instruments using an enzyme electrode requiring external application, such as cholesterol, uric acid, lactic acid, Measuring instruments that use enzyme electrodes for measuring NADH, urea, etc. are a common problem.

[0010]

According to the present invention, an electrode system of a working electrode and a counter electrode made of different materials is generated in place of a voltage applying means by an external power supply which is required in the measurement using the enzyme electrode of the prior art. It consists in using electromotive force. As a result, the current consumption of the measuring instrument can be minimized, the battery capacity to be built in can be reduced, the life of the battery can be extended, or the solar cell panel can be used as a drive power supply for the electronic circuit of the measuring instrument. At least one, and most preferably, all of the above requirements.

Accordingly, in a first aspect, the present invention provides:
An enzyme electrode having an immobilized phase of an enzyme and an electron mediator (mediator), wherein the working electrode and the counter electrode are made of mutually different electrode materials. In the present invention, the working electrode is an electrode in which the electron transfer medium is oxidized or reduced as described above, that is, a pole that plays a role in measuring the concentration of a target component, and a counter electrode is a pole for the working electrode. Used as meaning.

The electrode material that can be used to construct the enzyme electrode of the present invention may be any material that can be generally used as an electrode, provided that the materials of the working electrode and the counter electrode are different from each other. Good. Specific examples include carbon, metals, alloys, and various compounds of metals and alloys (eg, oxides, hydroxides, halides, sulfides, nitrides, and carbides). Further, combinations such as mixtures and composites of these electrode materials can also be used. In the present invention, a mixture means a material in which the electrode materials are mixed in a so-called micro order, and a composite means a material in which the electrode materials are mixed in a larger order than the mixture (accordingly, the material is mixed in a so-called macro order). And not as homogeneously mixed as a mixture) and combined as separate materials.

Preferred metals include silver, aluminum, gold, cobalt, barium, iron, manganese, nickel, lead, zinc, platinum, lithium, copper and the like. Preferred alloys include white copper, manganin,
Examples thereof include an aluminum-silicon alloy and a nickel-copper alloy. Particularly preferred metal compounds include Mn
O ₂ , Ag ₂ O, PbO ₂ , NiOOH, SOCl ₂ , V ₂ O ₅ ,
AgCl can be exemplified.

When carbon is used, graphite,
Various carbon materials such as pyrolytic carbon, glassy carbon, acetylene black and carbon black can be used. Of course, it is also possible to use a normal amorphous carbon material. When an electrode material is used as a mixture or a composite, a combination of MnO ₂ and acetylene black, platinum and graphite, silver and silver chloride, and the like can be exemplified.

[0015] In the enzyme electrode of the present invention, the structure of the electrode is not particularly limited, and various structures employed in the conventional enzyme electrode can be employed. The electrodes may be in the form of, for example, a line, a rod, or a sheet, and these electrodes are adapted to be connected to a suitable circuit (for example, a current measuring circuit) for measuring a current value. In a particularly preferred embodiment of the present invention, for example, a thin layer structure as shown in FIG. 1 can be adopted as the structure of the electrode.

Such an electrode can be manufactured by a conventional method for forming a thin-layer electrode. That is, a powder having a suitable size of a material for forming an electrode, a suitable binder (eg, polyvinyl chloride, epoxy, neoprene, cellulose, etc.) and a suitable solvent (eg, tetrahydrofuran, toluene, isopropyl alcohol, etc.) and, if necessary, A conductive material (eg, carbon powder, conductive polymer, etc.) is mixed to prepare an electrode material paste, and this paste is applied to a substrate material (eg, strip-like polyethylene terephthalate, ceramic substrate, etc.) by an appropriate method (eg, screen printing method). Etc.) to a suitable thickness (for example, 1
0 to 200 μm) and then dried, preferably by sintering to a thickness of 1 to 50 μm
Can be formed. Further, if necessary, the above-mentioned thin layer electrodes may be formed by laminating two or more layers.

In the enzyme electrode of the present invention, the enzyme and the electron carrier are present on the counter electrode and the working electrode as a solid phase uniformly mixed with each other, preferably as a solid phased thin layer. Preferably, the enzyme and the electron mediator coat the electrode so as to cover at least a portion of both the working and counter electrodes as a thin layer. Such a thin layer can be formed in the same manner as in the case of forming the above-mentioned electrode. That is,
A reagent paste comprising an enzyme, an electron carrier, a binder and a solvent is prepared, and is placed on the electrode so as to cover the electrode by an appropriate method (for example, by a dispenser), and then dried. Immobilize.

