JPS62232554A - Biosensor - Google Patents

Abstract

PURPOSE:To easily measure the concn. of the substrate in a biotic specimen without generating the irregularity of a measured value, by integrating an insulating base plate, an electrode system and a porous body supporting oxidoreductase and an electron acceptor and further covering the surface of an electrode with protein. CONSTITUTION:A carbon paste is printed on a polyethylene terephthalate base plate 1 to form an opposed electrode 2, a measuring electrode 3 and a reference electrode 4. Parts 2', 3', 4' are selectively left to cover other parts with an insulating layer 5 and an aqueous albumin solution is dripped and dried. A resin frame 6 having an opening is adhered to the insulating layer 5 and a porous body 7 is held in the opening and a resin cover having perforations having a diameter smaller than that of the porous body is adhered to the resin frame 6 to integrate the whole. The porous body is formed by impregnating a nylon nonwoven fabric with a solution prepared by dissolving glucose oxidase and potassium ferricyanate in a phosphate buffer solution with pH5.6 and drying the impregnated one. A glucose standard solution is dripped on the porous body and the potential of the measuring electrode is swept to an anode on the basis of the reference electrode. At this time, an oxidizing current is obtained and a current value corresponds to the concn. of glucose being a substrate.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a biosensor that can quickly and easily quantify specific components in various minute amounts of biological samples without diluting the sample liquid.

Conventional technology Traditionally, specific components in biological samples such as blood have been tested,
A biosensor as shown in FIG. 4 has been proposed as a method for highly accurate quantitative determination without operations such as diluting or stirring the liquid solution (for example, Japanese Patent Application Laid-Open No. 166852/1982). This biosensor has a lead 1 on an insulating substrate 9.
Measuring electrodes 10 made of platinum or the like, each having 2.13
and a counter electrode 11 are buried, and the exposed portions of these electrode systems are covered with a porous material carrying an oxidoreductase and an electron acceptor. When a sample solution is dropped onto a porous material, the oxidoreductase and electron acceptor in the porous material are dissolved in the sample solution, an enzymatic reaction proceeds with the substrate in the sample solution, and the electron acceptor is reduced. . After the enzymatic reaction is completed, the reduced electron acceptor is electrochemically oxidized, and the substrate concentration in the sample solution is determined from the oxidation current value obtained at this time.

Problems to be Solved by the Invention In such a conventional configuration, the porous body can be easily used for measurement by replacing it for each measurement, but the electrode system requires operations such as cleaning. On the other hand, if the electrode system, including the electrode system, could be disposed of after each measurement, the measurement operation would be extremely simple, but it would be extremely expensive due to the electrode materials such as platinum and the structure. do not have.

As a result of various studies on these points, the present invention has developed an inexpensive disposable type biotechnology that can extremely easily quantify specific components in biological samples quickly and with high precision by integrating an electrode system and a porous body. It provides a sensor.

Means to Solve the Problems In order to solve the above-mentioned problems, the present invention provides an electrode system consisting of at least a measurement electrode and a counter electrode on an insulating substrate, and detects changes in substance concentration during reactions between enzymes, electron acceptors, and sample liquids. In a biosensor that electrochemically detects substrate concentration in a sample solution by electrochemically detecting it with the electrode system, the surface of at least the measurement electrode of the electrode system is coated with protein in advance, and further coated with a redox enzyme and an electron acceptor. It is integrated with the supported porous body. This can prevent measurement variations due to adsorption of proteins, etc. in the sample solution, which may be expected in advance.

Effects According to the present invention, a disposable type biosensor including an electrode system can be constructed, and the substrate concentration can be extremely easily measured by adding a sample liquid to a porous body.

Moreover, by coating the surface of the electrode system with protein in advance, measurement variations due to adsorption of proteins in the sample solution are eliminated, making it possible to perform highly accurate measurements.

Example An embodiment of the present invention will be described below.

A glucose sensor will be described as an example of a biosensor. FIG. 1 shows an embodiment of a glucose sensor, and is an exploded view of the constituent parts. A counter electrode 2. is formed by printing a conductive carbon paste on an insulating substrate 1 made of polyethylene terephthalate by screen printing and drying it by heating. Measuring pole 3. An electrode system consisting of a reference electrode 4 is formed. 2' then partially covers the electrode system and becomes the electrochemically active part of each electrode.

An insulating paste is printed in the same manner as above so as to leave s/, 4/ (1 ffiIIP each), and heat treated to form an insulating layer 5.
Albumin (rs o mg/cc) was added to coat the exposed surfaces of this electrode system (2/, 3/, 4/) to form a
) was added dropwise and dried.

Next, a resin holding frame 6 with holes is glued to the insulating layer 6,
The porous body 7 is held in the hole so as to cover the electrode systems 2', 3', and 4'. Furthermore, a resin cover 8 having an opening having a diameter smaller than that of the porous body is adhered to integrate the entire body.

A cross-sectional view along the measurement electrode 3 of this integrated biosensor is shown in FIG. The porous body used above contained 100 mf of glucose oxidase as an oxidoreductase.
and potassium ferricyanide 150 mf as an electron acceptor.
This material was prepared by impregnating a nylon nonwoven fabric with a solution dissolved in 1 ml of phosphate buffer with a pH of 6.6 and drying it under reduced pressure.

