JPH01253648A - biosensor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

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 A biosensor described in Japanese Patent Application Laid-Open No. 61-294351 was proposed as a method that allows for easy quantitative determination of specific components in biological samples such as blood without diluting or stirring the sample solution. (Figure 5). This biosensor consists of electrode systems 8 (8'), 9 (9') made of carbon or the like, which are formed by screen printing or the like on an insulating substrate 1.
10 (10'), covered with a porous body 12 carrying an oxidoreductase and an electron acceptor, and integrated with a holding frame 11 and a cover 13. When the sample solution is dropped onto the porous material, the oxidoreductase and electron acceptor supported on the porous material are dissolved in the sample solution, and an enzymatic reaction proceeds with the substrate in the sample solution, causing the electron acceptor to be reduced. be done. After the reaction is completed, the reduced electron acceptor is electrochemically oxidized, and the substrate concentration in the sample liquid is determined from the oxidation current value obtained at this time.

Problems to be Solved by the Invention In such a conventional configuration, the wetting of the substrate surface including the electrode system is not necessarily uniform, so air bubbles remain between the porous body and the substrate, which may affect the response current. There were cases where the reaction rate decreased. In addition, the presence of substances that easily adsorb to the electrode or substances that are active on the electrode in the sample liquid affected the response of the sensor.

Means for Solving the Problems In order to solve the above problems, the present invention provides an electrode system consisting of at least a measuring electrode and a counter electrode mainly made of carbon on an insulating substrate, and a hydrophilic polymer and a hydrophilic polymer are used in the electrode system. It is equipped with an enzyme reaction layer consisting of oxidoreductase.

Furthermore, a two-silk electrode system consisting of at least a measuring electrode and a counter electrode, mainly made of carbon, was provided on an insulating substrate, and one electrode system was used for an enzymatic reaction consisting of a hydrophilic polymer and an oxidoreductase. A hydrophilic polymer layer or a layer consisting of a hydrophilic polymer and a deactivated oxidoreductase is provided on the other electrode system.

Effect of the Invention According to the present invention, it is possible to construct a disposable biosensor that can extremely easily and accurately measure substrate concentration and has excellent storage stability.

Example The present invention will be explained below using 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 produced as an example of the biosensor of the present invention, and FIG. 2 is a perspective view of the electrode portion used in the sensor production.

A silver paste is printed on an insulating substrate 1 made of polyethylene terephthalate by screen printing to form leads 2.
3 (3') is formed. Next, a conductive carbon paste containing a resin binder is printed and dried by heating to form an electrode system consisting of a measurement electrode 4 and a counter electrode 5. Furthermore, an insulating paste is printed so as to partially cover the electrode system, keep the area of the exposed part of the electrode constant, and cover unnecessary parts of the leads, and is heat-treated to form an insulating paste.

Next, the exposed portion of 4.5 (5') was polished and then heat treated in air at 100° C. for 4 hours. After constructing the electrode portion in this way, 0.0% of carboxymethyl cellulose (hereinafter abbreviated as CMC) was used as a hydrophilic polymer. 5wt
% aqueous solution is spread on the electrode and dried to form CMC5.

Next, a solution of glucose oxidase (COD) as an enzyme dissolved in a phosphate buffer solution was spread so as to cover this CMC layer, and dried to form a CMC-CoD layer 7.

In this case, CMC and COD are partially mixed into a thin film having a thickness of several microns.

CMC-GO of the glucose sensor configured as above
A 10 μm glucose standard solution was dropped as a sample solution onto the DH, and 1 minute after the drop, a pulse voltage of 1 μm was applied between the electrodes to polarize it in the direction of the anode, which is the measurement pole.

The added sample solution dissolved the enzyme and CMC and quickly spread over the electrode surface while becoming a viscous liquid, and no bubbles remained. This is considered to be due to improved wettability of the electrode surface due to the hydrophilic polymer layer previously formed on the electrode.

On the other hand, the added glucose in the sample solution reacts with oxygen by the action of glucose oxidase supported on the electrode to generate hydrogen peroxide. Therefore, by applying the voltage in the direction of the anode, an oxidation current of the generated hydrogen peroxide is obtained, and this current value corresponds to the concentration of glucose, which is the substrate.

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

(Example 2) In the same manner as in Example 1, two sets of electrode portions identical to those shown in FIG. 2 were formed close to each other on a single insulating substrate made of polyethylene terephthalate by screen printing. Next, C was applied on the two sets of electrode systems in the same manner as in Example 1.
After forming MCN, COD-CMCF' was formed in the same manner as above only on the CMC layer of one electrode system.

