JPH01156658A - Biosensor - Google Patents

Abstract

PURPOSE:To measure a measuring component with good accuracy up to a high-concn. region by installing an immobilized layer of oxidase on an electrode system and bringing a layer which consists of a water soluble mediator deposited in a dry state on a insoluble porous film into tight contact with the surface of said layer. CONSTITUTION:A measuring electrode 2, a counter electrode 3 and an insulating layer 4 are provided on an insulating substrate 1. An aq. soln. of 5% carboxymethyl cellulose is applied on this electrode system and is dried. Furthermore, a glucose oxidase soln. is coated as an enzyme soln. thereon and is dried to produce the enzyme immobilized layer 5. The mediator deposited layer 6 produced by impregnating a phosphate buffer soln. of potassium ferricyanide as the mediator to a porous cellulose film and drying the same is held in pressurized contact with the surface of the enzyme immobilized layer 5 by a holding frame 7. A large amt. of the potassium ferricyanide is thereby supplied into a sample liquid and the migration of the sample reaction liquid between the respective layers is smoothly executed at the time of measurement and, therefore, the objective component is measured with the good accuracy up to the high concn.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention provides a method for quickly and easily identifying specific components in various samples.
This invention relates to a biosensor that can perform quantitative measurements.

BACKGROUND OF THE INVENTION In recent years, biosensors have come into use that combine enzyme reactions and electrode reactions to measure specific components in samples.

A conventional biosensor will be explained below.

FIG. 4 is a cross-sectional view of a conventional biosensor, where 8 is an insulating substrate, and 9 and 10 are measurement electrodes and counter electrodes formed by screen printing conductive carbon paste on the insulating substrate 8. 11 is an insulating layer, and an insulating resin paste is applied to an insulating substrate 8. Measuring pole 9. The same printing as above was performed on the counter electrode 10 to form an electrode part. Reference numeral 12 denotes a mediator support layer prepared by applying a mediator to the surface of the electrode portion, and 1ait is an enzyme immobilization layer placed on the mediator support layer 12.

The operation of the biosensor configured as described above will be described below. When the sample liquid is dropped from above, first, in the enzyme immobilization layer 13, a specific component in the sample liquid reacts specifically with the oxidizing enzyme as shown in equation (1).

S + E (ox)'-+P + E (red)
-・”(1) S: Specific component, E (ox): Oxidized enzyme P
: product component, E(red) : reduced enzyme Further, in the mediator support layer 12, the mediator is reduced as shown in equation (2).

E (red)+M(0ri-+E (0ri+M(re)
d) -... (2) M (ox): Oxidized mediator - M (red): Reduced mediator - The reduced mediator produced here is transferred to the electrode system at 9°1
The above is oxidized as shown in formula (3), and M(red)−+M
(o, x) + e −−−−−−−−−−−(3)
e: Electron By measuring this oxidation current value, the concentration of a specific component in the sample liquid is detected.

Problems to be Solved by the Invention However, in the conventional configuration, when an oxidase is used, the reaction shown in the above equation (2) occurs in the mediator-carrying layer, and the dissolution in the sample liquid as shown in the equation (4) occurs. Since a competitive reaction with oxygen occurs, the production of reduced mediator is inhibited.

E (red) +02-)E (0ri +H2O2-
”・(→02: Acid However, when the enzyme immobilization layer 13 is formed on the mediator support layer 12, the enzyme and mediator are still immobilized in a wet manner.
The mutual redox reaction proceeds due to the coexistence of the mediator in a wet state, and the redox state of the mediator is unstable, resulting in the problem that accurate measurements cannot be performed.

The present invention solves the above-mentioned conventional problems, and provides a biosensor that can accurately measure a specific component up to a high concentration range by supplying a large amount of oxidized mediator and bringing each layer into close contact. The purpose is to

Means for solving the problem In order to achieve this objective, the biosensor of the present invention has an oxidase immobilized layer placed on an electrode consisting of a measurement electrode and a counter electrode, and a water-soluble mediator is further placed on top of the immobilized layer of oxidase. It has a structure in which a layer supported in a dry state is adhered to an insoluble porous membrane.

Function: With this configuration, the enzyme and the mediator do not coexist in a wet state during the biosensor formation process, and the oxidized mediator is stably retained and can be measured with high accuracy. In addition, when a sample solution is dropped from the top during measurement, it first passes through the water-soluble mediator support layer, and after a large amount of mediator is dissolved in the sample solution, an enzymatic reaction occurs, so dissolved oxygen in the sample solution and The influence of competitive reactions is reduced, making it possible to measure specific components at high concentrations.Furthermore, because the structure has high adhesion between each layer, the movement of the reaction liquid occurs smoothly, allowing for highly accurate measurements. It is possible.

EXAMPLE Hereinafter, a glucose sensor as an example of the present invention will be described with reference to the drawings.

FIG. 1 schematically shows a cross-sectional view of a glucose sensor according to an embodiment of the present invention. In Figure 1, 1
2 is an insulating substrate, 2 is a measurement electrode, 3 is a counter electrode, and 4 is an insulating layer, which have the same structure as the conventional example. 5 is an enzyme immobilization layer, and a KO-5% carboxymethylcellulose aqueous solution is applied on the electrode part 2,3°4 and dried at constant temperature at 45°C for 30 minutes. : A carboxymethyl cellulose membrane with a film thickness of 1 μm with good adhesion was obtained, and using this as a carrier, a glucose oxidase solution, which is an enzyme solution, was applied and dried to immobilize glucose oxidase.

