JPH09145665A - Oxygen sensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure the concentration of substrate in a specimen quickly and in high reproducibility and accuracy by constituting a sensor by an electrode system including a working electrode and a counter electrode which are made of graphite and glass carbon compound material, and an reaction layer in contact with the electrode system. SOLUTION: A working electrode is covered with an insulative imide resin 3 after a sectional part 1 of rod shape and a part 2 used for a lead among graphite and glass carbon compound material are masked. A counter electrode 4 and a counter lead 5 are formed by dipping a part of polyethylene terephthalate film to which a silver is applied through vacuum vapor deposition, in a potassium chloride aqueous solution so as to deposit a silver nitride, and by joining them in a manner to hold a sample solution on the outside of the working electrode covered with a resin 3. A reaction layer 6 includes enzymes, electron transfer body, and water-soluble high polymer, and is comprised of a plurality of layers.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD OF THE INVENTION The present invention is
The present invention relates to an enzyme sensor that measures the concentration of a specific component in a sample. More specifically, the present invention relates to an enzyme sensor using graphite and a glassy carbon composite material as a working electrode material.

[0002]

2. Description of the Related Art Recently, analytical methods utilizing the excellent substrate specificity of enzymes have been widely used in the fields of clinical analytical chemistry, food manufacturing, environmental chemistry and the like. In particular, in the field of clinical analytical chemistry, enzyme sensors capable of selectively detecting biological substrates are widely used.

[0003] They are substances to be measured by electrochemically detecting the amount of hydrogen peroxide, hydrogen ions, or consumed oxygen produced by an enzymatic reaction with a hydrogen peroxide electrode, a hydrogen ion electrode, or an oxygen electrode. This is to quantify the concentration. For example, in the case of a glucose sensor that uses an oxygen electrode, the sensor is constructed by immobilizing glucose oxidase, which is an oxidase, in the membrane, and mounting the enzyme membrane on the oxygen electrode. The sensor detects the amount of oxygen consumed when glucose, which is a substrate, is oxidized by the catalytic action of glucose oxidase at the oxygen electrode, and thereby the glucose concentration in the sample can be measured.

However, the enzyme sensor based on such a principle has the following problems. In other words, the substrate needs oxygen to react, but in the actual measurement,
For example, when measuring the blood glucose level of a diabetic patient, the amount of dissolved oxygen in the body fluid is insufficient, and it is necessary to supplement oxygen by some method.

On the other hand, in order to solve these problems, an enzyme sensor utilizing an electron carrier (mediator) for directly detecting electron transfer accompanying an enzymatic reaction has been developed. This is for coating the enzyme-immobilized layer and the electron carrier on the measurement electrode part, and as a method for coating the electron carrier, an artificial electron carrier is applied to the electrode surface in a thin film form, and further on top of it. A method of coating with a transparent film, a method of incorporating a poorly water-soluble electron carrier into an electrode, and a method of incorporating a water-soluble electron carrier into an electrode in advance and then forming an ionic polymer and an enzyme on the surface of the electrode A method of providing a thin film of the composition to prevent the electron carrier from eluting, a method of mixing the enzyme and the electron carrier with paraffin or the like to form a paste, and rubbing the paste on the electrode.

The measurement principle of the enzyme sensor using the electron carrier as described above is as follows. For example, in the case of a glucose sensor, when glucose, which is a substrate, is oxidized by glucose oxidase, which is an oxidase, the active center of the enzyme changes from an oxidized form to a reduced form, which reduces the electron carrier and the enzyme itself oxidizes again. Return to type. The electron carrier is electrolyzed by an appropriate applied potential on the working electrode,
The glucose concentration can be measured by measuring the oxidation current obtained at that time.