If necessary, another layer may be supplied between the electrode and the reagent layer or on the reagent layer. This additional layer is formed of a water-soluble polymer compound such as carboxymethylcellulose, polyvinyl alcohol and polyvinylpyrrolidone, and a surface-active substance such as phosphatidylcholine.
It may be about 0 μm. These layers have a function of smoothing the separator between the electrode and the sample or the suction of the sample.

The enzyme that can be used in the enzyme electrode of the present invention is not particularly limited, and the enzyme used in the conventional enzyme electrode can be used alone or in combination. Specifically, glucose oxidase, cholesterol oxidase, lactate oxidase, uricase, alcohol oxidase, NADH oxidase,
Diaphorase, urease, glucose dehydrogenase, lactate dehydrogenase, 3-hydroxybutyrate dehydrogenase, fructose dehydrogenase,
Alcohol dehydrogenase and the like can be used. still,
In the present specification, particularly, the case where the glucose concentration is measured is described as an example.However, the substance whose concentration can be measured by the enzyme electrode of the present invention is not limited to glucose, and is appropriately selected from the aforementioned enzymes used. The concentration of various substances can be measured by selecting the above.

For example, the concentration of lactic acid (for example, in blood or saliva) can be measured by using lactate oxidase as an enzyme, and the concentration of cholesterol (for example, in blood or saliva) can be measured by using uricase if cholesterol oxidase is used. If used, the concentration of uric acid (eg, in blood, urine), the concentration of NADH (eg, in blood) using NADH oxidase or diaphorase, or the concentration of NADH (eg, blood, urine) using fructose dehydrogenase. The concentration of fructose, the concentration of galactose (for example in blood) using galactose oxidase, and the concentration of alcohol (for example, in blood and saliva) using alcohol dehydrogenase and diaphorase together. Hydroxybutyrate dehydrogenase and NADH
If oxidase is used in combination, the concentration of 3-hydroxybutyric acid (for example, in blood) can be measured.

In the enzyme electrode of the present invention, any material which can be used as an electron carrier can be used, such as potassium ferricyanide, benzoquinone, phenazine methosulfate, thionine, ferrocene, naphthoquinone, methylene blue. And the like. As long as the electrode materials of the counter electrode and the working electrode are different from each other, any combination of the above-described electrode materials, electron carriers, and enzymes can be adopted. In particular, it is preferable to consider as a guide the type of enzyme used, the type or concentration of the component to be measured, the oxidation potential or reduction potential of the electron carrier, the electromotive force generated by the combination of electrodes, the production cost, and the stability. Depending on the nature or any combination of these factors, it can be appropriately selected, for example, by a try-and-error method. The enzyme electrode of the present invention may have a spacer and a cover on the immobilized phase containing the enzyme and the electron carrier formed as described above, as used in a conventional enzyme electrode. .

In a second aspect, the present invention provides a method for producing the enzyme electrode of the first aspect. That is, a counter electrode and a working electrode made of mutually different materials are formed on the substrate material, and on at least a part of both of these electrodes,
Provided is a method for producing an enzyme electrode, which comprises forming a solid phase containing an enzyme. Further, a spacer and a cover used for a conventional enzyme electrode may be placed on the solid phase containing the enzyme thus formed. The matters described in relation to the enzyme electrode described above can also be applied to the method for producing an enzyme electrode of the present invention.

In a third aspect, the present invention provides a measuring instrument for measuring the concentration of a target component in a liquid using the above-mentioned enzyme electrode. That is, a sample supply detection circuit,
It comprises a measurement timing control circuit, an arithmetic circuit, a display circuit, and a solar cell for a display unit for displaying concentration, and receives (or engages with) the enzyme electrode of the present invention;
Provided is a measuring instrument further comprising a member that enables a measurement timing control circuit to detect a current value generated according to the concentration of a component in an enzyme electrode.

In the present invention, the sample supply detecting circuit is
This circuit detects a change in impedance due to a sample being supplied from the sample suction port of the electrode, and automatically starts a measurement program. In the present invention, the measurement timing control circuit means that after a sample is supplied, an enzymatic reaction proceeds, and after a lapse of time sufficient to generate a product P,
This circuit detects the current flowing when the circuit is closed.

