JP3070818B2 - Biosensor and manufacturing method thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biosensor for easily and quickly performing high-precision quantification of a specific component in a biological sample using an enzyme reaction, and a method for producing the same.

[0002]

2. Description of the Related Art Conventionally, various biosensors have been proposed as a system for simply 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 system combining glucose oxidase and an enzyme electrode or a hydrogen peroxide electrode is generally known. Glucose oxidase selectively oxidizes β-D-glucose as a substrate to D-glucono δ-lactone using oxygen as an electron acceptor. With this reaction, oxygen is reduced to hydrogen peroxide. At this time, the amount of oxygen consumed is measured by an oxygen electrode, or the amount of generated hydrogen peroxide is measured by a hydrogen peroxide electrode using a platinum electrode or the like, so that glucose is quantified.

[0003] However, in the above-mentioned method, the measurement is greatly affected by the concentration of dissolved oxygen depending on the object to be measured, and the measurement becomes impossible under the condition without oxygen. Therefore, a glucose sensor has been developed which does not use oxygen as an electron acceptor but uses a metal complex or an organic compound such as potassium ferricyanide, a ferrocene derivative, or a quinone derivative as an electron acceptor. In this type of sensor, a reduced form of an electron acceptor generated as a result of an enzymatic reaction is oxidized at an electrode, and the glucose concentration is determined from the oxidation current. This technique is widely applied to the quantification of other substrates as well as glucose. The following glucose sensor is known as an example of this type of biosensor (Japanese Patent Application Laid-Open No.
-114747).

This sensor comprises an insulating substrate provided with an electrode system consisting of a measuring electrode and a counter electrode, a filter layer made of a polycarbonate porous membrane, an electron acceptor carrying layer, and an enzyme carrying layer spaced apart from the electrode system. , A buffer-carrying layer and a spreading layer made of a cellulose woven fabric. The above-mentioned carrier layer is obtained by impregnating an aqueous solution of an electron acceptor, an enzyme, and a buffer into a porous cellulose material and drying it. The operation of this glucose sensor is as follows. The sample solution dropped onto the developing 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
Reacts specifically with glucose oxidase in the enzyme-carrying layer. At the same time, the electron acceptor in the electron acceptor-supporting layer, for example, potassium ferricyanide is reduced by the electrons generated in the above reaction to produce potassium ferrocyanide. The amount of the 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 through 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 solution using an electrode system partially covered with an insulating layer.

In such a conventional configuration, since the wetting of the substrate surface including the electrode system is not always uniform, air bubbles remain between the porous body of the filtration layer and the substrate, which may affect the response current. there were. In addition, if a sample solution contains a substance that is easily adsorbed on the electrode or a substance that is active on the electrode, the response of the sensor may be affected. As a method for solving the above problem, the following biosensor has been proposed (Japanese Patent Application Laid-Open No. 2-062952). That is, an electrode system including a measurement electrode, a counter electrode, and a reference electrode is formed on an insulating substrate by a method such as screen printing, and a hydrophilic polymer, an oxidoreductase, and an electron An enzyme reaction layer containing a receptor and, if necessary, a buffering agent was formed. When a sample solution containing a substrate is dropped onto the enzyme reaction layer, the enzyme reaction layer dissolves and is adjusted to a pH at which the highest enzyme activity is obtained by the buffering action of the buffer, and the enzyme and the substrate react with each other. Is reduced. After the enzymatic reaction, the reduced electron acceptor is oxidized electrochemically,
The substrate concentration in the sample solution is determined from the oxidation current value obtained at this time.