By the way, what kind of electrode material is selected as a working electrode material of an enzyme sensor using an electron carrier has a great influence on whether or not the sensitivity, reproducibility and stability of the sensor can be quantitatively determined. Until now, as the electrode material, precious metal materials such as platinum, palladium and gold, semiconductor materials such as silicon, indium oxide and iridium oxide, glassy carbon (glassy carbon), carbon fiber,
Carbon materials such as HOPG (highly oriented pyrolytic graphite),
Carbon paste and the like have been studied. Among them, carbon materials can be measured in a wide range of positive and negative values, have excellent solvent resistance to organic solvents, acids and alkalis, thermal stability mechanical strength (flexural strength, elastic modulus, density), etc. Since it has various functional groups, especially quinone and hydroquinone groups, it has excellent properties as an electrode material for enzyme sensors, such as easy physical adsorption and chemical bonding with polymer membranes during sensor production. It is used a lot. However, since glassy carbon and carbon fiber have a smooth surface, the adherence of organic substances is poor, and the specific resistance is large. Therefore, the sensitivity is good and it is difficult to obtain stable data. HOPG has excellent adherence of organic substances to its edge surface, but has poor processability, is easily cleaved in scales, and is easily penetrated by the sample liquid. Therefore, it has good reproducibility and stable data. Hard to get. In addition, since carbon paste is a mixture of carbon powder and paraffin, it has high specific resistance and it is difficult to obtain an electrode surface with uniform characteristics. Therefore, it is difficult to obtain stable data with good sensitivity. have. Therefore, even with enzyme sensors using these electrode materials, sensitivity, reproducibility,
It is difficult to obtain a product with excellent stability.

[0008]

As described above, in the conventional enzyme sensor, an electrode material having excellent sensitivity, reproducibility and stability could not be obtained. I had to pay close attention to maintenance.

Therefore, the present invention is an enzyme sensor which uses an electron carrier and uses a carbon material as a working electrode material. The enzyme sensor can be easily adjusted without special pretreatment and the enzyme in the sample It is an object of the present invention to provide a highly reliable enzyme sensor capable of quantifying the concentration of a substance that can serve as a substrate with good reproducibility, high sensitivity and speed.

[0010]

The present inventors have used graphite and a glassy carbon composite material as a material for a working electrode of an enzyme sensor, and are in contact with an electrode system including this and a counter electrode, and the electrode system. When an enzyme sensor is composed of a reaction layer containing an enzyme, an electron carrier, and a water-soluble polymer, the concentration of a substance that can be a substrate of the enzyme in the sample can be measured with good reproducibility, and the sensor adjustment is easy and We have newly found here that it will be a highly reliable enzyme sensor that can quantify with high precision and speed because of its excellent adherence,
The present invention has been completed.

That is, according to the present invention, in order to quantify with good reproducibility, with high accuracy and speed, the working electrode material should have a graphite crystal in a glassy carbon matrix so that it has an electrode reaction activity specific to the graphite crystal. Characterized in that it is a composite material consisting of graphite and glassy carbon, which has a structure oriented in a direction, and which has electrolyte impermeability substantially equal to that of glassy carbon, and the reaction layer has an enzyme and an electron carrier. And an enzyme sensor comprising a water-soluble polymer and comprising at least two layers.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Next, the enzyme sensor of the present invention will be specifically described. The enzyme sensor of the present invention is an electrode system including a working electrode and a counter electrode made of graphite and a glassy carbon composite material, and a reaction layer which is in contact with the electrode system and contains an enzyme, an electron carrier and a water-soluble polymer. The enzyme sensor is composed of

The glass-like carbon composite material in which the graphite used for the working electrode of the present invention is composed of a glass-like carbon matrix having an impermeability of 40% or more and 95% or less of the material constitution,
If necessary, according to higher densification, etc., carbon residue is carbonized by carbonization to increase carbon residue and carbon residue yield of 75% or more and 95% or less is added and cocarbonized. It is characterized by doing. If the matrix carbon content is 40% or less of the total, defects or vacancies may occur in the material, the original impermeability may be lost, and the electrode characteristics may become unstable, which is not preferable. On the other hand, if the matrix carbon content exceeds 95%, the active electrode reaction characteristics of the graphite crystal are lost, which is not preferable.

Here, graphite is at least one selected from the group consisting of graphite whiskers, highly oriented pyrolytic graphite (HOPG), quiche graphite, natural graphite, and artificial graphite. In order to favorably carry out the electrode reaction in the present invention, it is important to prepare a composite carbon material having a textured orientation such that the crystal end faces (edge faces) of graphite are vertically aligned with the reaction face of the electrode. Therefore, powder such as Kish graphite or natural graphite is preferably used, and the maximum diameter is preferably several μm, although the particle size of the powder varies depending on the diameter of the target electrode.

