JP2502665B2 - Biosensor - Google Patents

Description

TECHNICAL FIELD The present invention relates to a biosensor capable of quickly and simply quantifying a specific component in various trace amounts of a biological sample without diluting the sample solution.

Conventional technology Conventionally, a biosensor disclosed in Japanese Patent Laid-Open No. 61-294351 is proposed as a method that can easily quantify a specific component in a biological sample such as blood without diluting or stirring the sample solution. (Fig. 5). In this biosensor, an electrode system 8 (8 '), 9 (9'), 10 (10 ') made of carbon or the like is formed on an insulating substrate 1 by a method such as screen printing, and the redox is formed on the electrode system. A porous body (12) carrying an enzyme and an electron acceptor is covered and a holding frame (11) and a cover (13) are integrated as a whole. When the sample solution is dropped on the porous body, the redox enzyme and the electron acceptor supported on the porous body are dissolved in the sample solution, and the enzyme reaction proceeds with the substrate in the sample solution to reduce the electron acceptor. To be done. After the reaction is completed, the reduced electron acceptor is electrochemically oxidized, and the concentration of the substrate 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, since wetting of the substrate surface including the electrode system is not always uniform, foaming remains between the porous body and the substrate, which affects the response current. In some cases, the reaction rate decreased. In addition, the presence of a substance that is easily adsorbed on the electrode or a substance that is active in the electrode in the sample solution has an effect on the sensor response.

Means for Solving the Problems In order to solve the above problems, the present invention provides an electrode system comprising at least a measurement electrode mainly composed of carbon and a counter electrode on an insulating substrate, and a hydrophilic polymer on the electrode system. It is provided with an enzyme reaction layer composed of a redox enzyme.

In addition, two sets of electrode systems mainly composed of carbon and composed of at least a measurement electrode and a counter electrode are provided on an insulating substrate, and an enzyme reaction layer composed of a hydrophilic polymer and a redox enzyme is provided on one electrode system. And a hydrophilic polymer layer or a layer comprising a hydrophilic polymer and a deactivated oxidoreductase on the other electrode system.

Effects According to the present invention, it is possible to construct a disposable type biosensor which can very easily and accurately measure the substrate concentration and is excellent in storage stability.

Examples Hereinafter, the present invention will be described with reference to Examples.

Example 1 A glucose sensor will be described as an example of a biosensor.

FIG. 1 is a cross-sectional view of a glucose sensor manufactured as an example of the biosensor of the present invention, and FIG. 2 is a perspective view showing an electrode portion used for manufacturing the sensor.

Insulating substrate 1 made of polyethylene terephthalate
Then, print the silver paste by screen printing and lead 2.
Form 3 (3 '). Next, a conductive carbon paste containing a resin binder is printed and heated and dried to form an electrode system including the measurement electrode 4 and the counter electrode 5. Further, an insulating paste is printed so as to partially cover the electrode system, keep the exposed area of the electrode constant, and cover unnecessary portions of the leads, and heat treatment is performed to form the insulating layer 6.

Next, after polishing the exposed parts of 4, 5 (5 '), in air
It heat-processed at 100 degreeC for 4 hours. After forming the electrode portion in this manner, a 0.5 wt% aqueous solution of carboxymethyl cellulose (hereinafter abbreviated as CMC) as a hydrophilic polymer is spread on the electrode and dried to form a CMC layer. Next, as an enzyme, glucose oxidase (GO
A solution obtained by dissolving D) in a phosphate buffer was developed and dried to form CMC-GOD layer 7. In this case, CMC and GOD are in the form of a thin film having a thickness of several microns in a partially mixed state.

10 μl of a glucose standard solution was dropped as a sample solution onto the CMC-GOD layer of the glucose sensor configured as described above,
The measurement electrode was polarized in the anode direction by applying a pulse voltage of 1 V between the electrodes 1 minute after the dropping.

The added sample solution rapidly spreads on the electrode surface while dissolving the enzyme and CMC into a viscous liquid, and no residual air bubbles were observed. It is considered that this is because the wettability of the electrode surface was improved by the hydrophilic polymer layer previously formed on the electrode.

On the other hand, glucose in the added sample liquid reacts with oxygen by the action of glucose oxidase carried on the electrode to generate hydrogen peroxide. Therefore, by applying a voltage in the direction of the above-mentioned anode, an oxidation current of hydrogen peroxide produced is obtained, and this current value corresponds to the concentration of glucose as a substrate.

FIG. 3 shows the relationship between the current value and the glucose concentration 5 seconds after the voltage application as an example of the response characteristics of the sensor having the above-mentioned configuration, and an extremely good response was obtained.

(Example 2) In the same manner as in Example 1, two sets of the same electrode portions as shown in Fig. 2 were formed in close proximity on a single insulating substrate made of polyethylene terephthalate by screen printing. Then, in the same manner as in Example 1 on the two sets of electrode systems
After forming the CMC layer, the GOD-CMC layer was formed in the same manner as above only on the CMC layer of one electrode system.

