JPS62108146A - Biosensor - Google Patents

Abstract

PURPOSE:To easily and quickly measure the specific component of a living body sample by providing a liquid retaining layer consisting of a hydrophilic porous body, filter layer and reaction layer contg. oxidoreductase and oxidizing type dye which conjugates therewith no electrode parts. CONSTITUTION:Measuring electrodes 3, 4, 5 are provided on a substrate 1. A space part 2 is opened thereon and the hydrophilic liquid retaining layer 6 consisting of rayon, etc., the filter layer 7 and the reaction layer 8 are provided thereon. Glucose oxidase and the potassium ferrocyanide which conjugates therewith are incorporated into the reaction layer 8 in the stage of detecting, for example, the glucose in blood. The glucose is oxidized and is reduced to the potassium ferricyanide when the sample is dropped onto the reaction layer 8. The blood cells are then filtered by the filter layer 7 and are penetrated into the liqiud retaining layer 6. The glucose in the blood is electrically measured by the measuring electrode 3. Since the filtrate is quickly penetrated into the filter layer and the liquid retaining layer in the above-mentioned manner, the trace sample is measured in a short time.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to biochemistry and is capable of detecting specific components in biological samples, 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 outcome 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, which was time-consuming and costly. Therefore, there is a need for a sensor that can easily perform measurements on the spot. As one such attempt, a multilayer analysis carrier as shown in FIG. 4 has been proposed. A reagent layer 12, a developing layer 13. Waterproof layer 14. It has a structure in which filtration layers 16 are laminated in order. When a blood sample is dropped from the top, solid components such as red blood cells and platelets in the blood are first removed by the filtration layer 15, and then passed through the small holes in the waterproof layer 14 to the expansion layer 13.
After completion of the reaction in which the substrate uniformly permeates into the reagent layer 12 and the reaction proceeds in the reagent layer 12, light is applied 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 that it can be easily measured by dropping a small amount of blood.

However, since it takes time for blood to permeate and react, a waterproof layer 14 is required to prevent the sample from drying out, and it is necessary to incubate at a high temperature to speed up the reaction.
There is a problem that the equipment and carrier become complicated.

Problems to be Solved by the Invention The purpose of the biosensor of the present invention is to avoid the above-mentioned problem of complicating the device and carrier, and to be able to quickly and accurately measure a substrate with a simple device and carrier.

Means to solve problems The biosensor of the present invention has a liquid retaining layer on top of the electrode part.

A filtration layer and a reaction layer are placed between the frames, and a liquid retaining layer is held in the space above the electrode section.

Function With such a configuration, when blood is dropped onto the reaction layer, 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. Further, the liquid holding layer, which does not support anything, promptly 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. 1A shows a mutually orthogonal schematic cross-sectional view of an embodiment of a BiC glucose sensor. The electrode part is made of an insulating substrate 1 made of polyvinyl chloride resin, with a groove 3.4 m wide and 0.15 m deep as a space 2 filled with platinum. Opposite 4. and a reference electrode 5, an electrode system was constructed. A reaction layer 8 and a filtration layer 7 are provided on the frames 9 and 10 so as to cover the electrode system. A measuring chip sandwiching the liquid retaining layer 6 is installed. The reaction layer 8 is a nonwoven fabric of the valve, glucose oxidase 20
0+115+ and potassium ferricyanide 40079 dissolved in phosphate buffer (pH 5, 6) IOC are impregnated, immersed in an organic solvent with high solubility in water such as ethanol, and dried under vacuum to remove glucose oxidase. and supports fine crystals of potassium ferricyanide at high density. The filtration layer 7 has a pore diameter of 1 μm.
This porous polycarbonate membrane removes solid components such as red blood cells from blood cells. As the liquid retaining layer 6, a band-shaped rayon paper with a width of 2 mm was used. Both ends of the rayon paper are fixed to the frame. FIG. 2B shows a view of the measurement chip from the electrode side, and FIG. 2C shows a top view of the electrode section. Furthermore, the rayon paper is held in such a position that it fits inside the groove with a width of 3.4 mm in the electrode part, and the filtration layer of the measuring chip is formed by the part other than the groove in the electrode part, as shown in cross-sectional view B in Figure 1. 7 is supported. Reaction layer 8 above. Filtration layer7. The liquid retaining layer 6 is fixed using frames 9 and 10 by pressure bonding or adhesive such as epoxy resin. FIG. 2A is an exploded perspective view of this sensor before assembly.

・After adding 30 pl of blood as a sample liquid onto the reaction layer 8 made of non-woven fabric of the valve and allowing it to fully penetrate, the reference electrode 5 was added.
The voltage of the measurement electrode 3 was changed in a sawtooth manner from O to +0.1 V at a rate of 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. Added glucose in the blood valve non-woven fabric 8
When oxidized by glucose oxidase supported on , 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.

Although it is difficult to filter a highly viscous and minute sample such as blood, it can be quickly filtered by placing a hydrophilic thin film such as rayon paper 6 underneath. Furthermore, the filtered reaction solution is uniformly spread over the band-shaped rayon, and the potassium ferricyanide in the 0 reaction solution supplied to the electrode section below is oxidized by sweeping the voltage of the measurement electrode 3, and the oxidized Measuring the current 0 This oxidation current is proportional to the amount of change in the dye, and if enough dye is present, the amount of change in the dye corresponds to the substrate concentration, so the concentration of glucose can be detected.

