JPH0816664B2 - Biosensor and manufacturing method thereof - Google Patents

Description

TECHNICAL FIELD The present invention relates to a biosensor capable of quantifying a specific component in various trace amounts of a biological sample quickly and simply without diluting a sample solution, and its production. Concerning the law.

2. Description of the Related Art Conventionally, the following biosensor has been proposed as a method for easily quantifying a specific component in a biological sample such as blood without diluting or stirring the sample solution (Japanese Patent Application No. 63- 38156, JP-A 1-212345).

This biosensor is one in which an electrode system is formed on an insulating substrate by a method such as screen printing, and a hydrophilic polymer layer and an enzyme reaction layer composed of a redox enzyme and an electron acceptor are formed on the electrode system. Is. When the sample solution is dropped on the enzyme reaction layer, the oxidoreductase and the electron acceptor are dissolved in the sample solution, the enzymatic reaction proceeds with the substrate in the sample solution, and the electron acceptor is reduced. After the completion of the enzymatic reaction, this reduced electron acceptor is electrochemically oxidized, and the concentration of the substrate in the sample solution is determined from the oxidation current value obtained at this time.

Problems to be Solved by the Invention In such a conventional configuration, since the oxidoreductase and the electron acceptor are already in contact with each other at the time when the biosensor is created, it is possible to perform the exposure in a high humidity atmosphere or under a light irradiation condition. When it was exposed, the enzyme activity was inactivated and a stable response could not be obtained.
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 has an effect on the sensor response.

Means for Solving the Problems In order to solve the above problems, the present invention provides an electrode system comprising at least a measurement electrode and a counter electrode on an insulating substrate, and a hydrophilic polymer and a redox compound are provided on the electrode system. The reaction layer comprises a first layer made of an enzyme, a second layer made of a hydrophilic polymer, and a third layer containing an electron acceptor.

Furthermore, after providing an electrode system consisting of at least a measurement electrode and a counter electrode on an insulating substrate, a first layer comprising a hydrophilic polymer and a redox enzyme is formed on the electrode system, and then a hydrophilic layer is formed. An organic solvent solution of a hydrophilic polymer is spread on the first layer to form a second layer made of a hydrophilic polymer, and an organic solvent dispersion liquid of an electron acceptor is further spread on the second layer. A biosensor is manufactured by forming a third layer containing an electron acceptor.

Effects According to the present invention, it is possible to construct a disposable type biosensor which can very easily and accurately measure the substrate concentration and is excellent in storage stability.

Examples Hereinafter, the present invention will be described with reference to Examples.

Example 1 A glucose sensor will be described as an example of a biosensor.

FIG. 1 is a cross-sectional view of a glucose sensor manufactured as an example of the biosensor of the present invention, and FIG. 2 is a perspective view showing an electrode portion used for manufacturing the sensor.

Insulating substrate 1 made of polyethylene terephthalate
Then, print the silver paste by screen printing and lead 2.
Forming 3 Next, a conductive carbon paste containing a resin binder is printed and heated and dried to form an electrode system including the measurement electrode 4 and the counter electrode 5. Furthermore, by partially covering the electrode system, the area of the exposed part of the electrode is made constant,
In addition, an insulating paste is printed so as to cover unnecessary portions of the leads, and heat treatment is performed to form the insulating layer 6.

Next, after polishing the exposed portions of 4 and 5, heat treatment was performed in air at 100 ° C. for 4 hours. After forming the electrode portion in this manner, a 0.5 wt% aqueous solution of carboxymethyl cellulose (hereinafter abbreviated as CMC) as a hydrophilic polymer is spread on the electrode and dried to form a CMC layer. Next, as an enzyme, glucose oxidase (hereinafter referred to as GO
CMC-GOD layer 7 was formed by developing a solution obtained by dissolving D) in a phosphate buffer (pH = 5.6) or water and drying it. In this case, CMC and GOD are in the form of a thin film having a thickness of several microns in a partially mixed state. In addition, this CMC
-A 1% ethanol solution of polyvinylpyrrolidone (hereinafter abbreviated as PVP) was developed so as to completely cover the GOD layer and dried to form the PVP layer 8. Onto this PVP layer,
A potassium ferricyanide-lecithin layer 9 was formed by adding dropwise a mixture of fine crystals of potassium ferricyanide as an electron acceptor to a 1% toluene solution of lecithin as a surfactant, developing and drying. Here, lecithin serves to uniformly disperse potassium ferricyanide in a toluene solvent. By using toluene, in which PVP is poorly soluble, as the solvent for forming this potassium ferricyanide-lecithin layer, the potassium ferricyanide-lecithin solution can be spread evenly on the PVP layer, and as a result, a uniform ferric A potassium cyanide-lecithin layer could be obtained.

