WO2007108388A1

WO2007108388A1 - Method for manufacturing biosensor and biosensor

Info

Publication number: WO2007108388A1
Application number: PCT/JP2007/055138
Authority: WO
Inventors: Hideki Tanaka; Nobukazu Tanabe; Takafumi Tanaka
Original assignee: Gunze Limited
Priority date: 2006-03-17
Filing date: 2007-03-14
Publication date: 2007-09-27
Also published as: JP4856697B2; JPWO2007108388A1

Abstract

To provide a biosensor exhibiting high storage stability in which variations in measurements due to storage temperature are extremely small, and to provide a manufacturing method of the biosensor. A method for manufacturing a biosensor (10) comprises a reactive portion forming step which comprises a first layer forming step for forming a first layer (28) on a working electrode (14) and a counter electrode (16) by applying and drying thereon a first coating liquid for the reactive portion containing an oxidation/reduction enzyme, and a second layer forming step for forming a second layer (30) on the first layer (28) by applying and drying thereon a second coating liquid for the reactive portion containing a hydrophilic polymer compound and an electron receptor.

Description

Specification

Biosensor manufacturing method and biosensor

Technical field

The present invention relates to a biosensor manufacturing method for measuring blood glucose level and the like, and a biosensor manufactured by the manufacturing method.

[0002] Conventionally, a biosensor and a manufacturing method thereof for measuring the blood sugar level or the like of the specimen are devised (e.g., Patent Documents 1 4 reference.) ₀ conventional biosensor 1 in FIG. 4 shows The This biosensor 1 is provided with a working electrode 3 and a counter electrode 4 in parallel proximity on an electrode insulating substrate 2, and a mask sheet 6 having a reaction cell 5 on the electrode insulating substrate 2, the working electrode 3 and the counter electrode 4. The reaction part 7 was formed by applying a reaction part coating solution containing a redox enzyme on the working electrode 3 and the counter electrode 4 in the reaction part cell 5 and drying them. Note that an electrically insulating spacer sheet 8 and a transparent cover sheet 9 are laminated on the mask sheet 6. According to the biosensor 1, the blood glucose level can be recognized by attaching the sample to the blood glucose level measurement display and taking the sample, and the blood glucose level measurement display displays the blood glucose level.

[0003] Here, it is generally known that the measurement value of a biosensor changes depending on the stored temperature due to its temperature dependence. For example, it is known that when a blood glucose level or the like is measured after being stored at a temperature higher than room temperature, it is detected higher than the actual level. For this reason, special considerations and storage devices were required for biosensor storage.

[0004] Patent Document 1: International Publication No. 2004Z017057 Pamphlet

Patent Document 2: Japanese Patent Publication No. 7-114705

Patent Document 3: Japanese Patent No. 3063442

Patent Document 4: Japanese Patent No. 3483314

Disclosure of the invention

Problems to be solved by the invention

[0005] Therefore, the present inventor has reached the present invention as a result of intensive studies to investigate the cause of such a problem and solve such a problem. [0006] That is, an object of the present invention is to provide a biosensor having a high storage stability in which a change in a measured value due to a stored temperature is minimized, and a method for manufacturing the same.

Means for solving the problem

[0007] The biosensor manufacturing method of the present invention includes a reaction part forming step of forming a reaction part having an oxidoreductase on a working electrode and a counter electrode provided on an electrically insulating substrate. The first reaction layer forming step includes applying a first reaction region coating solution having an oxidoreductase on the working electrode and the counter electrode, and drying to form a first layer; and A second layer forming step of forming a second layer by applying a coating solution for a second reaction part having a hydrophilic polymer compound and an electron acceptor on the first layer and drying the coating solution. Features.

[0008] In addition, the biosensor manufacturing method of the present invention is characterized in that, compared with the biosensor manufacturing method, the acid reductase is glucose oxidase.

[0009] In addition, the method for producing a biosensor of the present invention is characterized in that, compared with the method for producing a biosensor, the hydrophilic polymer compound is polybulurpyrrolidone.