Reference numeral 6 denotes a mediator-carrying layer, which was prepared by impregnating a porous cellulose membrane as a carrier in a phosphate buffer (pH 5, 6) containing potassium ferrinanide, which is a mediator, and then drying it. Reference numeral 7 denotes a holding frame for press-bonding and holding the mediator-carrying layer 6 produced as described above on the enzyme immobilization layer 6 with good adhesion.

The operation of the glucose sensor of this embodiment configured as described above will be described below. First, when a sample liquid is dropped onto the upper part of FIG. 1, water-soluble potassium ferricyanide is first dissolved in the sample liquid in the mediator-carrying layer 6, and a highly concentrated potassium ferricyanide solution is produced. Next, the solution quickly moves to the enzyme immobilization layer 5 that is in close contact with the mediator support layer 6, and in the enzyme immobilization layer 6, the reaction between glucose oxidase and potassium ferricyanide is carried out by the above equation (1), ? ), potassium ferrocyanide is produced. Then, the glucose concentration in the sample is detected by measuring the oxidation current value of potassium ferrocyanide on the electrode that is in close contact with the enzyme immobilization layer 5.

FIG. 2 shows the relationship between the oxidation current value measured by the glucose sensor and the glucose concentration of the glucose aqueous solution. A is formed by placing an enzyme immobilization layer on the electrode system of the present invention and then adhering the mediator support layer, and B is a conventional electrode system in which the mediator support layer is placed on the electrode system and then the enzyme immobilization layer is formed. It has a layer formed on it. In this diagram, the current value and glucose concentration are 5oor4/d at A.
It shows very good linearity up to t, but in contrast, in conventional example B, glucose (8 degrees is 2 degrees).

It can be seen that no linearity is observed in the response above d/dt. This is because in configuration B, the mediator is not sufficiently present in the sample solution, so the reaction of the dissolved enzyme (the above (4)
) is dominant, which is thought to be because the amount of mediator reduction does not correspond to the glucose concentration.

Figure 3 shows the same glucose measured with the glucose sensor mentioned above.
It shows the oxidation current value between each glucose sensor of v-r-x aqueous H. In the figure, A is the glucose sensor of the present invention, and B is the conventional glucose sensor. In this figure, A shows a stable measured value with small variations in current value between each glucose sensor. However, B
It can be seen that the average value is high, and that the current values between each glucose sensor vary widely and are not measured stably. This is because reduction of the mediator occurs in the wet state during the preparation of the enzyme immobilization layer, and reduced mediator is generated that does not depend on the amount of glucose in the sample solution, and the oxidized and reduced mediator forms in the mediator support layer. It is thought that because of the presence of mediators, the average of the measured values is large and the variation is also increased.

As described above, according to this example, an immobilized layer of an oxidizing enzyme such as glucose oxidase is installed on the electrode system, and potassium ferricyanide, which is a water-soluble mediator, is placed on top of the immobilized layer of an oxidizing enzyme, such as glucose oxidase, on the cellulose, which is an insoluble porous membrane. Due to the structure in which the dry supported layer is brought into close contact with the membrane,
It is possible to accurately measure the amount of glucose in a sample solution up to a high concentration. This is thought to be because a large amount of potassium ferricyanide is supplied to the sample solution during measurement, and the sample reaction solution moves smoothly between the layers.

In this example, potassium ferricyanide was used as the water-soluble mediator, but other dyes such as ferrician pluthenium red and mechelene blue may also be used.

Effects of the Invention As described above, according to the present invention, an oxidase immobilized layer is provided on an electrode system consisting of a measurement electrode and a counter electrode, and a water-soluble mediator is further placed on an insoluble porous body in a dry state on top of the oxidase immobilization layer. By having a structure in which the supported layers are in close contact with each other, it is possible to obtain the effect that a specific component can be measured with high accuracy up to a high concentration range.

[Brief explanation of the drawing]

FIG. 1 shows glucose concentration in one embodiment of the present invention.
FIG. 2 is a correlation characteristic diagram between glucose sensor concentration and response current, FIG. 3 is a relationship diagram between each glucose sensor and response current, and FIG. 4 is a sectional diagram of a conventional biosensor. 1...Insulating substrate, 2...Measurement electrode, 3...
Curved counter electrode, 4...Insulating layer, 6...Enzyme immobilization layer, 6...River/mediator support layer, 7...Mountain...
holding frame. Name of agent: Patent attorney Toshio Nakao and 1 other person1-
Insulation Sumakusa Figure 2 Silcourse No. (rq/a) Figure 3 Silcourse and Nsa No.

Claims (2)

[Claims] (1) An electrode system consisting of a measurement electrode and a counter electrode is provided, a layer of immobilized oxidase is placed on top of this electrode system, and a layer in which a mediator is supported in a dry state on an insoluble porous membrane is placed on top of the layer. A biosensor characterized by having a configuration. (2) The biosensor according to claim 1, wherein the mediator is a water-soluble mediator.