Using this glucose sensor, glucose at a high concentration of 400 μ/dl could be measured in a short time of 2 minutes.

This system allows the reaction to occur first, rather than through filtration as in conventional methods, and the enzyme and dye, which can handle high-concentration substrates, are supported in a state that is easy to dissolve, so it takes only a short time. It is thought that the reaction was completed within a few hours.

Furthermore, a hydrophilic thin rayon paper 6 is placed under the filtration layer 7.
By placing the electrode, it was possible to quickly filter a minute amount of blood, as small as 30) 11, and this made it possible to spread the reaction solution uniformly on the electrode and obtain a stable response current. The liquid retaining layer preferably covers at least each electrode and has a shape as small as possible.

When the liquid retaining layer 6 has the same shape as the filtration layer 7 and is incorporated into the frames 9 and 1o, blood is quickly filtered at the outer peripheral portion of the liquid retaining layer fixed by the frames 9 and 10, and collects there. It became difficult for the reaction solution to be supplied to the electrode section. By making the liquid retaining layer 6 into a band shape, the area of the outer periphery where it tends to accumulate was reduced, the rayon paper 6 was quickly wetted, and the reaction liquid could be supplied to the electrode part even with a small amount of blood. By reducing the size of the rayon paper that is the liquid-retaining layer in this way,
Even with a trace amount of sample of 15/Il, it became possible to intensively supply the reaction solution onto the electrode. When the rayon paper 6 covered the electrode grooves by adjusting the width of the electrode grooves in order of 1.5, the wrinkles that occurred when the reaction solution was supplied did not come off and adhered to the top of the measurement electrode, causing problems in the measurement. There were cases of interference. Therefore, when we made the width of the electrode groove wider than the width of the rayon paper so that the rayon paper 6 was set in the space 2 as shown in cross section B in Figure 1, no formation of bubbles was observed. The measurements were stable. This is thought to be because the gap between the rayon paper 6 and the groove of the electrode provides a passage for air and prevents formation of wrinkles. Furthermore, by making the depth of the groove provided in the electrode part larger than the thickness of the rayon paper that is the liquid retaining layer, the rayon paper 6 does not come into direct contact with the electrode surface, and the reaction area of the measurement electrode 3 is always maintained. A response with good reproducibility was obtained by keeping it constant. In the example, the thickness is 60 am
A thin film of rayon paper was used, but as the thickness increased, the amount of liquid it held increased, necessitating a larger amount of sample. Furthermore, when enzymes and pigments were supported on rayon paper, the number of points of contact with the filtration layer 7 decreased due to enzyme and pigment crystals, and filtration took a long time. From the above, it is desirable that the liquid retaining layer e be a hydrophilic thin film that does not support anything, and that it should cover the top of the electrode system with a minimum area smaller than the groove of the electrode. .

The biosensor of the present invention does not require a diluent other than the sample solution, so when the amount of blood added was varied from 15 to 100 μl, the same blood showed a constant value of 0 regardless of the amount added. For this reason, it is necessary to accurately add the amount of blood, but it has become possible to easily measure by adding only a small amount of blood. Furthermore, by using highly concentrated enzymes and oxidized dyes, the reaction can be completed in as little as 2 minutes, so there is no need for high-temperature incubation equipment or a waterproof layer to prevent evaporation. The device and carrier were able to measure with high accuracy.

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 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 can be used not only for glucose but also for 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 and xanthine oxidase were used.

Cholesterol oxidase and the like are also used. Note that the enzyme could be used even if it was immobilized with a crosslinking agent or the like.

Effects of the Invention As described above, according to the biosensor of the present invention, it was possible to measure a specific component with high accuracy in a short period of time simply by directly dropping a small amount of sample.

[Brief explanation of drawings]

1A and 1B are cross-sectional views of a glucose sensor according to an embodiment of the present invention, 2A is an exploded perspective view before assembly, 4B is a bottom view of the measuring chip, and 4C is a top view of the electrode section. , 3rd
The figure is a schematic diagram of a conventional biosensor. 1...Substrate, 2...Groove, 3...
・Measurement electrode, 4... Counter electrode, 5... Reference electrode, 6... Liquid retaining layer, 7... Filtration layer, 8...
...Reaction layer, 9.1o...Frame, 11.
... Support, 12 ... Reagent layer, 13 ...
...Development layer, 14...Filtering layer, 15...
...Waterproof layer. Name of agent: Patent attorney Toshio Nakao and 1 other person1-
11 groups frozen 2・-clear 3"-clear definite C-retaining liquid A 7---5 doors 1

Claims (1)

[Claims] A filtration layer consisting of a liquid retaining layer and a porous membrane, an oxidoreductase, and the enzyme are placed on an electrode part, which has an electrode system consisting of a measurement electrode, a counter electrode, and a reference electrode on an insulating substrate, through a space. A bio-reactive layer comprising a reaction layer containing a conjugated oxidized dye is placed between frames, and the liquid retaining layer is made of a hydrophilic porous material and has a shape that is retained at least within the space. sensor.