[0010] Further, the biosensor manufacturing method of the present invention is characterized in that, in the biosensor manufacturing method, the electron acceptor force S is ferricyan potassium.

[0011] Further, in the biosensor manufacturing method of the present invention, in the biosensor manufacturing method, the hydrophilic polymer compound is polyvinyl pyrrolidone, the electron acceptor force S ferricyanide potassium, and the ferricyanide. The weight ratio of polyvinyl pyrrolidone to potassium is 0.5 to 50%.

[0012] The biosensor of the present invention is manufactured by the above-described method for manufacturing a biosensor of the present invention.

The invention's effect

[0013] According to the biosensor manufacturing method and biosensor of the present invention, the first reaction part coating solution containing oxidoreductase is applied and dried to form the first layer, and the hydrophilic polymer compound is formed. In the manufactured biosensor, the oxidoreductase is present in the first layer because the second reaction part coating solution containing the product and the electron acceptor is applied and dried to form the second layer. The body is in the second layer. This separates the oxidoreductase from the electron acceptor. Therefore, long-term storage in a dry state can be improved. Further, since the first layer having the oxidation-reduction enzyme is covered with the second layer, it is possible to improve the storage stability of the acid-reductase that does not react unnecessarily. Actually, when the difference between the measured values of the biosensor manufactured and stored at high temperature and that stored at room temperature (25 ° C) was determined, the second layer was prepared using a solvent that did not dissolve the first layer. It was found that the difference was smaller when the film was formed than when the second layer was formed using a solvent that dissolves the first layer, and the effect of the storage temperature was less.

BEST MODE FOR CARRYING OUT THE INVENTION

Next, the biosensor manufacturing method and biosensor according to the present invention will be described in detail with reference to the drawings.

In FIG. 1 and FIG. 2, reference numeral 10 denotes a biosensor of the present invention. The manufacturing method of the biosensor 10 includes an electrode part forming step in which a working electrode 14 and a counter electrode 16 are provided in parallel and proximity on an electrically insulating substrate 12, and a mask step in which a mask sheet 20 having reaction part cells 18 is thermally bonded. A reaction part forming step for forming a reaction part 22 having a redox enzyme on the working electrode 14 and the counter electrode 16 in the reaction part cell 18, and an electrically insulating spacer sheet 24 and on the mask sheet 20. A laminating step of laminating a transparent cover sheet 26.

[0016] The reaction part forming step is a first layer forming step in which a first reaction part coating solution having an oxidoreductase is applied on the working electrode 14 and the counter electrode 16 and dried to form the first layer 28. And a second layer forming step of forming a second layer 30 by applying and drying a second coating liquid for a reaction part having a hydrophilic polymer compound and an electron acceptor on the first layer 28. In the first layer forming step, for example, oxidoreductase is dissolved in water and applied. In the second layer forming step, the hydrophilic polymer compound is dissolved in a solvent that does not dissolve the first layer 28 so that the first layer 28 does not dissolve. As this solvent, for example, ethanol solve ethanol can be mentioned. By such a reaction part forming step, as shown in FIG. 1, a reaction part 22 composed of a first layer 28 and a second layer 30 is formed.

[0017] Examples of the material of the electrical insulating substrate 12 include biodegradable polyester comprising polyethylene terephthalate (PET), polyethylene naphthalate, aliphatic units, and aromatic units. Examples thereof include polyester-based resin sheets such as tellurium, polyamidoimide sheets having superior heat resistance, chemical resistance and strength, plastic sheets such as polyimide film sheets, and inorganic substrates such as ceramics.

[0018] The working electrode 14 and the counter electrode 16 are formed on the electrically insulating substrate 12 by a good conductor such as platinum, gold, palladium, indium-tin oxide, or the like. As a formation method, hot stamping is considered, and vacuum deposition or sputtering is preferable because a fine electrode pattern can be formed quickly with high accuracy. In the case of sputtering, it can be formed at once by masking the outside of the bipolar electrode formation.

