JP2001249103A - Biosensor - Google Patents

Abstract

(57) [Summary] [Problem] To have good response characteristics up to a high concentration range,
And a biosensor having low blank response and high storage stability. SOLUTION: An electrode system including a working electrode and a counter electrode constituting an electrochemical measurement system in contact with a supplied sample liquid, an electrically insulating support for supporting the electrode system, and an electrode formed on the working electrode A first reagent layer, and a second reagent layer formed on the counter electrode, wherein the first reagent layer is mainly composed of an enzyme, and the second reagent layer is mainly composed of an electron carrier. It is.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biosensor for rapidly and accurately quantifying a substrate contained in a sample.

[0002]

2. Description of the Related Art As a quantitative analysis method for saccharides such as sucrose and glucose, a photometric method, a colorimetric method, a reductive titration method, and a method using various types of chromatography have been developed. However, none of these methods has a very high specificity for saccharides, and thus is inaccurate. Of these methods, the photometer method is easy to operate, but is greatly affected by the temperature during operation. Therefore, the photometer method is not suitable as a method for ordinary people to easily determine saccharides at home or the like. In recent years, various types of biosensors utilizing the specific catalytic action of enzymes have been developed. Hereinafter, a method for quantifying glucose will be described as an example of a method for quantifying a substrate in a sample. As an electrochemical glucose quantification method, an enzyme, glucose oxidase (E
A method using C1.1.3.4 (hereinafter abbreviated as GOD) and an oxygen electrode or a hydrogen peroxide electrode is generally known (for example, Shuichi Suzuki, "Biosensor" Kodansha). GOD selectively oxidizes β-D-glucose as a substrate to D-glucono-δ-lactone using oxygen as an electron carrier. In the presence of oxygen, oxygen is reduced to hydrogen peroxide during the oxidation reaction process by GOD. The oxygen electrode measures the decrease in oxygen, or the hydrogen peroxide electrode measures the increase in hydrogen peroxide. Since the amount of decrease in oxygen and the amount of increase in hydrogen peroxide are proportional to the content of glucose in the sample, glucose can be determined from the amount of decrease in oxygen or the amount of increase in hydrogen peroxide.

In the above method, glucose in a sample can be accurately quantified by utilizing the specificity of an enzyme reaction. However, as can be inferred from the reaction process, the measurement result has a disadvantage that it is greatly affected by the concentration of oxygen contained in the sample, and the measurement becomes impossible if oxygen does not exist in the sample. Therefore, a new type of glucose sensor using an organic compound such as potassium ferricyanide, a ferrocene derivative, a quinone derivative or a metal complex as an electron carrier without using oxygen as an electron carrier has been developed. In this type of sensor, the concentration of glucose contained in the sample is determined from the amount of oxidation current by oxidizing the reduced form of the electron carrier generated as a result of the enzyme reaction on the working electrode. At this time, on the counter electrode, the oxidized form of the electron carrier is reduced, and the reaction of generating the reduced form of the electron carrier proceeds. By using such an organic compound or metal complex as an electron carrier instead of oxygen, it is possible to form a reagent layer by accurately supporting a known amount of GOD and the electron carrier in a stable state on an electrode. This makes it possible to accurately determine glucose without being affected by the oxygen concentration in the sample. In this case, the reagent layer containing the enzyme and the electron mediator can be integrated with the electrode system in a state close to a dry state, so that a disposable glucose sensor based on this technology has attracted much attention in recent years. . A typical example is a biosensor disclosed in Japanese Patent No. 2517153. In a disposable glucose sensor, the glucose concentration can be easily measured by a measuring device simply by introducing a sample into a sensor detachably connected to the measuring device. Such a technique is applicable not only to the quantification of glucose but also to the quantification of other substrates contained in a sample.