The glassy carbon is a glassy non-graphitizable carbon obtained by firing an organic substance in an inert atmosphere or a non-oxidizing atmosphere, and an organic resin material having three-dimensional cross-linking, It is a carbonized material such as a natural organic material that solid-phase carbonizes. Specifically, one of organic polymer substances and their monomers / oligomers, tars / pitches, carbonized pitches, thermoplastic resins, thermosetting resins, prepolymers of thermosetting resins, or the like, or It is a carbonized product composed of a mixture of two or more kinds.

The reaction layer of the present invention comprises at least two layers containing an enzyme, an electron carrier and a water-soluble polymer. The water-soluble polymer layer and the layer containing the enzyme and the electron carrier may be two layers, but preferably, when the layer containing the enzyme and the layer containing the electron carrier are separated, the enzyme is activated by the electron carrier. Since it is not damaged, a highly reliable enzyme electrode whose performance does not deteriorate even if it is stored can be obtained, which is preferable. If a hydrophilic polymer film soluble in a solvent that does not dissolve the enzyme is provided between the enzyme layer and the electron carrier layer, the effect of separating the enzyme and the electron carrier is further increased. However, it is also important to make the reaction layer as small as possible. Compared with the multi-layer structure, the small-layer structure can simplify the manufacturing process.
This is because it is easy to dissolve in the sample solution, so that the response speed is shortened and the variation in response value can be reduced. Further, in order to reduce the influence of the pH of the sample solution and obtain a stable sensor response, it is preferable that the reaction layer contains a buffer solution or a reagent which is a component thereof in advance. In addition to the phosphate, phosphate-citric acid or acetic acid-acetate can be used as the reagent serving as a component of the buffer solution.

Here, the hydrophilic polymer is not limited to carboxymethylcellulose used as an example in the examples, and other cellulose type, vinyl alcohol type, vinylpyrrolidone type, gelatin type, acrylate type, Starch-based, maleic anhydride-based, acrylamide-based, etc. are used.

As the material that can be used as the electron carrier, any of the materials conventionally used can be used. For example, potassium ferricyanide, p-benzoquinone, ferrocene, phenazine methosulfate, thionine, pyrroloquinoline quinone, naphthoquinone, Examples thereof include methylene blue.

The enzyme that can be used in the present invention is not particularly limited, and the enzymes used in conventional enzyme sensors can be used alone or in combination.
Specifically, oxidases such as glucose oxidase, cholesterol oxidase, lactate oxidase, uricase, alcohol oxidase, NADH oxidase, galactose oxidase, pyruvate oxidase, and amine oxidase. Glucose dehydrogenase, cholesterol dehydrogenase, lactate dehydrogenase, 3-hydroxybutyrate dehydrogenase, fructose dehydrogenase, alcohol dehydrogenase, dehydrogenase such as glutamate dehydrogenase, diaphorase, urease and the like can be used.
As the substance whose concentration can be measured by the enzyme sensor of the present invention, the concentration of various substances can be measured by appropriately selecting from the above-mentioned enzymes to be used.

For example, glucose in a sample such as blood can be measured by using glucose oxidase as an enzyme, cholesterol concentration can be measured by using cholesterol oxidase, and lactic acid concentration can be measured by using lactate oxidase.

In the present invention, the structure of the sensor is not particularly limited, and various structures adopted in conventional enzyme sensors can be adopted. Therefore,
The shape of the sensor electrode and the working electrode may be linear, rod-shaped, or planar, and these electrodes are connected to an appropriate circuit (for example, a current measuring circuit) for measuring a current value. It is like this. In a particularly preferred embodiment of the present invention, a rod structure can be adopted as the structure of the electrode base material.

[0022]

