JPS61213663A - Biosensor - Google Patents

Abstract

PURPOSE:To simplify constitution and to measure quickly a substrate with high accuracy by providing electrodes to an insulating substrate and providing a liquid holding layer, filter layer and reaction layer contg. oxidation reduction enzyme, etc. on said electrodes. CONSTITUTION:The measuring electrode 2, counter electrode 3 and reference electrode 4 are provided on the substrate 1 consisting of a vinyl chloride resin, etc. The liquid holding layer 5 consisting of rayon paper, etc. is provided thereon so as to cover the electrode system and the filter layer 6 consisting of porous polycarbonate, etc. is provided thereon. The reaction layer 7 is provided thereon and in the case of, for example, a glucose sensor, the glucose oxidase of oxidation reduction enzyme and potassium ferrycyanide of the oxidation type dye conjugating therewith are used by impregnating the same in a non-woven pulp fabric. Sample blood is then dropped onto the reaction layer 7 and is brought into reaction with the oxidation reduction enzyme and dye. The red blood cells, etc. are filtered by the filter layer 6 and the filtration of the blood-plasma in the filtrate is accelerated in filtration by the liquid holding layer 5. The plasma is made to arrive at the electrode parts 2-4, by which the glucose is detected. Since the liquid holding layer is provided, the measurement of the specific component in a short period is made possible simply by dropping a slight amt. of the sample onto the sensor.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a biosensor that detects a specific component in a biological sample, and this biosensor can be widely applied to the medical field, food engineering, etc.

Conventional Technology With advances in medical technology, it has become possible to check health, determine disease status, and determine the effectiveness of treatment by measuring specific components in blood and urine. However, in the past, tests were carried out in hospital clinical laboratories using large machines and complicated methods.
The problem was that it was time consuming and costly. Therefore, there is a need for a sensor that can more easily measure on the spot. As one such attempt, a multilayer analytical carrier as shown in FIG. 3 has been proposed. It has a structure in which a reagent layer 10, a spreading layer 11, a waterproof layer 12, and a filtration layer 13 are laminated in this order on a transparent support 9. When a blood sample is dropped from above, solid components such as red blood cells and platelets in the blood are first removed by the filtration layer 13, uniformly permeates into the development layer 11 through the small holes 14 in the waterproof layer 12, and reacts in the reagent layer 1o. progresses. After the reaction is completed, light is applied from the direction of the arrow through the transparent support 9, and the substrate concentration is measured by spectroscopic analysis.

This method has the advantage that it can be easily measured by dropping a small amount of blood.

Problems to be Solved by the Invention However, in the above method, it takes time for the blood to permeate and react, so a waterproof layer 12 is required to prevent the sample from drying out, and incubation at high temperatures is required to speed up the reaction. There is a problem in that the equipment and the carrier are complicated.

Therefore, an object of the present invention is to provide a biosensor that can quickly and accurately measure a substrate with a simple configuration, avoiding the above-mentioned problems of complicating devices and carriers.

Means for Solving the Problems In the present invention, a liquid retaining layer, a filtration layer and a reaction layer are provided on the electrode section.

When the active blood is dropped, the oxidoreductase and the oxidized dye conjugated with the enzyme react promptly in the reaction layer. Red blood cells and platelets are then filtered in the filter layer. moreover,
The liquid holding layer, which does not support anything, quickly guides the filtered reaction liquid to the electrode section, where the amount of reaction is detected by the electrode reaction. In this way, the blood sample is reacted and filtered in a short time, making it possible to accurately measure the substrate using a simple device and carrier.

An example biosensor will be described using a glucose sensor as an example. Glucose oxidase was used as an oxidoreductase, and potassium ferricyanide was used as an oxidized dye conjugated with the oxidoreductase. FIG. 1 shows a schematic diagram of an embodiment of a glucose sensor. An electrode system consisting of a measurement electrode 2, a counter electrode 3, and a reference electrode 4 was constructed by embedding platinum in an insulating substrate 1 made of vinyl chloride resin. Rayon paper 5 was placed as a liquid retaining layer so as to cover the electrode system.

A porous polycarbonate membrane 6 with a pore diameter of 1 μm was placed thereon to serve as a filtration layer, and a pulp nonwoven fabric was placed on top as a reaction layer. This pulp nonwoven fabric 7 is impregnated in advance with a highly concentrated solution of glucose oxidase 200 Misaki and potassium ferricyanide 4001F dissolved in 100% phosphate buffer (pH 5°6), and an organic solvent with high solubility in water such as ethanol is used. After being immersed in the liquid, it is dried under vacuum to support fine crystals of enzymes and pigments at a high density.

30μ of blood as a sample liquid was placed on the nonwoven fabric T of this pulp.
After adding l and allowing it to penetrate sufficiently, measure the measuring electrode 2 using the reference electrode 4 as a reference.
The voltage of 0. It was varied at IV/sec. In this case, the potential of the reference electrode 4 made of platinum is determined by the concentration ratio of potassium ferricyanide and potassium ferrocyanide dissolved in the sample solution. When the added glucose in the blood is oxidized by glucose oxidase carried on the non-woven fabric of the pulp, potassium ferricyanide is reduced by an enzyme-dye coupling reaction to produce potassium ferrocyanide. Subsequently, when the reacted blood passes through the porous polycarbonate membrane 6, large solid components such as red blood cells are filtered out. It is difficult to filter a small sample with high viscosity like blood, but by placing a hydrophilic thin film like rayon paper 6 underneath, it can be filtered easily and reacts with the electrode. Plasma (filtrate) reaches the electrode portion, and the rayon paper 5 holds the reaction solution uniformly over the entire surface of the electrode section. Potassium ferrocyanide in the reaction solution is oxidized by sweeping the voltage of the measurement electrode 2, and an oxidation current flows at this time. This oxidation current is proportional to the amount of change in the dye, and if a sufficient amount of the dye is present, the amount of change in the dye corresponds to the substrate concentration. Therefore, by measuring the current value, the concentration of glucose, which is the substrate, can be detected. Using this glucose sensor, 4004/dl! This high concentration of glucose could be measured in just 2 minutes. This is a structure that allows the reaction to occur first, rather than filtering it as in the conventional case, and the enzyme and dye, which can handle high concentrations of substrates, are carried in an easily soluble state, so it can be carried out in a short time. It is considered that the reaction has finished.