[0006]

In such a conventional configuration, when a biosensor absorbs moisture, a buffer and an enzyme are partially mixed, and the enzyme activity is reduced by chemical interaction, thereby preserving the biosensor. There was a disadvantage that the properties deteriorated. The present invention solves the above problems,
It is an object of the present invention to provide a biosensor capable of quickly and easily quantifying a specific component contained in a biological sample with a simple operation. Another object of the present invention is to provide a biosensor that can withstand long-term storage and can be used for quality control of foods and clinical tests. It is another object of the present invention 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 at least a hydrophilic polymer and an enzyme.
Formed on the electrode system in contact with the electrode system
A water-soluble reaction layer, wherein the reaction layer is
It consists of at least two layers:
Wherein the enzyme and the buffer are at least the hydrophilic polymer.
It has a configuration which is separated by. In a preferred embodiment of the present invention, the reaction layer includes the buffer and the hydrophilicity-enhancing agent.
A layer composed of molecules, and an enzyme and a hydrophilic polymer in contact with the layer.
And at least two layers, both of which are hydrophilic
Conductive polymers are different substances and are formed on the top
The hydrophilic polymer of the layer to be formed
Soluble in an organic solvent that does not dissolve the polymer,
Element and buffer are separated by the amphiphilic polymer.
You. In another preferred aspect of the present invention, the reaction layer is made of the enzyme and a hydrophilic polymer, and is in contact with the electrode system.
Layer, comprising a buffer and an amphipathic lipid, formed on said layer.
And at least two layers, wherein the amphipathic
Lipids are soluble in organic solvents that do not dissolve the hydrophilic polymer
Wherein the enzyme and the buffer are combined with the amphipathic lipid.
Separated by hydrophilic polymer. Preferred of the present invention
In another embodiment, the reaction layer is hydrophilic with the buffer.
A layer made of a polymer and in contact with the electrode system;
From lipid-modifying enzymes soluble in organic solvents that do not dissolve molecules
From at least two layers with the layer formed on the above layer
And the enzyme and the buffer are separated by the hydrophilic polymer.
Separated .

[0008] In the biosensor of the present invention, the reaction layer has an electron receiving surface.
It is preferred to include a container. Further, in a preferred embodiment of the present invention, it has a layer that is located outside the layer containing a buffer and the layer containing an enzyme and is composed of a lipid, particularly an amphipathic lipid. Also, in other embodiments, contact with the electrode system, that have a layer consisting essentially of a hydrophilic polymer.

The method for producing a biosensor according to the present invention further comprises an electrode system having a measurement 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, in which the reaction layer comprises at least two layers of a layer comprising an enzyme and a hydrophilic polymer, and a layer comprising a buffer, wherein water is applied to the electrode system on the substrate in contact with the electrode system. Forming a first layer comprising an enzyme and a hydrophilic polymer as a medium, and forming a second layer containing a buffer on the first layer using an organic solvent which 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 an enzyme and a hydrophilic polymer are dissolved on a substrate and drying the substrate, and forming the second layer. Comprises developing a liquid in which a buffer is dispersed in a lipid organic solvent solution 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 substrate, and the step of forming the second layer is carried out. Developing a liquid in which a buffer is dispersed in an organic solvent solution of a hydrophilic polymer on the first layer, and drying.

Further, the present invention comprises an electrode system having a measurement 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 formed. A method for producing a biosensor comprising at least two layers of a layer comprising an enzyme and a hydrophilic polymer, and a layer comprising a buffer, wherein the buffer is in contact with the electrode system on the substrate using water as a medium. A step of forming a first layer made of a hydrophilic polymer, and a step of forming a second layer made of an enzyme and a hydrophilic polymer on the first layer using an organic solvent that does not dissolve the hydrophilic polymer as a medium A method for producing 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. Further, it is preferable that the method further includes a step of forming a third layer by further developing and drying an organic solvent solution of lipid on the second layer.

[0011]

According to the biosensor of the present invention, as described above, the reaction layer formed on the electrode system of the substrate in contact with the electrode system contains at least a hydrophilic polymer, an enzyme and a buffer ,
The buffer is based on hydrophilic polymers or even amphiphilic lipids.
Ri has the configuration that is separated. And, since the reaction layer contains a buffer, even when the pH of the sample solution does not match the pH at which the highest enzyme activity is obtained, the sample solution supplied to the sensor reaches the buffer in the reaction layer, The pH of the sample solution is automatically adjusted to a value at which the highest enzyme activity is obtained. Therefore, it is not necessary to adjust the pH of the sample solution with a buffer solution or the like in advance, and the measurement can be performed by a simple operation. Furthermore, by separating the enzyme layer and the buffer, partial mixing of the buffer and the enzyme due to moisture absorption of the biosensor,
In addition, it is possible to prevent a decrease in enzyme activity due to chemical interaction, and to keep the enzyme under stable conditions during storage of the biosensor.

