JPH08327580A - Biosensor and biosensor for measuring blood glucose level - Google Patents

Abstract

PURPOSE: To rapidly and easily quantitatively determine a substrate (specific component) in a simple with high accuracy by forming a reaction layer containing a pyranose oxidizing enzym, on a base or via the base and a space. CONSTITUTION: An aqueous solution of a hydrophilic polymer, e.g. 0.5wt.% carboxymethyl cellulose(CMC), is dropped onto an electrode system 8 (working electrode 4 and counter electrode 5) formed on an electrically insulating base 1, and is dried to form a CMC layer. Mixed solutions of pyranose oxidase(PyOx) as a pyranose oxidizing enzyme that oxidizes α-glucose and β-glucose at the same time and potassium ferricyanide as an electron acceptor are dropped onto the CMC layer and dried to form the reaction layer 7 of a glucose sensor. To measure the concentration of a substrate in a sample, the solution of the sample is dropped onto the reaction layer 7, then a voltage of +0.5V is applied to the working electrode with respect to the counter electrode 5 one minute thereafter, and a current value is measured after about five seconds.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a substrate (specific component) contained in a biological sample such as blood or urine, a raw material or a product in the food industry, and a sample such as fruit juice with high accuracy, quickly and easily. It relates to a quantifiable biosensor.

[0002]

2. Description of the Related Art As a quantitative analysis method for saccharides such as glucose and sucrose, a photometric method, a colorimetric method, a reduction titration method and a method using various chromatographies have been developed. However, none of these methods has high specificity for saccharides, and thus the accuracy is low. For example,
Although the operation of the above-described photometric method is simple, it is known that it is greatly affected by the temperature during the operation.

In recent years, there has been proposed a biosensor capable of easily quantifying a specific component (substrate) in a biological sample or food without diluting or stirring the sample solution.

Japanese Patent Laid-Open No. 3-202764 discloses that an electrode system is formed on an insulating substrate by a method such as screen printing, and a hydrophilic polymer, an oxidoreductase and an electron acceptor are contained on the electrode system. Disclosed is a biosensor having a reaction layer formed thereon. This biosensor can quantify the substrate concentration in the sample as follows: First, by dropping the sample solution on the reaction layer of the biosensor, the reaction layer is dissolved and the substrate in the sample solution is dissolved. An enzymatic reaction proceeds with the redox enzyme in the reaction layer, and then the electron acceptor in the reaction layer is reduced. After the completion of the enzymatic reaction, the reduced electron acceptor is electrochemically oxidized to determine the substrate concentration in the sample solution from the oxidation current value obtained at this time.

US Pat. No. 5,192,415 teaches that the pH of the sample solution can be optimized depending on the type of oxidoreductase contained in the reaction layer without first adjusting the pH of the sample solution. A biosensor having an ion concentration control layer is disclosed.

US Pat. No. 5,264,103 discloses a main electrode system formed on an electrically insulating substrate and having a working electrode and a counter electrode; a reaction layer containing a redox enzyme; and the main electrode system. Disclosed is a biosensor having a sub-electrode layer that is spaced apart and has a working electrode and a counter electrode.

Each of the biosensors described above can be selected, for example, from a glucose sensor, an alcohol sensor, a cholesterol sensor, and
And can be widely used as an amino acid sensor.

[0008]

Among such biosensors, glucose sensors are generally known to use glucose oxidase as an oxidoreductase. However, since glucose oxidase reacts only with β-glucose existing in a proportion of about 63% of glucose isomers in an equilibrium state, this glucose sensor has a low response current value (that is, detection sensitivity), Then, for example, in the case of quantifying a very small amount of glucose, there is a problem that a measurement error accompanying it becomes large.

Furthermore, when quantifying polysaccharides using the above glucose sensor, most of the glucose obtained by hydrolases is α-form, so α-glucose produced by hydrolysis should be measured before quantification. However, a step of once isomerizing β-form by mutarotase was necessary.

Japanese Patent Application No. 6-291401 discloses a biosensor using both the mutarotase and glucose oxidase. However, this biosensor cannot sufficiently improve the detection sensitivity of the glucose sensor when the reagent amount of the whole enzyme is small, and conversely, when the reagent amount of the whole enzyme is large, the manufacturing cost of the sensor becomes high. . Further, when the substrate concentration in the sample solution is relatively high, the biosensor containing this mutarotase and glucooxidase, as compared with the one not containing mutarotase,
Its detection sensitivity decreases.