For glucose sensors having two sets of electrode systems obtained as described above, glucose standard solutions (200 mg / dl) containing various concentrations of ascorbic acid were dropped onto each electrode system, and Similarly, after 1 minute, the voltage of 1 V was reduced, and the current value after 5 seconds was measured. Results are shown in FIG.
The output of the electrode system of the CMC-GOD layer is shown by A, and the output of the electrode system of only the CMC layer (blank output) is shown by B. As is clear from the figure, the output of A increases as the concentration of ascorbic acid increases, while the output of B also shows the same increase. This indicates that the sensitivity of each electrode system to ascorbic acid is almost equal. From this, a current value based on glucose can be obtained by detecting the difference (AB) between the outputs of both electrode systems. That is, by using two sets of electrode systems, the error due to the electrode active substance can be significantly reduced. Such an effect was observed not only for ascorbic acid but also for uric acid and the like.

In this way, by providing two sets of electrode systems, forming a hydrophilic polymer-enzyme layer on one electrode system and a hydrophilic polymer layer on the other electrode system, and configuring a sensor, an interfering substance can be obtained. It is possible to accurately measure the substrate concentration in the sample solution containing.

In the above, after forming the CMC-GOD layer on both electrode systems, local heating by laser irradiation only on one electrode system, deactivates GOD by performing ultraviolet irradiation, etc., as an electrode system for blank output. Is also good. By doing so, the configurations of both electrode systems are the same except for the enzyme activity, and the output currents due to the interfering substances of both electrode systems can be more closely matched, and the sensor detection accuracy can be improved.

Further, in the above embodiments, the electrode system has been described in which the electrode portion is composed of the two electrodes of the measurement electrode and the counter electrode. However, the accuracy is further improved by configuring the electrode system with three electrodes to which silver / silver chloride is added. be able to. As an example of a method for forming an electrode system, after printing three silver leads on a substrate, printing a carbon paste only on the tips of the two leads and coating an insulating layer, the silver is exposed. The surface of the remaining one lead tip is treated to form silver chloride to form a silver / silver chloride electrode.

As the hydrophilic polymer, gelatin, methylcellulose, etc. can be used in addition to CMC, and starch-based, carboxymethylcellulose-based, gelatin-based, acrylate-based, vinyl alcohol-based, vinylpyrrolidone-based, and maleic anhydride-based polymers are preferable. A hydrophilic polymer layer having a required film thickness can be formed on the electrode by applying a solution of these water-absorbing or water-soluble hydrophilic polymers in an appropriate concentration and drying.

Furthermore, the oxidoreductase is not limited to the glucose oxidase shown in the above examples, and various enzymes such as alcohol oxidase and cholesterol oxidase can be used.

Effect of the Invention As described above, the biosensor of the present invention is provided with an enzyme reaction layer composed of a hydrophilic polymer and a redox enzyme on the electrode system, and further, two sets of electrode systems are provided,
An enzyme reaction layer composed of a hydrophilic polymer and oxidoreductase was formed on one electrode system, and a hydrophilic polymer layer or a layer composed of a deactivated oxidoreductase and a hydrophilic polymer layer was formed on the other electrode system. By forming it, a highly reliable response can be obtained. Furthermore, since it is not necessary to carry an electron acceptor, it is possible to provide a biosensor that has a simple structure, is inexpensive, and has excellent storage stability.

[Brief description of drawings]

FIG. 1 is a sectional view of a biosensor which is an embodiment of the present invention, FIG. 2 is a perspective view of an electrode portion, FIGS. 3 and 4 are response characteristic diagrams of the biosensor, and FIG. 5 is a conventional biosensor. It is an exploded perspective view of a sensor. 1 ... Insulating substrate, 2,3,3 '... Lead, 4,9,9' ...
Measuring electrode, 5,5 ', 8,8' ... Counter electrode, 6 ... Insulating layer, 7 ... C
MC-GOD layer, 10,10 '…… Reference electrode, 11 …… Holding frame, 12 ……
Porous body, 13 …… Cover.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Takashi Iijima 1006 Kadoma, Kadoma City, Osaka Prefecture Matsushita Electric Industrial Co., Ltd. (56) Reference JP-A-63-58149 (JP, A) JP-A-1- 134244 (JP, A) JP-A 1-212345 (JP, A) JP-A-62-237348 (JP, A)

Claims (3)

(57) [Claims] 1. An electrode system comprising at least a measuring electrode mainly composed of carbon and a counter electrode is provided on an insulating substrate, and an enzyme reaction layer comprising a hydrophilic polymer and a redox enzyme is provided on the electrode system. A biosensor characterized by that. 2. An enzyme substrate comprising a hydrophilic polymer and an oxidoreductase on one electrode system, wherein two sets of electrode systems mainly composed of carbon and having at least a measuring electrode and a counter electrode are provided on an insulating substrate. A biosensor comprising a reaction layer, and a hydrophilic polymer layer or a layer comprising a hydrophilic polymer and an inactivated redox enzyme on the other electrode system. 3. The biosensor according to claim 1, wherein the electrode system is a reference electrode composed of a measurement electrode mainly composed of carbon, a counter electrode, and a silver / silver chloride reference electrode.