In the present invention, an arithmetic circuit refers to a substrate S based on an arithmetic expression (calibration curve) set in advance in a measuring instrument by the current value detected by the measurement timing control circuit.
Is a circuit for converting the density into In the present invention, the display circuit is a circuit that displays the density value converted by the arithmetic circuit on a display unit of the measuring device.

In the present invention, the solar cell is freely selected from silicon solar cells, amorphous solar cells, heterojunction solar cells, and other types, and all of them are emitted from lighting equipment such as sunlight or fluorescent lamps. It converts visible light into electrical energy. As the above-mentioned circuit of the measuring instrument of the present invention, a circuit using a conventionally used enzyme electrode can be used. For details thereof, reference can be made to, for example, a circuit described in Japanese Patent Application Laid-Open No. 4-357452.

Further, conventional solar cells can be used. For example, Electrochemical Handbook (4th edition published by Maruzen Co., Ltd.
61 to 468) can be referred to. The member for receiving the enzyme electrode of the present invention is the same as the member used conventionally, except that the means for applying to the enzyme electrode is omitted.

In a fourth aspect, the present invention provides a method for measuring the concentration of a target component using the above-mentioned enzyme electrode in a measuring instrument (ie, engaging with an electrode receiving member).

[0029]

According to the present invention, the working electrode (measurement electrode) of the enzyme electrode and the electrode material of the counter electrode are made different from each other,
An electromotive force is generated between the working electrode and the counter electrode without application from outside the enzyme electrode, and the current value can be measured by using this electromotive force for measuring the oxidized electrolytic current value. This electromotive force generation mechanism will be described below, for example, in the case of MnO ₂ .

MnO ₂ reacts as follows at the electrode. MnO ₂ + H ⁺ + e ⁻ ← → MnOOH The Nernst equation at this time is: E = E ⁰ +2.303 RT / F (loga _{MnO 2} / a _MnOOH ) +2.303 RT / Flog [H ⁺ ] Wherein, E ⁰ is the standard reduction potential, R represents gas constant, F is Faraday's constant, T is the temperature, a _MnO2, a _MnOOH
Is the activity of MnO ₂ and MnOOH, respectively, and [H ⁺ ] is the hydrogen ion concentration. As is clear from this equation, if MnO ₂ is sufficiently present, a constant electromotive force E is generated, and the present invention utilizes this electromotive force. Therefore, if the kinds of materials constituting the electrodes are different, in principle, no matter which electrode material is used, an electromotive force is generated, so that the enzyme electrode of the present invention can be constituted.

In the enzyme electrode of the present invention, a practically most preferable combination of electrode materials includes an embodiment in which an electrode is formed of graphite as a working electrode and an electrode is formed of MnO ₂ as a counter electrode. Other particularly preferred embodiments include the following combinations: Preferred electrode material combinations (counter electrode) (working electrode) (generated electromotive force, mV) Graphite AgCl -90 PbO ₂ glassy carbon +1300 Ag ₂ O Pt +530 NiOOH Ni ₂ O ₃ +30 Ag ₂ O Ag +460

Further, in consideration of the enzyme and the electron mediator, particularly preferred combinations for the enzyme electrode of the present invention are as follows: Preferred combinations (enzyme) (electron mediator) (counter electrode material) (working electrode material) glucose oxidase Potassium ferricyanide MnO ₂ graphite 3-hydroxybutyrate dehydrogenase 1-methoxy PMS AgCl graphite uricase methylene blue NiOHH Ni ₂ O ₃ cholesterol oxidase 1,4-benzoquinone Ag ₂ O Ag Ag lactate oxidase Potassium ferricyanide Ag ₂ O Pt oxidase -Naphthoquinone PbO ₂ Au fructose dehydrogenase 1,4-benzoquinone Ag ₂ O Ag alcohol dehydrogenase 1,4-naphthoquinone MnO ₂ Pt

[0033]

Next, embodiments of the present invention will be described in detail. In this embodiment, MnO ₂ or Ag ₂ O is used as a counter electrode material, but is not particularly limited to this. (Example 1 and Comparative Example 1) FIG. 2 shows a configuration of a cell which can be used for the enzyme electrode used in this example. In the cell, a glassy carbon electrode 10 (long diameter 5 mm) was used as a counter electrode,
The nO ₂ paste 11 and the collodion film 12 are solidified.

