JPH0452893B2 - - Google Patents

Description

[Detailed Description of the Invention] Industrial Application Field The present invention is a method for quickly and efficiently extracting specific components from various biological samples.
The invention also relates to a biosensor that can be easily quantified.

Configuration of conventional examples and their problems Recently, various biosensors that utilize the specific catalytic action of enzymes have been developed, and their application to the field of clinical testing is being attempted. Nowadays, as the number of test items and specimens increases, a bioseparator that can quickly and accurately perform measurements is desired.

Taking glucose sensors as an example, in order to measure and manage blood sugar levels in today's world where diabetes is rapidly increasing, it is no longer possible to centrifuge blood cells and other formed components and extract plasma from the blood, as in the past. Since it takes a very long time to measure, there is a need for a sensor that can measure whole blood. The simple type is similar to the test strips used in urine tests, and contains an enzyme that reacts only with sugar (glucose) and a dye that changes during the enzyme reaction or depending on the product of the enzyme reaction, on a stick-like support. The one with a carrier installed is required. This method involves adding blood to this carrier and measuring the change in pigment after a certain period of time by eye or light.
Accuracy is low due to interference from pigments in the blood.

Therefore, a multilayer analytical carrier as shown in FIG. 1 has been developed. A reagent layer 2 on a transparent support 1,
It has a structure in which a spreading layer 3, a waterproof layer 4, and a filtration layer 5 are laminated in this 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 5, uniformly permeates into the development layer 3 through the small holes 4a in the waterproof layer 4, and reacts in the reagent layer 2. progresses. After the reaction is completed, light is irradiated through the transparent support 1 in the direction of the arrow, and the substrate concentration is measured by spectroscopic analysis. Although it has a more complex structure than the conventional simple stick-shaped carrier, it has improved accuracy due to blood cell removal and other factors. However, since it takes time for blood to permeate and react, a waterproof layer 4 is required to prevent the sample from drying out, and it is necessary to incubate at a high temperature to speed up the reaction, making the equipment and carrier complex. be.

Recently, biosensors have been developed that measure substrate concentration by combining enzyme reactions and electrode reactions. Taking a glucose sensor as an example, as shown in Figure 2,
The glucose oxidase immobilized electrode 6 is placed in a container 7, filled with a buffer solution 8, and while stirring with a stirrer 9, a sample solution is added. A constant voltage is applied to the glucose oxidase immobilized electrode 6,
Hydrogen peroxide produced by reaction with glucose in the sample is detected, and a current flows to measure the glucose concentration. Using this method, measurements can be made quickly without being interfered with by pigments in the blood. However, since a stirring device is essential, there are problems in that bubbles occur and the turbulence of the liquid affects accuracy. Furthermore, since the method is diluted, precision is required in the amount of buffer solution and the amount of sample added, making the operation complicated.

OBJECTS OF THE INVENTION It is an object of the present invention to overcome the above-mentioned problems and to obtain a biosensor that can easily, quickly, and accurately measure a specific component in a biological sample.

Structure of the Invention The biosensor of the present invention has an electrode system consisting of at least a measurement electrode and a counter electrode on an insulating substrate, and the electrode system contains at least an oxidoreductase and an oxidized dye conjugated with the oxidoreductase. It is characterized in that it is laminated and coated with a reaction layer consisting of a membrane filter and a filtration layer consisting of a membrane filter having a pore size of 3 μm or less.

By using the biosensor of the present invention, biological samples can be measured easily and accurately.

Description of Examples A glucose sensor will be described as an example of one of the biosensors of the present invention. FIG. 3 shows a schematic diagram of an embodiment of a glucose sensor. Platinum is embedded in an insulating substrate 10 made of vinyl chloride resin to form a measurement electrode 11 and a counter electrode 12. A nylon nonwoven fabric 13 is placed so as to cover the electrode system. The mesh spacing of this nylon nonwoven fabric 13 is quite coarse, with a diameter of 6~
Although blood cells of 7 μm easily pass through, glucose oxidase 14 as an oxidoreductase and potassium ferricyanide 15 as an oxidized dye conjugated with the oxidoreductase are supported in a dry state after dissolution and impregnation. A filtration layer 16 made of porous polycarbonate (pore diameter: 1 μm) is installed on top of this nylon nonwoven fabric 13.

