JPH0640087B2 - Biosensor - Google Patents

Description

TECHNICAL FIELD The present invention relates to a biosensor for detecting a specific component in a biological sample, and the biosensor can be widely applied to the medical field, food engineering and the like.

2. Description of the Related Art With the advancement of medical technology, it has become possible to understand health checks, disease states, and therapeutic effects by measuring specific components in blood and urine. However, in the past, because clinical laboratory laboratories in hospitals use large machines and complicated methods,
There was a problem that it took time and money. Therefore, a sensor that can more easily perform on-site measurement is desired. As one of the attempts, a multi-layer type 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 an overlayer 13 are sequentially laminated on a transparent support 9. When a blood sample is dropped from the upper portion, solid components such as red blood cells and platelets in blood are first removed by the overlayer 13, and the development layer 11 is passed through the small holes 14 in the waterproof layer 12.
Uniformly penetrates into the reagent layer 10 and the reaction proceeds in the reagent layer 10.
After the reaction, the substrate concentration is measured by spectroscopic analysis by shining light from the direction of the arrow through the transparent support 9. This method has an 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 a long time for blood to permeate and react, so that a waterproof layer 12 for preventing the sample from drying is required, and it is necessary to incubate at a high temperature to accelerate the reaction. However, there is a problem that the device and the carrier are complicated.

Therefore, it is an object of the present invention to provide a biosensor capable of quickly and accurately measuring a substrate with a simple structure, avoiding the complication of the device and carrier which are the above problems.

Means for Solving the Problems In the present invention, a liquid retaining layer, an overlayer, and a reaction layer are sequentially installed on an electrode part.

Action When blood is dropped, the oxidoreductase and the oxidative dye conjugated with the enzyme react immediately in the reaction layer. Red blood cells and platelets are then passed in the overlayer. further,
The reaction liquid, which has passed the liquid-retaining layer on which nothing is supported, is promptly guided to the electrode portion, where the reaction amount is detected by the electrode reaction. As described above, since the blood sample is overreacted in a short time, the substrate can be accurately measured with a simple device and carrier.

Example A glucose sensor will be described as an example of one of biosensors. Glucose oxidase was used as the oxidoreductase, and potassium ferricyanide was used as the oxidative dye coupled with the oxidoreductase. FIG. 1 shows a schematic view of an embodiment of the glucose sensor. Platinum is embedded in an insulating substrate 1 made of vinyl chloride resin, and a measurement electrode 2 and a counter electrode 3
And an electrode system composed of the reference electrode 4 was constructed. Rayon paper 5 was placed as a liquid retaining layer so as to cover the electrode system. A polycarbonate porous film 6 having a pore diameter of 1 μm was placed thereon to form an overlayer, and a pulp nonwoven fabric 7 was placed as a reaction layer on the top. The pulp nonwoven fabric 7 was impregnated with a high-concentration solution of glucose oxidase 200 mg and potassium ferricyanide 400 mg dissolved in 1 cc of a phosphate buffer (pH 5.6) beforehand, and was then impregnated in an organic solvent having a high solubility in water such as ethanol. After being dipped in vacuum, it is vacuum-dried to carry fine crystals of enzyme and dye at high density.

30 μl of blood is used as a sample liquid on the pulp nonwoven fabric 7.
After adding 1 and allowing it to permeate sufficiently, the reference electrode 4 is used as a reference for the measurement electrode 2
The voltage was varied between 0 and + 0.1V in a sawtooth manner at 0.1V / 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 glucose in the added blood is oxidized by glucose oxidase carried on the pulp nonwoven fabric 7, potassium ferricyanide is reduced by an enzyme-dye coupling reaction to produce potassium ferrocyanide. When the subsequently reacted blood passes through the polycarbonate porous film 6, large solid components such as red blood cells are passed. In the case of a highly viscous and trace amount sample such as blood, it is difficult to pass, but by installing a hydrophilic thin film like rayon paper 5 underneath, it can be quickly passed and plasma reacted to the electrode part ( The excess liquid reaches, and the rayon paper 5 holds the reaction liquid uniformly on the entire surface of the electrode portion. The potassium ferrocyanide in the reaction solution is oxidized by sweeping the voltage of the measuring electrode 2, and an oxidation current flows at that time. This oxidation current is proportional to the amount of change in the dye, and if the dye is sufficiently present, the amount of change in the dye corresponds to the concentration of the substrate. Therefore, by measuring the current value, the concentration of glucose, which is the substrate, can be detected. Using this glucose sensor, a high-concentration curcose of 400 mg / dl could be measured in a short time of 2 minutes. This is a structure in which the reaction is performed first instead of passing it over as in the conventional example, and the enzyme and dye that can sufficiently support a high-concentration substrate are easily dissolved in a short time because they are carried in a state in which they are easily dissolved. It is considered that the reaction was completed. Further, by placing a hydrophilic thin rayon paper 5 under the overlayer, a minute amount of blood of only 30 μl was promptly applied and uniformly spread on the electrode, and a stable response current was obtained. .