[0019] The oxidoreductase is, for example, glucose oxidase when measuring glucose. Glucose oxidase reacts with glucose to produce darconic acid and hydrogen peroxide. When measuring alcohol levels, use alcohol oxidase or alcohol dehydrogenase. When measuring lactic acid, use lactate oxidase or lactate dehydrogenase. When measuring uric acid, use uricase. Application of acid reductase is performed by dissolving in water.

[0020] The hydrophilic polymer compound is a polymer compound soluble in water, alcohol and a mixed solvent thereof. An example of the hydrophilic polymer compound is polyvinyl pyrrolidone (PVP). Polybulylpyrrolidone as a hydrophilic polymer compound is used after being dissolved in a solvent such as ethylcete solve. In addition, examples of the hydrophilic polymer compound include hydroxypropyl cellulose and carboxyl vinyl polymer. By including the hydrophilic polymer compound in the second layer 30, the electron acceptor can be fixed.

[0021] The electron acceptor is generally an inorganic or organic fine powdery compound that promotes oxidation-reduction of an enzyme. For example, ferricyanium alkali metal salt (especially ferricyanium potassium metal salt is preferred), pheucene or its alkyl-substituted product, p-benzoquinone, methylene blue, β-naphthoquinone mono-4-sulfonate potassium, phenazine metho Examples thereof include sulfate, 2,6-dichlorophenol monoindophenol, and the like. Alkali metal ferricyanide, Phucene-based force Since it works stably as an electron transfer medium and is well soluble in water, alcohols, and aqueous solvents such as these mixed solvents, it is effective as an electron acceptor. Use. Polyvinylpyrrolidone is selected as the hydrophilic polymer compound, and as an electron acceptor If you select the Ferishiani spoon potassium, weight iti of Poribyurupi opening pyrrolidone for potassium ferricyanide or 0.5 to 50 ^_0/0 (0.005 to 0.5) force preferably! /ヽ. When 0.5 ^_0/0 below in are too small, it is impossible to fix the electron acceptor. On the other hand, if it exceeds 50%, the dissolution rate in blood is too slow.

[0022] The force described above for the method of manufacturing the biosensor 10 of the present invention. The biosensor 10 manufactured in this way is used as appropriate after being stored in a dry state. According to the method for producing biosensor 10, the first reaction part coating solution having oxidoreductase is applied and dried to form the first layer 28, and the second layer having the hydrophilic polymer compound and the electron acceptor. In the manufactured biosensor 10, the oxidoreductase is present in the first layer 28 and the electron acceptor is in the second layer 30. Exists. Thereby, since the oxidoreductase and the electron acceptor are present separately, the long-term storage stability in a dry state can be improved.

[0023] The measurement of blood glucose level by the biosensor 10 will be described below. The blood glucose level is measured by attaching the biosensor 10 to the sensor cover 34 and attaching it to the blood glucose level measurement display 36 as shown in FIG. 2, and bringing the tip of the biosensor 10 into contact with the blood 38. Do by. The blood 38 that has contacted the tip of the biosensor 10 contacts the second layer 30 of the reaction layer 22 as shown in FIG. When the blood 38 comes into contact with the second layer 30, an enzymatic reaction occurs while penetrating into the second layer 30 and the first layer 28, and the electrochemical change at that time is detected at the working electrode 14 and the counter electrode 16, and the blood glucose level is reduced. Measured.

[0024] Although the embodiments of the present invention have been described with reference to the drawings, the present invention is not limited to the illustrated ones. For example, the application of the present invention is not limited to the measurement of blood glucose level, and may be a wide range of medical, biological, chemical examinations or tests. In addition, blood tests other than blood sugar levels may be used. Further, the shape of the biosensor is not particularly limited as long as the effects of the present invention are produced.

[0025] In addition, the technical scope of the present invention includes embodiments in which various improvements, modifications, and variations are added based on the knowledge of those skilled in the art without departing from the spirit of the present invention. In addition, within a range where the same action or effect is produced, any invention-specific matter may be replaced with another technique. Example

[0026] As described below, the biosensor 10 of the present invention was actually manufactured and the blood glucose level was measured, and the difference in the measured value depending on the storage temperature was evaluated.

