JPH0660882B2 - Biosensor - Google Patents

Description

TECHNICAL FIELD 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 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, there is a demand for a sensor that can measure on the spot most easily.
As one of the attempts, a multi-layer type analytical carrier as shown in FIG. 5 has been proposed. Reagent layer 12 on transparent support 11,
It has a structure in which a spreading layer 13, a waterproof layer 14, and a filtration layer 15 are sequentially stacked. When a blood sample is dropped from the top,
First, solid components such as red blood cells and platelets in blood are removed by the filter layer 15, and the solid components uniformly penetrate into the spreading layer 13 through the small holes in the waterproof layer 14, and the reaction proceeds in the reagent layer 12. After completion of the reaction, light is applied from the direction of the arrow through the transparent support 11 to measure the substrate concentration by spectroscopic analysis. 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 time for blood to permeate and react, so that the waterproof layer 14 that prevents 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.

SUMMARY OF THE INVENTION It is an object of the present invention to avoid the complication of the device and the carrier, which are the above problems, and to enable the substrate to be measured quickly and accurately with a simple device and carrier.

Means for Solving the Problems The present invention provides an insulating substrate with an electrode layer having a measuring electrode, a counter electrode, and a reference electrode, and a hydrophilic liquid-retaining layer and a porous membrane on the electrode layer. And a reaction layer containing a redox enzyme and an oxidation dye that is conjugated with oxygen are sequentially laminated, and the liquid retaining layer has a smaller shape than the filtration layer.

Further, the present invention is a biotechnology in which a protrusion is provided on the peripheral portion of the liquid retaining layer, a hollow frame is provided between the electrode portion and the liquid retaining layer, and the protrusion of the liquid retaining layer is locked to the peripheral portion of the frame body. It is a sensor.

Further, specifically, a liquid retaining layer having protrusions on the periphery thereof, a filter layer disposed on the liquid retaining layer, and a reaction layer disposed on the filter layer have a bottom with an opening. It is housed in a hollow frame, the projection of the liquid retaining layer is locked to the edge of the bottom of the hollow frame, and the electrode layer is provided in the opening of the bottom through a space. .

Action When blood is dropped on the biosensor of the present invention, the oxidoreductase and the oxidative dye conjugated with this enzyme react immediately in the reaction layer. Red blood cells and platelets are then filtered in the filtration layer. The filtered reaction liquid reaches the lower liquid retaining layer. Since the liquid-retaining layer has a notched outer peripheral portion to form a space, the reaction solution spreads over the entire surface of the liquid-retaining layer and fills the space where the electrode is exposed. That is, when the shape of the liquid retaining layer is smaller than the outer shape of the filtration layer as in the present invention, the volume of the liquid retaining layer also becomes smaller and the amount of the test liquid per unit volume in the liquid retaining layer increases. Further, in the present invention, only a part of the liquid-retaining layer or the protruding portion is pressurized between the edge around the opening at the bottom of the hollow frame and the frame arranged above, but the opening at the bottom of the hollow frame is The part facing to will not be pressurized. Therefore, when the entire periphery of the liquid retaining layer is pressurized, the test liquid from the filtration layer accumulates around the liquid retaining layer, but in the present invention, the entire periphery of the liquid retaining layer is not pressurized. There is no liquid pool around the liquid retention layer. Therefore, the test liquid from the filtration layer is supplied to the entire liquid retention layer through the protrusions of the liquid retention layer, and the central part of the liquid retention layer does not become thin, and the liquid retention layer is removed from the filtration layer. The absorbed liquid can be quickly supplied to the electrode surface. In this way, the reaction liquid filtered by the liquid retaining layer is promptly guided to the electrode portion, where the reaction amount is detected by the electrode reaction. Since the blood sample reacts and is filtered in such 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 the configuration of an embodiment of the glucose sensor. A groove 2 having a depth of 0.1 mm is formed on the upper end surface of a cylindrical insulating substrate 1 made of vinyl chloride resin, and platinum is embedded so as to be exposed in the groove 2, and the measuring electrode 3, counter electrode 4, and reference electrode are formed. 5 constitutes an electrode system. A measuring chip sandwiching the reaction layer 8, the filtration layer 7 and the liquid retaining layer 6 with the frames 9 and 10 is placed so as to cover the electrode system.

The reaction layer 8 is made of pulp non-woven fabric, and glucose oxidase 200 mg and potassium ferricyanide 400 mg
A high-concentration solution of H5.6 in phosphate buffer (1 cc) was impregnated, immersed in an organic solvent having a high solubility for water such as ethanol, and then vacuum dried to obtain fine crystals of glucose oxidase and potassium ferricyanide at high density. It is carried on. 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. The liquid-retaining layer 6 is made of rayon paper, for example, and has a disk shape having a smaller diameter than the filtration layer 7 as shown in FIG. 2, but has a protruding portion 6a at its end and only the protruding portion is fixed to the frame body. I am trying to do it. FIG. 3 shows a view of the measurement chip viewed from the liquid retaining layer 6 side. The shape having a space around the liquid-retaining layer means the shape of the liquid-retaining layer such that a portion without the liquid-retaining layer is formed below the filtration layer 7 in FIG. The reaction layer 8, the filtration layer 7, and the liquid-retaining layer are fixed on the frames 9 and 10 by pressure bonding or epoxy resin.

