JPH10104192A - Biosensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To highly precisely measure substrate concentration of sample liquid by adding enzyme promoting isomerization of glucose oxidase, pyranose oxidase and glucose in a reaction layer. SOLUTION: Mixture of glucose oxidase, pyranose oxidase, mutarotase and potassium cyanide is dropped on a CMC (carboxylmethylcellulose) layer formed on an electrode system (measuring electrode 4, counter electrode 5) to form a reaction layer 7 by prescribed treatment. When glucose water solution of sample liquid is supplied from a sample supply hole 10, the sample liquid reaches an air hole 11 by a capillary phenomenon, when the reaction layer 7 is dissolved in the sample liquid, electrons are moved by oxidation reaction caused by enzyme, and potassium ferricyanide is reduced to potassium ferrocyanide. After constant time of sample liquid supply, pulse voltage of 40. 5V is applied to the measuring pole 4 by making the counter electrode 5 reference, and a current value is measured after 5 seconds. The current value corresponds to concentration of glucose which is substrate.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a biosensor for quantifying a specific component in a sample by utilizing an enzyme reaction.

[0002]

2. Description of the Related Art Conventionally, the following biosensors are known as a method for simply quantifying a specific component in a sample without diluting or stirring the sample solution. In this biosensor, an electrode system is formed on an insulating substrate by a method such as screen printing, and a reaction layer comprising a hydrophilic polymer, an oxidoreductase, and an electron acceptor is formed on the electrode system. The change in substrate concentration during the reaction between the oxidoreductase, the electron acceptor, and the sample solution is electrochemically detected by the electrode system,
It measures the substrate concentration.

[0003] Among these biosensors, glucose oxidase is generally used as an enzyme for quantifying glucose. This enzyme reacts only with β-glucose among glucose isomers and does not react with α-glucose. Therefore, in order to further improve the responsiveness, a biosensor using an enzyme having the ability to promote the isomerization reaction between α-glucose and β-glucose together with glucose oxidase, and furthermore, α-glucose and β-glucose Biosensors using pyranose oxidase that react to both, and biosensors using a mixture of pyranose oxidase and glucose oxidase have been proposed.

[0004]

SUMMARY OF THE INVENTION In the prior art,
When glucose oxidase and mutarotase are used at the same time, there is a problem that the responsiveness is lower than in the case where mutarotase is not added, particularly in a region where the substrate concentration is relatively high. Further, when pyranose oxidase and glucose oxidase are used in combination, there is a problem that a response line depending on the substrate concentration cannot be obtained in a wide range.

[0005]

According to the present invention, there is provided a biosensor comprising an insulating substrate, an electrode system including a measuring electrode and a counter electrode formed on the substrate, and a reaction layer. Wherein the reaction layer contains glucose oxidase and pyranose oxidase, and an enzyme that promotes glucose isomerization.

[0006]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In the present invention, a synthetic resin substrate such as polyethylene terephthalate is used as an electrically insulating substrate. An electrode system including a working electrode and a counter electrode is provided on this substrate by using a known method. For example, after forming the leads on the substrate, a working electrode and a counter electrode are provided so as to be connected to each lead and insulated from each other. Known conductive materials are used as the materials for the leads and electrodes. Examples are carbon,
Silver, platinum, gold, palladium and the like.

[0007] The reaction layer is composed of glucose oxidase (EC 1.1.
3.4, hereinafter referred to as GOD) and pyranose oxidase (EC 1.1.1.310, hereinafter referred to as PyOx). Further, as an enzyme which promotes the isomerization of glucose, for example, mutarotase (EC 5.1.3.3, hereinafter referred to as EC) Mut)
It contains. Α-glucose, β-
Of glucose, GOD oxidizes only β-glucose, and PyOx is an enzyme that simultaneously oxidizes both α-glucose and β-glucose. Mut is an enzyme that promotes the isomerization of β-glucose and α-glucose, which are usually present in an aqueous solution at a ratio of 63:37, whereby the time to reach equilibrium is short. The preferred content of GOD is 1 to 1 cm 2 of the reaction layer.
40 units. The preferred content of PyOx is 0.1 to 2 units per square centimeter of the reaction layer, and more preferably 0.1 to 1 unit. Mu
The preferred content of t is from 10 to 200 units per square centimeter of the reaction layer. If the GOD content exceeds 40 units per square centimeter of the reaction layer, the reaction current tends to fluctuate due to, for example, the formation of the reaction layer during the production of the reaction layer. When PyOx and Mut exceed 2 units and 200 units per square centimeter of the reaction layer, respectively, the stability during storage tends to decrease.

Further, the reaction layer can contain various hydrophilic polymers. Examples include carboxymethylcellulose (CMC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HP
C), polyamino acids such as methylcellulose, ethylcellulose, ethylhydroxyethylcellulose, carboxymethylethylcellulose, polyvinylpyrrolidone, polyvinylalcohol, polylysine, polystyrenesulfonic acid, gelatin and its derivatives, acrylic acid and its salts, methacrylic acid and its salts, starch And its derivatives, maleic anhydride and its salts. Particularly, CMC is preferable.

