JPH0783872A - Biosensor and manufacture thereof - Google Patents

Abstract

PURPOSE:To improve the preservation characteristic of a sensor and ensure a measurement with high accuracy by applying the constitution where a reaction layer contains a hydrophilic macromolecule and a cushioning agent, and enzyme is separated from the cushioning agent. CONSTITUTION:Leads 2 and 3, measurement and counter electrodes 4 and 5, and an insulation layer 6 are respectively laid on a substrate 1. Also, the layer 7 of sodium salt (CMC) composed of enzyme fructose dehydrogenase-potassium ferricyanide-hydrophilic macromolecule carboxyl methyl cellulose is formed on an electrode system. In addition, a cushioning agent (KHPO3 and K2PO3)- lecitin layer 8 is formed on the layer 7. Also, solvent toluene used for forming the layer 8 does not dissolve CMC as an underlayer. The ingress of sample liquid to a reaction layer from the outside is facilitated by forming amphipatic lipid layer like lecitin on the surface of the reaction layer. Consequently, the reaction layer of a fructose sensor is formed. Furthermore, a manufacturing process can be simplified by dropping all at once a mixed solution of hydrophilic macromolecule, enzyme and an electron receptor and then drying the solution.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biosensor for performing a quick and highly accurate quantification of a specific component in a biological sample by utilizing an enzymatic reaction, and a method for producing the same.

[0002]

2. Description of the Related Art Conventionally, various biosensors have been proposed as a method for easily quantifying a specific component in a sample without diluting or stirring the sample solution. First, a glucose sensor will be described as an example of a biosensor. As a method for quantifying glucose using an enzyme electrode,
A method in which glucose oxidase is combined with an enzyme electrode or a hydrogen peroxide electrode is generally known. Glucose oxidase selectively oxidizes the substrate β-D-glucose into D-glucono δ-lactone by using oxygen as an electron acceptor. With this reaction, oxygen is reduced to hydrogen peroxide. Glucose is quantified by measuring the oxygen consumption at this time with an oxygen electrode or the hydrogen peroxide production amount with a hydrogen peroxide electrode using a platinum electrode or the like.

However, according to the above method, the dissolved oxygen concentration is greatly influenced depending on the object to be measured, and the measurement becomes impossible under the condition of no oxygen. Therefore, a type of glucose sensor that does not use oxygen as an electron acceptor but uses a metal complex such as potassium ferricyanide, a ferrocene derivative, a quinone derivative or an organic compound as an electron acceptor has been developed. In this type of sensor, the reduced form of the electron acceptor generated as a result of the enzymatic reaction is oxidized at the electrode, and the glucose concentration is obtained from the oxidation current. This method is widely applied not only to glucose but also to quantification of other substrates. The following glucose sensor is known as an example of this type of biosensor (Japanese Patent Application Laid-Open No. HEI-1).
-114747).

This sensor comprises an insulating substrate provided with an electrode system consisting of a measuring electrode and a counter electrode, which is spaced apart from the electrode system by a filtration layer made of a polycarbonate porous membrane, an electron acceptor carrying layer and an enzyme carrying layer. A buffer layer and a spreading layer made of cellulose woven fabric are sequentially laminated. In addition, the above-mentioned supporting layer is obtained by impregnating an aqueous solution of an electron acceptor, an enzyme, and a buffer into a cellulose porous body and drying it. The operation of this glucose sensor is as follows. The sample solution dropped on the spreading layer is adjusted to a pH at which the highest enzyme activity is obtained by the buffering action of the buffer in the buffer-supporting layer. Next, glucose in the sample solution is
It reacts specifically with glucose oxidase in the enzyme carrying layer. At the same time, the electron acceptor of the electron acceptor supporting layer, for example, potassium ferricyanide is reduced by the electrons generated in the above reaction to form potassium ferrocyanide. The amount of potassium ferrocyanide is proportional to the glucose concentration in the sample solution. Next, after a substance having a large molecular weight such as a protein that interferes with the reaction at the electrode is filtered by the filtration layer, the sample solution reaches the electrode system provided on the insulating substrate. In order to prevent erroneous measurement, the glucose concentration can be detected by measuring the oxidation current value of potassium ferrocyanide in the sample liquid with an electrode system partially covered with an insulating layer.