Glucose standard solution is dropped as a sample solution into the porous body of the glucose sensor configured as above, and 2 minutes after dropping, the potential of the measurement electrode is changed to o, 1v toward the anode with reference to the reference electrode.
/second. In this case, the added glucose reacts with potassium ferricyanide by the action of glucose oxidase supported on the porous material to produce potassium ferrocyanide. Therefore, by the above-mentioned sweep toward the anode, an oxidation current based on the concentration of potassium ferrocyanide produced is obtained, and this current value corresponds to the concentration of glucose, which is the substrate.

A serum sample was dropped onto the noglucose sensor, and the peak current value obtained 2 minutes later had very good reproducibility as shown in Figure 3.

B in FIG. 3 was measured in the same manner as humans when no protein was coated on the surface of the electrode system, and the peak current value varied widely. This is thought to be because proteins in serum were adsorbed on the surface of the carbon paste of the electrode material, and the degree of adsorption varied with each measurement, causing the peak current to vary.

However, if the carbon paste is treated with albumin in advance, all of the adsorption sites on the surface of the carbon paste will have albumin adsorbed, which will prevent the measured values from varying due to adsorption by proteins in the sample solution, resulting in a response with good reproducibility. can get.

Instead of albumin, an aqueous solution of glucose oxidase (100 mg/cc) was used to coat the surface of the electrode system. In this case as well, a response with good reproducibility was obtained as with albumin. Furthermore, like albumin, the glucose oxidase coating made the electrode surface hydrophilic and improved wetting, so that even a small amount of sample liquid quickly spread over the electrode surface and a response was obtained. In addition, since it is very easy to wet, no wrinkles are formed on the electrode surface. moreover,
By coating the electrode system with glucose oxidase, the amount of redox enzyme involved in the enzymatic reaction increases, and a sufficient amount of electron acceptor (potassium ferricyanide) is transferred to the porous material 7.
If it was supported on the membrane, it became possible to measure a higher glucose concentration than when coated with albuman. If glucose oxidase is supported at a high density on the electrode surface, if only potassium ferricyanide is supported on the porous body 7 and a sample solution is dropped, the sample solution in which potassium ferricyanide is dissolved can react with glucose oxidase on the electrode surface. .

The protein to be coated is not limited to albumin or glucose oxidase. Further, the coating method is not limited to the method of dropping an aqueous protein solution and drying, but may also be immersion in an aqueous protein solution followed by washing with water and drying, or immobilization of enzymes and the like with a crosslinking agent.

Screen printing as a method for forming electrode systems can produce disposable biosensors with uniform characteristics at low cost, and in particular, it is possible to fabricate electrodes using carbon, which is an inexpensive and stable electrode material. This is a convenient way to form.

The method of integration in the biosensor of the present invention is as follows:
The present invention is not limited to the shapes and combinations of frames, covers, etc. shown in the examples. In addition, the porous body used is
In addition to nylon nonwovens, porous bodies made of cellulose, rayon, ceramic, polycarbonate, etc. can be used alone or in combination. Further, the combination of oxidoreductase and electron acceptor is not limited to the above examples, and any combination can be used as long as it meets the gist of the present invention. On the other hand, in the above embodiment, a three-electrode force type electrode system is described, but measurement can also be performed using a two-electrode force type consisting of a counter electrode and a measurement electrode.

Effects of the Invention As described above, the biosensor of the present invention extremely easily adjusts the substrate concentration in a biological sample by integrating an insulating substrate, an electrode system, and a porous body carrying an oxidoreductase and an electron acceptor. can be measured, and the electrode surface is coated with protein to improve reproducibility.

[Brief explanation of drawings]

Fig. 1 is an exploded perspective view of a biosensor that is an embodiment of the present invention, Fig. 2 is a longitudinal cross-sectional view thereof, Fig. 3 is a response characteristic diagram of the biosensor, and Fig. 4 is a longitudinal cross-section of a conventional biosensor. It is a diagram. 1...Substrate, 2...Counter electrode, 3...
...Measurement pole, 4...Reference, pole, 6...
Insulating layer, 6... Holding frame, 7... Porous body, 8... Cover. Name of agent: Patent attorney Toshio Nakao and 1 other person 2nd
Figure 3 Figure 4

Claims (5)

[Claims] (1) An insulating substrate is provided with an electrode system consisting of at least a measurement electrode and a counter electrode, and the electrode system electrochemically detects a change in substance concentration during the reaction between an enzyme, an electron acceptor, and a sample liquid. A biosensor for measuring the substrate concentration of a biosensor, characterized in that the surface of at least the measuring electrode of the electrode system is coated with a protein in advance and is further integrated with a porous body carrying an oxidoreductase and an electron acceptor. biosensor. (2) The biosensor according to claim 1, wherein the electrode system includes a measurement electrode, a counter electrode, and a reference electrode. (3) The biosensor according to claim 1 or 2, wherein the electrode system is made of a carbon-based material formed by screen printing on an insulating substrate. (4) The biosensor according to claim 1, wherein the protein covering the surface of the electrode system is an oxidoreductase used in an enzyme reaction. (5) The biosensor according to claim 1, wherein the protein covering the surface of the electrode system is albumin.