After 30 μm of blood was added as a sample solution onto the reaction layer 8 and was sufficiently permeated, the voltage of the measurement electrode 3 was set to 0 with reference to the reference electrode 5.
The voltage was changed in a sawtooth shape between +0.1 V and 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. When glucose in the added blood is oxidized by glucose oxidase supported on the reaction layer 8, potassium ferricyanide is reduced by the enzyme-dye coupling reaction to produce potassium ferrocyanide. When the reacted blood subsequently passes through the polycarbonate porous film 7, large solid components such as red blood cells are filtered. It is difficult to filter a sample with a high viscosity and a small amount like blood, but by installing a hydrophilic thin film like rayon paper as a liquid retaining layer underneath, it is possible to filter quickly and to the electrode part. The reacted plasma (filtrate) reaches and the reaction liquid is uniformly held on the entire surface of the electrode portion by the liquid retaining layer 6. Measuring electrode for potassium ferrocyanide in reaction solution 3
It is oxidized by sweeping the voltage of, and at that time, an oxidation current flows. 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. With this glucose sensor, 400mg
High-concentration glucose of / dl could be measured in a short time of 2 minutes. This is a configuration in which the reaction is performed first instead of being filtered to carry out the reaction as in the conventional example, and the enzyme and the dye capable of sufficiently responding to a high-concentration substrate are easily supported in a short time because they are carried in a easily melted state. It is considered that the reaction was completed. Further, by placing a thin hydrophilic liquid retaining layer 6 under the filter layer, a very small amount of blood of only 30 μm can be quickly filtered and uniformly spread on the electrode to obtain a stable response current. . However, when the liquid retaining layer 6 is incorporated into the frames 9 and 10 with the same shape as the filtration layer 7, the filtered blood is fixed at the outer peripheral portion of the liquid retaining layer 6 by the frames 9 and 10. Since the blood is rapidly filtered and blood plasma accumulates in that portion, the groove 2 of the electrode portion may not be filled with the liquid.
Therefore, if a space is provided in the outer peripheral portion where the liquid is easily accumulated as shown in FIG. 1, the filtered blood quickly fills the entire surface of the liquid holding layer 6 with the liquid. It was By providing this groove 2, it is possible to prevent the measuring tip from coming into direct contact with the electrode surface, so that the reaction area of the measuring electrode 3 is always kept constant and a response with good reproducibility is obtained. Further, the area of the liquid retaining layer 6 was reduced by forming the space portion, and the amount of blood added could be further reduced to 20 μm. If the liquid retaining layer 6 is removed, the plasma cannot be filtered immediately,
It took time for an insufficient amount of reacted plasma (reaction solution) to penetrate onto the electrode. Further, since the spread of the reaction liquid on the electrode is non-uniform, the variation of the response current becomes large and the measurement cannot be performed accurately.

In this example, a thin film of rayon paper having a thickness of 50 μm was used as the liquid retaining layer. However, as the thickness is increased, the amount of retained liquid increases, and it takes time to allow a sufficient amount of the reaction liquid to permeate into the electrode. Needed a lot too. When an enzyme or dye was carried on the rayon paper, the contact surface on the contact surface with the filter layer 7 was reduced due to the crystals of the enzyme or dye and the filtration took time.
From the above, it is desirable that the liquid retaining layer 6 is a hydrophilic thin film and is a porous body on which nothing is supported. Further, the shape of the liquid retaining layer other than the shape shown in FIG. 2 can be used as shown in FIG. 4, and can be variously changed according to the shape of the substrate 1 and the shapes of the frames 9 and 10. It is possible.

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 30 to 100 μ, 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, a hydrophilic and thin porous film is desirable in order to quickly spread a small amount of liquid from the filter layer onto the electrode. In addition to rayon paper, it was possible to use litter paper or nylon non-woven fabric.

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, a specific component can be accurately measured in a short time simply by dropping a small amount of sample directly.

[Brief description of drawings]

FIG. 1 is a longitudinal sectional view of a glucose sensor according to an embodiment of the present invention, FIG. 2 is its exploded perspective view, and FIG. 3 is FIG. 1 III-II.
FIG. 4 is a plan view showing a modified example of the liquid retaining layer,
FIG. 5 is a vertical sectional 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 and 10 ...... Frame body, 11 ...... Support body, 12 ...... Reagent layer, 13 ...... Development layer, 14 ... filtration layer, 15 ... waterproof layer.

Claims (2)

[Claims] 1. An insulating base is provided with an electrode layer having a measuring electrode, a counter electrode and a reference electrode, and a hydrophilic liquid-retaining layer, a filtration layer made of a porous membrane, and redox are provided on the electrode layer. A biosensor comprising a reaction layer containing an enzyme and an oxidation dye that is conjugated with the enzyme, which are sequentially laminated, and the shape of the liquid retaining layer is smaller than the shape of the filtration layer. 2. A hollow having a bottom with an opening, a liquid-retaining layer having protrusions on its periphery, a filter layer disposed on the liquid-retaining layer, and a reaction layer disposed on the filter layer. It is housed in a frame body, the projection of the liquid retaining layer is locked to the edge portion of the bottom portion of the hollow frame body, and the electrode layer is provided in the opening portion of the bottom portion through a space portion. The biosensor according to claim 1.