JPH01134246A - Biosensor - Google Patents

Abstract

PURPOSE:To easily measure the substrate concentration in an organic body by integrating an insulating substrate, an electrode system, a porous body which carries oxidation reducing enzyme, a porous body film, and a porous body which carries an electron acceptor. CONSTITUTION:The electrode system consisting of a counter electrode 2, a measuring electrode 2, and a reference electrode 4 is formed on the insulating substrate 1 and an insulating layer 5 is formed covering said part partially except electrochemical operating parts 2'-4'. Then a water-absorptive high polymer layer is provided on the surface of electrode systems 2'-4', a groove is formed on the electrode system with both-sided adhesive tapes 7, and tapes of cellulose carrying oxidation reducing enzyme are formed on both sides as guide layers 8. Then a polycarbonate porous material film 9 is adhered as the filter film on a holding frame 10 and fixed with the both-sided adhesive tapes 7 so that the electrode systems 2'-4' are covered. Further, the porous body 11 which carries the electron acceptor is placed at the hole part of the holding frame 10 and a resin-made cover 12 is adhered to integrate the entire body.

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 6 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. Ta. (For example, Japanese Unexamined Patent Application Publication No. 61-2943151) This biosensor is manufactured by printing a conductive carbon paste on an insulating substrate 1 by thread printing and drying it by heating, thereby forming a counter electrode 2. An electrode system consisting of a measurement electrode of 3 degrees and a reference electrode of 4 is formed. 2', 3' then partially cover the electrode system and become the electrochemically active part of each electrode.
, Print the insulating paste in the same way as above so as to leave 4',
The insulating layer 5 is formed by heat treatment. Next, a holding frame 9 made of resin with holes is glued to the insulating layer 6, and the electrode system 2',
A porous body 1o is held in the hole so as to cover 3' and 4', and a resin cover 11 having an opening having a diameter smaller than that of the porous body is bonded to integrate the whole. The porous body carries an oxidoreductase and an electron acceptor, and when a sample solution containing a substrate is added to the porous body, an enzymatic reaction proceeds and the electron acceptor is reduced. After the enzymatic reaction is completed, the reduced electron acceptor is electrochemically treated with acid (1,-
Then, 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 With this conventional configuration, substrate concentration can be measured accurately and in a short time by adding only a trace amount of a sample with low viscosity such as urine or serum; , when blood cells are mixed,
Blood cells adhered to the electrode surface, greatly reducing the response.Furthermore, due to the high viscosity, the enzymatic reaction was slow, and it took a long time to complete the 5-minute collection reaction, resulting in variations in measured values. Furthermore, when an oxidoreductase and an electron acceptor are supported on the same porous material, the initial properties may not be maintained due to the influence of humidity and light.

As a result of various studies on these points, the present invention has been developed by providing a water-absorbing polymer layer on the surface of the filtration membrane and the electrode system and integrating it with the porous body, and furthermore, providing an induction layer under the filtration membrane and adding oxidation to this layer. By supporting reductase, we provide a disposable biosensor that can be stored for a long period of time and is supported separately from the electron acceptor in the porous material, allowing for extremely easy, rapid, and highly accurate quantification of specific components in biological samples. It is something to do.

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 substrate concentration in a sample solution by electrochemically detecting it with the electrode system, a water-absorbing polymer layer is provided on the surface of the electrode system, and a redox enzyme is applied so as to sandwich the electrode from the front and back. A supported induction layer is arranged, and a filtration membrane is placed to cover the electrode.
Furthermore, a porous body carrying an electron acceptor is placed on top of the porous body, thereby integrating each element.

According to the present invention, a disposable biosensor 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 carrying the enzyme and electron acceptor separately, it is possible to obtain a long-term stable response without being affected by light or humidity during production and storage. Furthermore, since the guiding layer was installed to sandwich the electrode system, the sample liquid filtered through the filtration membrane was reliably guided onto the electrode system, and the enzyme reaction was carried out. The water-absorbing polymer on the electrode system improves wettability and prevents adsorption of proteins in the sample, allowing highly accurate measurements. moreover,
Since only the low-viscosity sample solution filtered through the filtration membrane reacts with the enzyme, the reaction can be completed in a short time, and the amount of enzyme carried can be small.

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 screen 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. 2' then partially covers this electrode system and becomes the electrochemically active part of each electrode.

An insulating paste is printed in the same manner as above so as to leave s', 4' (1 rtm each!) and heat treated to form an insulating layer 6.0 of this electrode system (2/, 3/, 4/). A 0.5% aqueous solution of auae (carboxymethyl cellulose), a type of cellulosic water-absorbing polymer, was applied to the surface.
It was dried at 6°C for 30 minutes.

Next, as shown in FIG. 1, grooves are made on the electrode system using double-sided adhesive tape 7, and the guide layer 8 is placed on both sides so as to sandwich the electrodes from the front and back.
Install cellulose tape as a protection. This cellulose tape was impregnated with a solution in which 10 mg of glucose oxidase as an oxidoreductase was dissolved in a phosphate buffer (PHs, e), and then dried. Next, a porous polycarbonate membrane 9 with a pore diameter of 1 μm is adhered as a filtration membrane to the resin holding frame 10 with holes, and the electrode systems 2', 3
′, 4′ are fixed with double-sided adhesive tape 7 so as to cover them. Further, a porous body 11 is placed in the opening of the holding frame 10, and a resin cover 12 having an opening having a diameter smaller than that of the porous body is adhered to integrate the whole. The porous body 11 is a nylon nonwoven fabric containing potassium ferricyanide as an electron acceptor at pH 5.6.
It was prepared by impregnating with a solution dissolved in phosphate buffer and drying under reduced pressure. A cross-sectional view along the measurement electrode 3 of this integrated biosensor is shown in FIG.