In such a conventional structure, the wetting of the substrate surface including the electrode system is not always uniform, so that bubbles may remain between the porous body of the filtration layer and the substrate, which may affect the response current. there were. In addition, the presence of a substance that is easily adsorbed on the electrode or a substance that is active in the electrode in the sample solution may affect the response of the sensor. As a method for solving the above problems, the following biosensor has been proposed (JP-A-2-062952). That is, an electrode system consisting of a measuring electrode, a counter electrode and a reference electrode is formed on an insulating substrate by a method such as screen printing, and on this electrode system, a hydrophilic polymer, a redox enzyme and an electron are brought into contact with the electrode system. An enzyme reaction layer containing a receptor and a buffer added as necessary is formed. When the sample solution containing the substrate is dropped onto the enzyme reaction layer, the enzyme reaction layer is dissolved and the pH is adjusted to the highest enzymatic activity by the buffering action of the buffer, the enzyme reacts with the substrate, and the electron acceptor Is reduced. After completion of the enzymatic reaction, the reduced electron acceptor is electrochemically oxidized,
The substrate concentration in the sample solution is determined from the oxidation current value obtained at this time.

[0006]

In such a conventional structure, when the biosensor absorbs moisture, the buffer and the enzyme are partially mixed, and the chemical activity lowers the enzyme activity, so that the biosensor is stored. There was an inconvenience that the property deteriorated. The present invention is to solve the above problems,
It is an object of the present invention to provide a biosensor capable of quantifying a specific component contained in a biological sample with high speed and simple operation with high accuracy. Another object of the present invention is to provide a biosensor that can be stored for a long period of time and can be used for food quality control and clinical examination. Further, the present invention aims to provide a method for producing a biosensor in which an enzyme and a buffer are not mixed even during production.

[0007]

The biosensor of the present invention comprises:
An insulating substrate, an electrode system having a measurement electrode and a counter electrode provided on the substrate, and a reaction layer formed on the electrode system in contact with the electrode system, wherein the reaction layer is at least a hydrophilic polymer. And an enzyme and a buffer, and the enzyme is separated from the buffer. In a preferred embodiment of the present invention, the reaction layer is composed of at least two layers, one layer is in contact with the electrode system, and is composed of an enzyme and a hydrophilic polymer, and the other layer contains a buffer. The other layer preferably consists of lipids. In another preferred embodiment of the present invention, the reaction layer is composed of at least two layers, one layer is in contact with the electrode system, and is composed of a buffering agent and a hydrophilic polymer, and the other layer is carrying an enzyme. . The other layer is
It is preferable that the hydrophilic polymer is soluble in an organic solvent that does not dissolve the hydrophilic polymer. In the biosensor of the present invention, the reaction layer preferably contains an electron acceptor.

The present invention also provides a biosensor in which the reaction layer comprises at least three layers, one layer comprises a buffer and a hydrophilic polymer, and the other layer comprises an enzyme and a hydrophilic polymer. The layer containing the enzyme preferably further contains an electron acceptor. Furthermore, in a preferred embodiment of the present invention,
Located outside the layer containing the buffer and the layer containing the enzyme,
It has a layer of lipids, especially amphipathic lipids. In another aspect, it has a layer in contact with the electrode system and consisting essentially of a hydrophilic polymer. In still another aspect, a layer composed of a buffer and a hydrophilic polymer and a layer composed of an enzyme and a hydrophilic polymer are in contact with each other, and the hydrophilic polymers of both layers are different substances, and The polymer of the layer formed in 1) is soluble in an organic solvent that does not dissolve the polymer formed underneath.