In the above structure, except for the rayon paper 5, which is the liquid retaining layer, the blood cannot be smoothly passed, and it took time for a sufficient amount of the reaction liquid to permeate the electrodes. Further, since the spread of the reaction solution on the electrode is non-uniform, the variation of the response current becomes large and the measurement cannot be performed accurately.

In the above example, thin rayon paper with a thickness of 50 μm was used. However, if the thickness is increased, the amount of retained liquid increases and it takes time to permeate a sufficient amount of the reaction liquid into the electrodes, and the blood volume also increases. Needed. When an enzyme or dye was carried on rayon paper, the contact surface with the overlayer was crystallized by the enzyme or dye, and the number of contact points decreased. From the above,
The liquid-retaining layer is preferably a porous thin film that is hydrophilic and does not carry anything.

A carrier consisting of a liquid-retaining layer for measurement, an overlayer, and a reaction layer may be directly placed on the surface of the electrode part.However, when the carrier is heavy or is placed under pressure, the carrier of the measurement carrier is directly attached to the electrode surface. When a part of them come into contact with each other, the electrode reaction area becomes small, and the current value becomes small and easily varies. Therefore, in such a case, as shown in FIG. 2, it is necessary to provide a groove 8 in the electrode portion so that the measuring carrier and the electrode surface do not come into contact with each other.

A liquid retaining layer is necessary in order to allow a sufficient amount of a perfusion of a small amount of blood to permeate the electrode portion having the above shape. Furthermore, by making the shape of the liquid retaining layer cover only the upper part of the groove 8, the excess liquid can be concentrated in the groove 8 and the groove 8 can be effectively filled with a small amount of liquid. It was This makes it possible to perform measurements without directly contacting the measurement carrier with the electrode surface,
A reproducible response current 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, the addition amount was changed to 30 to 100 μl and the measurement was performed, and the same blood showed a constant value regardless of the addition amount. Therefore, it is not necessary to accurately add the amount, and the measurement can be easily performed by adding a very small amount of blood. Furthermore, since the reaction is completed in a short time of 2 minutes by using a high concentration of the enzyme and the oxidative dye, a device for incubating at a high temperature and a waterproof layer for preventing evaporation are unnecessary, and a simple device and a carrier are provided. I was able to measure accurately.

Although rayon paper was used as the liquid retaining layer in the above example, a hydrophilic and thin porous film is desirable in order to quickly spread a small amount of liquid from the overlayer onto the electrode. In addition to rayon paper, paper and nylon non-woven fabric could also be used.

As the dye, potassium ferricyanide used in the above-mentioned examples is suitable because it reacts stably, but when P-benzoquinone is used, the reaction rate is fast, and therefore it is suitable for speeding up.
Further, 2,6-dichlorophenolindophenol, methylene blue, phenazine methosulfate, potassium β-naphthoquinone 4-sulfonate and the like can also be used.

The sensor in the above-mentioned examples is not limited to glucose, but can be used in systems involving oxidoreductase such as alcohol sensor and cholesterol sensor. Glucose oxidase was used as the oxidoreductase, but other enzymes such as alcohol oxidase, xanthine oxidase, and cholesterol oxidase can also be used.

EFFECTS OF THE INVENTION According to the sensor of the present invention, it is possible to accurately measure a specific component in a short time by directly applying a small amount of sample.

[Brief description of drawings]

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 electrode, 3 ... Counter electrode, 4 ... Reference electrode, 5 ... Liquid retaining layer, 6 ... Overlayer, 7 ... Reaction layer, 8 ...
…groove.

Claims (2)

[Claims] 1. A superlayer composed of a liquid-retaining layer and a porous membrane, an oxidoreductase, and the above enzyme are conjugated on an electrode portion provided with an electrode system comprising a measuring electrode, a counter electrode and a reference electrode on an insulating substrate. A biosensor characterized in that reaction layers containing an oxidizing dye are sequentially installed. 2. The biosensor according to claim 1, wherein the liquid retaining layer is made of a hydrophilic porous material.