[0011]

The biosensor of the present invention solves the above-mentioned drawbacks by providing an electrically insulating substrate, an electrode system formed on the substrate and having a working electrode and a counter electrode, A biosensor having a reaction layer arranged on a substrate or with a space between the substrate, wherein the reaction layer contains a pyranose oxidase.

The present invention also contains pyranose oxidase and glucose oxidase in the reaction layer.

Further, according to the present invention, a pyranose oxidase and a polysaccharide degrading enzyme are contained in the reaction layer.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a synthetic resin plate such as polyethylene terephthalate may be used as the electrically insulating substrate.

An electrode system having a working electrode and a counter electrode can be provided on this substrate using known methods. For example,
After forming the leads on the substrate, a working electrode and a counter electrode are provided so as to be connected to the leads and insulate each other. A known conductive material can be used as the material of the lead and the electrode. For example, carbon,
Included are silver, platinum, gold, and palladium.

The reaction layer contains pyranose oxidase. Pyranose oxidase is an enzyme that simultaneously oxidizes both α-glucose and β-glucose. Examples of such a pyranose oxidase include pyranose oxidase (EC 1.1.3.10; PyOx).

The content of PyOx is preferably 1 to 200 units, and more preferably 2 to 50 units per 1 cm 2 of the reaction layer. Here, the term “unit” used in the present specification refers to the amount of oxidoreductase that oxidizes 1 μmol of a substrate in 1 minute. P
When the content of yOx is less than 1 unit per 1 cm 2 of the reaction layer, a time of several minutes or more is required at the time of measurement, and the vaporization of the sample solution generated during that time easily affects the measured response current value. When the content of PyOx exceeds 200 units per square centimeter of the reaction layer, the manufacturing cost becomes high, and the reaction layer is easily cracked during the formation of the reaction layer, and the response current value tends to vary.

The reaction layer may further contain glucose oxidase (GOD) together with the pyranose oxidase in order to further improve the detection sensitivity of glucose in the sample solution and respond to a wider range of glucose concentrations. A suitable GOD content is preferably 1 to 200 units per square centimeter of the reaction layer. In this case, the content of PyOx is preferably 0.1 to 200 units, and more preferably 0.2 to 40 units per 1 cm 2 of the reaction layer.

Further, the reaction layer may contain various hydrophilic polymers. Examples include carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC),
Polyamino acids such as hydroxypropyl cellulose (HPC), methyl cellulose, ethyl cellulose, ethyl hydroxyethyl cellulose, carboxymethyl ethyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, polylysine, polystyrene sulfonic acid, gelatin and its derivatives, acrylic acid and its salts, methacrylic acid and Mention may be made of its salts, starch and its derivatives, maleic anhydride and its salts. Particularly, CMC is preferable.

The reaction layer hydrolyzes the polysaccharide to produce α-
It may contain a polysaccharide degrading enzyme to produce glucose. The polysaccharide degrading enzyme is an enzyme having the ability to hydrolyze a polysaccharide such as sucrose or maltose to produce glucose. Examples of polysaccharide degrading enzymes include sucrose hydrolases such as invertase (INV),
Examples thereof include maltose hydrolase such as maltase and lactose hydrolase such as β-galactosidase. The content of the polysaccharide-degrading enzyme is preferably 1 to 400 units, more preferably 2 to 200 units, per 1 cm 2 of the reaction layer.

When the sample solution containing glucose is supplied to the reaction layer of the biosensor, α-glucose and β-glucose in the sample solution are oxidized by the pyranose oxidase. At the same time, the dissolved oxygen in the sample solution is reduced to hydrogen peroxide. Here, when a voltage is applied, hydrogen peroxide is oxidized. Since the response current generated at this time is proportional to the concentration of hydrogen peroxide produced, that is, the substrate concentration in the sample solution, the substrate concentration in the sample solution can be obtained by measuring the response current value.

By incorporating an electron acceptor in the reaction layer instead of producing hydrogen peroxide simultaneously with the oxidation reaction of the substrate, a reduced form of the electron acceptor can be formed simultaneously with the enzymatic reaction. Examples of such electron acceptors include ferricyanide, p-benzoquinone and its derivatives, phenazine methosulfate, methylene blue, ferrocene and its derivatives. As the electron acceptor, one or more of these may be used. In particular, it is preferable to use ferricyan ion.

