JPS63317095A - Biosensor - Google Patents

Abstract

PURPOSE:To obtain a biosensor capable of readily measuring, having high precision and useful in measurement of biopolymer ingredient, etc., by treating surface of a specific electrode system with ozone, coating the surface with a water-absorbing polymer and integrating the electrode system together with a porous body supporting enzyme, etc., and electron acceptor. CONSTITUTION:A counter pole 2, measuring pole 3 and reference pole 4 are subjected to screening printing on an insulating substrate 1 such as polyethylene terephthalate to form electrode system. Then an insulating paste is printed in state left in 2', 3' and 4' part of each electrode to form an insulating layer 5 and then an aqueous solution of water-absorbing high polymer such as CMC is applied to the surface in the 2', 3' and 4' part of each electrode and the applied electrode is dried and then a retaining frame 7 having opening and made of resin is bonded to the insulating layer 5. Then after a porous body 8 consisting of oxidation-reduction enzyme such as glucose oxidase and electron acceptor such as ferricyanide is charged into the opening part of the retaining frame 7, a cover 9 made of resin and having opening of smaller diameter than that of the porous body 8 is bonded to the porous body 8 to integrate the whole.

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 3 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 Laid-Open No. 59-166852). This biosensor has a lead 1 on an insulating substrate 10.
Measuring electrodes 11 made of platinum or the like, each having 3.14
and a counter electrode 12 are buried, and the exposed portions of these electrode systems are covered with a porous body 16 carrying an oxidoreductase and an electron acceptor. When the sample solution is dropped onto the porous material, the oxidoreductase and electron acceptor in the porous material are dissolved in the sample solution.
An enzymatic reaction progresses 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 it were possible to use only the electrode system for 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. I don't get it.

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 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, and detects changes in substance concentration during reactions between enzymes, electron acceptors, and sample liquids. In a biosensor that electrochemically detects with the electrode system and measures the substrate concentration in a sample solution, the surface of the electrode system is exposed in advance in an ozone atmosphere and coated with a water-absorbing polymer,
Furthermore, it is integrated with a porous material carrying an oxidoreductase and an electron acceptor.

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.

Furthermore, by exposing the surface of the electrode system in advance in an ozone atmosphere and coating it with a water-absorbing polymer, the wettability of the electrode is improved, and the water-absorbing polymer adsorbs proteins and air bubbles in the sample solution to the electrode surface. This enables 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 conductive carbon paste is printed by screen printing on an insulating substrate 1 made of polyethylene terephthalate color, and then heated and dried to form a counter electrode 2. 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.

3/, 4/<1H each) is printed in the same manner as described above, and heat-treated to form the insulating layer 5.

By exposing the surface of this electrode system (2/, 3/, 4/) in an ozone atmosphere for 10 minutes (hereinafter referred to as ozone treatment), dirt on the electrode surface is decomposed and it becomes easier to wet. An aqueous solution of CMC6 (carboxymethyl cellulose), a type of cellulose-based water-absorbing polymer, was completely applied onto the ozone-treated electrode and dried at 46°C for 1 hour. When CMG is applied to an electrode that is not subjected to ozone treatment, it is difficult to form a CMC film on the electrode because it is repelled by the insulating paste and the CMG film peels off during drying at 46°C. Ta.

Next, a resin holding frame 7 with holes is glued to the insulating layer 6,
The porous body 8 is held via the space so as to cover the electrode systems 2't3', , *'. Furthermore, a resin cover 9 having an opening having a diameter smaller than that of the porous body is adhered to integrate the entire body. For this integrated biosensor, measuring electrode 3
A cross-sectional view along line 1C is shown in FIG. The porous body used above contained glucose oxidase 100 as an oxidoreductase.
mg and potassium ferricyanide as electron acceptor 150
It was prepared by impregnating a nylon nonwoven fabric with a solution of vag dissolved in 1 m/ml of phosphate buffer solution of pH 5.6 and then drying it under reduced pressure.