The method of manufacturing a biosensor of the present invention further comprises an electrode system having a measuring electrode and a counter electrode provided on an insulating substrate, and a reaction layer formed on the electrode system in contact with the electrode system, A method for producing a biosensor, wherein the reaction layer comprises at least two layers of a layer comprising an enzyme and a hydrophilic polymer, and a layer containing a buffer, wherein water is used as a medium in contact with the electrode system on the substrate. And a step of forming a second layer containing a buffer on the first layer using an organic solvent that does not dissolve the hydrophilic polymer as a medium. Have. Here, in a preferred embodiment, the step of forming the first layer comprises spreading an aqueous solution in which the enzyme and the hydrophilic polymer are dissolved on a substrate and drying the solution to form the second layer. The method comprises developing a liquid in which a buffer is dispersed in an organic solvent solution of lipids on the first layer and drying. In another aspect, the step of forming the first layer comprises spreading an aqueous solution in which an enzyme and a hydrophilic polymer are dissolved on a substrate and drying the solution to form the second layer. , A solution in which a buffering agent is dispersed in an organic solvent solution of a hydrophilic polymer is spread on the first layer and dried.

Further, the present invention comprises an electrode system having a measuring electrode and a counter electrode provided on an insulating substrate, and a reaction layer formed on the electrode system in contact with the electrode system, wherein the reaction layer is A method for producing a biosensor comprising at least two layers of a layer comprising an enzyme and a hydrophilic polymer, and a layer containing a buffer, wherein the electrode system is in contact with the substrate on the substrate, and a buffer is used with water as a medium A step of forming a first layer composed of a hydrophilic polymer, and a step of forming a second layer composed of an enzyme and a hydrophilic polymer on the first layer using an organic solvent which does not dissolve the hydrophilic polymer as a medium. There is provided a method for manufacturing a biosensor having: Here, it is preferable that the aqueous solution in which the enzyme and the hydrophilic polymer are dissolved further dissolves the electron acceptor. In addition, it is preferable to further include a step of forming a third layer on the second layer by further developing an organic solvent solution of a lipid and drying the solution.

[0011]

As described above, in the biosensor of the present invention, the reaction layer formed on the substrate electrode system in contact with the electrode system contains at least a hydrophilic polymer, an enzyme and a buffer, and the enzyme is buffered. It has a structure separated from the agent. Since the reaction layer contains a buffer, the sample solution supplied to the sensor reaches the buffer in the reaction layer even when the pH of the sample solution does not match the pH at which the highest enzyme activity is obtained. , The pH of the sample solution is automatically adjusted to the value that gives the highest enzyme activity. Therefore, it is not necessary to previously adjust the pH of the sample solution with a buffer solution or the like, and the measurement can be performed by a simple operation. Furthermore, by separating the enzyme layer and the buffer, it is possible to prevent partial mixing of the buffer and enzyme due to moisture absorption of the biosensor and the resulting decrease in enzyme activity due to chemical interaction. At times the enzyme can be kept under stable conditions.

In the biosensor of the present invention, even when the layer containing the buffer and the layer containing the enzyme are in contact with each other, the hydrophilic polymers in both layers are different from each other and the layer formed on the upper side. By making the polymer formed underneath it soluble in an organic solvent that does not dissolve the polymer formed underneath it, direct contact between the buffer agent and the enzyme during the production of the biosensor can be eliminated. . The biosensor having such a configuration can be obtained by the following manufacturing method. One of them is a step of contacting an electrode system on an insulating substrate to form a first layer composed of an enzyme and a hydrophilic polymer using water as a medium, using an organic solvent that does not dissolve the hydrophilic polymer as a medium. It is a manufacturing method including a step of forming a second layer containing a buffering agent on the first layer. Further, the other one is a step of contacting the electrode system on the insulating substrate to form a first layer composed of a buffer and a hydrophilic polymer using water as a medium,
The production method comprises a step of forming a second layer composed of an enzyme and a hydrophilic polymer on the first layer using an organic solvent which does not dissolve the hydrophilic polymer as a medium.

In the biosensor of the present invention, it is preferable that the surface of the reaction layer has a layer containing a lipid that facilitates penetration of the sample solution into the reaction layer. As the lipid, in addition to lecithin used in the following examples, phospholipids such as phosphatidylserine and phosphatidylethanolamine, and amphipathic lipids are preferable. Further, as the hydrophilic polymer for forming the reaction layer, in addition to carboxymethyl cellulose and polyvinyl pyrrolidone used in the following examples, polyvinyl alcohol, water-soluble cellulose derivative, particularly ethyl cellulose, hydroxypropyl cellulose; gelatin,
Polyacrylic acid and salts thereof, starch and derivatives thereof, maleic anhydride and salts thereof, polyacrylamide, methacrylate resins, poly-2-hydroxyethyl methacrylate and the like can be used.