A glucose standard solution was dropped as a sample solution into the porous body of the glucose sensor configured as described above, and after 2 minutes, the reference electrode 4'
With reference to , the potential of the measuring electrode 3' is set to +0.
Apply a 5v pulse voltage and measure the current after 5 seconds. In this case, potassium ferricyanide supported on the porous body is dissolved by the added glucose standard solution. After passing through the porous polycarbonate membrane, the liquid is guided by cellulose tape placed between the front and back of the electrode system and reaches the top of the electrode system. At this time, glucose oxidase supported on the cellulose tape dissolves and oxidizes glucose, and potassium ferricyanide is simultaneously reduced to produce potassium ferrocyanide. Therefore, by applying the pulse voltage described above, an oxidation current is obtained based on the concentration of the generated potassium ferrocyanide, which corresponds to the concentration of glucose, which is the substrate. Good linearity of the oxidation current was obtained up to a glucose concentration of -r o m f/ell.

Blood sample 20μ for the above glucose sensor! When the response current was measured 2 minutes after dropping the solution, a response with very good reproducibility was obtained. In the case of blood, it has a high viscosity due to the presence of blood cells, making it extremely difficult to carry out enzymatic reactions quickly. Conventionally, operations such as centrifugation and stirring were indispensable.

In addition, a phenomenon in which proteins adhered to the electrode surface caused variations in response was observed. However, when a porous polycarbonate membrane with a pore size of 1 μm is used as a filtration membrane, the viscosity decreases because blood cells are filtered through 'f5 and then react with glucose oxidase.
The enzymatic reaction could be carried out quickly. Furthermore, in order to supply the liquid on the polycarbonate porous membrane onto the electrode, the cellulose tape placed between the electrodes as a guide layer allows the filtrate to be guided quickly and efficiently, and 30 minutes after dropping the blood. The reaction solution could be supplied onto the electrode system in seconds. If you don't have cellulose tape,
It took some time for the reaction solution to reach the electrode system. When the height of the groove was lowered, the reaction solution arrived faster, but when the groove was lowered by 100 μm, air bubbles remained and the response current varied due to the influence of the porous polycarbonate membrane. When a cellulose tape is placed over the electrode,
Although the reaction solution quickly reached the electrode, bubbles were sometimes generated, and the response was low and varied. It has been found that if the cellulose tape is placed close to the electrode so as not to cover it, the reaction solution can be effectively and quickly supplied onto the electrode and a stable response can be obtained. In addition to cellulose, thin hydrophilic porous materials such as rayon and pulp could be used.

Although measurement is possible by supporting glucose oxidase and potassium ferricyanide on the porous body 11 made of nylon nonwoven fabric, it was found that glucose oxidase and potassium ferricyanide react with each other due to the influence of humidity and light during preparation and storage. Therefore, it is considered easier to prepare and more effective for long-term storage if they are supported separately. Furthermore, the amount of filtered liquid is about 〃～Akira of the dropped liquid, so
The amount of enzyme supported was also reduced to a very small amount.

By applying CMC to the electrode surface, the wettability of the electrode was improved, and even only 2 to 3 μl of the reaction solution was able to sufficiently cover the electrode system. In addition, proteins in the reaction solution were prevented from adhering to the electrode surface, and a response with good reproducibility was obtained. Water-absorbing polymers include carboxymethyl cellulose and gelatin.

Acrylate-based, vinyl alcohol-based, vinylpyrrolidone-based, and maleic anhydride-based materials are preferred.

The method of integration in the biosensor of the present invention is not limited to the shapes and combinations of the frame, cover, etc. shown in the embodiments. Moreover, the combination of oxidoreductase and electron acceptor is not limited to the above embodiments, 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 As described above, the biosensor of the present invention can be manufactured very easily by integrating an insulating substrate, an electrode system, a porous body carrying an oxidoreductase, a porous membrane, and a porous body carrying an electron acceptor. It is possible to measure the substrate concentration in biological samples, and by supporting the enzyme and electron acceptor through a porous membrane, it enables rapid measurement and long-term storage. Furthermore, a water-absorbing polymer was applied to the electrode surface to prevent interfering substances from adsorbing to the electrode, increasing measurement accuracy.

[Brief explanation of the drawing]

FIG. 1 is an exploded perspective view of a biosensor according to an embodiment of the present invention, FIG. 2 is a vertical sectional view thereof, and FIG. 3 is an exploded perspective view of a conventional biosensor. 1... Insulating substrate, 2... Counter electrode, 3.
...Measuring pole, 4...Reference pole, 6...
...Insulating layer, 6...CMC17...Double-sided adhesive tape, 8...Induction layer, 9...
P membrane, 1o...Holding frame, 11...Porous body, 12...Cover〇Name of agent Patent attorney Toshi Nakao and 1 other person! −
1 [Residence 1

Claims (2)

[Claims] (1) In a biosensor that is equipped with an insulating substrate provided with an electrode system consisting of at least a measurement electrode and a counter electrode, and that measures the substrate concentration during the reaction between an enzyme, an electron acceptor, and a sample solution, the surface of the electrode system has a water-absorbing surface. A polymer layer is provided, a dielectric layer supporting an oxidoreductase is provided between the front and back of the electrode system, a filtration membrane is placed to cover the electrode, and a porous material supporting an electron acceptor is placed on top of the filtration membrane. A biosensor characterized in that each of the above-mentioned elements is integrated. (2) Claim 1 in which the water-absorbing polymer is one of the group consisting of carboxymethyl cellulose, gelatin, acrylic acid, vinyl alcohol, vinylpyrrolidone, and maleic anhydride, or a mixture thereof.
Biosensor as described in section.