In the biosensor of the present invention, even when the layer containing a buffer and the layer containing an enzyme are in contact with each other, the hydrophilic polymers in both layers are different from each other, and the layers formed on the upper side are different. The polymer can be dissolved in an organic solvent that does not dissolve the polymer formed thereunder, thereby eliminating direct contact between the buffer and the enzyme during the production of the biosensor. . The biosensor having such a configuration can be obtained by the following manufacturing method. One is a step of forming a first layer made of an enzyme and a hydrophilic polymer using water as a medium by contacting the electrode system on an insulating substrate, using an organic solvent that does not dissolve the hydrophilic polymer as a medium. A manufacturing method including a step of forming a second layer containing a buffer agent on the first layer. Another one is a step of forming a first layer made of a buffer and a hydrophilic polymer by using water as a medium, in contact with the electrode system on the insulating substrate;
A production method comprising a step of forming a second layer comprising an enzyme and a hydrophilic polymer on a first layer using an organic solvent which does not dissolve the hydrophilic polymer as a medium.

The biosensor of the present invention preferably has, on the surface of the reaction layer, a layer containing a lipid that facilitates the 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 amphiphilic lipids are preferable. Further, as the hydrophilic polymer for forming the reaction layer, in addition to carboxymethylcellulose and polyvinylpyrrolidone used in the following examples, polyvinyl alcohol, water-soluble cellulose derivatives, particularly ethylcellulose, hydroxypropylcellulose; gelatin,
Polyacrylic acid and its salts, starch and its derivatives, maleic anhydride and its salts, polyacrylamide, methacrylate resins, poly-2-hydroxyethyl methacrylate, and the like can be used.

In the following embodiments, a bipolar electrode system having only a measurement electrode and a counter electrode will be described. However, 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, a buffer such as a buffer using citrate in addition to phosphate can be used as a buffer to obtain the highest enzyme activity of the enzyme.
A buffer for obtaining H can be used freely.

Further, the present invention provides a fructose sensor,
In addition to lactic acid sensors and glucose sensors, alcohol sensors, scroll sensors, and cholesterol sensors can be widely used in enzyme-related reaction systems. In this case,
As the enzyme, besides fructose dehydrogenase, lactate oxidase and glucose oxidase, alcohol oxidase, lactate dehydrogenase, cholesterol oxidase, cholesterol dehydrogenase, xanthine oxidase, amino acid oxidase and the like can be used according to the specific substance to be quantified. The biosensor of the present invention can easily and quickly perform high-precision quantification of specific components in various biological samples, and can withstand long-term storage. Its utility value is extremely large.

[0016]

The present invention will be described below 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 longitudinal sectional view of a fructose sensor manufactured as one embodiment of the biosensor of the present invention, excluding a cover and a spacer, and FIG. 2 is an exploded perspective view of the fructose sensor excluding a reaction layer. Reference numeral 1 denotes an insulating substrate made of polyethylene terephthalate. On the substrate 1, leads 2 and 3 are formed by printing 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 measurement electrode 4 and the counter electrode 5, and an insulating layer 6 is formed by printing an insulating paste. . The insulating layer 6 keeps the areas of the exposed portions of the measurement electrode 4 and the counter electrode 5 constant and partially covers the leads.