Further, in the following embodiments, a bipolar electrode system having only a measurement electrode and a counter electrode will be described, but a three-electrode system including a reference electrode enables more accurate measurement. Further, as the electron acceptor, p-benzoquinone, phenazine methosulfate, ferrocene and the like can be used in addition to potassium ferricyanide shown in the following examples. On the other hand, as a buffering agent, in addition to phosphate, a buffering agent using citrate, etc., which provides the highest enzyme activity of the enzyme, p
A buffer for obtaining H can be freely used.

Further, the present invention provides a fructose sensor,
In addition to lactic acid sensor and glucose sensor, it can be widely used in reaction systems involving enzymes such as alcohol sensor, scroll sensor, cholesterol sensor.
As the enzyme, in addition to fructose dehydrogenase, lactate oxidase, glucose oxidase, alcohol oxidase, lactate dehydrogenase, cholesterol oxidase, cholesterol dehydrogenase, xanthine oxidase, amino acid oxidase and the like can be used depending on the specific substance to be quantified. The biosensor of the present invention, for specific components in various biological samples, can perform rapid and highly accurate quantification easily and can withstand long-term storage, so in food quality control and clinical testing. Its utility value is extremely high.

[0016]

EXAMPLES The present invention will now be described in more detail with reference to examples. [Example 1] A fructose sensor will be described as an example of a biosensor. FIG. 1 is a vertical cross-sectional view of a fructose sensor manufactured as an example of the biosensor of the present invention with a cover and a spacer removed, and FIG. 2 is an exploded perspective view of the fructose sensor without a reaction layer. Reference numeral 1 denotes an insulating substrate made of polyethylene terephthalate. The leads 2 and 3 are formed on the substrate 1 by printing a silver paste by screen printing. On the substrate 1, a conductive carbon paste containing a resin binder is further printed to form a detection electrode system including the measuring electrode 4 and the counter electrode 5, and an insulating paste is printed to form the insulating layer 6. . The insulating layer 6 keeps the exposed areas of the measurement electrode 4 and the counter electrode 5 constant and partially covers the leads.

After the electrode portion is prepared in this manner, the enzyme fructose dehydrogenase (EC
1.1.99.11; hereinafter abbreviated as FDH) and potassium ferricyanide which is an electron acceptor are dissolved in a 0.5 wt% aqueous solution of sodium salt of hydrophilic polymer carboxymethyl cellulose (hereinafter abbreviated as CMC) to prepare a mixed aqueous solution. FDH-potassium ferricyanide-CMC layer 7 was added dropwise and dried in a warm air drier at 40 ° C. for 10 minutes.
Has been formed. On this FDH-potassium ferricyanide-CMC layer 7, 0.5 wt% of lecithin was used as a dispersant.
A buffer solution-lecithin layer 8 is formed by dropping a dispersion of microcrystals of potassium phosphate-dipotassium phosphate as a buffer agent in a toluene solution and drying it. The solvent toluene used for forming the layer 8 is the lower layer CMC.
There is no direct contact between the buffer of layer 8 and the enzyme of layer 7 as it does not dissolve. Further, by forming a layer of amphipathic lipid such as lecithin on the surface of the reaction layer, it is possible to facilitate the intrusion of the sample solution into the reaction layer from the outside.
The reaction layer of the fructose sensor is formed as described above. As described above, the manufacturing process can be simplified by dropping the mixed solution of the hydrophilic polymer, the enzyme and the electron acceptor all at once and drying. Further, as a temperature range during drying, a range of 20 ° C. to 80 ° C., which is a temperature at which oxygen deactivation is not observed and which can be dried in a short time, is suitable.

