JPS61241651A - Biosensor - Google Patents

Abstract

PURPOSE:To make the quick and accurate measurement of a substrate with a simple instrument and carrier possible by constituting the title sensor in such a manner that a blood sample reacts quickly and is filtered. CONSTITUTION:Oxido-reductase and oxidation type dye which conjugates with the enzyme react quickly in a reaction layer when the blood is dropped to the biosensor. The red blood cells and platelet are then filtered in a filter layer 7 and the filtered reaction liquid arrives at the lower liquid holding layer 6. A liquid holding layer 6 is notched in the outside peripheral part to have a space part and therefore the reaction liquid spreads over the entire surface of the liquid holding layer 6 and is filled in the space part where the electrode part consisting of a counter electrode 4 and reference electrode 5 is exposed. The reaction liquid filtered by the liquid holding layer 6 is guided quickly to the electrode part in the above-mentioned manner and the reacting weight is detected by the electrode reaction. Since the blood sample reacts and filtered in the short period in the above-mentioned manner, the accurate measurement of the substrate with the simple instrument and carrier is made possible.

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 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 took time and cost. Therefore, there is a need for a sensor that can more easily measure on the spot. As one such attempt, a multilayer analysis carrier as shown in FIG. 6 has been proposed. It has a structure in which a reagent layer 12, a spreading layer 13, a waterproof layer 14, and a filtration layer 15 are laminated in this order on a transparent support 11. When a blood sample is dropped from the top, solid components such as red blood cells and platelets in the blood are first removed from the filtration layer 16, and then passed through the small holes in the waterproof layer 14 to the expansion layer 13.
The reaction proceeds in the reagent layer 12.

After the reaction is completed, light is irradiated from the direction of the arrow through the transparent support 11, and the substrate concentration is measured by spectroscopic analysis.

This method has the advantage of being able to easily measure by dropping a minute amount of blood.2 Problems that the invention aims to solveHowever, in the above method, it takes time for the blood to permeate and react. A waterproof layer 14 is required to prevent drying, and incubation at a high temperature is required to speed up the reaction, making the apparatus and carrier complicated.

An object of the present invention is to avoid the above-mentioned problem of complicating devices and carriers, and to enable rapid and accurate measurement of substrates using simple devices and carriers.

Means to Solve the Problems The present invention provides an electrode system consisting of a measurement electrode, a counter electrode, and a reference electrode on an insulating substrate, and a hydrophilic porous material is placed above the electrode part with a space therebetween. A filtration layer consisting of a liquid retaining layer and a porous membrane, and a reaction layer containing an oxidoreductase and an oxidized dye conjugated with the enzyme are placed between frames, and a space is further formed around the filtration layer. It is something to do.

Effect: When blood is dropped onto the biosensor of the present invention, the redox enzyme and the oxidized dye conjugated with this enzyme rapidly react in the reaction layer. Red blood cells and platelets are then filtered in the filter layer. This filtered reaction solution reaches the lower liquid retaining layer. Since the outer periphery of the liquid retaining layer is cut out to form a space, the reaction liquid spreads over the entire surface of the liquid retaining layer and fills the exposed space of the electrode. In this way, the liquid retaining layer quickly guides the filtered reaction liquid to the electrode section, where the amount of reaction is detected by the electrode reaction. Since the blood sample is reacted and filtered in such a short time, it has become possible to accurately measure the substrate using a simple device and carrier.

EXAMPLE A glucose sensor will be explained as an example of a biosensor. Glucose oxidase was used as the oxidoreductase, and potassium ferricyanide was used as the oxidized dye conjugated with the oxidoreductase. FIG. 1 shows the configuration of an embodiment of a glucose sensor.

A groove 2 with a depth o and 1 mm is formed in the upper end surface of a cylindrical insulating substrate 1 made of vinyl chloride resin, and platinum is buried in the groove 2 so as to be exposed. An electrode system consisting of a counter electrode 4 and a reference electrode 5 is configured. A reaction layer 8. is formed by frames 9 and 10 so as to cover the electrode system. Filtration layer7. A measuring chip 6-pa sandwiching the liquid retaining layer 6 is installed.

The reaction layer 8 is made of a nonwoven pulp fabric, and contains 200 mg of glucose oxidase and 400 mg of potassium ferricyanide. n
of glucose oxidase and potassium ferricyanide. Supports crystals at high density. The filtration layer 7 is a porous polycarbonate membrane with a pore diameter of 1 μm, and is used to remove solid components such as red blood cells from blood. The liquid retaining layer 6 is made of rayon paper, for example, and has a disk shape with a diameter smaller than that of the filtration layer 7 as shown in FIG. I'm trying to keep it fixed.

FIG. 3 shows a diagram of the measurement chip viewed from the liquid retaining layer 6 side. The shape that creates a space around the liquid retaining layer means the shape of the liquid retaining layer such that there is a part without the liquid retaining layer under the filtration layer 7 in FIG. 3. Reaction layer 8 above. Filtration layer7. The liquid retaining layer 6 is fixed to the frames 9 and 10 by pressure bonding or epoxy resin.