As described above, the present invention uses the graphite and glassy carbon composite material as the material for the working electrode of the enzyme sensor, the electrode system including this and the counter electrode, and the enzyme which is in contact with the electrode system, It is characterized in that an enzyme sensor is composed of a reaction layer containing an electron carrier and a water-soluble polymer. To quantify the concentration of a substance that can be a substrate for an enzyme in a sample solution using the enzyme sensor of the present invention, the sample solution is supplied to the reaction layer in contact with the electrode system. This sample solution dissolves the hydrophilic polymer in the reaction layer, and the enzyme and electron carrier contained in the reaction layer come into contact with the sample, and the sample is oxidized by the enzyme. An electron carrier such as potassium ferricyanide is reduced to potassium ferrocyanide by the electrons transferred by the oxidation reaction. The reductant of the electron carrier generated by the oxidation reaction by the oxidase is then oxidized by the voltage application with the working electrode as the anode based on the counter electrode, and at that time, it is composed of graphite and glassy carbon composite material having excellent electrode characteristics. With the working electrode, the response current can be measured quickly, with high accuracy, and with good reproducibility. In addition, the reaction layer contains a hydrophilic polymer, and this polymer dissolves in the sample solution to provide a field for reaction between the enzyme or electron carrier contained in the reaction layer and the sample. Since it is possible to substantially prevent the polymer from using the graphite and glassy carbon composite electrode materials, which have excellent adhesion properties with, to make contact with the electrode system surface such as enzymes and electron mediators, Adsorption of proteins on the surface of the electrode system and changes in the characteristics of the electrode system due to the chemical action of substances having an oxidizing ability such as electron acceptors are less likely to occur. As a result, the current response can be obtained with higher accuracy.

Therefore, in the present invention, a graphite and glassy carbon composite material having excellent electrode characteristics such as high electrode reaction activity, large potential window, low specific resistance, high stability without deterioration over time, and the like are used. Therefore, an enzyme sensor with higher accuracy, reproducibility, and stability can be obtained as compared with the conventional one using a carbon material. Moreover, it has become possible to simplify the maintenance of sensor characteristics.

[0024]

The present invention will be described in more detail with reference to the following examples.

[Embodiment 1] In this embodiment, a glucose sensor will be described. First, a method for producing graphite and glassy carbon composite material for a working electrode will be described. The graphite and glassy carbon composite materials are 35% by weight of chlorinated vinyl chloride resin (T-742 manufactured by Nippon Carbide Co.) and furan resin (Hitafuran VF-302 manufactured by Hitachi Chemical Co., Ltd.) as matrix carbon (glassy carbon) raw materials. 50% by weight of a mixed resin system, 15% by weight of natural graphite fine powder (Nippon Graphite Co., Ltd. CSSP-B average particle size 1 μm) and 20% by weight of diallyl phthalate monomer as a plasticizer were mixed with a Henschel mixer. After mixing / dispersing, kneading with two rolls for mixing, extruding this kneaded product with a screw type extruder using a die of 1.25 mm in diameter while degassing, fixing this to a frame, and heating to 180 ° C. The pre-cursor (carbon precursor) wire rod was treated for 10 hours in the air oven. Next, this is 500 in nitrogen gas.
Up to ℃ at a heating rate of 50 ℃ / hour, then 1500
The temperature was raised to 100 ° C./hour, the temperature was maintained at 1500 ° C. for 3 hours, and then naturally cooled to complete the firing. The obtained rod-shaped carbon material for a working electrode was graphite and glassy carbon = 32
It was / 68 and the diameter was 1.0 mm. The electrode characteristics are the Yanamoto P-1 polarographic analyzer (YANACO P-1).
100) was used to evaluate the redox reactivity of the ferro / ferricyan ion in the system 5 × 10 ⁻³ M Fe (CN) ₆ ⁴⁻ and 1M KC1. as a result,
The blank current was small without performing a special pretreatment, and sharp peaks of the oxidation wave and the reduction wave were obtained, and the peak potential difference ΔEp was also close to the theoretical value. No increase in current value was observed with the invasion of the electrolytic solution, and it was substantially as impervious to the electrolytic solution as glassy carbon. The resistivity of this material is 5
It was × 10 ^-4 Ωcm. From these, it was found that the electrode characteristics and the specific resistance of this material were superior to those of the existing carbon-based electrode materials such as glassy carbon and carbon fiber / carbon paste materials.

Next, a method and structure for manufacturing a glucose sensor using the graphite and glassy carbon material manufactured by the above method as working electrodes will be described. Of the graphite and the glassy carbonaceous material, a rod-shaped cross-section portion 1 used as a working electrode and a portion 2 used as a lead were masked and then covered with an insulating resin (imide resin) 3. Further, a part of a film made of polyethylene terephthalate (thickness 12 μm) on which silver is vacuum-deposited is immersed in a 1 M potassium chloride aqueous solution, 0.34 V is applied with SCE as a reference electrode to deposit silver chloride, and this is coated with the above imide resin. An electrode system was prepared by joining the counter electrode 4 and the counter lead 5 so that the sample solution can be held outside the working electrode.