After forming the reaction layer as described above, the cover 14 and the spacer 13 are adhered to each other in a positional relationship shown by an alternate long and short dash line in FIG. 2 to complete the fructose sensor. In this biosensor, the sample solution is simply introduced into the reaction layer portion by a simple operation of bringing the sample solution into contact with the sample supply hole 15 at the tip of the sensor. The supply amount of the sample solution depends on the space volume generated by the cover 14 and the spacer 13, and therefore it is not necessary to quantify it in advance. Furthermore, evaporation of the sample liquid during measurement can be minimized, and highly accurate measurement can be performed. Note that FIG.
Inside, 16 is an air hole provided in the cover 14. here,
When a transparent polymer material is used for the cover 14 and the spacer 13, the state of the reaction layer and the introduction state of the sample solution can be easily observed from the outside.

In the fructose sensor thus manufactured,
3 μl of fructose standard solution was supplied as a sample solution from the sample supply hole, and after 2 minutes, a pulse voltage of +0.5 V was applied in the anode direction to the measurement electrode based on the counter electrode, and the current value after 5 seconds was measured. When the sample solution reaches the reaction layer, the buffer-
The lecithin layer 8 is dissolved to reach the desired pH, and the F
The DH-potassium ferricyanide-CMC layer 7 was dissolved. Fructose in the sample solution is oxidized by FDH, and the electrons transferred therein reduce potassium ferricyanide to potassium ferrocyanide. Next, by applying the above-mentioned pulse voltage, an oxidation current of the generated potassium ferrocyanide is obtained, and this current value corresponds to the concentration of fructose which is the substrate. The enzyme used for this fructose sensor shows an almost maximum enzyme activity at pH = 4.5 (37 ° C.), but since the fructose standard solution is almost neutral, the sample solution is in the buffer-lecithin layer 8. When the pH of the sample solution reaches to 4.5, the enzyme activity can be maximized. In addition, the storage characteristics of the sensor can be improved by separating the buffer agent and the enzyme.

The response to the fructose standard solution obtained by the fructose sensor thus produced showed linearity with respect to the fructose concentration, and the above characteristics could be maintained even after long-term storage. In the above example, instead of the buffer-lecithin layer 8, a hydrophilic polymer soluble in an organic solvent that does not dissolve CMC in the lower layer,
For example, a buffer-hydrophilic polymer layer may be formed by developing a solution in which a buffer is dispersed in an ethanol solution of polyvinylpyrrolidone and drying it.

[Example 2] An electrode system consisting of a measuring electrode 4 and a counter electrode 5 is formed by screen printing on an insulating substrate made of polyethylene terephthalate in the same manner as in Example 1. A 0.5 wt% aqueous solution of CMC is dropped onto this electrode system and dried to form a CMC layer. Next, this CM
An aqueous solution of the enzyme FDH and the electron acceptor potassium ferricyanide is developed so as to cover the C layer and dried to form the FDH-potassium ferricyanide-CMC layer 7. In this case, CMC, FDH, and potassium ferricyanide are in a partially mixed state to form a thin film having a thickness of several microns. That is, when the aqueous solution is dropped onto the CMC layer, the first formed CMC layer is once dissolved, and the layer 7 is formed in a form mixed with an enzyme or the like in the subsequent drying process. However, since it is not accompanied by stirring or the like, it is not in a completely mixed state, and the electrode system surface is in a state of being covered only with CMC. Thus, since the enzyme and electron acceptor do not come into contact with the surface of the electrode system, the adsorption of proteins on the surface of the electrode system and the change in the characteristics of the electrode system due to the chemical action of substances having an oxidizing ability such as potassium ferricyanide occur. It may be difficult, and as a result, a sensor having a highly accurate sensor response can be obtained.

Further, the hydrophilic polymer polyvinylpyrrolidone (hereinafter abbreviated as PVP) 0.5 is formed so as to completely cover the FDH-potassium ferricyanide-CMC layer 7.
Phosphate-potassium as a buffer in a wt% ethanol solution,
Drop the dispersed fine crystals of dipotassium phosphate,
The buffer-PVP layer 10 is formed by drying. The ethanol used for forming the layer 10 is CMC in the lower layer.
There is no direct contact between the enzyme of layer 7 and the buffer of layer 10 since it does not dissolve. A lecithin layer 9 is formed by dropping a 0.5 wt% toluene solution of lecithin onto the buffer-PVP layer 10 and drying. The reaction layer of the fructose sensor is formed as described above. The structure of the fructose sensor thus obtained is shown in FIG.

Example 1 was formed on the substrate on which the above reaction layer was formed.
Similarly to the above, the fructose sensor is completed by combining the spacer 11 and the cover 12. When a liquid such as fruit juice containing a solid component such as pulp is used as a sample liquid by providing the buffer-PVP layer 10, the buffer-PVP layer causes the solid component to be adsorbed on the electrode surface. It is possible to prevent it from affecting the response of the above, and at the same time, it is possible to set the pH of the sample solution to a pH at which the enzyme activity becomes maximum. The fructose sensor manufactured in this way shows a quick and accurate response,
Further, as in Example 1, the retention property is excellent because the buffer and the enzyme are separated.

[Example 3] As an example of a biosensor,
The glucose sensor will be described. On the insulating substrate made of polyethylene terephthalate, the same electrode system as the electrode portion shown in FIG. 1 is formed by screen printing in the same manner as in Example 1. After the electrode portion was prepared in this way, a solution of monopotassium phosphate and dipotassium phosphate as a buffer in a 0.5 wt% aqueous solution of CMC was dropped and dried to form a buffer-CMC layer. Form. Next, a lipid-modified glucose oxidase (hereinafter abbreviated as lipid-modified GOD) is used as an enzyme so as to cover the buffer-CMC layer.
A lipid-modified GOD is prepared by developing a solution in which ethanol and potassium ferricyanide as an electron acceptor are dispersed in ethanol, and drying the solution.
Forming a potassium ferricyanide layer; After forming the reaction layer as described above, it is integrated with the spacer 13 and the cover 14 in the same manner as in Example 1 to form a glucose sensor. The above lipid-modified GOD is an amphipathic lipid DC-
Glucose oxidase (manufactured by Toyobo Co., Ltd.) was added to a solution prepared by dispersing 3-12 L in water, allowed to stand at 4 ° C. for 1.5 days, and then freeze-dried. This lipid modified GOD
Can be easily dispersed in an organic solvent without agglomeration,
Moreover, it is also soluble in water.

[Example 4] As an example of a biosensor,
The glucose sensor will be described. On the insulating substrate made of polyethylene terephthalate, the same electrode system as the electrode portion shown in FIG. 1 is formed by screen printing in the same manner as in Example 1. After preparing the electrode portion in this manner, a solution prepared by dissolving monopotassium phosphate, dipotassium phosphate as a buffer, and potassium ferricyanide as an electron acceptor in a 0.5 wt% CMC aqueous solution was dropped and dried, A buffer-potassium ferricyanide-CMC layer is formed. Then this buffer-potassium ferricyanide-C
Lipid modified GOD as an enzyme so as to cover the MC layer
The benzene solution is spread and dried to form a lipid-modified GOD layer. After forming the reaction layer as described above, it is integrated with the spacer 13 and the cover 14 in the same manner as in Example 1 to form a glucose sensor.

When the electrode portion in the above embodiment is formed of platinum by sputtering or the like, the potassium ferricyanide used as the electron acceptor is not necessary, and in this case, oxygen in the substrate solution becomes lactic acid ( Alternatively, hydrogen peroxide produced by being reduced in proportion to the glucose concentration can be detected by the platinum electrode and the lactic acid (or glucose) concentration can be quantified.

[0027]

As described above, according to the present invention, it is possible to automatically set the optimum hydrogen ion concentration according to the characteristics of the enzyme without adjusting the hydrogen ion concentration of the sample solution in advance. As a result, it is possible to obtain a biosensor capable of highly accurate measurement in a shorter time. In addition, by separating the enzyme and the buffer, it is possible to prevent partial mixing of the buffer and enzyme due to moisture absorption of the biosensor and the resulting decrease in enzyme activity due to chemical interaction, resulting in long-term storage. After that, the initial activity can be maintained.