When blood is added to this sensor, large molecules such as red blood cells are passed through the filtration layer, and when glucose in the blood is oxidized by glucose oxidase 14 in the reaction layer made of nylon nonwoven fabric 13, potassium ferricyanide 15 is conjugated and reduced. and potassium ferrocyanide is produced. This potassium ferrocyanide is oxidized by sweeping the potential of the measurement electrode 11 from 0V to +0.5V at a rate of 0.1V/sec with respect to the counter electrode 12. The oxidation current obtained at this time is proportional to the concentration of potassium ferrocyanide, and since potassium ferrocyanide is produced in proportion to the substrate concentration, the concentration of glucose, which is the substrate, can be detected by measuring the oxidation current. When the obtained current value was measured using a glucose standard solution, it showed good linearity with the glucose concentration up to 800 mg/dl. The reaction layer and filtration layer consisting of enzyme and oxidized dye were replaced after each measurement.
Reproducibility was good for both standards and blood samples. Also, the amount of blood added should be 20 to 140μ.
However, since the amount of oxidized dye and enzyme was sufficient, it showed a constant value regardless of the amount added.

By using a porous polycarbonate material having a large number of fine pores with a pore diameter of 3 μm or less as the filtration layer 16, blood cells and viscous substances in the blood can be filtered out in advance, and staining of the electrodes can be reduced. Platinum is a very stable material, making it ideal for electrodes. However, if there is no filtration layer that allows liquid components in the blood to pass through but blocks solid components such as blood cells from passing through, blood cells will adhere to the electrodes over long periods of use, resulting in a decrease in the current value obtained. Therefore, it was necessary to wash the electrodes with alcohol, but with the filtration layer, the response can now be maintained with good reproducibility just by washing the electrodes with water. Also, when a porous polycarbonate body is used as a surfactant, for example, by immersing it in a 1% solution of polyethylene glycol alkyl phenyl ether (trade name: Triton X) and then drying it,
Blood filtration became faster and reproducibility was further improved. Since blood is quite viscous, there was a problem that the filtration rate was slow. However, by using a filtration layer treated with a surfactant, filtration is quick, and the reaction with enzymes and dyes is quick and uniform, allowing sample The reaction was completed within a short time of about 1 minute after the addition. If no surfactant is used,
It took about 1 minute and 30 seconds for the reaction to complete after blood was added, so this had a great effect on speeding up the measurement.

When measuring with a two-electrode system using platinum for the measurement electrode and the counter electrode, when the area of the counter electrode was made sufficiently larger than that of the measurement electrode, the polarization of the counter electrode was reduced and a good response was obtained. Furthermore, by using silver silver chloride as the counter electrode, the potential becomes stable and the area of the counter electrode can be reduced, making it possible to miniaturize the device.

As shown in FIG. 4, platinum is embedded in a vinyl chloride resin substrate 10, and a measurement electrode 11 and a counter electrode 12 are embedded.
The electrode system was composed of three electrodes including a reference electrode 17 and a reference electrode 17. By using three electrodes using a reference electrode,
The potential became stable and response reproducibility improved. Also,
As mentioned above, there is no need to increase the area of the opposite electrode, and the size can be reduced. To form the electrode system, platinum may be embedded in the resin as described above, but the electrode system may also be formed by forming a platinum layer on the substrate by vapor deposition or sputtering.

The reaction layer consisting of an oxidized dye and an enzyme is desirably a hydrophilic porous membrane so that it can quickly absorb a sample liquid and carry out an enzymatic reaction. For example, when filter paper, pulp nonwoven fabric, porous ceramic material, porous glass material, etc. were used, the sample liquid penetrated uniformly and quickly, and the reproducibility was also good. Furthermore, in the nylon nonwoven fabric treated with the above-mentioned surfactant, the sample liquid permeated more quickly than in the untreated fabric, which was effective in speeding up the measurement.

After finely pulverizing and mixing enzymes and oxidized pigments, pressurized living bodies were used as the reaction layer, and the liquid components of blood quickly dissolved the enzymes and oxidized pigments to mix uniformly, greatly contributing to speeding up the reaction. .
Furthermore, when press molding the oxidized dye and enzyme, if a small amount of SiO ₂ or the like is mixed as a binder, the strength of the molded product will increase, making it easier to handle. As the binder, a hydrophilic binder that is unrelated to enzyme reactions and electrode reactions is suitable.

It is desirable that the oxidized pigment and enzyme be in a state where they dissolve as quickly as possible in the liquid components of blood. Therefore, when a nylon nonwoven fabric was impregnated with an aqueous solution of the oxidized pigment and then dried with hot air, the fabric became much finer crystals than those dried in vacuum, making them easier to dissolve in liquids.
Furthermore, when a nylon nonwoven fabric soaked in an aqueous solution of an oxidized dye was immersed in an organic solvent with high solubility in water, such as ethanol, and then vacuum-dried, even finer crystals could be supported. Since enzymes are sensitive to heat, vacuum drying was performed after impregnation.