The reaction layer hydrolyzes a polysaccharide to form α-
A polysaccharide-degrading enzyme for producing glucose can be contained. A polysaccharide-decomposing enzyme is an enzyme capable of hydrolyzing polysaccharides such as sucrose and maltose to produce glucose. Examples of polysaccharide degrading enzymes include sucrose hydrolases such as invertase (INV), maltose hydrolases such as maltase, and lactose hydrolases such as β-galactosidase.

When a sample solution containing glucose is supplied to the reaction layer of the biosensor, α-glucose and β-glucose in the sample solution are oxidized by the GOD, PyOx, and Mut, respectively. At the same time, the dissolved oxygen in the sample solution is reduced to hydrogen peroxide. Here, when a voltage is applied, hydrogen peroxide is oxidized. Since the response current value generated at this time is proportional to the generated hydrogen peroxide concentration, that is, the substrate concentration in the sample solution, the glucose concentration in the sample solution can be obtained by measuring the response current value.

In addition, instead of generating hydrogen peroxide simultaneously with the oxidation reaction of the substrate, the reaction layer may contain an electron acceptor, and a reduced form of the electron acceptor may be formed simultaneously with the enzymatic reaction. Examples of such electron acceptors include ferricyan ion, p-benzoquinone and its derivatives, phenazine methosulfate, methylene blue, ferrocene and its derivatives, and the like. As the electron acceptor, at least one or more selected from the group consisting of these is used. In particular, it is preferable to use ferricyan ions. The content of the ferricyan ion ranges from 0.23 to 3 / cm 2 per 1 cm 2 of the reaction layer.
30 mg is preferred. When the content of the ferricyan ion is less than 0.21 mg per 1 cm 2 of the reaction layer, the measurable glucose concentration range tends to be extremely narrow. When the content of the ferricyan ion exceeds 3.30 mg per 1 cm 2 of the reaction layer, the reaction layer is cracked, causing a variation in the response current value, and furthermore, the reliability during storage tends to decrease.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS A biosensor according to an embodiment of the present invention will be described below. Embodiment 1 FIG. 1 is a sectional view of a glucose sensor manufactured as one embodiment of the biosensor of the present invention. FIG. 2 is an exploded perspective view of FIG. However, the reaction layer is not shown. Hereinafter, a method for manufacturing the glucose sensor will be described. Silver paste was printed by screen printing on an insulating substrate 1 made of polyethylene terephthalate to form leads 2 and 3. Next, using a conductive carbon paste containing a resin binder, the measuring electrode 4 of the electrode system was used.
Then, an insulating layer 6 made of an insulating paste was formed by printing. The insulating layer 6 keeps the area of the exposed portion of the measuring electrode 4 constant and partially covers the leads 2 and 3. Finally, the counter electrode 5 is formed by printing using the same carbon paste as the measurement electrode, and the measurement electrode 4 and the measurement electrode 5 are formed.
Was formed. The area of the electrode system was 0.1 square centimeter.

Next, a CMC aqueous solution of 0.5% by weight as a hydrophilic polymer was dropped on the electrode system (measurement electrode 4 and counter electrode 5) and dried to form a CMC layer. Subsequently, a mixed solution of GOD and PyOx as enzymes having the ability to oxidize glucose on the CMC layer, Mut as an enzyme that promotes isomerization between α-glucose and β-glucose, and potassium ferricyanide as an electron acceptor Was dropped and dried in a warm air dryer at 50 ° C. for 10 minutes to form a reaction layer 7. The content of various reagents contained in the reaction layer 7 is GOD per square centimeter.
10 units, 1 unit of PyOx, 100 units of Mut, and 0.65 mg of ferricyan ion. After the formation of the reaction layer 7 as described above, the cover 9 and the spacer 8 were bonded in a positional relationship as indicated by a dashed line in FIG. Even if the cover 9 and the spacer 8 are not used, the effect of the present invention is not affected at all. However, when the cover 9 is attached, the sample liquid is supplied to the sample supply hole 10 at the tip of the sensor due to the capillary action in the space created by the cover 9 and the spacer 8.
It is easily introduced into the reaction layer portion by a simple operation of just contacting the reaction layer. In order to smoothly supply the sample solution, it is preferable that a toluene solution of lecithin is spread on the surface from the sample supply section to the reaction layer and dried as needed.

3 μl of an aqueous glucose solution was supplied from the sample supply hole 10 as a sample solution to the glucose sensor prepared as described above. The sample liquid quickly reached the air hole 11 by capillary action, and the reaction layer 7 on the electrode system was dissolved. When the reaction layer 7 is dissolved in the aqueous glucose solution, glucose in the sample solution is oxidized by the enzyme, and potassium ferricyanide is reduced to potassium ferrocyanide by the electrons transferred by the oxidation reaction. Next, the counter electrode 5 of the electrode system
When a pulse voltage of +0.5 V is applied to the measurement electrode 4 in the anode direction with reference to the above, an oxidation current of the generated potassium ferrocyanide is obtained. This current value corresponds to the concentration of glucose as a substrate. Therefore, a pulse voltage of +0.5 V is applied to the measurement electrode 4 in the anode direction with reference to the counter electrode 5 of the electrode system after a certain period of time from the supply of the sample solution, and the current value after 5 seconds is measured. A calibration curve proportional to the glucose concentration in the solution was obtained. This calibration curve is shown in FIG.