Regarding the glucose sensor having two sets of electrode systems obtained as described above, a glucose standard solution containing various concentrations of ascorbic acid (200 mg/kg) was poured onto each electrode system.
dl) was added dropwise, and as in Example 1, after 1 minute, 1■
A voltage of 5 seconds was applied, and the current value was measured 5 seconds later. The results are shown in Figure 4. The output of the electrode system of the CMC-C0D layer is A,
Further, the output (blank output) of the electrode system including only the CMC layer is indicated by B. As is clear from the figure, the output of B increases with increasing concentration of ascorbic acid, while the output of B also shows a similar increase. This indicates that the sensitivity of each electrode system to ascorbic acid is approximately equal. From this, when the difference (A-B) between the outputs of both electrode systems is detected, a current value based on glucose can be obtained. That is, by using a two-dark blue electrode system, errors caused by electrode active substances can be significantly reduced. Such effects were observed not only with ascorbic acid but also with uric acid.

In this way, two sets of electrode systems are provided, one electrode system is formed with a hydrophilic polymer-enzyme layer, and the other electrode system is formed with only a hydrophilic polymer layer to form a sensor. It is possible to accurately measure the substrate concentration in a sample solution containing .

In the above, after forming a CMC-C0D layer on both electrode systems, only one electrode system is subjected to local heating by laser irradiation, ultraviolet irradiation, etc. to deactivate the COD, and then used as an electrode system for blank output. Also good. In this case, the configurations of both electrode systems are the same except for the enzyme activity, and the output currents of the two electrode systems due to the interfering substance can be roughly matched, and the sensor detection accuracy can be improved.

Furthermore, in the above embodiments, an electrode system was described in which the electrode part consisted of two electrodes, a measurement electrode and a counter electrode, but the accuracy could be further improved by configuring the electrode system from three electrodes including silver/silver chloride. be able to. An example of a method for constructing an electrode system is to print three silver leads on a substrate, print carbon paste only on the -L ends of the two leads, coat with an insulating layer, and then apply silver to the substrate. The surface of the remaining exposed lead tip may be treated to form silver chloride to form a silver/silver chloride electrode.

In addition to CMC, gelatin, methylcellulose, and the like can be used as the hydrophilic polymer, and starch, carboxymethylcellulose, gelatin, acrylate, vinyl alcohol, vinylpyrrolidone, and maleic anhydride are preferred.

By coating and drying a solution of these water-absorbing or water-soluble hydrophilic polymers at an appropriate concentration, a hydrophilic polymer layer with a required thickness can be formed on the electrode.

Furthermore, the oxidoreductase is not limited to the glucose oxidase shown in the above example, and various enzymes such as alcohol oxidase and cholesterol oxidase can be used. By forming an enzyme reaction layer consisting of a hydrophilic polymer and an oxidoreductase on an electrode system, and furthermore, by providing two electrode systems, a hydrophilic polymer and a oxidation-reduction enzyme are placed on one electrode system. A highly reliable response can be obtained by forming an enzyme reaction layer consisting of an enzyme and a hydrophilic polymer layer or a layer consisting of a hydrophilic polymer and an inactivated redox enzyme on the other electrode system. Can be done. Furthermore, since there is no need to carry an electron acceptor, the structure can be simplified, and a biosensor that is inexpensive and has excellent storage stability can be provided.

[Brief explanation of the drawing]

Fig. 1 is a cross-sectional view of a biosensor that is an embodiment of the present invention, Fig. 2 is a perspective view of the electrode portion, Figs. 3 and 4 are response characteristic diagrams of the biosensor, and Fig. 5 is a conventional biosensor. FIG. 3 is an exploded perspective view of the sensor. 1...Insulating substrate, 2. 3. 3'...
...Lead, /L, 9. 9'...Measurement pole, 5. 5', 8. 8'・・・opposite, 6
...Insulating layer, 7...CMC-(';O
D layer, 10.10'...Reference electrode, 11...
... Holding frame, 12 ... Porous body, 13 ...
·cover. Name of agent Patent attorney Toshio Nakao 1 person Figure 3 Figure 4 Figure 5 0tθ π 30 41) Askolhin Oyuwanbin η〃9 18・

Claims (3)

[Claims] (1) An electrode system consisting of at least a measurement electrode and a counter electrode mainly made of carbon is provided on an insulating substrate, and an enzyme reaction layer consisting of a hydrophilic polymer and an oxidoreductase is provided on the electrode system. A biosensor featuring: (2) Two sets of electrode systems consisting of at least a measurement electrode and a counter electrode mainly made of carbon are provided on an insulating substrate, and an enzyme reaction layer consisting of a hydrophilic polymer and an oxidoreductase is placed on one electrode system. A biosensor comprising: a hydrophilic polymer layer or a layer consisting of a hydrophilic polymer and an inactivated redox enzyme on the other electrode system. (3) The biosensor according to claim 1 or 2, wherein the electrode system is a reference electrode consisting of a measurement electrode and a counter electrode mainly made of carbon, and a silver/silver chloride reference electrode.