Therefore, we tried a sensor with the configuration shown in FIG. The electrode system is the same as that shown in FIG. 4, with a filtration layer 16 made of a porous polycarbonate membrane on top of the filtration layer 16, then a nylon nonwoven fabric 18 carrying glucose oxidase 14, and potassium ferricyanide 15 on top of it.
A nylon nonwoven fabric 19 is installed, which is impregnated, dipped in ethanol, dried, and supported. Note that the polycarbonate porous membrane and the nylon nonwoven fabric were previously treated with the above-mentioned surfactant.

When blood is added to this sensor, it quickly permeates the nylon nonwoven fabric layer, potassium ferricyanide 15 and glucose oxidase 14 are dissolved, and while the reaction progresses, only the liquid component of the blood passes through the filtration layer 16 and reaches the electrode system. Since potassium ferricyanide is supported in a fine crystalline state, it can be quickly dissolved and reacted by conjugating with the enzyme, and the reaction time can be shortened to within about 1 minute. The filtration layer may be placed on the electrode, as shown in FIG. 5, or on top of the reaction layer. Moreover, the dye-supporting layer 19 and the enzyme-supporting layer 18
You can put it in between. The permeation of the liquid was fastest when the filtration layer was placed below the reaction layer, and the reaction time was shortest. However, if a filtration layer is installed above the reaction layer, the solid components in the blood can be filtered out first, so there is no interference from blood cells in the reaction layer, so the reaction proceeds smoothly, resulting in high precision and Ta. Porous materials such as nonwoven fabric, chemical fibers, paper (filter), and glass can be considered as the filtration layer, but a membrane filter with a pore size of 3 μm or less is required to filter out the formed components of blood cells. Therefore, a membrane filter may be used as a single layer, or the above-mentioned nonwoven fabric, chemical fiber, paper, etc. may be laminated thereon.

As the surfactant, in addition to the above examples, polyoxyethylene glycerin fatty acid ester, polyoxyethylene alkyl ether, polyethylene glycol fatty acid ester, etc. can also be used. By treating not only the filtration layer but also the dye and enzyme with a surfactant, the filtration and dipping speeds can be further increased, and the reaction can also be faster.

Potassium ferricyanide used above is suitable as a dye because it reacts stably, but P-
If benzoquinone is used, the reaction rate is fast, so it is suitable for speeding up the reaction. Further, 2,6-dichlorophenol, indophenol, methylene blue, phenazine methosulfate, potassium β-naphthoquinone 4-sulfonate, etc. can also be used.

Note that the sensor in the above embodiments is not limited to glucose, and can be used in systems involving redox enzymes, such as alcohol sensors and cholesterol sensors. Furthermore, by supporting the enzyme in an immobilized state, the activity can be stably maintained even during long-term storage.

Effects of the Invention According to the sensor of the present invention, it is possible to directly impregnate a sample liquid and measure a trace amount of a characteristic component simply, quickly, and accurately. Furthermore, the filter layer allows the electrode to be stably maintained for a long period of time. Furthermore, surfactants can speed up penetration and shorten reaction time.

[Brief explanation of the drawing]

FIGS. 1 and 2 are schematic diagrams showing the configuration of a conventional glucose sensor, and FIGS. 3, 4, and 5 are schematic diagrams of a glucose sensor according to an embodiment of the present invention. 10... Substrate, 11... Measurement electrode, 12... Counter electrode, 13... Porous body (reaction layer), 14... Enzyme,
15...Dye, 16...Filtering layer, 17...Reference electrode.

Claims (1)

[Scope of Claims] 1. An electrode system consisting of at least a measurement electrode and a counter electrode is provided on an insulating substrate, and this electrode system is connected to a reaction layer and a filtration layer containing an oxidized-reductase and an oxidized dye conjugated with the oxido-reductase. A biosensor coated with a layer of 2. The biosensor according to claim 1, wherein the filtration layer is treated with a surfactant. 3. The biosensor according to claim 1, wherein the measuring station is platinum. 4. The biosensor according to claim 1, wherein the counter electrode is platinum or silver silver chloride. 5. The biosensor according to claim 1, wherein the reaction layer and the filtration layer are hydrophilic porous membranes. 6. The biosensor according to claim 5, wherein the oxidoreductase and the oxidized dye are held in a dry state in a hydrophilic porous membrane.