[0004]

However, in the above-described conventional biosensor, in the case of a sample containing a high concentration of a substrate, the enzymatic reaction proceeds even on the counter electrode, so that the electron carrier is supplied to the counter electrode. Is insufficient, and the counter electrode reaction is rate-determining, so that there is a problem that a current response proportional to the substrate concentration cannot be obtained and the substrate cannot be quantified. In recent years, there has been a demand for a biosensor that has a low response when the substrate concentration is zero (hereinafter abbreviated as a blank response) and has excellent storage stability.

[0005]

According to the present invention, there is provided a biosensor comprising: an electrode system including a working electrode and a counter electrode which constitute an electrochemical measurement system by contacting a supplied sample liquid; and an electric system for supporting the electrode system. An insulating support, a first reagent layer formed on the working electrode, and a second reagent layer formed on the counter electrode, wherein the first reagent layer is a main component of an enzyme, The second reagent layer is characterized in that the main component is an electron carrier. It is preferable that the first reagent layer does not contain an electron carrier and the second reagent layer does not contain an enzyme.
In a preferred embodiment, the support is made of one electrically insulating substrate, and the working electrode and the counter electrode are formed on the substrate. In another preferred embodiment,
The support includes an electrically insulating substrate and an electrically insulating cover member that forms a sample liquid supply path between the substrate, the working electrode is formed on the substrate, and the counter electrode is the cover member. Are formed facing the working electrode on the inner surface of the. The cover member is preferably formed of a sheet member having a curved surface portion bulging outward for forming a sample liquid supply path or a sample liquid storage section between the cover member and the substrate.
More preferably, the cover member includes a spacer having a slit forming the sample liquid supply path and a cover covering the spacer. At least the first reagent layer preferably contains a hydrophilic polymer.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION A biosensor according to a preferred embodiment of the present invention comprises an electrically insulating substrate, a working electrode and a counter electrode formed on the substrate, a first reagent layer formed on the working electrode, And a second reagent layer formed on the counter electrode, wherein the first reagent layer is mainly composed of an enzyme, and the second reagent layer is mainly composed of an electron carrier. . In this biosensor, since the main component of the second reagent layer on the counter electrode is an electron carrier, the enzymatic reaction hardly proceeds on the counter electrode particularly in the case of a sample containing a high concentration of the substrate. Therefore, the frequency of collision between the enzyme and the electron carrier in the solution in which the reagent is dissolved in the sample liquid is reduced.
Therefore, the slope of the response line decreases. However, since the electron carrier sufficient to react on the counter electrode is retained, the counter electrode reaction does not become rate-determining, and as a result, the response linearity can be maintained up to the high concentration region of the substrate.

A biosensor according to another preferred embodiment of the present invention includes an electrically insulating substrate, an electrically insulating cover member for forming a sample liquid supply passage between the substrate and the working electrode, and a working electrode formed on the substrate. A counter electrode formed on the inner surface of the cover member so as to face the working electrode, a first reagent layer formed on the working electrode, and a second reagent layer formed on the counter electrode. The cover member is a sheet member having a curved surface portion bulging outward for forming a sample liquid supply path or a sample liquid storage section between the cover member and the substrate. More preferably, the cover member includes a spacer having a slit forming the sample liquid supply path and a cover covering the spacer. In such a biosensor, since the first reagent layer and the second reagent layer are formed on different members, respectively, the first reagent layer and the second reagent layer having different compositions from each other.
And the reagent layer can be easily separated and formed. Further, since the working electrode and the counter electrode are formed at positions facing each other, ion movement between the electrodes becomes smooth, so that the current response can be further increased.

In a biosensor in which the cover member is composed of a spacer and a cover, the physical strength of the cover increases, so that the first reagent layer and the second reagent layer come into contact with each other by external physical pressure. And the enzyme activity can be prevented from fluctuating due to contact between the enzyme and the electron carrier. In any of the above forms of biosensor, it is preferable that at least the first reagent layer contains a hydrophilic polymer. Since the adsorption of proteins and the like to the working electrode is suppressed by the hydrophilic polymer, the current response sensitivity is further improved. In addition, at the time of measurement, the viscosity of the sample solution is increased by the hydrophilic polymer dissolved in the sample solution, so that the influence of physical impact or the like on the current response is reduced, and the stability of the current response is improved.