After the electrode portion has been prepared in this way, the enzyme fructose dehydrogenase (EC
1.1.99.11; hereinafter abbreviated as FDH) and a mixed aqueous solution obtained by dissolving potassium ferricyanide as an electron acceptor in a 0.5 wt% aqueous solution of a sodium salt of a hydrophilic polymer carboxymethylcellulose (hereinafter abbreviated as CMC). The FDH-potassium ferricyanide-CMC layer 7 is dropped and dried for 10 minutes in a warm air dryer at 40 ° C.
Is formed. On this FDH-potassium ferricyanide-CMC layer 7, 0.5 wt% of lecithin as a dispersant
A buffer-lecithin layer 8 is formed by dropping a dispersion of microcrystals of phosphate-potassium phosphate and dipotassium phosphate, which are buffer agents, in a toluene solution and drying. The solvent toluene used for forming the layer 8 was CMC of the lower layer.
There is no direct contact between the buffer of layer 8 and the enzyme of layer 7 Further, by forming an amphipathic lipid layer such as lecithin on the surface of the reaction layer, it is possible to easily invade the sample solution from the outside into the reaction layer.
As described above, the reaction layer of the fructose sensor is formed. As described above, the manufacturing process can be simplified by dripping the mixed solution of the hydrophilic polymer, the enzyme, and the electron acceptor at once and drying. Further, as a temperature range for drying, a range of 20 ° C. to 80 ° C., which is a temperature at which no deactivation of oxygen is observed and which can be dried in a short time, is suitable.

After forming the reaction layer as described above, the fructose sensor is completed by bonding the cover 14 and the spacer 13 in a positional relationship shown by a dashed line in FIG. In this biosensor, the sample liquid is easily introduced into the reaction layer portion by a simple operation of merely bringing the sample liquid into contact with the sample supply hole 15 at the sensor tip. Since the supply amount of the sample liquid depends on the space volume generated by the cover 14 and the spacer 13, it is not necessary to determine the supply amount in advance. Furthermore, evaporation of the sample liquid during measurement can be minimized, and highly accurate measurement can be performed. Note that FIG.
Reference numeral 16 denotes 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 state of introduction of the sample solution can be easily observed from the outside.

The fructose sensor thus manufactured has
3 μl of a standard fructose 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 to the measurement electrode in the anode direction with reference to the counter electrode, and the current value was measured after 5 seconds. When the sample solution reaches the reaction layer, a buffer-
The lecithin layer 8 is dissolved to reach a desired pH, and
The DH-potassium ferricyanide-CMC layer 7 was dissolved. Fructose in the sample solution is oxidized by FDH, and the transferred electrons reduce potassium ferricyanide to potassium ferrocyanide. Next, by the application of the pulse voltage, an oxidation current of the generated potassium ferrocyanide is obtained, and this current value corresponds to the concentration of fructose as a substrate. The enzyme used in this fructose sensor shows almost the maximum enzyme activity at pH = 4.5 (37 ° C.), but the fructose standard solution is almost neutral. When the pH of the sample solution reaches 4.5, the enzyme activity can be maximized. Further, by separating the buffer from the enzyme, the storage characteristics of the sensor can be improved.

The response to the fructose standard solution obtained by the fructose sensor produced in this way showed linearity with respect to the fructose concentration, and the above characteristics could be maintained 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 the lower layer CMC,
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 the solution.

Example 2 An electrode system consisting of a measuring electrode 4 and a counter electrode 5 is formed on an insulating substrate made of polyethylene terephthalate by screen printing in the same manner as in Example 1. A 0.5 wt% aqueous solution of CMC is dropped on 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 partially mixed to form a thin film having a thickness of several microns. That is, when the aqueous solution is dropped on the CMC layer, the CMC layer formed first is dissolved once, and the layer 7 is formed in a state of being mixed with an enzyme or the like in a subsequent drying process. However, since no stirring or the like is involved, a completely mixed state is not obtained, and the surface of the electrode system is in a state covered with only CMC. In this way, since the enzyme and electron acceptor do not come into contact with the electrode system surface, adsorption of proteins to the electrode system surface and changes in the characteristics of the electrode system due to the chemical action of oxidizing substances such as potassium ferricyanide occur. As a result, a sensor having a highly accurate sensor response can be obtained.

Further, the FDH-potassium ferricyanide-CMC layer 7 is completely covered with 0.5 μm of a hydrophilic polymer polyvinylpyrrolidone (hereinafter abbreviated as PVP).
wt-% ethanol solution in buffer-potassium phosphate,
Dropping a dispersion of dipotassium phosphate microcrystals,
By drying, the buffer-PVP layer 10 is formed. The ethanol used to form layer 10 was the CMC of the lower layer.
There is no direct contact between the enzyme in layer 7 and the buffer in layer 10. A lecithin 0.5 wt% toluene solution is dropped on the buffer-PVP layer 10 and dried to form the lecithin layer 9. As described above, the reaction layer of the fructose sensor is formed. FIG. 3 shows the structure of the fructose sensor thus obtained.

On the substrate on which the above-mentioned reaction layer was formed, Example 1
Similarly, the fructose sensor is completed by combining the spacer 11 and the cover 12. When a liquid such as juice containing solid components such as pulp is used as a sample liquid by providing the buffer-PVP layer 10, the buffer-PVP layer allows the solid components to be adsorbed to the electrode surface and the like to be used as a sensor. Of the sample solution can be prevented, and at the same time, the pH of the sample solution can be adjusted to the pH at which the enzyme activity is maximized. The fructose sensor produced in this way shows a quick and accurate response,
As in Example 1, the buffer and the enzyme have excellent retention characteristics due to the separation.

Embodiment 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 preparing the electrode portion in this manner, a solution in which monopotassium phosphate and dipotassium phosphate as a buffer are dissolved in a 0.5 wt% aqueous solution of CMC is dropped and dried, and the buffer-CMC layer is formed. Form. Next, lipid-modified glucose oxidase (hereinafter abbreviated as lipid-modified GOD) is used as an enzyme so as to cover the buffer-CMC layer.
And a solution in which potassium ferricyanide as an electron acceptor is dispersed in ethanol is developed and dried to obtain lipid-modified GOD.
Forming a potassium ferricyanide layer; After forming the reaction layer as described above, the glucose sensor is integrated with the spacer 13 and the cover 14 as in the first embodiment. The above lipid-modified GOD is an amphiphilic lipid DC-
It is obtained by adding glucose oxidase (manufactured by Toyobo) to a solution in which 3-12 L is dispersed in water, leaving it at 4 ° C. for 1.5 days, and then freeze-drying. This lipid-modified GOD
Can be easily dispersed without aggregation in organic solvents,
Moreover, it is soluble in water.

Embodiment 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 way, a solution in which monopotassium phosphate and dipotassium phosphate as buffering agents and potassium ferricyanide as an electron acceptor are dissolved in a 0.5 wt% aqueous solution of CMC is dropped and dried. Form buffer-potassium ferricyanide-CMC layer. Next, the buffer-potassium ferricyanide-C
Lipid-modified GOD as an enzyme so as to cover the MC layer
Is developed and dried to form a lipid-modified GOD layer. After forming the reaction layer as described above, the glucose sensor is integrated with the spacer 13 and the cover 14 as in the first embodiment.

When the electrode portion in the above embodiment is formed of platinum by sputtering or the like, potassium ferricyanide used as an electron acceptor is not required. In this case, oxygen in the substrate solution is reduced by glucose reaction due to enzymatic reaction.
Hydrogen peroxide produced by reduction in proportion to the degree can be detected by the platinum electrode, and the 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 previously adjusting the hydrogen ion concentration of the sample solution. As a result, a biosensor capable of performing highly accurate measurement in a shorter time can be obtained. In addition, by separating the enzyme and the buffer, it is possible to prevent a partial mixing of the buffer and the enzyme due to the moisture absorption of the biosensor, thereby preventing a decrease in the enzyme activity due to a chemical interaction. Thereafter, the initial activity can be maintained.

[Brief description of the drawings]

FIG. 1 is a longitudinal sectional view illustrating a 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 excluding a reaction layer.

FIG. 3 is a longitudinal sectional view illustrating a configuration of a main part of a biosensor according to a second embodiment of the present invention.

