JPH0640089B2 - Biosensor - Google Patents

Description

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

Conventional technology With the advancement of medical technology, it has become possible to understand the health check, disease state, and therapeutic effect by measuring specific components in blood and urine. However, conventionally, there is a problem that it takes time and cost because the examination is performed in a clinical laboratory of a hospital with a large machine or a complicated method. 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. 2 has been proposed. It has a structure in which a reagent layer 13, a spreading layer 14, a waterproof layer 15, and a filtration layer 16 are sequentially laminated on a transparent support 12. When a blood sample is dropped from the upper part, first, solid components such as red blood cells and platelets in blood are removed by the filtration layer 16, and the development layer 14 is passed through the small holes in the waterproof layer 15.
Uniformly penetrates into the reagent layer 13 and the reaction proceeds in the reagent layer 13.
After completion of the reaction, light is applied from the direction of the arrow through the transparent support 12 and the substrate concentration is measured by spectroscopic analysis.

Problems to be Solved by the Invention This method has an advantage that measurement can be easily performed by dropping a small amount of blood. However, since it takes time for blood to permeate and react, a waterproof layer 15 for preventing the sample from drying is required, and it is necessary to incubate at a high temperature to accelerate the reaction, which complicates the device and the carrier. is there.

An object of the biosensor of the present invention is to avoid the problem that the device and the carrier are complicated and to measure the substrate quickly and accurately with a simple device and carrier.

Means for Solving Problems The biosensor of the present invention has a liquid retaining layer, a filtration layer, a reaction layer, and a sample addition layer provided on the electrode portion in order to achieve the above object.

Operation When blood is dropped on such a sensor, it spreads evenly in the sample addition layer and is quickly supplied to the reaction layer. In the reaction layer, the oxidoreductase and the oxidative dye conjugated with the enzyme react immediately. Red blood cells and platelets are then filtered in the filtration layer. Furthermore, the liquid-retaining layer on which nothing is supported promptly guides the filtered reaction liquid to the electrode portion, where the reaction amount is detected by the electrode reaction. As described above, since the blood sample reacts and is filtered in a short time, the substrate can be accurately measured with a simple device and carrier.

Example A glucose sensor will be specifically described below as one of the 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 in cross-sectional views A and B of planes orthogonal to each other. The electrodes are formed on the insulating substrate 1 made of polyvinyl chloride resin by forming a groove having a width of 3.4 mm and a depth of 0.15 mm as the space 2, and burying platinum, and the measurement electrode 3, the counter electrode 4, and An electrode system including the reference electrode 5 was constructed. A measurement chip sandwiching the sample addition layer 9, the reaction layer 8, the filtration layer 7, and the liquid retaining layer 6 is placed on the frames 10 and 11 so as to cover the electrode system. The sample addition layer 9 is made of Heisegase (manufactured by Asahi Chemical Industry Co., Ltd.), which is a cellulose woven fabric. The reaction layer 8 is made of pulp non-woven fabric, and glucose oxidase (200 mg) and potassium ferricyanide (400 mg) are respectively added to the phosphate buffer solution (pH).
5.6) Impregnate a high-concentration solution dissolved in 1 cc, immerse it in an organic solvent having a high solubility for water such as ethanol, and then vacuum-dry it to support fine crystals of glucose oxidase and potassium ferricyanide in high density. There is. The filtration layer 7 is a polycarbonate porous film having a pore diameter of 1 μm and removes solid components such as red blood cells in blood. As the liquid retaining layer 6, band-shaped rayon paper having a width of 2 mm was used. The face end of rayon paper is fixed to the frame, and the width of the electrode part is 3.4.
It is held in a retractable position inside the mm groove. The filter layer 7 of the measuring chip is supported by the portion other than the groove of the electrode portion as shown in the section B of FIG. The sample addition layer 9, the reaction layer 8, the filtration layer 7, and the liquid retaining layer 6 are used as the frame 1
It is fixed by pressure bonding using 0 and 11 or by an adhesive such as an epoxy resin.