[0027] (Example 1)

1 μl of glucose oxidase 74 mg (containing 50% stabilizer) dissolved in 4.9 g of water is dropped on the surface of the electrode system and dried in a conveyor drying oven at 40 ° C for 6 minutes. Layer 28 was formed. Next, 240 mg of PVP (polybulurpyrrolidone) with a molecular weight of about 40,000 was dissolved in 2.16 g of ethylceous sorb (2-ethoxyethanol) and then mixed with 0.8 g of finely divided ferricyanium potassium. Then, 0.81 was dropped on the first layer 28 and dried in a conveyor drying furnace at 40 ° C. for 6 minutes to form the second layer 30. In the above composition, the weight ratio of PVP to potassium ferricyanide is 30%.

[Example 2]

A solution of 50 mg glucose oxidase (including 50% stabilizer) dissolved in 3.3 g of water is dropped on the surface of the electrode system by 1 μ1 and dried in a conveyor drying oven at 40 ° C for 6 minutes. Layer 28 was formed. Next, a solution obtained by dissolving 22 mg of PVP (polybulurpyrrolidone) having a molecular weight of about 40,000 in 1.77 g of ethylceous solve (2-ethoxyethanol) and then mixing 0.6 g of finely divided ferricyanium potassium, 0.81 was dropped on the first layer 28 and dried in a conveyor drying furnace at 40 ° C. for 6 minutes to form the second layer 30. In the above composition, the weight ratio of PVP to potassium ferricyanide is 3.7%.

[0029] (Example 3)

1 μl of glucose oxidase 50 mg (containing 50% stabilizer) dissolved in 3.3 g of water is dropped on the surface of the electrode system and dried in a 40 ° C conveyor drying oven for 6 minutes. Layer 28 was formed. Next, a solution obtained by dissolving 45 mg of PVP (polybulurpyrrolidone) having a molecular weight of about 40,000 in 1.75 g of ethethyl solvate (2-ethoxyethanol), and then mixing 0.6 g of finely divided ferricyanium potassium, 0.81 was dropped on the first layer 28 and dried in a conveyor drying furnace at 40 ° C. for 6 minutes to form the second layer 30. In the above composition, the weight ratio of PVP to potassium ferricyanide is 7.5%.

[0030] (Example 4) A solution of 50 mg glucose oxidase (including 50% stabilizer) dissolved in 3.3 g of water is dropped on the surface of the electrode system by 1 μ1 and dried in a conveyor drying oven at 40 ° C for 6 minutes. Layer 28 was formed. Next, 112 mg of PVP (polybulurpyrrolidone) with a molecular weight of about 40,000 was dissolved in 1.68 g of ethylceous solve (2-ethoxyethanol), and then mixed with 0.6 g of finely divided ferricyanium potassium. Then, 0.81 was dropped on the first layer 28 and dried in a conveyor drying furnace at 40 ° C. for 6 minutes to form the second layer 30. In the above composition, the weight ratio of PVP to potassium ferricyanide is 18.7%.

[0031] (Example 5)

1 μl of glucose oxidase 50 mg (containing 50% stabilizer) dissolved in 3.3 g of water is dropped on the surface of the electrode system and dried in a 40 ° C conveyor drying oven for 6 minutes. Layer 28 was formed. Next, a solution obtained by dissolving 225 mg of PVP (polybulurpyrrolidone) with a molecular weight of about 40,000 in 1.57 g of ethyl acetate sorb (2-ethoxyethanol), and then mixing 0.6 g of finely divided ferricyanium potassium. Then, 0.81 was dropped on the first layer 28 and dried in a conveyor drying furnace at 40 ° C. for 6 minutes to form the second layer 30. In the above composition, the weight ratio of PVP to potassium ferricyanide is 37.5%.

[Example 6]

1 μl of glucose oxidase 77 mg (including 50% stabilizer) dissolved in 5. lg of water is dropped on the surface of the electrode system and dried in a conveyor drying oven at 40 ° C for 6 minutes. Layer 28 was formed. Next, 71 mg of PVP (polybulurpyrrolidone) with a molecular weight of about 360,000 was dissolved in 5.38 g of ethethyl solvate (2-ethoxyethanol) and then mixed with 2.05 g of finely divided ferricyanium carbonate. 0.81 was dropped on the first layer 28 and dried in a conveyor drying furnace at 40 ° C. for 6 minutes to form the second layer 30. In the above composition, the weight ratio of PVP to ferricyanide is 3.5%.

[0033] (Example 7)

A solution of 52 mg of glucose oxidase (containing 50% stabilizer) dissolved in 3.2 g of water is dropped on the surface of the electrode system by 1 μ1 and dried in a conveyor drying oven at 40 ° C for 6 minutes. Layer 28 was formed. Next, 9.30 g of ethyl acetate sorb (2-ethoxyethanol) was added to a polyethylene oxide (PEO) -polypropylene oxide (PPO) block copolymer with a molecular weight of about 10 250 After dissolving 750 mg of coalescence (trade name: New Pole PE-78), 1.01 g of a mixture of 0.81 g of finely divided Felicyan シ potassium was dropped on the first layer 28, and the temperature was 40 ° C. The second layer 30 was formed by drying for 6 minutes in a compare drying oven. In the above composition, the weight ratio of -Eupol to potassium ferricyanide is 92.6%.

[Example 8]

1 μl of glucose oxidase 49 mg (containing 50% stabilizer) dissolved in 3.lg of water is dropped on the surface of the electrode system and dried in a conveyor oven at 40 ° C for 6 minutes. Layer 28 was formed. Next, 600 mg of polyethylene oxide (PEO) -polypropylene oxide (PPO) block copolymer (trade name: New Pole PE-75) with a molecular weight of about 4100 is dissolved in 2.40 g of ethyl acetate solve (2-ethoxyethanol). After that, 0.81 g of a solution in which 0.88 g of finely divided potassium ferricyanide was mixed was dropped on the first layer 28 and dried for 6 minutes in a 40 ° C compare drying oven, and then the second layer 30 Formed. In the above composition, the weight ratio of -Eupol to potassium ferricyanide is 75%.

[0035] (Example 9)

1 μl of glucose oxidase 75 mg (containing 50% stabilizer) dissolved in 4.9 g of water is dropped on the surface of the electrode system and dried in a conveyor drying oven at 40 ° C for 6 minutes. Layer 28 was formed. Next, 360 mg of polyethylene oxide (PEO) -polypropylene oxide (PPO) block copolymer (trade name: New Pole PE-75) with a molecular weight of about 4100 is dissolved in 3.24 g of ethyl acetate solve (2-ethoxyethanol). After that, a mixture of 1.20 g of finely divided potassium ferricyanide was added dropwise to the first layer 28 with 0.8 1 and dried in a 40 ° C compare drying oven for 6 minutes to form the second layer 30 Formed. In the above composition, the weight ratio of -Eupol to potassium ferricyanide is 30%.

[0036] (Comparative example)

1 μl of glucose oxidase 5 lmg (containing 50% stabilizer) dissolved in 3.2 g of water is dropped on the surface of the electrode system and dried in a conveyor drying oven at 40 ° C for 6 minutes. One layer was formed. Next, 240 mg of polyethylene oxide (PEO) -polypropylene oxide (PPO) block copolymer (trade name: New Pole PE-75) with a molecular weight of about 4100 was dissolved in 2.98 g of ethylceous sorb (2-ethoxyethanol). Later, crystalline cellulose (trade name: (Ceras cream, water content 90%) 1. A solution of 12 g and finely divided ferricyanium potassium (0.81 g) was dropped on the first layer, 1.3 1 drops, in a conveyor drying oven at 40 ° C for 6 minutes. A second layer was formed by drying. In the above composition, the weight ratio of eupol to ferricyanium potassium is 29.6%. The weight ratio of crystalline cellulose to potassium ferricyanide is 13.8%. The coating solution used in this comparative example contained 19.6% water. Therefore, a part of ferricyanium potassium is dissolved, and when it is dropped on the first layer, it is dissolved and mixed with a part of the GOD of the first layer.