5 μl of a 0.5 wt% aqueous solution of carboxymethyl cellulose (hereinafter abbreviated as CMC) as a hydrophilic polymer was dropped into the obtained electrode system and dried to obtain CMC.
Form a layer. On the other hand, glucose oxidase (Type II-S, EC1.1.3.4, Aspergillus niger: manufactured by Sigma, hereinafter GO)
(Abbreviated as D) 1000 U and 25 mg of potassium ferricyanide as an electron acceptor in a phosphate buffer solution (0.1 M: KH
₂ PO ₄ -0.1 M: Na ₂ HPO ₄ ; pH = 7.0)
Dissolve in 1 ml. Add this mixed solution to the CMC layer 5
μl is dropped and dried in a dryer at 40 ° C. for 20 minutes to form a reaction layer 6. The reaction layer portion of the obtained glucose sensor was substantially circular with a diameter of 3 mm.

In the above reaction layer forming step, when a mixed solution of phosphate, glucose oxidase and potassium ferricyanide is dropped on the CMC layer, the CMC layer composed of the hydrophilic polymer is once dissolved and the enzyme is dried in the subsequent drying process. And the like to form a composite layer. But,
Of the graphite and glassy carbon materials, the cross-section where the graphite edges are integrated has excellent adherence to the hydrophilic polymer, and thus is not in a completely mixed state, and the electrode system surface is covered with only CMC. It becomes a state. That is, since the enzyme and the electron acceptor do not come into contact with the electrode surface, adsorption of protein on the electrode system surface, potassium ferricyanide, K
It is difficult for the characteristics of the electrode system to change due to the chemical action of a substance having an oxidizing ability such as H ₂ PO ₄ . as a result,
A biosensor having a highly accurate sensor response can be obtained.

Next, the measurement will be described. To the glucose sensor manufactured as described above, 10 μl of glucose aqueous solution was supplied as a sample solution, and after a certain time, a pulse voltage of +0.4 V was applied to the working electrode in the anode direction with reference to the counter electrode of the electrode system. When the current value after 2 seconds was measured,
As shown in FIG. 2, a response current value proportional to the glucose concentration in the sample solution was obtained.

[Comparative Example 1] A glucose sensor was prepared and measured in the same manner as in Example 1 except that a glassy carbon material was used as the working electrode material. The result is shown in FIG. Although the working electrode area was the same as in Example 1, the response current value was small, and the response current value proportional to the glucose concentration could not be obtained. Further, when the aqueous glucose solution was supplied to the glucose sensor, peeling of the reaction layer was observed.

[Embodiment 2] In this embodiment, a lactic acid sensor will be described. Graphite and glassy carbon composite materials are 50% by weight of furan resin (Hitafuran VF302 manufactured by Hitachi Chemical Co., Ltd.), 30% by weight of carbonization pitch (KH manufactured by Kureha Chemical Industry Co., Ltd.) as a matrix carbon (glassy carbon) raw material, and natural materials. Graphite fine powder (Nippon Graphite Co., Ltd. CSSP-B
20% by weight of average particle size 1 μm) is mixed and dispersed by a Henschel mixer, and then kneaded by a twin roll for mixing, and this kneaded product is extruded by a screw type extruder with a die having a diameter of 1.3 mm while performing deaeration. It was Hereinafter, Example 1
The same operation as described in (1) above was performed to obtain the target carbonaceous material for electrodes and to evaluate the electrode characteristics. The obtained rod-shaped carbon material for working electrode was graphite and glassy carbon = 30/70,
It had a diameter of 1.0 mm. In addition, the blank current was small and sharp peaks of the oxidation wave and the reduction wave were obtained without any special pretreatment, and the electrode characteristics with the peak potential difference ΔEp close to the theoretical value were obtained. No increase in current value was observed with the invasion of the electrolytic solution, and it was substantially as impervious to the electrolytic solution as glassy carbon. The resistivity of this material is 6 × 10 ^-4 Ω
cm. From these, it was found that the electrode characteristics and the specific resistance of this material are also superior to those of the existing carbon-based electrode materials such as glassy carbon and carbon fiber / carbon paste materials.

