JPH01134245A - Biosensor - Google Patents

Abstract

PURPOSE:To easily measure the substrate concentration in an organic sample by adhering an insulating substrate, an electrode system, and a filter film with a water-absorptive high polymer layer, and integrating them with a porous body. CONSTITUTION:The electrode system consisting of a counter electrode 2, a measuring electrode 3, and a reference electrode 4 is formed on the insulating substrate 1, and an insulating layer 5 is formed covering the electrode system partially except pats 2'-4' which operate electrochemically. A holding frame 7 is adhered on the electrode system, a water-absorptive high polymer layer is provided on the surface of electrode systems 2'-4' in the holding frame 7, and a porous film is provided thereupon as a filter film 8 and fixed covering the electrode systems. Then nylon nonwoven fabric is coated with liquid obtained by dissolving oxidation reducing enzyme and an electron acceptor and dried, the porous body 9 is placed at the hole part of the holding frame 7, and a resin-made cover 10 with a hole part having a smaller diameter than the porous body 9 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 Publication No. 61-294361) This biosensor is manufactured by printing a conductive carbon paste on an insulating substrate 1 by screen printing, heating and drying it, and then forming a counter electrode 2. Measuring pole 3. An electrode system consisting of a reference electrode 4 is formed. 2 1 , which then partially covers the electrode system and becomes the electrochemically active part of each electrode.
An insulating paste is printed in the same manner as described above so as to leave s/ and 4/, and heat treated to form an insulating layer 5. next,
A holding frame 7 made of resin with holes is glued to the insulating layer 6, and a porous body 9 is held in the hole so as to cover the electrode systems 2', 3', and 4'. A resin cover 10 having an opening is bonded to integrate the whole. The above-mentioned porous material carries an oxidoreductase and an electron acceptor.
When a sample solution containing a substrate is added to the porous body, an enzymatic reaction proceeds and electron acceptors are reduced. After the enzymatic reaction is completed, the reduced electron acceptor is electrochemically oxidized using the electrode, 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 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, resulting in a sharp drop in response.Furthermore, due to the high viscosity, the enzyme reaction was slow, and it took more than 5 minutes to complete the reaction, causing measurement values to vary.

As a result of various studies on these points, the present invention has been developed by bonding an electrode system and a filtration membrane with a water-absorbing polymer and further integrating it with a porous material, thereby making it possible to absorb liquids in highly viscous biological samples such as blood. The present invention provides an inexpensive disposable biosensor that can extremely easily quantify specific components quickly and with high precision.

Means for Solving the Problems In order to solve the above problems, the present invention provides an electrode system consisting of at least a measurement electrode and a counter electrode on an insulating substrate, allows an enzyme, an electron acceptor, and a sample solution to react, and during the reaction, In a biosensor that electrochemically detects changes in the substance concentration using the electrode system and measures the substrate concentration in the sample solution, the electrode system and the filtration membrane are bonded with a water-absorbing polymer, and this is further bonded with a porous material. It is integrated.

According to the present invention, a disposable type biosensor including an electrode system can be constructed, and by adding it to a sample liquid and a porous body, the substrate concentration can be measured very easily.

Moreover, by bonding the electrode system and the filtration membrane with a water-absorbing polymer, it can be manufactured easily, and even highly viscous samples such as blood can be quickly filtered from the filtration membrane onto the electrode, preventing the formation of bubbles and protein. This prevents interfering substances from adsorbing on the electrode surface, allowing 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 counter electrode 2. is formed by printing a conductive carbon paste on an insulating substrate 1 made of polyethylene terephthalate by screen printing and drying it by heating. Measuring pole 3. An electrode system consisting of a reference electrode 4 is formed. Next, partially cover the electrode system and
2' which becomes the electrochemically active part of each electrode.

An insulating base plate is printed in the same manner as described above so that 3' and 4' (1H2 each) are left, and an insulating layer is formed by heat treatment.

A holding frame 7 made of resin with holes drilled therein was adhered onto this electrode system. Next, the electrode systems 2', 3' in the holding frame T.

The surface of 4' is coated with 0.5% of 0M06 (carboxymethylcellulose), a type of cellulosic water-absorbing polymer.
An aqueous solution is applied, and a polycarbonate porous membrane (pore diameter: 1 μm) is placed thereon as a filtration membrane 8, and is air-dried to be fixed so as to cover the electrode system.

Next, a solution prepared by dissolving glucose oxidase as an oxidoreductase and potassium ferricyanide as an electron acceptor in a phosphate buffer solution (PH5, 6) was applied to a nylon nonwoven fabric, and the porous body 9 was dried under reduced pressure and placed in the opening of the holding frame. A resin cover 10 having an opening having a diameter smaller than that of the porous body is bonded to integrate the whole body. At this time, the thickness of the holding frame 7 is adjusted so that the resin cover 10 does not press the porous body directly, but there is a gap of at least 200 μm between the resin cover 10 and the porous body. 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 was
′ as a reference, the potential of the measurement electrode 3′ is +0 towards the anode.
．． Apply a 6v pulse voltage and measure the current after 5 seconds. In this case, the added glucose standard solution causes the porous body to
Glucose oxidase and potassium ferricyanide supported on the ferrocyanide dissolve, oxidize glucose, and potassium ferricyanide is simultaneously reduced to produce potassium ferrocyanide. This reaction liquid passes through the porous polycarbonate membrane, wets and swells the CMC layer, and forms a liquid film on the electrode system. Therefore, by applying the above pulse voltage,
An oxidation current is obtained based on the concentration of potassium ferrocyanide produced, which corresponds to the concentration of the substrate glucose. Glucose concentration is roorng/dI! Good linearity was obtained up to

When 20 μl of a blood sample was dropped onto the above glucose sensor and the response current was measured 2 minutes later, 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 was observed in which proteins adhered to the electrode surface and the response varied. However, when a porous polycarbonate membrane with a pore size of 1 μm is used as a filtration membrane, the blood cells are filtered and the p-liquid quickly penetrates into the CMC layer, allowing the reaction to proceed in as little as 1 minute without being affected by the blood cells. The reaction ended. In addition, since the 0M0 layer swelled, proteins were prevented from adhering to the electrode surface, allowing accurate measurements.

As for the configuration of the sensor, instead of simultaneously supporting the enzyme and electron acceptor on the porous material, good measurements can be made by separately supporting glucose oxidase on the polycarbonate porous membrane and potassium ferricyanide on the porous material and integrating them.

Furthermore, by supporting them separately, there is no risk of potassium ferricyanide and glucose oxidase reacting due to the effects of light or moisture during preparation and storage, making it easier to prepare senna and improving reliability in long-term storage. .

As water-absorbing polymers, carboxymethyl cellulose,
Gelatin-based, 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. Further, the combination of oxidoreductase and electron acceptor is not limited to the above-mentioned 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 embodiment, a three-electrode blade type electrode system was described, but a two-electrode blade type consisting of a counter electrode and a measurement electrode can also be used for measurement.

Effects of the Invention As described above, the biosensor of the present invention has an insulating substrate, an electrode system, and a filtration membrane bonded together with a water-absorbing polymer layer, and is integrated with a porous body, thereby making it extremely easy to adjust the substrate concentration in a biological sample. was able to be measured. Furthermore, the water-absorbing polymer layer prevents interfering substances in the sample solution from adsorbing to the electrode surface, improving 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 longitudinal sectional view thereof, and FIG. 3 is a longitudinal sectional view of a conventional biosensor. 1... Insulating substrate, 2... Counter electrode, 3
...Measuring pole, 4...Reference pole, 6...
...Insulating layer, 6...Water-absorbing polymer, 7...
...Holding frame, 8...Filtration membrane, 9...
Porous body, 1o...Cover. Name of agent: Patent attorney Toshio Nakao and 1 other person/-
- Itsutsu #a substrate 6 - Water-absorbing appeal molecule 3 - Fuchisada pancreas
8-Filtration, filtration membrane 4--One piece only 9-----Shishi・
Koshiine 5-Insulation/1 to 6 Par Fig. 1 Fig. 2 Fig. 3

Claims (2)

[Claims] (1) In a biosensor that electrochemically measures changes in substrate concentration during a reaction between an enzyme, an electron acceptor, and a sample solution, the biosensor is equipped with an insulating substrate provided with an electrode system consisting of at least a measurement electrode and a counter electrode. A biosensor characterized in that a system and a filtration membrane are bonded together using a water-absorbing polymer, and this is further integrated with a porous body that retains at least an electron acceptor. (2) 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. The biosensor according to item 1.