The content of ferricyan ion is determined by the reaction layer 1
It is preferably 0.21 to 3.30 mg, and more preferably 0.3 to 2.59 mg per square centimeter. When the content of the ferricyan ion is less than 0.21 mg per 1 cm 2 of the reaction layer, the measurable glucose concentration range tends to be extremely narrow. When the content of the ferricyan ion exceeds 3.30 mg per 1 cm 2 of the reaction layer, the reaction layer is cracked and the response current value varies, and further, the reliability during storage tends to decrease.

Next, a preferred embodiment of the biosensor manufacturing method of the present invention will be described with reference to FIGS.

First, a conductive material such as silver paste is printed on the electrically insulating substrate 1 by screen printing,
The leads 2 and 3 are formed. Then, a conductive material containing a resin binder is printed on the substrate 1 to form the working electrode 4. The working electrode 4 is in contact with the lead 2.

Then, an insulating paste is printed on the substrate 1 to form an insulating layer 6. The insulating layer 6 is the working electrode 4
The outer peripheral portion of the working electrode 4 is covered therewith, so that the area of the exposed portion of the working electrode 4 is kept constant. As shown in FIG. 1, the insulating layer 6
Also covers part of the leads 2, 3. Further, a ring-shaped counter electrode 5 is formed on the outer peripheral portion of the working electrode 4 by using a conductive material containing a resin binder. The counter electrode 5 is in contact with the lead 3. In this way, the electrode system 8 having the working electrode 4 and the counter electrode 5 is formed on the substrate 1.

Further, in the biosensor of the present invention, in order to further stabilize its accuracy, a three-electrode system having a reference electrode in addition to the working electrode 4 and the counter electrode 5 may be formed on the substrate 1.

The reaction layer can be arranged on the substrate 1 as follows. First, the aqueous solution of the hydrophilic polymer is dropped on the electrode system 8 and dried to form a hydrophilic polymer layer. Then, PyOx is formed on the hydrophilic polymer layer.
Then, an aqueous solution containing the above-mentioned electron acceptor and / or polysaccharide degrading enzyme is added dropwise, and dried. The biosensor of the present invention can also be cross-linked and immobilized on the hydrophilic polymer layer so that it can be repeatedly used, using a glutaraldehyde and a pyranose oxidase and optionally a polysaccharide degrading enzyme, Alternatively, they may be entrapped and fixed together with the polymer material. Further, if necessary, the electron acceptor may be chemically fixed using a polymer material. In this way, the electrode system 8 shown in FIG.
A reaction layer 7 covering the whole is formed.

Alternatively, the reaction layer may be disposed on the substrate 1 with a space. In this case, as shown in FIG. 3, the biosensor has a substrate 1 and a cover 30 arranged on the substrate 1 with a spacer 20 interposed therebetween. The cover 30 has a hole 31 and a reaction layer 37 on one surface thereof. The substrate 1 and the cover 30 may be arranged so that the reaction layer 37 and the electrode system 8 face each other. Such a substrate 1
The method of forming the reaction layer 37 through the space is, for example,
It is described in Japanese Patent Laid-Open No. 1-114747. In this biosensor, when the sample solution supplied from the sample solution supply port 38 reaches between the reaction layer 37 and the electrode system 8, as in the biosensor shown in FIG. Hydrogen oxide or a reduced form of an electron acceptor can be measured with the electrode system 8.

(Example 1) As an example of the biosensor of the present invention, a glucose sensor was produced as follows. FIG. 1 is a schematic plan view in which a reaction layer of a glucose sensor manufactured as an example of the present invention is removed. Figure 2
It is a schematic sectional drawing of the glucose sensor.

As shown in FIG. 1, a silver paste was printed by screen printing on an electrically insulating substrate 1 made of polyethylene terephthalate to form leads 2 and 3.
Then, a conductive carbon paste containing a resin binder was printed on the substrate 1 to form the working electrode 4. The working electrode 4 was in contact with the lead 2.

Next, an insulating paste was printed on the substrate 1 to form an insulating layer 6. The insulating layer 6 is the working electrode 4
The outer surface of the working electrode 4 is covered with this, so that the area of the exposed portion of the working electrode 4 is kept constant.

Next, a conductive carbon paste containing a resin binder was printed on the substrate 1 so as to come into contact with the leads 3 to form a ring-shaped counter electrode 5.