The electrode was made of MnO ₂ powder (average particle diameter of 0.1%).
9 g of acetylene black powder (average particle size: 0.1 μm) was mixed with 9 g of 0.1 M phosphate buffer (pH 7.0).
0) MnO ₂ paste 11 kneaded into a paste with 50 ml
Is placed on the glassy carbon electrode 10 in an amount of 50 mg.
To prevent the MnO ₂ paste 11 from being detached, 7 μl of a collodion solution diluted 25-fold with ethanol was dropped thereon, and the mixture was naturally dried. Here, acetylene black powder is mixed to give MnO ₂ good conductivity, and another material such as carbon black, carbon powder, or a conductive polymer such as a conductive polymer may be used. Absent. The working electrode has a diameter of 0.
A coil 13 of a 5 mm platinum wire was used.

In the cell of FIG. 2, reference numeral 14 denotes a glass filter, and the working electrode chamber 16 and the counter electrode chamber 17 have a capacity of 0.1 mm.
Filled with 1M phosphate buffer. Using this cell,
First, the electromotive force generated between the electrodes was measured (Example 1). For comparison, the electromotive force generated was also measured for the electrode using the same platinum as the working electrode instead of the MnO ₂ electrode of the counter electrode 15 (Comparative Example 1).

FIG. 3 shows the measurement results of the electromotive force. As can be seen from FIG. 3, by using MnO ₂ for the counter electrode, about +3
An electromotive force of 80 mV was obtained. This electromotive force is
It is generated by the potential difference between MnO ₂ and platinum, and different electromotive force can be obtained when another electrode material is used. In the case of platinum-platinum, generation of electromotive force was not recognized. Therefore, by appropriately selecting different electrode materials, it is possible to freely obtain a potential required for measurement.

Example 2 and Comparative Example 2 The glucose concentration was actually measured using the electrode system (MnO ₂ -platinum) of FIG. 0.25M of Na ₂ SO ₄ to the working electrode chamber 16 in FIG. 2,
5 mM K ₃ Fe (CN) ₆ and each were dissolved to a glucose oxidase concentration of 166 mg / dl.
1M phosphate buffer was filled, and the counter electrode chamber 17 was filled with 0.1M.
Filled with 1M phosphate buffer. Next, when the working electrode 13 and the counter electrode 15 are connected in series with a resistance of 1 Mohm, glucose is added to the working electrode chamber 16 in various amounts, and the glucose concentration is changed, the current value flowing through this resistance is changed. Was measured (Example 2). For comparison, the current value was also measured when platinum was used for the counter electrode (Comparative Example 2). FIG. 4 shows the results obtained by plotting the current value against the glucose concentration.

In Example 2, a response current varying according to the glucose concentration was obtained, whereas in Comparative Example 2, no response current was obtained at any glucose concentration. This is because the use of MnO ₂ as the counter electrode allows K ₄
This is an effect achieved by utilizing an electromotive force generated between the electrodes without applying a voltage required for the constant potential electrolysis of Fe (CN) ₆ from outside the cell.

(Examples 3 and 4 and Comparative Example 3) The enzyme electrode of the present invention was produced by a printing method. FIG.
FIG. 4 is a diagram schematically showing an enlarged cross section along a line AA ′ of a portion of a working electrode 1 and a counter electrode 2 of a printed electrode substrate 8 shown in FIG.
A silver paste 9 (including silver particles having an average particle size of 5 μm and a binder) is printed on an insulating substrate 8 made of polyethylene terephthalate by a screen printing method, and dried by heating (the thickness of the silver layer is about 10 μm). Further, a graphite paste 18 (including graphite particles having an average particle size of 0.5 μm and a binder) is printed by screen printing, and heated to 75 ° C. and dried (the thickness of the dried graphite layer is about 10 μm).

On top of that, an MnO ₂ paste 19 comprising the following components was screen-printed only on the counter electrode portion and air-dried (the thickness of the dried MnO ₂ layer was about 10 μm).
An electrode system composed of the counter electrode 2 and the working electrode 1 was formed. Polyvinyl chloride (degree of polymerization 1100) 10 g MnO ₂ 9 g acetylene black 1 g tetrahydrofuran 100 ml

The electromotive force generated by this electrode system was measured by the same measuring method as in Example 1 (Example 3).
An electromotive force of 250 mV was generated. The generated electromotive force (+250 mV) is caused by a potential difference between graphite and MnO ₂ . Next, the glucose concentration was measured using this electrode system.