In the present invention, as the substrate, the spacer, and the cover, any material having electrical insulation and sufficient rigidity during storage and measurement can be used. Examples thereof include thermoplastic resins such as polyethylene, polystyrene, polyvinyl chloride, polyamide, and saturated polyester resins, and thermosetting resins such as urea resins, melamine resins, phenol resins, epoxy resins, and unsaturated polyester resins. Among them, polyethylene terephthalate is preferred from the viewpoint of adhesion to the electrode. As the working electrode, any conductive material that is not itself oxidized when oxidizing the electron carrier can be used. As the counter electrode, any commonly used conductive material such as palladium, silver, platinum, and carbon can be used. As the enzyme, an enzyme corresponding to the substrate to be measured contained in the sample is used. For example, fructose dehydrogenase, glucose oxidase, alcohol oxidase, lactate oxidase, cholesterol oxidase,
Xanthine oxidase, amino acid oxidase and the like can be mentioned.

Examples of the electron carrier include potassium ferricyanide, p-benzoquinone, phenazine methosulfate, methylene blue, ferrocene derivatives and the like. Also, a current response can be obtained when oxygen is used as the electron carrier. One or more of these electron carriers are used. Various kinds of hydrophilic polymers can be used. For example, hydroxyethylcellulose, hydroxypropylcellulose, methylcellulose, ethylcellulose, ethylhydroxyethylcellulose, carboxymethylcellulose, polyvinylpyrrolidone, polyvinylalcohol, polyamino acids such as polylysine, polystyrenesulfonic acid, gelatin and its derivatives, polyacrylic acid and its salts, polymeta Examples thereof include polymers of acrylic acid and salts thereof, starch and derivatives thereof, and maleic anhydride or salts thereof. Among them, carboxymethyl cellulose, hydroxyethyl cellulose and hydroxypropyl cellulose are preferred.

[0011]

The present invention will be described in more detail with reference to the following examples. << Example 1 >> A glucose sensor will be described as an example of a biosensor. FIG. 1 is a longitudinal sectional view of the glucose sensor of the present embodiment, and FIG. 2 is an exploded perspective view of the glucose sensor excluding a reagent layer and a surfactant layer.
First, silver paste was printed by screen printing on an electrically insulating substrate 1 made of polyethylene terephthalate to form leads 2, 3 and a base for electrodes to be described later.
Next, a conductive carbon paste containing a resin binder was printed on the substrate 1 to form the working electrode 4. The working electrode 4 is in contact with the lead 2. Further, on the substrate 1,
The insulating paste was printed to form the insulating layer 6. The insulating layer 6 covers the outer peripheral portion of the working electrode 4, thereby keeping the area of the exposed portion of the working electrode 4 constant. Next, the counter electrode 5 was formed by printing a conductive carbon paste containing a resin binder so as to be in contact with the leads 3.

The first reagent layer 7 is formed by dropping a first aqueous solution containing the enzyme GOD and not containing the electron mediator on the working electrode 4 of the substrate 1 and then drying.
The second reagent layer 8 was formed by dropping a second aqueous solution containing potassium ferricyanide, which is an electron carrier, and not containing an enzyme, on the counter electrode 5 of the substrate 1, followed by drying. Furthermore, for the purpose of smoothly supplying the sample,
A layer 9 containing lecithin as a surfactant was formed so as to cover the first reagent layer 7 and the second reagent layer 8. Finally, the glucose sensor was manufactured by bonding the substrate 1, the cover 12, and the spacer member 10 in a positional relationship as indicated by a dashed line in FIG. The spacer member 10 sandwiched between the substrate 1 and the cover 12 has a slit 11, and the slit 11 forms a sample liquid supply path between the substrate 1 and the cover 12. Cover 12
Since the air hole 14 communicates with the sample liquid supply path, the sample supply port 13 formed at the open end of the slit 11
When the sample is brought into contact with the sample solution, the sample easily reaches the first reagent layer 7 and the second reagent layer 8 in the sample liquid supply path by capillary action.