[Brief description of drawings]

FIG. 1 is a vertical cross-sectional view showing the configuration of a main part of a biosensor according to a first embodiment of the present invention.

FIG. 2 is an exploded perspective view of the biosensor without a reaction layer.

FIG. 3 is a vertical cross-sectional view showing the configuration of a main part of a biosensor according to a second embodiment of the present invention.

[Explanation of symbols]

 1 Insulating Substrate 2, 3 Lead 4 Measurement Electrode 5 Counter Electrode 6 Insulation Layer 7 FDH-Potassium Ferricyanide-CMC Layer 8 Buffer-Lecithin Layer 9 Lecithin Layer 10 Buffer-PVP Layer 13 Spacer 14 Cover 15 Sample Supply Hole 16 Air Hole

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Satoko Fujisawa 1006 Kadoma, Kadoma City, Osaka Prefecture Matsushita Electric Industrial Co., Ltd. (72) Inventor Shiro Nankai 1006 Kadoma, Kadoma City, Osaka Matsushita Electric Industrial Co., Ltd.

Claims (10)

[Claims] 1. An insulating substrate, an electrode system having a measuring electrode and a counter electrode provided on the substrate, and a reaction layer formed on the electrode system in contact with the electrode system, the reaction layer comprising: A biosensor containing at least a hydrophilic polymer, an enzyme, and a buffer, and the enzyme is separated from the buffer. 2. The reaction layer comprises at least two layers,
The biosensor according to claim 1, wherein one layer is in contact with the electrode system and comprises an enzyme and a hydrophilic polymer, and the other layer contains a buffer. 3. The biosensor according to claim 2, wherein the other layer comprises an amphipathic lipid. 4. The reaction layer comprises at least three layers,
The bio according to claim 1, wherein one layer comprises a buffer and a hydrophilic polymer, the other layer comprises an enzyme and a hydrophilic polymer, and the other layer is located outside the bilayer and comprises a lipid. Sensor. 5. The biosensor according to claim 2, wherein one layer contains an electron acceptor. 6. The layer composed of the buffer and the hydrophilic polymer and the layer composed of the enzyme and the hydrophilic polymer are in contact with each other, and the hydrophilic polymers of both layers are different substances and the upper side The polymer of the layer formed in the above is soluble in an organic solvent which does not dissolve the polymer formed thereunder.
The biosensor described. 7. An electrode system having a measuring electrode and a counter electrode provided on an insulative substrate, and a reaction layer formed on the electrode system in contact with the electrode system, wherein the reaction layer is enzyme and hydrophilic. A method for producing a biosensor comprising at least two layers of a layer comprising a hydrophilic polymer and a layer containing a buffer, comprising contacting the electrode system on the substrate with water as a medium from an enzyme and a hydrophilic polymer. And a step of forming a second layer containing a buffer on the first layer using an organic solvent that does not dissolve the hydrophilic polymer as a medium. 8. An electrode system having a measuring electrode and a counter electrode provided on an insulating substrate, and a reaction layer formed on the electrode system in contact with the electrode system, wherein the reaction layer is an enzyme and a hydrophilic layer. A method for manufacturing a biosensor comprising at least two layers of a layer made of a conductive polymer and a layer containing a buffer, comprising a buffer and a hydrophilic polymer in water in contact with the electrode system on the substrate. And a step of forming a second layer composed of a hydrophilic polymer carrying an enzyme on the first layer using an organic solvent which does not dissolve the hydrophilic polymer as a medium. Biosensor manufacturing method. 9. The method for producing a biosensor according to claim 7, further comprising the step of forming a third layer on the second layer by further developing an organic solvent solution of lipid and drying the solution. 10. An electrode system having a measuring electrode and a counter electrode provided on an insulating substrate, and a reaction layer formed on the electrode system in contact with the electrode system, wherein the reaction layer is hydrophilic with an enzyme. A method for manufacturing a biosensor comprising a polymer and a buffer, comprising: a first step of developing an aqueous solution containing a hydrophilic polymer and a buffer on the substrate and drying the organic solvent, and then an organic solvent containing at least an enzyme. A method for manufacturing a biosensor, which has a second step of developing and drying.