A glucose standard solution is dropped as a sample solution into the porous body of the glucose sensor configured as described above, and after 2 minutes of dropping, the potential of the measurement electrode is changed to +0.7 points toward the anode with reference to the reference electrode.
Apply a v pulse voltage and measure the current after 5 seconds. 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 applying the pulse voltage described above, an oxidation current based on the concentration of the generated potassium ferrocyanide is obtained, and this current value corresponds to the concentration of glucose, which is the substrate. When a standard solution of glucose was added dropwise and the response current was measured, it was found that 700
Good linearity was obtained up to a high concentration of ml/dl.

When a drop of 20 μl of a blood sample was added to the above glucose sensor and the response current was measured 2 minutes later, a response with very good reproducibility was obtained. When the electrode surface was not coated with CMG, the blood response was lower and more variable than when CMG was coated on the electrode surface. This is thought to be because blood cell components and proteins in blood are adsorbed to the surface of the carbon electrode and interfere with the response. Ozone treatment cleans dirt on the electrode surface, and hydrophilic treatment forms a CMG layer that adheres to the electrode surface, preventing blood cells from being adsorbed to the electrode surface, resulting in good reproducibility. You can get a response. A surfactant may also be used as a hydrophilic treatment for the electrode surface, but during measurement, the dissolved surfactant affected the response and was a cause of variation. Since ozone treatment can be performed in a dry state and in a short time, it is considered to be very advantageous when manufacturing disposable type sensors in large quantities. C as a water-absorbing polymer applied to the electrode surface
Besides MC, gelatin, methylcellulose, etc. can also be used. De/pun type, carboxymethyl cellulose type, gelatin type, acrylate type.

Vinyl alcohol-based, vinylpyrrolidone-based, and maleic anhydride-based materials are preferred. Since these polymers can be easily made into an aqueous solution, a thin film of a required thickness can be formed on the electrode by applying an aqueous solution of an appropriate concentration and drying.

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 embodiments, a three-electrode blade type electrode system was described, but measurements can also be made with a two-electrode blade type consisting of a counter electrode and a measurement electrode.

Effects of the Invention As described above, the biosensor of the present invention can extremely easily adjust the substrate concentration in a biological sample liquid by integrating an insulating substrate, an electrode system, and a porous body carrying an oxidoreductase and an electron acceptor. Furthermore, the electrode surface is treated with ozone and then coated with a water-absorbing polymer to improve wettability and prevent interfering substances from adsorbing to the electrode surface, increasing measurement accuracy.

[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 sectional view thereof, and FIG. 3 is a longitudinal sectional view of a conventional biosensor. 1...Substrate, 2...Counter electrode, 3...
...Measurement pole, 4...Reference pole. Name of agent: Patent attorney Toshio Nakao and 1 other person/-
--J color ball/gender j slope 2 ~ Opposite pole 3-/5x11 fixed pole-DMC 7-Guardian frame Figure 2 Otsu Figure 3/,<

Claims (3)

[Claims] (1) An insulating substrate is provided with an electrode system consisting of at least a measurement electrode and a counter electrode, and the change in substance concentration during the reaction between an enzyme, an electron acceptor, and a sample liquid is electrochemically detected by the electrode system, and the substrate concentration is A biosensor for measuring , wherein the surface of the electrode system is exposed in advance in an ozone atmosphere, coated with a water-absorbing polymer, and further integrated with a porous body carrying an oxidoreductase and an electron acceptor. biosensor. (2) The water-absorbing polymer applied to the surface of the electrode system is one of the group consisting of starch, carboxymethylcellulose, gelatin, acrylate, vinyl alcohol, vinylpyrrolidone, and maleic anhydride. The biosensor according to claim 1, which is a mixture thereof. (3) The biosensor according to claim 1, wherein the electrode system is made of a carbon-based material formed by screen printing on an insulating substrate.