As a comparative example, a glucose sensor was manufactured in the same manner as in this example except for the step of forming a reagent layer. FIG. 7 is a longitudinal sectional view of a glucose sensor of a comparative example. The reagent layer 30 was formed by dropping an aqueous solution containing GOD and potassium ferricyanide on the working electrode 4 and the counter electrode 5 and then drying. Further, a layer 9 containing lecithin as a surfactant was formed on the reagent layer 30.

Next, with respect to the glucose sensors of Example 1 and Comparative Example, the glucose concentration was measured using a solution containing a fixed amount of glucose as a sample. The sample is supplied from the sample supply port 13 to the sample liquid supply path, and after a certain time,
A voltage of 500 mV was applied to the working electrode 4 based on the counter electrode 5. By interposing the spacer member 10 between the cover 12 and the substrate 1, the strength of the sensor against physical pressure increases. Therefore, the volume of the sample liquid supply path is easily kept constant, and the influence of the physical pressure on the current response is reduced. When a current value flowing between the working electrode 4 and the counter electrode 5 due to the application of the voltage was measured, a current response proportional to the glucose concentration in the sample was observed in both Example 1 and Comparative Example. When the sample comes into contact with the first reagent layer 7 and the second reagent layer 8, potassium ferricyanide, which is an oxidized electron carrier, dissociates into ferricyanide ions and potassium ions. GOD reacts with glucose in the sample, ferricyanide ions dissolved in the sample from the second reagent layer 8. As a result, glucose is oxidized to gluconolactone, and oxidized ferricyanide ions are reduced to reduced ferrocyanide ions. Working electrode 4
On the above, a reaction in which ferrocyanide ions are oxidized to ferricyanide ions proceeds, and on the counter electrode 5, a reaction in which ferricyanide ions are reduced to ferrocyanide ions proceeds. Since the concentration of the ferrocyanide ion is proportional to the concentration of glucose, the glucose concentration can be measured based on the oxidation current of the ferrocyanide ion.

In the glucose sensor of the present embodiment, higher linearity was observed up to a higher glucose concentration than in the glucose sensor of the comparative example. This is for the following reason. That is, since GOD and potassium ferricyanide are separated on the working electrode and the counter electrode, the enzyme reaction hardly proceeds on the counter electrode. For this reason, the concentration of potassium ferricyanide at the counter electrode is maintained, which has the effect of preventing the counter electrode reaction from becoming rate-determining even for a high-concentration sample.As a result, the response linearity can be maintained up to the high-concentration region. is there. In the sensor of the comparative example, the ferricyan ion on the counter electrode is reduced to ferrocyanide ion by the same degree of enzymatic reaction progressing on the counter electrode as on the working electrode. For this reason, the shortage of necessary ferricyan ions at the counter electrode limits the counter electrode reaction. Furthermore, the glucose sensor of the present example had a lower blank response than the glucose sensor of the comparative example, and the fluctuation of the current response before and after long-term storage was suppressed. This is because GOD and potassium ferricyanide are separated from each other, so that they can be prevented from contacting each other and causing an interaction, thereby suppressing an increase in blank response and suppressing fluctuations in enzyme activity during long-term storage. This is because it could be suppressed.