<< Comparative Example 1 >> Mut from the reaction layer of Example 1
A glucose sensor was configured in the same manner as in Example 1 except for the above, and an experiment was performed in the same manner. As a result, a calibration curve as shown in FIG. 3B was obtained.

Comparative Example 2 PyO was prepared from the reaction layer of Example 1.
A glucose sensor was constructed in the same manner as in Example 1 except for x and Mut, and an experiment was conducted in the same manner.
A calibration curve as shown in (c) was obtained.

The GOD of this embodiment reacts with only β-glucose among glucose isomers, while PyOx reacts with α-glucose.
Reacts with both glucose and β-glucose isomers. Therefore, a sensor containing only GOD as an enzyme is shown in FIG.
As shown in (c), the lowest response value is shown because the concentration of β-glucose alone is reflected. When GOD and PyOx were included, as shown in FIG. 3B, high response was exhibited for some reason, particularly in a high concentration range. So GOD and Py
It has been found that by including Mut in the reaction layer in addition to Ox, a high response value can be obtained in a wide concentration range as shown in FIG.

Embodiment 2 As an example of a biosensor,
The sucrose sensor will be described. In the same manner as in Example 1, on an insulating substrate 1, FIG.
The electrode system including the leads 2 and 3 and the measurement electrodes 4 and 5 shown in FIG. Next, the measurement electrode 4 and the counter electrode 5
An aqueous solution of 0.5% by weight of CMC as a hydrophilic polymer was spread on an electrode system of 0.1 square centimeter consisting of and dried to form a CMC layer. Subsequently, INV as an enzyme capable of decomposing a polysaccharide on the CMC layer to generate glucose, GOD and PyOx as enzymes capable of oxidizing glucose, and isomerization between α-glucose and β-glucose. A mixed aqueous solution of Mut as an enzyme for promoting the reaction and potassium ferricyanide as an electron acceptor was developed and dried in a hot-air drier to form a reaction layer 7. The content of the various reagents contained in this reaction layer was 10 INV per square centimeter.
Unit, GOD is 30 units, PyOx is 1 unit, Mut is 10 units, and ferricyan ion is 1. unit.
3 mg. In the same manner as in Example 1, 3 μl of a sucrose aqueous solution was supplied as a sample solution to the sucrose sensor manufactured as described above. After a certain time, the counter electrode 5 of the electrode system
When a pulse voltage of +0.5 V was applied to the working electrode 4 in the anode direction with reference to and the current value after 5 seconds was measured,
A response current value proportional to the sucrose concentration in the sample solution was obtained.

In the above embodiment, a method was employed in which the enzyme and the electron acceptor were dissolved in the sample solution. However, the present invention is not limited thereto. The method can also be applied. In the above embodiment, a two-electrode system including a measurement electrode and a counter electrode has been described. However, a three-electrode method including a reference electrode enables more accurate measurement.

[0020]

As described above, according to the present invention, a biosensor capable of measuring the substrate concentration of a sample solution with high accuracy can be obtained.

[Brief description of the drawings]

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

FIG. 2 is an exploded perspective view of the sensor viewed obliquely from above except for a reaction layer.

FIG. 3 is a response characteristic diagram of a glucose sensor.

[Explanation of symbols]

 REFERENCE SIGNS LIST 1 insulating substrate 2, 3 lead 4 measuring electrode 5 counter electrode 6 insulating layer 7 reaction layer 8 spacer 9 cover 10 sample supply hole 11 air hole

Claims (6)

[Claims] An electrode system including a measurement electrode and a counter electrode formed on an electrically insulating substrate, and a reaction layer formed in contact with the electrode system, wherein the reaction layer has at least glucose oxidase; A biosensor comprising pyranose oxidase and an enzyme that promotes isomerization of glucose. 2. The biosensor according to claim 1, wherein the enzyme that promotes glucose isomerization is mutarotase. 3. The biosensor according to claim 1, wherein the reaction layer further contains an electron acceptor. 4. The biosensor according to claim 1, wherein the reaction layer further contains an enzyme capable of hydrolyzing a polysaccharide to generate glucose. 5. The biosensor of claim 2, wherein the reaction layer contains 1 to 40 units of glucose oxidase per square centimeter, 0.1 to 2 units of pyranose oxidase, and 10 to 200 units of mutarotase. . 6. A reaction layer comprising 1 to 40 units of glucose oxidase per square centimeter, 0.1 to 2 units of pyranose oxidase and 10 to 200 units of mutarotase, and ferricyan ion as an electron acceptor. 4. The biosensor according to claim 3, containing 0.21 mg to 3.30 mg.