[Explanation of symbols]

 Reference Signs List 1 Insulating substrate 2, 3 Lead 4 Measurement electrode 5 Counter electrode 6 Insulating 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

──────────────────────────────────────────────────の Continuing on the front page (72) Inventor Riko Fujisawa 1006 Kadoma Kadoma, Osaka Prefecture Inside Matsushita Electric Industrial Co., Ltd. (72) Inventor Shiro Nankai 1006 Odaka Kadoma Kadoma City, Osaka Matsushita Electric Industrial Co., Ltd. (56) References JP-A-1-114747 (JP, A) JP-A-6-94672 (JP, A) JP-A-5-196595 (JP, A) JP-A-5-119013 (JP, A) JP-A-6-109692 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 27/327

Claims (10)

(57) [Claims] 1. An insulating substrate, an electrode system having a measurement electrode and a counter electrode provided on the substrate, and at least a hydrophilic substrate
A polymer, an enzyme and a buffer, and an electrode system on the electrode system.
A water-soluble reaction layer formed in contact with the reaction layer;
Is a layer comprising the buffer and a hydrophilic polymer, and the layer
At least the enzyme and the layer composed of the hydrophilic polymer in contact with
It consists of two layers, and the hydrophilic polymers in both layers are different materials
And the hydrophilic polymer of the layer formed on the upper side is
Organic solvent that does not dissolve the hydrophilic polymer formed underneath
Soluble in water and the enzyme and buffer
Biosensor separated by aqueous polymer . 2. An insulating substrate, provided on the substrate.
An electrode system having a measuring electrode and a counter electrode, and at least hydrophilic
A polymer, an enzyme and a buffer, and an electrode system on the electrode system.
A water-soluble reaction layer formed in contact with the reaction layer;
Is composed of the enzyme and a hydrophilic polymer and is in contact with the electrode system.
Layer, comprising a buffer and an amphipathic lipid, formed on said layer.
And at least two layers, wherein the amphipathic
Lipids are soluble in organic solvents that do not dissolve the hydrophilic polymer
And the enzyme and the buffer are the amphiphilic fats.
Biosensors separated by quality and hydrophilic polymer . 3. An insulating substrate provided on the substrate.
An electrode system having a measuring electrode and a counter electrode, and at least hydrophilic
A polymer, an enzyme and a buffer, and an electrode system on the electrode system.
A water-soluble reaction layer formed in contact with the reaction layer;
Is composed of the buffer and a hydrophilic polymer and is in contact with the electrode system.
Layer and an organic solvent that does not dissolve the hydrophilic polymer.
A layer comprising a soluble lipid-modifying enzyme and formed on said layer
And at least two layers, and said enzyme and buffer
Is separated by the hydrophilic polymer . 4. The reaction layer is located outside the two layers.
The biosensor according to claim 1, further comprising a layer made of lipid . 5. A method according to claim 1, wherein one layer contains an electron acceptor .
4. The biosensor according to any one of 3 . 6. An electrode system having a measurement 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 formed of an enzyme and a hydrophilic material. A method for producing a biosensor comprising at least two layers of a layer made of a hydrophilic polymer and a layer containing a buffer, comprising: contacting the electrode system on the substrate with water and using an enzyme and a hydrophilic polymer as a medium. A method for producing a biosensor, comprising: forming a first layer, and 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. 7. An electrode system having a measurement 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 formed of an enzyme and a hydrophilic material. A method for producing a biosensor comprising at least two layers of a layer made of a hydrophilic polymer and a layer containing a buffer, wherein the buffer and the hydrophilic polymer are in contact with the electrode system on the substrate using water as a medium. Forming a first layer consisting of an enzyme and a hydrophilic polymer on the first layer using an organic solvent that does not dissolve the hydrophilic polymer as a medium. Manufacturing method. To wherein said second layer on, further expand the organic solvent solution of lipids, a manufacturing method of a biosensor according to claim 6 or 7 comprising the step of forming a third layer by drying. 9. An electrode system having a measurement 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 has an enzyme and a hydrophilic property. A method for producing a biosensor comprising a polymer and a buffer, wherein an aqueous solution containing a hydrophilic polymer and a buffer is developed on the substrate, and a first step of drying is performed. A method for producing a biosensor, comprising a second step of developing and drying. 10. The method according to claim 10, wherein the enzyme is a lipid-modifying enzyme.
Item 10. The method for producing a biosensor according to Item 9.