Furthermore, by placing a thin hydrophilic rayon paper 5 under the filtration layer, it became possible to quickly filter a minute amount of blood, only 30 μl, and spread it uniformly on the electrode, resulting in a stable response current. .

In the above configuration, if the rayon paper 5 serving as the liquid retaining layer was removed, blood could not be filtered smoothly, and it took a long time for a sufficient amount of the reaction liquid to permeate onto the electrode. Furthermore, since the reaction solution was spread unevenly on the electrode, the response current varied greatly, making it impossible to measure accurately.

In the above example, a thin film of rayon paper with a thickness of 50 μm was used, but as the thickness increases, the amount of liquid retained increases, and it takes time to infiltrate a sufficient amount of reaction liquid into the electrode, and the amount of blood increases. I needed it. Furthermore, when enzymes and pigments were supported on rayon paper, the contact surface with the filtration layer was reduced by crystals of enzymes and pigments, reducing the number of points of contact, making filtration time-consuming. From the above,
As the liquid retaining layer, a porous body that is a hydrophilic thin film and does not support anything is desirable.

A carrier consisting of a liquid retaining layer, a filtration layer, and a reaction layer for measurement may be placed directly on the surface of the electrode, but if the carrier is heavy or installed under pressure, the measurement carrier may be placed directly on the electrode surface. Due to the partial contact, the electrode reaction area becomes smaller and the current value becomes smaller and more likely to vary. Therefore, in such a case, as shown in FIG. 2, it is necessary to provide a groove 8 in the electrode portion to prevent the measurement carrier from coming into contact with the electrode surface.

A liquid retaining layer is required to allow a sufficient amount of minute amount of blood filtrate to permeate into the electrode portion having the above shape. Furthermore, by changing the shape of the liquid retaining layer so that it covers only the upper part of the groove 8, the filtrate can be concentrated and permeated into the groove 8, and the groove 8 can be effectively filled with a small amount of liquid. Ta. This allows measurement without direct contact of the measurement carrier with the electrode surface.
A response current with good reproducibility was obtained. Instead of forming a groove in the electrode portion, a spacer may be provided around the electrode.

Since the biosensor of the present invention does not require a diluent other than the sample solution, when the amount added was varied from 30 to 100 .mu.l, the same blood showed a constant value regardless of the amount added. Therefore, there is no need to make the addition amount accurate, and measurement can be easily performed by simply adding a small amount of blood. moreover.

By using highly concentrated enzymes and oxidized dyes, the reaction is completed in as little as 2 minutes, so there is no need for high-temperature incubation equipment or a waterproof layer to prevent evaporation.
Accurate measurement was possible using a simple device and carrier.

Rayon paper was used on the upper side as the liquid retaining layer, but in order to quickly spread a small amount of liquid from the filtration layer onto the electrode, it is desirable to use a hydrophilic and thin porous membrane. In addition to rayon paper, other materials such as Okigami and nylon non-woven fabrics could also be used.

As the dye, potassium ferricyanide used in the above examples is suitable because it reacts stably, but P-benzoquinone is suitable for increasing the reaction rate because it has a fast reaction rate.

Further, 2,6-cyclophenol indophenol, methylene blue, phenazine methosulfate, potassium β-naphthoquinone 4-sulfonate, etc. can also be used.

Note that the sensor in the above embodiments is not limited to glucose, but can be used in systems involving redox enzymes, such as alcohol sensors and cholesterol sensors. Although glucose oxidase was used as the oxidoreductase, other enzymes such as alcohol oxidase, xanthine oxidase, cholesterol oxidase, etc. may also be used.

Effects of the Invention According to the sensor of the present invention, a specific component can be accurately measured in a short time by simply dropping a small amount of sample directly.

[Brief explanation of the drawing]

1 and 2 are schematic cross-sectional views of a glucose sensor according to an embodiment of the present invention, and FIG. 3 is a schematic cross-sectional view of a conventional biosensor. 1...Substrate, 2...Measurement pole, 3...
... counter electrode, 4 ... reference electrode, 6 ... liquid retention layer, 6 ... filtration layer, 7 ... reaction layer,
8...Groove. Name of agent: Patent attorney Toshio Nakao and 1 other person No. 1
Figure 2! ...Basic handling 2...Sokusanbashi 3...Pairing 4...4. Terumi 5... A system liquid layer t2). Filtration layer 7...Metal layer I...琲

Claims (2)

[Claims] (1) On the electrode part, which has an electrode system consisting of a measurement electrode, a counter electrode, and a reference electrode on an insulating substrate, there is a filtration layer consisting of a liquid retaining layer and a porous membrane, an oxidoreductase, and an oxidation conjugate with the enzyme. A biosensor characterized by having a reaction layer containing a dye. (2) The biosensor according to claim 1, wherein the liquid retaining layer is made of a hydrophilic porous material.