(Evaluation methods)

The sensor chip was packed in aluminum together with about 25 mg of desiccant (activated alumina), and then stored at 25 ° C and 40 ° C for 1 month for thermal stability evaluation. The measurement was performed using whole blood adjusted to glucose concentrations of 30,50 and 13 OmgZdl, and the measurement result of the sensor stored at 25 ° C was subtracted from the measurement result of the sensor stored at 40 ° C, which was used as an index of thermal stability. The difference in measurement results is shown in Table 1 for the biosensor 10 manufactured according to Examples 1-6, Table 2 for the biosensor 10 manufactured according to Examples 7-9, and for the biosensor manufactured according to the comparative example. Table 3 shows.

[table 1]

[Table 2]

[Table 3]

For Examples 1 to 6, the average absolute value of the difference in the measurement results was 4. Og / dl for a glucose concentration of 30 mgZd 1 4. For a glucose concentration of 50 mgZdl 4. For a 7 mg / dU glucose concentration of 130 mgZdl, it was 4.2 mgZdl. It was. For Examples 7 to 9, the average difference in measurement results was 21.3 mgZdl for a glucose concentration of 30 mgZdl, 18.7 mgZdl for a glucose concentration of 50 mgZdl, and 15.7 mgZdl for a glucose concentration of 130 mgZdl. On the other hand, in the comparative examples, they were 97, 96, and 71 mg Zdl, respectively, and the difference in the measurement results was stronger when the second layer 30 was applied using a solvent that did not dissolve the first layer 28. In particular, the highest stability was obtained when polyvinylpyrrolidone was used.

Industrial applicability

[0038] The biosensor manufacturing method of the present invention can improve the storage stability by reducing the influence of the storage temperature of the biosensor. Therefore, it can be widely used for manufacturing various biosensors.

Brief Description of Drawings

[0039] [FIG. 1] A diagram showing a biosensor of the present invention, in which (a) is a plan view and (b) is A

FIG.

FIG. 2 is a front view showing a usage state of the biosensor of FIG.

FIG. 3 is a diagram showing the usage state of the biosensor of FIG. 1, in which FIG. (A) is a cross-sectional view taken along the line AA showing the moment of blood contact, and FIG. FIG. 5 is a cross-sectional view taken along the line AA showing a state in which is reacted.

FIG. 4 is a view showing a conventional biosensor, in which FIG. 4 (a) is a plan view and FIG. 4 (b) is a cross-sectional view taken along line AA.

Explanation of symbols

[0040] 10: Biosensor : Electrical insulating substrate: Working electrode

: Counter electrode

: Reaction unit cell: Mask sheet: Reaction unit

: Spacer sheet: Cover sheet: First layer

: Second layer

Claims

The scope of the claims

[1] In a biosensor manufacturing method including a reaction part forming step of forming a reaction part having an oxidoreductase on a working electrode and a counter electrode provided on an electrically insulating substrate, the reaction part forming step includes:

A first layer forming step of forming a first layer on the working electrode and the counter electrode by applying and drying a first reaction part coating solution having an oxidoreductase; and

On the first layer, a second layer forming step of forming a second layer by applying and drying a second reaction part coating solution having a hydrophilic polymer compound and an electron acceptor;

A method for producing a biosensor comprising:

2. The method for producing a biosensor according to claim 1, wherein the acid reductase is glucose oxidase.

[3] The method for producing a biosensor according to claim 1 or 2, wherein the hydrophilic polymer compound is polyvinylpyrrolidone.

[4] The biosensor manufacturing method according to any one of [1] to [3], wherein the electron acceptor is potassium fricyanide.

[5] The hydrophilic polymer compound is polyvinylpyrrolidone, the electron acceptor is ferri-cyan potassium, and a weight ratio of the polyvinyl pyrrolidone to ferricyan potassium is 0.5 to 50%. Or the manufacturing method of the biosensor of Claim 2.

[6] A biosensor manufactured by the method for manufacturing a biosensor according to any one of claims 1 to 5.