JPS633249A - Biosensor - Google Patents

Abstract

PURPOSE:To permit the easy measurement of the substrate concn. in a vital sample by integrating an insulating substrate, electrode system and porous body and further to improve reproducibility by preliminarily adsorbing a protein to the electrode surface. CONSTITUTION:A conductive carbon paste is printed on a substrate 1 and is dried by heating to form the electrode system consisting of a counter electrode 2, a measuring electrode 3 and a reference electrode 4. An insulating paste is then printed thereon so as to partially cover the electrode system and to leave 2'-4' which are the electrochemically working parts of the respective electrodes. The printing is then subjected to a heating treatment to form an insulating layer 5. An aq. soln. of albumin is dropped onto the substrate to cover the surface of the electrode systems 2'-4' and is rested for about 5-10 minutes, then the substrate is rinsed to remove the excess alubmin and is dried. The albumin is thereby adsorbed on the respective electrode surfaces. A holding frame 6 is then adhered to the insulating layer 5 and the porous body 7 is held in a hole so as to cover the electrode systems 2'-4' and further, a cover 8 is adhered thereto to integrate the entire part. The specific components in the vital sample are thus quantitatively determined with high accuracy.

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 In the past, a biosensor as shown in Figure 4 has been proposed as a method for quantifying specific components in biological samples such as blood with high precision without performing operations such as diluting or stirring the sample solution. (For example, Japanese Patent Application Laid-Open No. 59-166852)
. This biosensor has leads 12.
A measuring electrode 10 and a counter electrode 11 made of platinum or the like having 13 are buried, and the exposed portions of these electrode systems are covered with a porous body 14 carrying an oxidoreductase and an electron acceptor. When the sample liquid is dropped onto the porous body 14, the oxidoreductase and electron acceptor in the porous body are dissolved in the sample liquid,
The enzymatic reaction progresses with the substrate in the sample solution, the electron acceptor is reduced, and after the b0 enzymatic reaction is completed, the reduced electron acceptor is electrochemically oxidized, and the sample is determined from the oxidation current value obtained at this time. Determine the substrate concentration in the solution.

Problems to be Solved by the Invention In such a conventional configuration, the porous body can be easily used for measurement by being replaced after each measurement, but the electrode system requires operations such as cleaning. be. If it were possible to discard the negative electrode system after each measurement, it would greatly simplify the measurement operation, but it would be extremely expensive due to the electrode materials such as platinum and the structure. I don't get it.

As a result of various studies on these points, the present invention integrates an electrode system and a porous body to improve biological testing.

The object of the present invention is to provide an inexpensive disposable type biosensor that can extremely easily, quickly, and highly accurately quantify a specific component in a food.

Means for Solving 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, allows the enzyme, electron acceptor, and sample solution to react, and In a biosensor that electrochemically detects a change in substance concentration during a reaction using the electrode system and measures the substrate concentration in a sample solution, a protein is adsorbed in advance on at least the surface of the measurement electrode of the electrode system, and then oxidized. The electrode system is covered with a porous body carrying a reductase and an electron acceptor, and the porous body is integrated with the electrode system and the substrate.

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

Furthermore, by adsorbing proteins on the surface of the electrode system in advance, fluctuations in measured values due to new adsorption of proteins, etc. in the sample solution are eliminated, making it possible to measure substrate concentrations with high precision and good reproducibility. It became.

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 conductive carbon paste is printed on an insulating substrate 1 made of polyethylene terephthalate by Nuclean printing and dried by heating to form a counter electrode 2. Measuring pole 3. An electrode system consisting of a reference electrode 4 is formed. Next, partially cover the electrode system,
2' which becomes the electrochemically active part of each electrode.

An insulating paste is printed in the same manner as described above so that 3' and 4' (each 1-) remain, and heat treated to form an insulating layer 5.

A 60 mji/ml aqueous solution of albumin was dropped to cover the surface of this electrode system (2', 3', 4'), left for 5 to 10 minutes, and then washed with water to remove excess albumin. dry. Through this operation, albumin is adsorbed onto the surface of each electrode.

Next, a resin holding frame 6 with holes is glued to the insulating layer 5,
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.

FIG. 2 shows a cross-sectional view along the measurement electrode 3 of this biosensor. The porous body used above contained 1 oomg of glucose oxidase as an oxidoreductase.
and potassium ferricyanide as electron acceptor 1 somg
It was prepared by impregnating a nylon non-woven fabric with a solution obtained by dissolving the above in 1 ml of a phosphate buffer solution with a pH of 5,6, and then drying it under reduced pressure.

A glucose standard solution was dropped as a sample solution into the porous body of the glucose sensor configured as described above, and 2 minutes after dropping, a pulse voltage of 700 mV was applied to the measurement electrode with respect to the reference electrode to polarize it toward the anode. . In this case, the added glucose reacts with potassium ferricyanide to produce potassium ferrocyanide by the action of glucose oxidase supported on the porous material. Therefore, by applying the pulse voltage in the direction of the anode, an oxidation current proportional to 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 containing approximately 90 mg/al of glucose was dropped onto each of the 10 glucose measurement sensors configured as described above, a pulse voltage of 700 m'i was applied after 2 minutes, and the current value was measured 10 seconds after application. As a result, good reproducibility was shown as shown in Figure 3. - On the other hand, if a glucose sensor is constructed in the same manner as above using an electrode system that is not subjected to albumin adsorption treatment, and measurements are taken in the same manner as in humans, the result will be as shown in Figure 3B.
As shown in Figure 3, the variation in response current was greater in dogs than in A. A.

Panel B shows the response for 10 glucose sensors that were each manufactured in the same way, but the difference in reproducibility due to such adsorption treatment is due to the degree of adsorption of adsorbed substances such as proteins in the serum sample. This is thought to be due to the difference. As shown above, it is thought that this effect can be prevented by first performing sufficient adsorption treatment.

In addition to albumin, an aqueous solution of glucose oxidase 1
When the same treatment as described above was performed using an oomg/ml aqueous solution, response characteristics with good reproducibility were obtained.

The protein to be adsorbed to the electrode is not limited to the albumin and glucose oxidase shown in the above examples. Furthermore, if at least a measured % of the electrode system is subjected to adsorption treatment, the same effect as described above can be obtained.

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 was 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 By integrating an insulating substrate, an electrode system, and a porous body carrying an oxidoreductase and an electron acceptor, the biosensor of the present invention can extremely easily measure the substrate concentration in a biological sample. Furthermore, reproducibility can be improved by adsorbing proteins on the electrode surface in advance.

[Brief explanation of the drawing]

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. DESCRIPTION OF SYMBOLS 1...Substrate, 2...Counter electrode, 3...Measurement electrode, 4...Reference tube, 5--Insulating layer, 6-Holding frame, 7... - Porous body, 8...Cover. Name of agent: Patent attorney Toshio Nakao and 1 other person f-
Kuruma color (shi) King's ear JJG - 悸树J卆7-ru 訃, δ-11ka7, - Fig. 2 Fig. 3

Claims (4)

[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. In a biosensor that measures the substrate concentration of a liquid, proteins are adsorbed in advance on the surface of at least the measurement electrode of the electrode system, and the electrode system is covered with a porous material carrying an oxidoreductase and an electron acceptor. A biosensor characterized in that it is integrated with an electrode system and the substrate. (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 is at least either albumin or an oxidoreductase used in an enzyme reaction.