After adding 30 μl of blood as a sample liquid onto the reaction layer 8 and allowing it to penetrate sufficiently, the voltage of the measurement electrode 3 is set to 0 to 1000 with reference to the reference electrode 5.
The voltage was changed in a sawtooth manner between 10 and I VO at 1 V/sec. In this case, the potential of the reference electrode 5 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 the glucose oxidase supported on the reaction layer 8, 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 7, large solid components such as red blood cells are filtered out. It is difficult to filter a small sample with high viscosity such as blood, but it can be quickly filtered by placing a hydrophilic thin film like rayon paper underneath as a liquid retaining layer. The reacted plasma (filtrate) reaches the stain, and the liquid retaining layer 6 holds the reaction liquid uniformly over the entire surface of the stain. Potassium ferrocyanide in the reaction solution is oxidized by sweeping the voltage of the measurement electrode 3, 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. With this glucose sensor, 400 m
g/d/? High concentrations of glucose could be measured in just 2 minutes. This is a structure in which the reaction is carried out first, rather than by filtration as in the conventional case, and the enzyme and dye, which can handle high concentrations of substrates, are carried in an easily dissolvable 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 liquid retaining layer 6 under the filtration layer, a minute amount of blood, only 30 μl, can be quickly filtered and spread uniformly on the electrode, making it possible to obtain a stable response current. . However, when the liquid retaining layer 6 is assembled into the IT frames 9 and 10 having the same shape as the filtration layer 7, the filtered blood flows through the frames 9 and 10 at the outer periphery of the liquid retaining layer 6.
Since the part fixed by 10 is quickly filtered and plasma accumulates there, the groove 2 of the electrode part may not be filled with liquid. Therefore, if the shape of the liquid retaining layer 6 is made as shown in Fig. 1 and a space is provided at the outer periphery where the liquid tends to accumulate, the filtered blood will quickly spread over the entire surface of the liquid retaining layer 6 and the liquid will also flow into the grooves 2. Satisfied. By providing this groove 2, it was possible to prevent the measurement chip from coming into direct contact with the electrode surface, so that the reaction area of the measurement electrode 3 was always kept constant and a response with good reproducibility was obtained.

In addition, the area of the liquid retaining layer 6 becomes smaller when a space is created.
The amount of blood added could also be further reduced to 20 μl. When the liquid retaining layer 6 is removed, the blood cannot be filtered out quickly, and a sufficient amount of reacted plasma (reaction liquid) is deposited on the electrode.
It took a while for it to sink in. Furthermore, the reaction solution spread unevenly onto the electrode, resulting in large variations in the response current, making it impossible to measure accurately.

In this example, a thin film of rayon paper with a thickness of 60 μm was used as the liquid retaining layer, 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. It also requires a large amount of blood.

In addition, when enzymes and pigments were supported on rayon paper, 9″
- The number of contact points with the filtration layer 7 was reduced due to enzyme and pigment crystals, and filtration took a long time. From the above, it is desirable that the liquid retaining layer 6 be a hydrophilic thin film and a porous body that does not support anything. Moreover, the shape of the liquid retaining layer can be changed to the shape shown in FIG. 4 other than the shape shown in FIG. It is possible.

Since the biosensor of the present invention does not require any diluent other than the sample solution, when the amount of blood added was varied from 30 to 100 μl, the same blood showed a constant value regardless of the amount added. For this reason, it is necessary to accurately add the amount of blood, and it has become possible to easily measure by adding only a small amount of blood. Furthermore, by using high concentrations of enzymes and oxidized dyes! Since the reaction was completed in a short time of 112 minutes, there was no need for equipment for high temperature incubation or a waterproof layer to prevent evaporation, and accurate measurements could be made using simple equipment and carriers.

Rayon paper was used 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, filter paper and nylon nonwoven fabric 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 speed because it has a fast reaction rate. Also, 2,6-cyclophenol indophenol, methylene blue, phenazine methosulfate, potassium β-naphthoquinone-4-nurphonate, etc. were used to kill the virus.

Incidentally, the sensor in the above practical example can be used for threads in which redox enzymes are involved, such as glucose bilayer, alcohol sensor, and cholenuterol sensor. Although glucose oxidase was used as the oxidoreductase, other enzymes such as alcohol oxidase, xanthine oxidase, cholesterol oxidase, etc. may also be used.

Effect of the invention 9 According to the sensor of the present invention, it is possible to directly collect a small amount of sample 11
By simply dropping a drop onto a page, specific components can be measured quickly and accurately.

[Brief explanation of drawings]

Fig. 1 is a longitudinal cross-sectional view of a glucose sensor according to an embodiment of the present invention, Fig. 2 is an exploded perspective view thereof, Fig. 3 is a cross-sectional view taken along the line 1-1 in Fig. 1, and Fig. 4 is a deformation of the liquid retaining layer. A plan view showing an example, and FIG. 6 is a longitudinal sectional view of a conventional biosensor. 1...Base body, 2...Groove, 3...
・Measurement electrode, 4...Counter electrode, 5...Reference electrode, e...Liquid retaining layer, 7...Filtering layer, 8...
...Reaction layer, 9 and 10... Frame, 11...
... Support, 12 ... Reagent layer, 13 ...
...Development layer, 14...Filtration layer, 15...
・Waterproof layer.

Claims (1)

[Claims] A liquid retaining layer made of a hydrophilic porous material, a filtration layer made of a porous film, and an oxidation layer are placed on the electrode part, which has an electrode system consisting of a measurement electrode, a counter electrode, and a reference electrode on an insulating base, through a space. A biosensor in which a reaction layer containing a reductase and an oxidized dye conjugated with the enzyme is sandwiched between frames, and a space is formed around the filtration layer.