JPH0652249B2 - 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.

2. Description of the Related Art Conventionally, there is a biosensor as shown in FIG. 3 as a method for quantifying a specific component in a biological sample such as blood with high precision without performing operations such as dilution and stirring of a sample solution. In this biosensor, electrode systems 12 and 13 are formed on an insulating substrate 11 by a method such as screen printing, a hydrophilic polymer layer 15 is formed on the electrodes, and the hydrophilic polymer layer 15 is oxidized. The enzyme reaction layer 18 composed of a reductase and an electron acceptor is formed. When the sample solution is dropped onto the enzyme reaction layer 18, the enzyme reaction layer 1 is added to the sample solution.
The oxidoreductase in 8 and the electron acceptor are dissolved, an enzymatic reaction proceeds with the substrate in the sample solution, and the electron acceptor is reduced. After the completion of the enzymatic reaction, this 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 According to such a conventional biosensor, since the oxidoreductase and the electron acceptor in the enzyme reaction layer are in contact with each other, a reaction occurs between the two in the presence of water and a response is generated. To vary,
It was difficult to obtain a stable response, and it became necessary to control the water content during manufacturing and storage.

Means for Solving the Problems In order to solve the above problems, the present invention includes an insulating substrate provided with an electrode system consisting of at least a measurement electrode and a counter electrode, and a hydrophilic polymer layer is provided on the surface of the electrode system. An oxygen reaction layer containing an electron acceptor and a redox enzyme independently of each other is formed on top of it, and a change in the substance concentration during the reaction between the enzyme, the electron acceptor and the sample solution is detected electrochemically by the electrode system. Then, the substrate concentration in the sample solution is measured, and preferably, the electrode system and the hydrophilic polymer layer and the oxygen reaction layer are respectively formed by screen printing.

When added to the oxygen reaction layer of the sample solution, the oxidoreductase and electron acceptor in the reaction layer are independent of each other when measuring the substrate concentration in the sample solution, and no interaction occurs due to water. Therefore, the response does not vary and a stable response is obtained, and the measurement accuracy is improved. Also,
Since it is possible to manufacture the sensor by screen printing without the need to control the water content at the time of manufacture or during the existence, it is possible to mass-produce disposable biosensors including the electrode system.

Example One example of the present invention will be described below.

As an example of a biosensor, an example of a glucose sensor will be described with reference to FIG.

On the insulating substrate 1 made of polyethylene terephthalate,
An electrically conductive carbon paste is printed by screen printing and dried by heating to form an electrode system composed of the counter electrode 2a and the measurement electrode 3a. Next, an insulating paste is printed in the same manner as above so as to cover the electrode system partially and leave the electrode systems 2b and 3b which become the electrochemically acting portions of the respective electrodes, and heat-treated. The insulating layer 4 is formed. An aqueous solution of CMC (carboxymethyl cellulose), which is a kind of cellulosic hydrophilic polymer, is printed by screen printing so as to cover the surfaces of the electrode systems 2b and 3b.
CMC layer 5 was formed by drying at C for 1 hour.
On the obtained CMC layer 5, glucose oxidase (GOD) as a redox enzyme 6 dissolved in water was formed by screen printing as shown in FIG. 2, and dried at room temperature. Furthermore, fine crystals of potassium ferricyanide mixed with ethanol together with lecithin are formed as an electron acceptor 7 on the CMC layer 5 by screen printing, and the ferricyanide is converted into glucose oxidase by allowing it to stand at room temperature to vaporize ethanol. The separated oxygen reaction layer 8 was formed. Since potassium ferricyanide is insoluble in ethanol, it cannot be dispersed as it is, but when lecithin is present, it can be dispersed as fine particles of potassium ferricyanide due to its amphipathic structure, and
Since ethanol rapidly vaporized, a uniform layer having a high dissolution rate could be formed. When potassium ferricyanide is formed on glucose oxidase as in the conventional example,
If left as it is, the response will be high and it will flutter, so it was necessary to maintain the dry state using silica gel or the like, but by considering the effect of water by separating the oxidoreductase 6 and the electron acceptor 7 as in the present invention. Even without it, a stable response was obtained.