Embodiment 2 FIG. 3 is a longitudinal sectional view of a glucose sensor in this embodiment, and FIG. 4 is an exploded perspective view of the glucose sensor excluding a reagent layer and a surfactant layer. The working electrode 4 and the lead 2 were formed by sputtering palladium on the electrically insulating substrate 21. Next, the working electrode 4 and the terminal portion to be inserted into the measuring instrument were defined by attaching the insulating sheet 23 on the substrate 21. On the other hand, the counter electrode 5 was formed by sputtering palladium on the inner wall surface of the curved portion 24 of the electrically insulating cover member 22 having the curved portion 24 bulging outward. Curved surface 24
Has an air hole 14 at its end. The first reagent layer 7
The first aqueous solution containing the enzyme GOD and not containing the electron mediator was dropped on the working electrode 4 of the substrate 21 and then dried. The second reagent layer 8 was formed by dropping a second aqueous solution containing potassium ferricyanide, which is an electron carrier, and not containing an enzyme, on the counter electrode 5 of the cover member 22, followed by drying. Furthermore, the first
On the reagent layer 7, a layer 9 containing lecithin as a surfactant was formed. Finally, the substrate 21 and the cover member 22 were attached to each other to produce a glucose sensor. Thus, the working electrode 4 and the counter electrode 5 are arranged at positions facing each other via a space formed between the substrate 21 and the curved surface 24 of the cover member. This space serves as a sample accommodating portion. If the sample is brought into contact with the open end of the space, the sample easily moves to the air hole 14 side by capillary action, and the first reagent layer 7 and the second reagent Contact layer 8.

Next, the glucose concentration was measured in the same procedure as in Example 1. As a result, a current response proportional to the glucose concentration in the sample was observed. However, for the counter electrode 5, electrical continuity was obtained by sandwiching the end of the curved surface portion 24 with a clip. The glucose sensor according to the second embodiment includes:
Since only potassium ferricyanide was contained in the second reagent layer 8, higher linearity was observed up to a higher glucose concentration, as in Example 1, as compared with the glucose sensor of the comparative example. Further, since GOD and potassium ferricyanide are separated from each other, similar to Example 1,
Compared with the comparative example, the blank response was reduced, and the fluctuation of the current response before and after long-term storage was suppressed. In addition, an increase in the response value was observed as compared with the comparative example. This is because the formation of the working electrode 4 and the counter electrode 5 at positions facing each other facilitates the movement of ions between the electrodes.

Embodiment 3 FIG. 5 is a longitudinal sectional view of a glucose sensor of the present embodiment, and FIG. 6 is an exploded perspective view of the glucose sensor excluding a reagent layer and a surfactant layer. First, a silver paste was printed by screen printing on an electrically insulating substrate 31 made of polyethylene terephthalate to form leads 2. Next, a conductive carbon paste containing a resin binder was printed on the substrate 31 to form the working electrode 4. The working electrode 4 is in contact with the lead 2. Further, an insulating paste was printed on the substrate 31 to form the insulating layer 6. The insulating layer 6 covers the outer peripheral portion of the working electrode 4, thereby keeping the area of the exposed portion of the working electrode 4 constant. Next, the lead 3 is formed by printing a silver paste on the back surface of the electrically insulating cover 32, then the conductive carbon paste is printed to form the counter electrode 5, and the insulating paste is printed by printing the insulating paste. Was formed. Air holes 14 were formed in the cover 32. A first aqueous solution containing the enzyme GOD and not containing the electron mediator is dropped on the working electrode 4 of the substrate 31 and then dried to form the first reagent layer 7, and potassium ferricyanide as the electron mediator is formed. And a second aqueous solution containing no enzyme was dropped on the counter electrode 5 of the cover 32 and then dried to form a second reagent layer 8. Further, a layer 9 containing lecithin as a surfactant was formed on the first reagent layer 7. Finally, the glucose sensor was manufactured by bonding the substrate 31, the cover 32, and the spacer member 10 in a positional relationship as indicated by a dashed line in FIG.

The spacer member 10 provided so as to be sandwiched between the substrate 31 and the cover 32 has a slit 11, which forms a sample liquid supply path between the substrate 31 and the cover 32. . Working electrode 4 and counter electrode 5
Are arranged so as to face each other in a sample liquid supply path formed in the slit 11 of the spacer member 10. Since the air hole 14 of the cover 32 communicates with the sample liquid supply path, if the sample is brought into contact with the sample supply port 13 formed at the open end of the slit 11, the sample is easily removed by capillary action. The first reagent layer 7 and the second reagent layer 8 in the supply path are reached. Next, glucose was measured in the same procedure as in Example 1. As a result of the measurement, a current response proportional to the glucose concentration in the sample was observed.