Then, 0.5% by weight of an aqueous solution of carboxymethyl cellulose (CMC) was dropped onto the electrode system 8 (working electrode 4 and counter electrode 5) and dried to obtain CMC.
A layer was formed. A mixed solution of pyranose oxidase (PyOx) and potassium ferricyanide was dropped on the CMC layer and dried to form a reaction layer 7. The amounts of PyOx and potassium ferricyanide contained in the reaction layer 7 were 10 units and 1.3 mg, respectively, per 1 cm 2 of the reaction layer.

On the reaction layer 7 of the glucose sensor produced as described above, a 90 mg / dl glucose aqueous solution was dropped as a sample solution. Then, 1 minute after dropping, a voltage of +0.5 V was applied to the working electrode 4 with respect to the counter electrode 5 of the electrode system 8 and the current value after 5 seconds was measured. In this way, the response current value to the glucose aqueous solution was measured 12 times in total using a new glucose sensor for each measurement. The variation in the obtained response current value was small.

Further, the response current values to the glucose aqueous solutions of 180 and 360 mg / dl were measured 12 times in the same manner as above. The obtained response current value tended to increase depending on the increase in glucose concentration, and the rate of change was large.

As a comparative experiment, the response current value for each glucose concentration was measured 12 times in the same manner as above except that a glucose sensor containing glucose oxidase instead of PyOx was prepared. The obtained response current values varied among the same glucose concentrations. Furthermore, these response current values tended to increase depending on the increase in glucose concentration, but the rate of change was small.

Example 2 As an example of the biosensor of the present invention, a sucrose sensor was produced as follows.

In the same manner as in Example 1, the leads 2 and 3, the electrode system 8 (working electrode 4 and counter electrode 5) and the insulating layer 6 were formed on the electrically insulating substrate 1 made of polyethylene terephthalate. Then, 0.5% by weight of an aqueous solution of carboxymethyl cellulose (CMC) was dropped onto the electrode system 8 having the working electrode 4 and the counter electrode 5 and dried to form C.
The MC layer was formed.

A mixed aqueous solution of PyOx, invertase (INV) and potassium ferricyanide was dropped on the CMC layer and dried to form a reaction layer 7. The amounts of PyOx, INV and potassium ferricyanide contained in the reaction layer 7 were 10 units, 40 units and 1.
It was 3 mg.

When a 171 mg / dl aqueous solution of sucrose was dropped as a sample solution onto the reaction layer 7 of the sucrose sensor manufactured as described above, the reaction layer 7 was formed by the sample solution.
Dissolved. Then, 3 minutes after dropping, the counter electrode 5 of the electrode system 8
On the other hand, a voltage of +0.5 V was applied to the working electrode 4, and the current value after 5 seconds was measured.

Further, the response current values to the sucrose aqueous solutions of 342 and 684 mg / dl were measured in the same manner as above, using a new sucrose sensor for each measurement.

The obtained response current value showed a tendency to increase depending on the increase of the sucrose concentration, and the rate of the change was large.

As a comparative experiment, the same effects as described above were obtained also with a maltose sensor or a lactose sensor prepared by using maltose hydrolase or lactose hydrolase instead of INV.

(Example 3) As an example of the biosensor of the present invention, a glucose sensor was produced as follows. FIG. 4 is a graph showing the correlation between various glucose concentrations and the response current of the glucose sensor showing an example of the biosensor of the present invention; where the curve (a) in FIG.
The change in the response current value when the reaction layer contains pyranose oxidase (PyOx) and glucose oxidase (GOD) is shown, and the curve (b) shows the change in the response current value when only GOD is contained, and Curve (c)
Represents the change in the response current value when only PyOx is contained.

In the same manner as in Example 1, the leads 2 and 3, the electrode system 8 (working electrode 4 and counter electrode 5) and the insulating layer 6 were formed on the electrically insulating substrate 1 made of polyethylene terephthalate. Then, 0.5% by weight of an aqueous solution of carboxymethyl cellulose (CMC) was dropped onto the electrode system 8 having the working electrode 4 and the counter electrode 5 and dried to form C.
The MC layer was laminated.

A mixed aqueous solution of PyOx, glucose oxidase (GOD) and potassium ferricyanide was added dropwise to this CMC layer and dried to form a reaction layer 7.
The amounts of PyOx, GOD and potassium ferricyanide contained in this reaction layer 7 were 1 unit, 10 units and 1.3 mg, respectively, per 1 cm 2 of the reaction layer.

On the reaction layer 7 of the glucose sensor manufactured as described above, a 90 mg / dl glucose aqueous solution was dropped as a sample solution. Then, 1 minute after dropping, a voltage of +0.5 V was applied to the working electrode 4 with respect to the counter electrode 5 of the electrode system 8 and the current value after 5 seconds was measured.

Furthermore, the response current values to the glucose aqueous solutions of 180 and 360 mg / dl were measured for each measurement using a new glucose sensor and in the same manner as above. The response characteristic between the obtained glucose concentration and the response current value is shown by the curve (a) in FIG.

As a comparative experiment, of the glucose sensors, a sensor not containing PyOx and a sensor not containing GOD were prepared, and the response current values were measured in the same manner as above. These results are shown in FIG.
The curves (b) and (c) are shown inside.

As shown in FIG. 4, GOD was used as an enzyme.
Glucose sensor containing only (curve (b) in FIG. 4)
Reflects on the concentration of only β-glucose contained in the sample solution, and the response current value was the lowest. On the other hand, the glucose sensor containing only PyOx as the enzyme (curve (c) in FIG. 4) reflects on the sum of the concentrations of α-glucose and β-glucose contained in the sample solution.
Particularly in the region where the glucose concentration is low, the curve (c) in FIG.
The response current was higher than that. However, the response current value of the glucose sensor containing only PyOx was lower than that of the curve (b) in FIG. 4 in the high glucose concentration region. As a result, the glucose sensor containing both PyOx and GOD (curve (a) in FIG. 4) always had a high response current value in the widest concentration range.

(Example 4) As an example of the biosensor of the present invention, a sucrose sensor was manufactured as follows.

In the same manner as in Example 1, the leads 2 and 3, the electrode system 8 (working electrode 4 and counter electrode 5) and the insulating layer 6 were formed on the electrically insulating substrate 1 made of polyethylene terephthalate. Then, 0.5% by weight of an aqueous solution of carboxymethyl cellulose (CMC) was dropped onto the electrode system 8 having the working electrode 4 and the counter electrode 5 and dried to form C.
The MC layer was formed.

A mixed aqueous solution of PyOx, GOD, invertase (INV) and potassium ferricyanide was added dropwise to this CMC layer and dried to form a reaction layer 7. The amounts of PyOx, GOD, invertase, and potassium ferricyanide contained in this reaction layer 7 are determined by the reaction layer 1
1 unit per square centimeter, 10 each
Units, 40 units and 1.3 mg.

When a 171 mg / dl sucrose aqueous solution was dropped as a sample solution onto the reaction layer 7 of the sucrose sensor manufactured as described above, the reaction layer 7 was formed by the sample solution.
Dissolved. Then, 3 minutes after dropping, the counter electrode 5 of the electrode system 8
On the other hand, a voltage of +0.5 V was applied to the working electrode 4, and the current value after 5 seconds was measured.

Furthermore, the response current values for the 342 and 684 mg / dl sucrose aqueous solutions were measured in the same manner as above, using a new sucrose sensor for each measurement.

The obtained response current value showed a tendency to increase depending on the increase of sucrose concentration, and had a high value in a wide concentration range.

As a comparative experiment, a sucrose sensor was prepared in the same manner as above except that the reaction layer 7 did not contain GOD, and the response current value was measured. The obtained response current value was always lower than that of the sucrose sensor containing the GOD.

The same effects as described above were obtained also with a maltose sensor or a lactose sensor prepared by using maltose hydrolase or lactose hydrolase instead of INV.

Example 5 A biosensor was produced in the same manner as in Examples 1 to 4 except that the reaction layer 7 did not contain potassium ferricyanide. Sample liquids having the same substrate concentrations as in Examples 1 to 4 were dropped on these biosensors, and a voltage of +1.0 V was applied to the working electrode 4 with respect to the counter electrode 5 of the electrode system 8 after a certain period of time. , And the current value after 5 seconds was measured.

The obtained response current value showed a tendency to increase depending on the increase of each substrate concentration. Example 6 A glucose sensor was manufactured in the same manner as in Example 3. Whole blood having a glucose concentration of 95 mg / dl was dropped as a sample solution onto the reaction layer 7 of the glucose sensor. One minute after the dropping, a voltage of +0.5 V was applied to the working electrode 4 with respect to the counter electrode 5 of the electrode system 8 and the current value after 5 seconds was measured.

Furthermore, the response current values for whole blood having glucose concentrations of 170 and 320 mg / dl were measured using a new glucose sensor for each measurement and in the same manner as above. The response characteristic between the obtained glucose concentration and the response current value is shown by the curve d in FIG.

As a comparative experiment, of the glucose sensors, a sensor not containing PyOx and a sensor not containing GOD were prepared, and the response current value was measured in the same manner as above. These results are shown in the curve e and the curve f in FIG. As shown in FIG. 5, the glucose sensor containing only GOD as an enzyme (the curve e in FIG. 5 reflects only the β-glucose concentration in whole blood, and therefore its response current value was the lowest. On the other hand, the glucose sensor containing only PyOx as the enzyme (curve f in FIG. 5) reflects the sum of the concentrations of α-glucose and β-glucose in whole blood, and the response current is always higher than that in FIG. 5e. As a result, the glucose sensor containing both PyOx and GOD (curve d in FIG. 5) always had a high response current value in the widest concentration range.

[0064]

As is apparent from the above description, according to the present invention, a substrate (specific component) contained in a biological sample such as blood or urine, a raw material or a product in the food industry, and a sample such as fruit juice. It is possible to obtain a biosensor capable of quantifying rapidly with high accuracy.

[Brief description of drawings]

FIG. 1 is a schematic plan view of a biosensor according to an embodiment of the present invention.

FIG. 2 is a schematic cross-sectional view of a biosensor according to an embodiment of the present invention in which a reaction layer is arranged in direct contact with a substrate.

FIG. 3 is a schematic cross-sectional view of a biosensor according to an embodiment of the present invention in which a reaction layer is arranged on a substrate with a space in between.

FIG. 4 is a graph showing the correlation between glucose concentration and response current in an example of the biosensor of the present invention.

FIG. 5 is a graph showing the correlation between glucose concentration and response current in an example of the biosensor of the present invention.

[Explanation of symbols]

1 Electrically Insulating Substrate 2,3 Lead 4 Working Electrode 5 Counter Electrode 6 Insulating Layer 7,37 Reaction Layer 8 Electrode System 20 Spacer 30 Cover 31 Hole 38 Sample Supply Hole a Pyranose oxidase (PyOx) and glucose oxidase (GOD) in the reaction layer ) And the change in the response current value when containing only b b The change in the response current value when only containing GOD c The change in the response current value when only containing PyOx d The pyranose oxidase (PyOx) and glucose oxidase in the reaction layer (GOD) and change in response current value when containing e Change in response current value when only containing GOD f Change in response current value when only containing PyOx

Claims (8)

[Claims] 1. An electrically insulating substrate, an electrode system having a working electrode and a counter electrode formed on the substrate, and a reaction layer arranged on the substrate or with the substrate interposed in space. A biosensor in which the reaction layer contains pyranose oxidase. 2. The biosensor according to claim 1, wherein the pyranose oxidase is pyranose oxidase (EC 1.1.3.10). 3. The biosensor according to claim 1, wherein the reaction layer further contains at least one of glucose oxidase and an electron acceptor. 4. The biosensor according to claim 1, 2 or 3, wherein the reaction layer further contains one selected from sucrose hydrolase, maltose hydrolase and lactose hydrolase. 5. The biosensor according to claim 3, wherein the electron acceptor is a ferricyan ion. 6. An electrically insulating substrate, an electrode system having a working electrode and a counter electrode formed on the substrate, and a reaction layer disposed on the substrate or with the substrate interposed in space. The reaction layer contains pyranose oxidase and ferricyan, and the reaction layer contains 1 to 200 units of the pyranose oxidase and 0.21 mg to 3.30 m 2 per square centimeter.
A biosensor containing g of ferricyanide. 7. An electrically insulating substrate, an electrode system having a working electrode and a counter electrode formed on the substrate, and a reaction layer arranged on the substrate or with the substrate interposed in space. Then, the reaction layer contains pyranose oxidase, glucose oxidase and ferricyan, and the reaction layer contains 0.1 to 200 units of the pyranose oxidase and 1 unit from 1 unit per square centimeter. A biosensor containing 200 units of glucose-oxidase and 0.21 mg to 3.30 mg of ferricyan ion. 8. A biosensor for measuring blood glucose level, which has the structure according to any one of claims 1 to 7.