A glucose standard solution (10 μm) was dropped as a sample solution onto the glucose sensor configured as described above, and a pulse voltage of +0.6 V was applied to the measurement electrode in the anode direction 2 minutes after the dropping, and the current was measured 5 seconds later. The glucose standard solution dissolves potassium ferricyanide and glucose oxidase in the oxygen reaction layer 8 to oxidize glucose, at which time potassium ferricyanide is reduced to potassium ferrocyanide. Then, by applying the above-mentioned pulse voltage, an oxidation current based on the concentration of the produced potassium ferrocyanide is obtained, and this current value corresponds to the concentration of glucose as a substrate. When a standard solution of glucose was dropped and the response current was measured, good linearity was obtained up to a high concentration of 700 mg / d. Add 10 blood samples to the above glucose sensor
When the response current was measured 2 minutes after μ was dropped, a very reproducible response was obtained. When the oxygen-reactive layer was formed directly on the electrode, blood cells and the like were adsorbed on the electrode surface, and the response was considerably low, and there were variations. However, by forming the CMC layer 5,
It was possible to prevent adsorption. In addition to CMC, gelatin, methyl cellulose, etc. can be used as the hydrophilic polymer, and starch-based, carboxymethylcellulose-based, gelatin-based, acrylate-based, vinyl alcohol-based, vinylpyrrolidone-based, maleic anhydride-based ones can be used. preferable. Since these polymers can be easily made into an aqueous solution, a thin film having a required thickness can be formed on the electrode by printing and drying an aqueous solution having an appropriate concentration.

In addition to lecithin, glycerophospholipids, sphigophospholipids, fatty acids and the like can be used as the polymer having an amphipathic structure, and stearic acid, sphingomyelin, cyclodextrin and the like can be used. As the solvent, those which have little effect on the GOD activity and the printed electrode are preferable, and toluene, petroleum ether and the like are suitable.

Screen printing as a method for forming the sensor portion can inexpensively produce a disposable type biosensor having uniform characteristics, and in particular, the electrode is formed using carbon, which is a cheap and stable electrode material. It is a convenient method to form.

Moreover, since the shapes of the CMC layer and the oxygen reaction layer can be freely formed by using screen printing, the oxidoreductase and the electron acceptor could be easily and independently carried. In the above embodiment, the case of the two-electrode system as the electrode system has been described, but the measurement can be performed with three electrodes including the counter electrode, the measurement electrode, and the reference electrode. The places where potassium ferricyanide and glucose oxidase are carried are shown in Fig. 2 (a).
Although it can be freely changed by screen printing in addition to that shown in (b), the reaction was more rapid when the oxidoreductase and the electron acceptor were carried closer to each other, so that the response was stable. The carrying pattern is not limited to the examples.

The biosensor of the present invention is not limited to the glucose sensor shown in the above examples, but can be used in systems involving oxidoreductase such as alcohol sensor and cholesterol sensor. Glucose oxidase was used as the oxidoreductase in the examples, but other enzymes such as alcohol oxidase, cholesterol oxidase, xanthine oxidase and the like can be used. Further, as the electron acceptor, potassium ferricyanide used in the above-mentioned examples is suitable because it reacts stably, but when P-benzoquinone is used, the reaction rate is high, and therefore it is suitable for speeding up. Furthermore, 2.6-dichlorophenol indophenol, methylene blue, phenazine methosulfate, β
-Naphthoquinone 4-sulfonate potassium, ferrocene and the like can be used.

EFFECTS OF THE INVENTION As described above, the biosensor of the present invention has the oxygen reaction in which the electrode system and the hydrophilic polymer layer are formed on the insulating substrate, and the oxidoreductase and the electron acceptor are independently contained on the electrode system. Since the layer is formed, the interaction does not occur in water, so that the response does not vary and a stable response can be obtained. Therefore, the substrate concentration in the biological sample can be measured accurately and easily, and storage stability is also simplified.
Moreover, since the sensor can be manufactured by screen printing, there are advantages such as easy mass production.

[Brief description of drawings]

FIG. 1 is a perspective view of a biosensor according to an embodiment of the present invention, FIG. 2 (a) is a schematic view of an essential part of the biosensor from above, and FIG. 2 (b) is an essential part of the biosensor. FIG. 3 is a schematic view of the side view of FIG. 3, and FIG. 3 is a vertical sectional view of a conventional biosensor. 1 ... Insulating substrate, 2 ... Counter electrode, 3 ... Measuring electrode, 4 ...
Insulating layer, 5 ... CMC layer, 6 ... Redox enzyme, 7 ...
Electron acceptor, 8 ... Enzyme reaction layer.

Claims (2)

[Claims] 1. An insulating substrate provided with an electrode system comprising at least a measurement electrode and a counter electrode, a hydrophilic polymer layer provided on the surface of the electrode system, and an electron acceptor and a redox enzyme independent from each other on the upper side thereof. To form an oxygen-reactive layer containing it, and electrochemically detect the change in the substance concentration during the reaction of the enzyme, electron acceptor and sample solution with the electrode system to measure the substrate concentration in the sample solution. Characteristic biosensor. 2. The biosensor according to claim 1, wherein the electrode system, the hydrophilic polymer layer and the oxygen reactive layer are formed by screen printing.