Next, a method and structure for manufacturing a lactic acid sensor using the graphite and glassy carbon material manufactured by the above method as working electrodes will be described. In the same manner as in Example 1, of the graphite and glassy carbon materials, the rod-shaped cross-section portion 1 used as the working electrode and the portion 2 used as the lead were masked, and then coated with an insulating resin (furan resin) 3. In addition, a part of a polyethylene terephthalate film obtained by vacuum-depositing silver is converted into silver chloride, and the electrode is formed by joining the counter electrode 4 and the counter electrode lead 5 so that the sample solution can be held outside the working electrode coated with the furan resin. A system was made.

5 μl of a 0.5 wt% aqueous solution of CMC as a hydrophilic polymer was dropped into the obtained electrode system and dried to form a CMC layer. On the other hand, lactate oxidase (produced by Boehringer Mannheim, Pediococcus sp.
A phosphate buffer (0.1% LOD) containing 100 U and 25 mg of potassium ferricyanide as an electron acceptor (0.1
M: KH ₂ PO ₄ -0.1 M: Na ₂ HPO ₄ ; pH =
7.0) Dissolve in 1 ml. This mixed solution is
5 μl is dropped on the layer and dried in a drier at 40 ° C. for 20 minutes to form a reaction layer 6. The reaction layer portion of the obtained lactic acid sensor was substantially circular with a diameter of about 3 mm.

In the above reaction layer forming step, when a mixed solution of phosphate, lactate oxidase and potassium ferricyanide is dropped on the CMC layer, the CMC layer made of the hydrophilic polymer is once dissolved and the enzyme is dried in the subsequent drying process. And the like to form a composite layer. However, the cross-section of the graphite and glassy carbon materials where the graphite edges are accumulated has excellent adherence with the hydrophilic polymer, so that it is not in a completely mixed state, and the electrode system surface is covered with only CMC. It will be in the state of being. That is, since the enzyme and the electron acceptor do not come into contact with the electrode surface, protein adsorption onto the electrode system surface, potassium ferricyanide, KH ₂ P
It is difficult for the characteristics of the electrode system to change due to the chemical action of a substance having an oxidizing ability such as O ₄ . As a result, a biosensor having a highly accurate sensor response can be obtained.

Next, the measurement will be described. An aqueous glucose solution 10 was used as a sample solution in the lactate sensor manufactured as described above.
After a certain period of time from the supply of μl, a pulse voltage of +0.5 V was applied to the working electrode in the direction of the anode with the counter electrode of the electrode system as a reference, and the current value after 10 seconds was measured. As shown in FIG. Moreover, a response current value proportional to the lactic acid concentration in the sample solution was obtained.

[0036]

INDUSTRIAL APPLICABILITY As described above, the enzyme sensor of the present invention uses the graphite and glassy carbon composite material having excellent electrode characteristics as the working electrode material, so that no special pretreatment or manufacturing control is required when manufacturing the enzyme sensor. At the same time, the concentration of the substrate present in the sample can be measured rapidly, with good reproducibility, and with high accuracy. That is, in addition to overcoming the drawbacks of the conventional enzyme sensor using a carbon material, an enzyme sensor having remarkably excellent characteristics was obtained.

[Brief description of the drawings]

FIG. 1 is a cross-sectional view (schematic diagram) of an enzyme sensor according to the present invention.

FIG. 2 shows the relationship between glucose concentration and current value obtained by the glucose sensors of Example 1 and Comparative Example 1.

FIG. 3 shows the relationship between the lactic acid concentration and the current value obtained by the lactic acid sensor of Example 2.

[Explanation of symbols]

 1 ... Working electrode (end face) 2 ... Working electrode lead 3 ... Insulating resin layer 4 ... Counter electrode 5 ... Counter electrode lead 6 ... Reaction layer 7 ... Polyethylene terephthalate film

Front page continuation (72) Inventor Makiko Seki 1091 Tateishi, Fujioka, Gunma Mitsubishi Pencil Co., Ltd. Gunma R & D Center

Claims (3)

[Claims] 1. An enzyme sensor comprising an electrode system including a working electrode and a counter electrode made of graphite and a glassy carbon composite material, and a reaction layer in contact with the electrode system. 2. The working electrode has a structure in which graphite crystals are unidirectionally oriented in a glassy carbon matrix, and only graphite and glassy carbon having substantially the same impermeability of electrolyte as glassy carbon. The enzyme sensor according to claim 1, which is a composite material comprising: 3. The enzyme sensor according to claim 1, wherein the reaction layer contains an enzyme, an electron carrier and a water-soluble polymer and is composed of at least two layers.