Since the glucose sensor of the third embodiment contains only potassium ferricyanide in the second reagent layer 8, it is similar to the glucose sensor of the comparative example in the same manner as in the first embodiment.
High linearity was observed up to higher glucose concentrations. Further, since the working electrode 4 and the counter electrode 5 were formed at positions facing each other, the current response was increased as in Example 2 as compared with the comparative example. Further, since GOD and potassium ferricyanide were separated from each other, the blank response was lower than in the comparative example, and the fluctuation of the current response before and after long-term storage was suppressed. Further, since the spacer member 10 is sandwiched between the substrate 31 and the cover 32, the strength with respect to the physical pressure is increased, so that the first reagent layer 7 and the second
No contact was caused by the physical pressure of the reagent layer 8, and the current response fluctuation due to the fluctuation of the enzyme activity due to the contact between GOD and potassium ferricyanide was eliminated.
Further, since the volume of the sample liquid supply path was easily kept constant, the stability of the current response was improved as compared with the second embodiment.

Example 4 In this example, a glucose sensor was manufactured in the same manner as in Example 3, except for the step of forming the first reagent layer 7 and the second reagent layer 8. The first reagent layer 7 contains GOD as an enzyme and carboxymethylcellulose as a hydrophilic polymer, and a first aqueous solution containing no electron carrier is dropped on the working electrode 4 of the substrate 31 and then dried. The second reagent layer 8 was formed by dropping a second aqueous solution containing potassium ferricyanide as an electron carrier and carboxymethylcellulose and not containing an enzyme on the counter electrode 5 of the cover 32 and then drying. . Further, a layer 9 containing lecithin as a surfactant was formed on the first reagent layer 7. Next, glucose was measured in the same procedure as in Example 1. As a result of the measurement, a current response proportional to the glucose concentration in the sample was observed.

In the glucose sensor of this embodiment, since the second reagent layer 8 contains only potassium ferricyanide,
As compared with the glucose sensor of the comparative example, similar to Example 1, higher linearity was observed up to a higher glucose concentration. Further, since the working electrode 4 and the counter electrode 5 were formed at positions facing each other, an increase in the response value was observed as compared with the comparative example. Further, since GOD and potassium ferricyanide were separated from each other, the blank response was lower than in the comparative example, and the fluctuation of the current response before and after long-term storage was suppressed. Spacer member 1 between substrate 31 and cover 32
Since 0 is sandwiched between the GOD and the potassium ferricyanide, the current response fluctuation due to the fluctuation of the enzyme activity caused by the contact is eliminated. Further, since the volume of the sample liquid supply path was easily kept constant, the stability of the current response was improved as compared with the second embodiment. Further, the current response was further increased as compared with Examples 2 and 3. This is because the presence of carboxymethylcellulose in the first reagent layer 7 eliminates the adsorption of proteins and the like to the surface of the working electrode 4 and the electrode reaction at the working electrode 4 proceeds smoothly. Further, since the viscosity of the sample increases at the time of measurement, the influence of a physical impact applied to the sensor is reduced, and the variation of the sensor response is reduced.

In the above embodiment, the voltage applied to the working electrode 4 is set to 500 mV with reference to the counter electrode 5, but the present invention is not limited to this. Any voltage may be used as long as the electron carrier reduced by the enzymatic reaction is oxidized. In the above embodiment, the second reagent layer formed on the counter electrode contains only the electron carrier. However, the counter electrode reaction does not limit the rate, and the influence on the blank response and the storage stability is reduced. Components other than the electron carrier may be contained to the extent that they can be almost ignored. Further, in the above embodiment, the first reagent layer formed on the working electrode was configured to include only the enzyme or the enzyme and the hydrophilic polymer. However, the effect on the blank response and the storage stability was almost ignored. May be contained to the extent possible. In the above embodiment, one type of electron carrier is used, but two or more types of electron carriers may be used.