After adding 30 μl of blood to the sample solution addition layer 9 and allowing it to sufficiently permeate, the voltage of the measurement electrode 3 is set to 0 to +0.1 with reference to the reference electrode 5.
The voltage was changed in a sawtooth manner at 0.1 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. The added blood is Hize Gauze 9
Is spread over the entire surface and supplied to the reaction layer 8. When glucose in blood is oxidized by glucose oxidase supported on the nonwoven fabric 8 of pulp, potassium ferricyanide is reduced by an enzyme-dye coupling reaction to produce potassium ferrocyanide. Subsequently, when the reacted blood passes through the polycarbonate porous film 7, large solid components such as red blood cells are filtered. It is difficult to filter a very small amount of sample with high viscosity like blood,
By installing a hydrophilic thin film such as rayon paper 6 underneath, filtration can be performed quickly. Further, the filtered reaction liquid is spread evenly on a band-shaped rayon paper and is supplied to the electrode portion thereunder. The potassium ferrocyanide in the reaction solution is oxidized by sweeping the voltage of the measuring electrode 3, and the oxidation current flowing at that time is measured. This oxidation current is proportional to the change amount of the dye, and if the dye is sufficiently present, the change amount of the dye corresponds to the substrate concentration, so that the glucose concentration can be detected. Using this glucose sensor, glucose with a high concentration of 400 mg / dl could be measured in a short time of 2 minutes. This is a structure in which the reaction is first carried out instead of being filtered to carry out the reaction as in the conventional example, and the enzyme and the dye capable of sufficiently supporting a high-concentration substrate are carried in an easily melted state. It is considered that the reaction was completed in a short time. Further, since the high-gasease 9 having a high affinity for blood is installed on the reaction layer 8, even high-viscosity blood is quickly spread over the entire surface and supplied to the reaction layer 8, so that even a small amount of blood of 15 μl is stable. The resulting response current is obtained. In the case where the hezegase 9 is not present, when the blood has a high viscosity, the spread on the reaction layer 8 is poor, and it may take about 1 minute for the reaction solution to reach the electrode part.
When the Hize Gauze 9 was placed on the top, the added blood was spread over the entire surface and supplied to the reaction layer within 1 second, and the reaction solution of most of the blood sample reached the electrode portion in about 30 seconds. When the reaction layer 8 is loaded with a surfactant, the spread and penetration of blood is improved, but depending on the concentration of the surfactant, hemolysis may occur, which may cause a measurement error. However, by using Hizegase 9, it is not necessary to carry a surfactant on the reaction layer 8, hemolysis is not observed, and blood uniformly penetrates into the reaction layer, and a response with good reproducibility was obtained. Furthermore, when blood is added and blood is directly measured by inserting a needle into the finger, if the reaction layer 8 directly touches the finger, enzymes or dyes may be dissolved in the wound and there is a risk of contamination. By doing so, it became possible to safely add blood.

As the sample addition layer 9, hizegase was used in the examples. This porous membrane is a non-woven fabric made of defatted cotton long fibers, and has high affinity with blood because it is a highly pure cellulose. In addition, since it is a nonwoven fabric processed from long fibers, the added blood spreads over the entire surface in less than 1 second if the area has a diameter of 5 mm, so even a highly viscous blood can effectively supply a small amount of sample to the reaction layer. We were able to. Furthermore, since the non-woven fabric having a large porosity, the added blood was retained in the sample addition layer 9 and the supply to the reaction layer was not disturbed. In addition to Hize Gauze, non-woven fabric made of cotton, non-woven fabric of pulp, etc. could also be used.

In the embodiment, the sample addition layer 9 is incorporated into the frame bodies 10 and 11 together with the reaction layer 8 and the like, but after the liquid retention layer 6, the filtration layer 7 and the reaction layer 8 are incorporated into the frame body, the sample liquid retention layer 9 is dropped. It may be installed in a stowed place.

Since the biosensor of the present invention does not require a diluting liquid other than the sample liquid, when the blood addition amount was changed to 15 to 100 μl, 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.

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 be used.

Incidentally, the sensor in the above embodiment is not limited to glucose, such as an alcohol sensor or a cholesterol sensor,
It can be used in a system involving oxidoreductase. Glucose oxidase was used as the oxidoreductase,
Other enzymes such as alcohol oxidase, xanthine oxidase, cholesterol oxidase and the like can also be used. The enzyme could be used even after being immobilized with a crosslinking agent or the like.

EFFECTS OF THE INVENTION As described above, according to the biosensor of the present invention, it is possible to accurately measure a specific component in a short time by simply dropping a small amount of sample.

[Brief description of drawings]

1A and 1B are cross-sectional schematic views of the glucose sensor of one embodiment of the present invention taken along a plane orthogonal to each other, and FIG. 2 is a schematic view of a conventional biosensor. 1 ... Substrate, 2 ... Groove, 3 ... Measuring electrode, 4 ... Counter electrode, 5
...... Reference electrode, 6 ...... Retaining layer, 7 ...... Filtration layer, 8 ...... Reaction layer, 9 ...... Sample addition layer, 10, 11 ...... Frame body, 12 ...... Support body, 13 ...... Reagent layer , 14 ... Development layer, 15 ... Filtration layer, 16 ... Waterproof layer.

Claims (2)

[Claims] 1. A filter layer comprising a liquid-retaining layer and a porous membrane and an oxidoreductase on an electrode portion having an electrode system comprising a measuring electrode, a counter electrode and a reference electrode provided on an insulating substrate through a space. And a reaction layer containing an oxidative dye that is conjugated with the enzyme is sandwiched between frames, and a sample addition layer made of a hydrophilic porous body is further provided on the reaction layer. 2. The biosensor according to claim 1, wherein the sample addition layer is made of cellulose.