In a beaker, 20 mM K ₃ Fe (CN) ₆ , 5
The electrode was immersed in a 0.1 M phosphate buffer containing these components so as to give a glucose oxidase concentration of 00 mg / dl. The working electrode and the counter electrode were connected with a resistance of 1 Mohm, and the current flowing through this resistance was measured in the same manner as in Example 1 (Example 4). For comparison, the counter electrode is the same electrode material as the working electrode (ie, graphite paste)
(Comparative Example 3). FIG. 6 shows the obtained results.
As is apparent from FIG. 6, only the example in which MnO ₂ was printed on the counter electrode showed a current value responding to various glucose concentrations.

Example 5 In order to apply the enzyme electrode of Example 3 to a disposable glucose electrode, a reagent layer (FIG. 5) was formed on the printed electrodes (1 and 2) shown in FIG. (Corresponding to 1 of 3)) and immobilized. At first,
0.1M with 0.25% carboxymethylcellulose
4 μl of a phosphate buffer (pH 7.0) solution was dispensed so as to cover the counter electrode 2 and the working electrode 1 of the electrode, and dried by heating to form a carboxymethyl cellulose layer. This carboxymethyl cellulose layer mainly functions as an electrode, a sample, and a separator.

Next, 4 μl of a 0.1 M phosphate buffer solution containing 0.5% of carboxymethylcellulose, 3.0% of K ₃ Fe (CN) ₆ and 500 mg / dl of glucose oxidase was allowed to act on the counter electrode 2 of the electrode. Dispense to cover pole 1,
By heating and drying, the reagent layer 3 was formed. Furthermore,
4 μl of a toluene solution containing 0.5% phosphatidylcholine (lecithin) was dispensed in an amount of 4 μl so as to cover the counter electrode 2 and the working electrode 1 of the electrode, and the mixture was air-dried to form a lecithin layer. This mainly has a function of smoothly aspirating the sample.

After the layer on the electrode was sufficiently dried, a spacer 4 and a cover 5 as shown in FIG. 1 were bonded on the electrode with a double-sided tape or the like. Samples of whole blood, plasma, urine, saliva, etc. come into contact with the sample inlet 6 and are automatically sucked by discharging air from the outlet 7 to dissolve the reagent layer and proceed with the enzymatic reaction. I do. When the glucose concentration was measured using such an enzyme electrode, a predetermined response current to each glucose could be obtained without applying a voltage from outside.

(Example 6) A glucose concentration measuring instrument which can use the enzyme electrode produced in Example 5 was produced. FIG. 7 schematically shows the appearance of this measuring instrument. In the illustrated embodiment, the measuring device includes a solar cell panel unit 20 and a liquid crystal display (LCD) 22, and the enzyme electrode 21 of the present invention is inserted into the measuring device via the electrode receiving unit 23. . A sample supply detection circuit, a measurement timing control circuit, an arithmetic circuit, and a display circuit are connected in a predetermined manner in the measuring instrument.

In a conventional enzyme electrode which does not generate an electromotive force, a constant voltage of 0.3 to 0.6 V is externally applied to the electrolytic oxidation of the electron carrier. In this case, the maximum current consumption of the measuring instrument is as follows. It was about 4-7 mA. However, in the measuring instrument using the enzyme electrode of the present invention prepared in Example 5, the externally applied power required for electrolysis can be omitted.
Current consumption can be minimized. That is, the circuit of the measuring instrument includes a sample detection circuit, a measurement timing control circuit, an arithmetic circuit, an LCD display unit including a display circuit, and the like. The total current consumption of these circuits is 0.2 mA or less. A solar cell can be used as a power source, and a semi-permanent measuring instrument that does not require any battery replacement can be constructed.

(Example 7) In the same manner as in Example 1, Ag ₂ O (1
1) was mounted thereon and solidified with a collodion film. Working electrode 13
Was measured using a measurement system as shown in FIG. 2, and an electromotive force of about +530 mV was obtained. Further, as a counter electrode material, lead oxide (PbO ₂ ), nickel oxyhydroxide (NiOOH), thionyl chloride (SOC)
l ₂ ), using vanadium pentoxide (V ₂ O ₅ ) and various combinations using silver / silver chloride electrode and glassy carbon electrode as working electrodes, the electromotive force was measured to be about -0.2.
An electromotive force in the range of ~ + 1.2 V was obtained.