When a solvent in which the hydrophilic polymer forming the second layer is sparingly soluble is used as the solvent for developing the third layer containing the electron acceptor as described above, the highly uniform third layer is obtained. Can be formed. However, in this case, since the third layer is formed on the entire surface of the second layer, it is difficult to strictly control the amount of electron acceptors on the electrode system to the minimum amount required for the enzymatic reaction. .

10 μl of glucose standard solution was dropped as a sample solution into the glucose sensor configured as described above, and 1 minute later, a pulse voltage of +0.6 V was applied to the measurement electrode in the anode direction with the counter electrode as a reference, and the current after 5 seconds Was measured. When the sample solution is dropped, first, the potassium ferricyanide-lecithin layer is dissolved in the glucose standard solution. When the sample solution reaches the PVP layer, it is instantly dissolved and reaches the CMC-GOD layer to oxidize glucose, and at the same time potassium ferricyanide is reduced to potassium ferrocyanide. Then, by applying the above pulse voltage, an oxidation current based on the concentration of the produced potassium ferrocyanide was obtained, and this current value corresponded to the concentration of glucose as the substrate. Two types of the glucose sensor prepared by the above method and one having no second layer made of a hydrophilic polymer among the glucose sensors were subjected to a storage test at 35 ° C. for 30 days in a dry state.
Use glucose standard solution (90 mg / dl) as the sample solution
Looking at the sensor response after a day, the one without the second layer showed a variation in the response and its CV value was 5.3, but the one with the second layer made of PVP had a CV value of 2.5.
And an extremely good value was obtained.

When the response current was measured by dropping a glucose standard solution, a good linear relationship was obtained up to a high concentration of 900 mg / dl (0.05 mol / l) or more. Furthermore, when 10 μl of a blood sample was dropped on the glucose sensor and the response current was measured after 1 minute, a very reproducible response was obtained.

Example 2 On the insulating substrate made of polyethylene terephthalate, the same electrode part as shown in FIG. 2 was formed by screen printing in the same manner as in Example 1. Further, a CMC-GOD layer and a PVP layer were formed in the same manner as in Example 1.
On this PVP layer, a mixture of 0.5% ethanol solution of lecithin, which is a surfactant, with microcrystals of potassium ferricyanide, which is an electron acceptor, was added dropwise, developed, and dried to form a potassium ferricyanide-lecithin layer. Formed. By using ethanol, in which PVP is easily dissolved, as the solvent for forming the potassium ferricyanide-lecithin layer, the potassium ferricyanide-lecithin layer could be concentrated on one point on the PVP layer. This is because ethanol, which is a dispersion medium for potassium ferricyanide and lecithin, which was added later was promptly absorbed in the PVP layer, so potassium ferricyanide and lecithin did not spread on the PVP layer and a layer was concentratedly formed at the dropping point. Because. In this way, it was possible to form a uniform potassium ferricyanide-lecithin layer by concentrating on the measurement pole of the sensor, and it was possible to create a sensor that could obtain a stable response by developing the minimum necessary amount.

When a solvent in which the hydrophilic polymer forming the second layer is easily soluble is used as the solvent for developing the third layer containing the electron acceptor as described above, it is aimed at the electrode system and the like. It is possible to form a uniform third layer by concentrating on the surface, and the development amount can be minimized. However, in this case, since the second layer needs to absorb the developing solvent of the third layer, if the amount of the solvent that can be absorbed is exceeded, a uniform third layer cannot be obtained. .

10 μl of a glucose standard solution was dropped as a sample solution onto the glucose sensor configured as described above, and a pulse voltage of +0.6 V was applied between the electrodes 1 minute after the dropping, and the current was measured after 5 seconds. A response current value corresponding to the glucose concentration was obtained in the same manner as in Example 1, and also for this, 900 mg / dl (0.
A good linear relationship was obtained up to a concentration as high as 05 mol / l) or higher. A very reproducible response was obtained when blood was used as the sample solution.

Furthermore, in the same manner as in Example 1, two types of samples, one provided with a second layer made of a hydrophilic polymer and the other not provided, were subjected to a storage test at 35 ° C. for 30 days in a dry state. The response after 30 days was observed using whole blood as a sample solution, and it was found that the CV with the second layer of PVP was extremely good.
The value was obtained.