The first reagent layer 7 and the second reagent layer 8 are
By immobilizing this on the working electrode 4 or the counter electrode 5, the enzyme or the electron carrier may be insolubilized. In the case of immobilization, it is preferable to use a cross-linking immobilization method or an adsorption method. Further, they may be mixed in the electrode. A surfactant other than lecithin may be used. Further, in the above embodiment, the surfactant layer 9 is formed only on the first reagent layer 7 or on the first reagent layer 7 and the second reagent layer 8, but is not limited thereto. Alternatively, it may be formed at a position facing the sample station supply path, such as the side surface of the slit 11 of the spacer member 10. In the above embodiment, the two-electrode system having only the working electrode and the counter electrode has been described.
If a three-electrode system with a reference electrode is used, more accurate measurement is possible. It is preferable that the first reagent layer 7 and the second reagent layer 8 are separated from each other via a space without contacting each other. As a result, the effects of suppressing an increase in blank response and improving storage stability can be further enhanced.

[0025]

As described above, according to the present invention, a biosensor having good current response characteristics up to a high concentration range can be obtained. In addition, a biosensor further having a low blank response and high storage stability can be obtained.

[Brief description of the drawings]

FIG. 1 is a longitudinal sectional view of a glucose sensor according to one embodiment of the present invention.

FIG. 2 is an exploded perspective view of the glucose sensor from which a reagent layer and a surfactant layer are removed.

FIG. 3 is a longitudinal sectional view of a glucose sensor according to another embodiment of the present invention.

FIG. 4 is a perspective view of the glucose sensor from which a reagent layer and a surfactant layer are removed.

FIG. 5 is a longitudinal sectional view of a glucose sensor according to still another embodiment of the present invention.

FIG. 6 is an exploded perspective view of the glucose sensor from which a reagent layer and a surfactant layer are removed.

FIG. 7 is a longitudinal sectional view of a glucose sensor in a comparative example.

[Explanation of symbols]

 1, 21, 31 Substrate 2, 3 Lead 4 Working electrode 5 Counter electrode 6 Insulating layer 7 First reagent layer 8 Second reagent layer 9 Surfactant layer 10 Spacer 11 Slit 12, 22, 32 Cover 13 Sample supply port 14 Air hole 23 Insulating sheet 24 Curved surface

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 27/30 353B 27/46 338

Claims (7)

[Claims] 1. An electrode system including a working electrode and a counter electrode, which make up an electrochemical measurement system in contact with a supplied sample liquid, an electrically insulating support for supporting the electrode system, and a support formed on the working electrode. A first reagent layer, and a second reagent layer formed on the counter electrode, wherein the first reagent layer is mainly composed of an enzyme, and the second reagent layer is mainly composed of an electron transfer layer. A biosensor characterized by being a body. 2. The method according to claim 1, wherein the first reagent layer does not contain an electron carrier,
The biosensor according to claim 1, wherein the second reagent layer does not contain an enzyme. 3. The biosensor according to claim 1, wherein the support comprises a single electrically insulating substrate, and the working electrode and the counter electrode are formed on the substrate. 4. The support member comprises an electrically insulating substrate and an electrically insulating cover member forming a sample liquid supply path or a sample liquid storage section between the substrates, and the working electrode is provided on the substrate. The biosensor according to claim 1, wherein the counter electrode is formed, and the counter electrode is formed on an inner surface of the cover member so as to face the working electrode. 5. The sheet member having a curved surface portion bulging outward for forming a sample liquid supply path or a sample liquid storage section between the cover member and the substrate.
The biosensor as described. 6. The biosensor according to claim 4, wherein the cover member comprises a spacer having a slit forming the sample liquid supply path, and a cover covering the spacer. 7. The biosensor according to claim 1, wherein the first reagent layer contains a hydrophilic polymer.