[0049]

As described above, according to the present invention, the applied voltage required for the potentiostatic electrolysis of the oxidizing or reducing compound produced by the reaction of the substance to be measured with the enzyme is controlled by the action of different materials. Since it is obtained from the electromotive force generated by the electrode system of the pole and the counter electrode, an external power supply required for measurement is not required, and the current consumption of the measurement system can be greatly reduced. Thereby, the power supply of the sample supply detection circuit, the measurement timing control circuit, the arithmetic circuit, the display circuit, and the display unit required for the measurement system using the enzyme electrode of the present invention can be obtained from the solar cell, as in the conventional case. This eliminates the need to use a dry battery, thus making it possible to construct a measurement system that does not require battery replacement.

[Brief description of the drawings]

FIG. 1 is a schematic exploded perspective view of an enzyme electrode for measuring glucose concentration.

FIG. 2 is a diagram schematically showing the structure of a measurement cell used in Example 1.

FIG. 3 is a graph showing an electromotive force generated in the electrode system of Example 1.

FIG. 4 is a graph showing a glucose response measured by the electrode system of Example 2.

FIG. 5 is a diagram schematically showing a cross section taken along line A-A ′ of FIG. 1;

FIG. 6 is a graph showing a glucose response in the electrode system of Example 4.

FIG. 7 is a diagram schematically showing the appearance of a measuring instrument using the enzyme electrode of the present invention.

[Explanation of symbols]

1 working electrode, 2 counter electrode, 3 reagent layer, 4 spacer
5 ... Cover, 6 ... sample suction port, 7 ... exhaust port, 8 ... substrate, 9 ... silver connector portion, 10 ... glassy carbon electrode, 11 ... MnO ₂ paste, 12 ... collodion film, 13
... Platinum electrode (working electrode), 14 ... Glass filter, 15
... MnO ₂ electrode (counter electrode), 16 ... working electrode chamber, 17 ... counter electrode chamber, 18 ... graphite electrode, 19 ... MnO ₂ electrode, 20
... Solar cell panel part, 21 ... Battery integrated enzyme electrode, 22
... LCD display section, 23 ... Enzyme electrode receiving section.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Katsumi Hamamoto 57 Katsumi Higashikujo Nishida, Minami-ku, Kyoto-shi, Kyoto Prefecture Kyoto Daiichi Kagaku Co., Ltd. 57 Akita-cho, Kyoto Daiichi Kagaku Co., Ltd. (72) Inventor Shinzo Yoshida 57, Higashi-Kujo, Nishi-kujo, Minami-ku, Kyoto, Kyoto Prefecture Kyoto Daiichi Kagaku Co., Ltd.Examiner Jun Koriyama (58) Int.Cl. ⁷ , DB name) G01N 27/327

Claims (7)

(57) [Claims] An enzyme electrode having an immobilized phase of an enzyme and an electron carrier and used for measuring the concentration of a specific component in a liquid, wherein a working electrode and a counter electrode are formed of electrode materials different from each other. An enzyme electrode which is made and uses an electromotive force generated between these electrodes as a power source for measurement. 2. The enzyme electrode according to claim 1, wherein the electrode material is selected from carbon, metals, alloys, compounds of metals and alloys, and mixtures and combinations thereof. 3. The enzyme electrode according to claim 1, wherein the electrode material is selected from carbon, platinum and manganese dioxide. 4. The method of claim 1, wherein one electrode material comprises graphite and the other electrode material comprises platinum.
An enzyme electrode according to any one of the above. 5. The enzyme electrode according to claim 1, wherein the electrode material is obtained by solidifying a mixture further comprising a conductive powder and a binder. 6. The enzyme electrode according to claim 1, wherein the enzyme is glucose oxidase. 7. An enzyme electrode having an immobilized phase of an enzyme and an electron carrier and used for measuring the concentration of a specific component in a liquid, wherein a working electrode and a counter electrode are formed of electrode materials different from each other. A method of manufacturing an enzyme electrode using an electromotive force generated between these electrodes as a power source for measurement, wherein a counter electrode and a working electrode made of mutually different materials are formed on a substrate material, and both of them are formed. A method for producing an enzyme electrode, comprising forming a phase comprising an enzyme on at least a portion of the electrode.