(Example 3) In the sensor manufactured by the method of Example 1 or 2,
I put on the cover. An exploded perspective view of this cover is shown in FIG. The cover 13 and the spacer 10 are made of a polymer material,
The cover, the spacer, and the substrate on which the electrode system was printed were adhered in a positional relationship shown by a broken line in the figure. In the sensor with a cover thus configured, the sample solution can be introduced through the end opening 12 of the spacer or the circular hole 14 of the cover.

When the inlet at the tip of the glucose sensor configured as described above is brought into contact with the sample liquid (human whole blood), the sample liquid is promptly introduced into the interior through the opening, and the space 11 surrounded by the cover and the spacer is The sample liquid was filled without generating bubbles. Furthermore, by pre-treating the surfaces of the members constituting the space, such as the cover and the spacer, with a surfactant to make them hydrophilic, the sample liquid can be introduced more smoothly. As a hydrophilic treatment method, for example, the following method was also effective enough such that the liquid absorption was completed in 0.5 to 1 second. That is, the first to third layers are formed on the insulating substrate made of polyethylene terephthalate as in Example 1 or 2, and the insulating substrate corresponding to the end openings 12 is formed on the third layer. Until lecithin
It was possible to perform hydrophilic treatment from the end opening to the surface of the reaction layer by dropping, developing and drying a 0.5 wt% toluene solution.

This effect was also obtained when the cover was made of a hydrophilic material or a spacer made of a hydrophilic material was used.

By providing the cover in this way, evaporation of the sample liquid in the cover could be minimized. As a result, it has become possible to measure glucose concentration easily, quickly and with high accuracy without being affected by ambient humidity and the like. Furthermore, by reducing the volume inside the cover, the amount of sample liquid and the amount of substances carried in the sensor can be made minute.

Example 4 On the insulating substrate made of polyethylene terephthalate, the same electrode portion as shown in FIG. 2 was formed by screen printing in the same manner as in Example 1. Further, a CMC-GOD layer and a PVP layer were formed in the same manner as in Example 1.
On top of this PVP layer, a mixture of 0.5% ethanol of lecithin, a surfactant, and electron microparticles of potassium ferricyanide, which is an electron acceptor, and PVP was added dropwise, spread, and dried to form potassium ferricyanide-lecithin. -PVP layer formed. Although potassium ferricyanide is dispersed by lecithin in an ethanol solvent, it is difficult to obtain a uniform solvent as it is. Therefore, by adding a hydrophilic polymer such as PVP to this solvent, the homogeneity of the solution was further improved, and a constant concentration potassium ferricyanide solution could be developed by a simple operation. Furthermore, by adding this hydrophilic polymer, it is possible to form a thin film that is stronger and does not easily peel off in a dry state, and it is possible to create a stable sensor.

A cover was attached to the glucose sensor configured as described above in the same manner as in Example 3, 10 μl of a glucose standard solution was dropped as a sample solution, and 1 minute after the dropping, a pulse voltage of +0.6 V was applied between the electrodes, and 5 The current after 2 seconds was measured. A response current value corresponding to the glucose concentration was obtained in the same manner as in Example 1, and also for this, a good linear relationship was obtained up to a high concentration of 900 mg / dl (0.05 mol / l) or more. A very reproducible response was obtained when blood was used as the sample solution.

In the traditional configuration, the GO was already in place when the sensor was created.
Since the D-CMC layer was in contact with the potassium ferricyanide-lecithin layer, it was difficult to improve the storage performance.
However, the hydrophilic polymer layer composed of PVP used in Example 1, 2 or 3 plays a role of completely separating the GOD-CMC layer and the potassium ferricyanide-lecithin layer in a dry state. This has a great effect on the improvement of the storage stability of the sensor.

Furthermore, this hydrophilic polymer layer is extremely effective in ensuring a stable sensor response even when a substance that is easily adsorbed on the electrode or a substance that is active in the electrode exists in the sample liquid. Even when the glucose concentration was measured by the glucose sensor using blood as the sample solution, a stable sensor response was obtained regardless of the viscosity of the sample solution.

In the first, second, third, or fourth embodiment, CMC is used in the first layer.
PVP was used as the hydrophilic polymer in the second layer and the third layer, respectively, but not limited to these, vinyl alcohol-based, cellulose-based, specifically hydroxyethyl cellulose, hydroxypropyl cellulose,
Similar effects can be obtained by using methyl cellulose, ethyl cellulose, ethyl hydroxyethyl cellulose, carboxymethyl ethyl cellulose, and further vinylpyrrolidone type, gelatin type, acrylate type, starch type,
Similar effects were obtained by using maleic anhydride type, acrylamide type, methacrylate resin, etc., respectively. A hydrophilic polymer layer having a required film thickness can be formed on the electrode by applying a solution of these water-absorbing or water-soluble hydrophilic polymers in an appropriate concentration and drying.

Although lecithin was used to disperse the electron acceptor in the above-mentioned examples, in addition to this, polyethylene glycol alkylphenyl ether, oleic acid, polyoxyethylene glycerin fatty acid ester, cyclodextrin, etc. that do not affect the enzyme activity. If there is no particular restriction.

Further, in the above embodiment, the bipolar electrode system having only the measurement electrode and the counter electrode was described, but more accurate measurement can be performed by using the three-electrode system including the reference electrode. The electrode constituent material is not limited to the carbon shown in the above embodiment, but a noble metal such as platinum or gold or a metal oxide can also be used. On the other hand, as the electron acceptor, p-benzoquinone, phenazine methosulfate, ferrocene and the like can be used in addition to potassium ferricyanide shown in the above examples.

Furthermore, if an alcohol oxidase, a cholesterol oxidase, or the like is used as the enzyme in addition to the glucose oxidase of the above-mentioned examples, it can be used for an alcohol sensor, a cholesterol sensor, or the like.

Effects of the Invention As described above, the biosensor of the present invention has the effect of further improving the storage stability of the sensor by completely separating the oxidoreductase layer and the electron acceptor layer in the dry state. Furthermore, stable and more accurate measurement can be performed without being affected by substances such as proteins that affect the electrode response when applied to biological components.

[Brief description of drawings]

FIG. 1 is a sectional view of a biosensor which is an embodiment of the present invention, FIG. 2 is a perspective view of an electrode portion, and FIG. 3 is an exploded perspective view of a biosensor with a cover. 1 ... Insulating substrate, 2, 3 ... Lead, 4 ... Measuring electrode,
5 ... Counter electrode, 6 ... Insulating layer, 7 ... CMC-GOD layer, 8 ...
PVP layer, 9 ... Potassium ferricyanide-lecithin layer, 1
0 …… Spacer, 11 …… Space part, 12 …… End opening, 13
…… Cover, 14 …… Circular hole in the cover.

Front page continuation (72) Mayumi Otani Mayumi Otani 1006 Kadoma, Kadoma City, Osaka Prefecture Matsushita Electric Industrial Co., Ltd. (72) Inventor Takashi Iijima 1006 Kadoma, Kadoma City, Osaka Matsushita Electric Industrial Co., Ltd. (56) Reference Documents JP-A 1-23153 (JP, A) JP-A 2-62952 (JP, A)

Claims (7)

[Claims] 1. An electrode system comprising at least a measuring electrode and a counter electrode provided on an insulating substrate, and a reaction layer provided on the electrode system, wherein the reaction layer is a hydrophilic polymer and oxidoreductase. A biosensor comprising: a first layer made of a hydrophilic polymer; a second layer made of a hydrophilic polymer; and a third layer containing an electron acceptor. 2. The biosensor according to claim 1, wherein the third layer containing an electron acceptor comprises at least an electron acceptor and a hydrophilic polymer. 3. The biosensor according to claim 1, wherein a cover is installed on the electrode system so as to include the enzyme reaction layer inside. 4. The hydrophilic polymer is selected from cellulose, vinylpyrrolidone, gelatin, acrylate, vinyl alcohol, starch, maleic anhydride, acrylamide, and methacrylate resins. Item 4. The biosensor according to Item 1, 2 or 3. 5. An electrode system comprising at least a measurement electrode and a counter electrode is provided on an insulating substrate, and then a first layer comprising a hydrophilic polymer and a redox enzyme is formed on the electrode system. Next, a solution of a hydrophilic polymer in an organic solvent is spread on the first layer to form a second layer of the hydrophilic polymer, and an organic solvent dispersion liquid of an electron acceptor is further deposited on the second layer. And a third layer containing an electron acceptor to form a third layer. 6. The solvent for developing the third layer containing an electron acceptor is a solvent in which the hydrophilic polymer forming the second layer is hardly soluble or easily soluble. 5. The method for manufacturing the biosensor according to 5. 7. An electron acceptor is dispersed in an organic solvent in which a hydrophilic polymer is dissolved, and this is spread on a second layer to form a third layer containing the electron acceptor and the hydrophilic polymer. The method for manufacturing a biosensor according to claim 5, wherein: