JPH01223338A - Biosensor - Google Patents

Abstract

PURPOSE:To obtain an inexpensive disposable type sensor with a high reservation stability, by providing an electrode system comprising at least a measuring pole and an opposed pole on an insulation substrate to cover the surface of the electrode system with an organic high polymer beforehand. CONSTITUTION:A conducting carbon paste is printed on an insulation substrate 1 by a screen printing and heated to form an electrode system comprising an opposed pole 2, a measuring pole 3 and a reference pole 4. The electrode system is coated with a photosensitive resin and after dried, it is exposed to light through a mask to form an organic high polymer layer 5 at a part thereof by development. Then, a holding frame 6 bored is bonded on the high polymer layer 5, a porous body 7 fixed on a window of the holding frame 6 and a protective frame 8 is bonded to integrate totally. This enables the production of an inexpensive disposable type sensor with a high preservation stability.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention 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.

Conventional technology Conventionally, 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, enzymes and proteins with substrate selectivity have been used to quantify specific components in biological samples such as blood. There is a sensor consisting of a transducer that measures changes in the concentration and heat of a substance consumed or produced by a reaction between a selective substance and a substrate. The transducer used here is an electrochemical device using a platinum electrode, a carbon electrode, a gas electrode, an ion-selective electrode, or the like. Among these, in particular, in the method using carbon electrodes, an oxidoreductase and an electron acceptor are combined, and the electron acceptor reduced by the reaction between the substrate and the enzyme is electrochemically oxidized, and the oxidation current value obtained at this time is A disposable biosensor that measures substrate concentration has been proposed. A conventional example of such a disposable biosensor is the biosensor shown in FIG.
Publication No. 54). This biosensor bar has an electrode system consisting of a counter electrode 10, a measurement electrode 11, and a reference electrode 12 on an insulating substrate 9, and a protein (albumin, etc.)
The holding frame 1 is provided with a porous body 14 carrying an oxidoreductase and an electron acceptor on the top of the coating layer.
6 and a protective frame 16. When the sample liquid is dropped onto the porous body, the oxidoreductase and electron acceptor in the porous body are dissolved in the sample liquid, an enzymatic reaction proceeds with the substrate in the sample liquid, and the electron acceptor is reduced. Substrate concentration has been measured from the oxidation current when reduced electron acceptors are oxidized.

Problems to be Solved by the Invention In such a conventional configuration, a disposable type biosensor has been proposed and a sensor that is inexpensive and has good reproducibility has been provided, but in terms of storage stability, it is necessary to store it in N2. Not much progress was seen.

The present invention provides an inexpensive disposable biosensor with good storage stability.

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, and coats the surface of the electrode system with an organic polymer having a crosslinked structure in advance. Further, the insulating substrate is integrated with a porous body carrying an oxidoreductase and an electron acceptor.

According to the present invention, it is possible to construct a biosensor that is of a disposable type and has good storage stability, including an electrode system, and the substrate concentration can be extremely easily measured by adding a sample liquid to a porous body. be able to.

Examples Examples of organic polymers that can be used in the present invention include -vinyl pyrrolidone, vinyl alcohol, 2-hydroxylethyl acrylate, 2-hydroxylethyl methacrylate, and acrylamide. A photosensitive organic polymer or a vinyl group obtained by adding a photocrosslinking agent to a water-soluble organic polymer obtained by polymerizing at least one of the monomers mentioned above and a non-aqueous monomer (such as methyl methacrylate). Photosensitive organic polymers containing monomers modified with photosensitive groups, such as organic polymers polymerized from monomers with polymerizable double bonds such as acrylates and methacrylates, photosensitive organic polymers such as glycidyl groups, chalcone groups, etc. Cross-linked organic polymers having electron acceptor permeability, such as photosensitive organic polymers containing functional groups and photosensitive organic polymers containing silicon, can be used. As a method for forming a crosslinked structure, a method of photocrosslinking the photosensitive polymer or a method of thermosetting the photosensitive polymer can be used.

An embodiment of the present invention will be described below with reference to the drawings.

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. By printing conductive carbon paste on the insulating substrate 1 by screen printing and heating and drying it, a counter electrode 2, a measuring electrode 3,
An electrode system consisting of a reference electrode 4 is formed. A photosensitive resin consisting of a 6% aqueous solution of 100 parts of polyvinylpyrrolidone (K-90) and 6 parts of a photocrosslinking agent Mu-066 (manufactured by Shinko Giken) was applied to this electrode system, and after drying, it was exposed to light through a mask. By development, an organic polymer layer 6 is formed on a part of the electrode system. Next, a resin holding frame 6 with holes drilled therein was adhered to the organic polymer layer 6, and glucose oxidase 1o was added as an oxidoreductase.
Potassium ferricyanide 150 as Oda and electron acceptor
After impregnating a nylon nonwoven fabric with a solution of 11i1 dissolved in 1 cc of a phosphate buffer solution of pH 6.6, the porous body 7 prepared by drying under reduced pressure is fixed to the window of the holding frame 6, the protective frame 8 is glued, and the whole is assembled. Unify.

A glucose standard solution is dropped as a sample solution into the porous body of the glucose sensor configured as described above, and two minutes after the drop, the potential of the measurement electrode is adjusted to 0. I
It was swept at a speed of V/sec. In this case, the added glucose reacts with potassium ferricyanide by the action of glucose oxidase supported on the porous material to produce potassium ferrocyanide. Therefore, by the above-mentioned sweep toward the anode, an oxidation current based on the concentration of potassium ferrocyanide produced is obtained, and this current value corresponds to the concentration of glucose, which is the substrate.

A serum sample was dropped onto the above glucose sensor, and the peak current value observed 2 minutes later had good reproducibility as shown in Figure 2. This sensor has a temperature of 26 months for 6 months.
Fig. 3B shows the peak current value measured in the same manner as in the previous example after storage under conditions of 60% temperature and humidity.

For comparison, FIG. 3G shows a sample coated with protein instead of the organic polymer and subjected to the same experiment as B. This is considered to be because the protein was denatured under the above storage conditions, causing a drift in the peak current.

However, the use of organic polymers increases the shelf life of the sensor and provides responses with good reproducibility.

The method of integration in the biosensor of the present invention is as follows:
The present invention is not limited to the shapes and combinations of the frames shown in the examples. Further, the combination of an oxidoreductase and an electron acceptor is not limited to the above examples, and any combination can be used as long as it meets the spirit of the present invention. Furthermore, any organic polymer can be used as long as it has the ability to pass through an electron acceptor. On the other hand, in the above embodiments, a three-electrode system was used as the electrode system, but a two-electrode system consisting of a counter electrode and a measuring electrode can also be used for measurement.

Effects of the Invention As described above, since the biosensor of the present invention can be manufactured at extremely low cost, it is possible to provide a disposable sensor, and it is also possible to extremely easily measure the substrate concentration in a biological sample.

Furthermore, it has become possible to suppress deterioration of the characteristics of the sensor during storage.

[Brief explanation of the drawing]

FIG. 1 is an exploded perspective view of a biosensor that is an embodiment of the present invention, and FIGS. 2 and 3 are diagrams showing response examples of the sensor.
FIG. 4 shows an exploded perspective view of a conventional biosensor. DESCRIPTION OF SYMBOLS 1...Insulating substrate, 2...Counter electrode, 3...Measurement electrode, 4...Reference electrode, 6...Organic polymer layer, 6 . . . Holding frame, 7 . . . Porous body, 8 . . . Protection frame. Name of agent: Patent attorney Toshio Nakao and 1 other person
1ffl Figure 2 Zumanri] 2 C] Huan Figure 3

Claims (1)

[Claims] An electrode system consisting of at least a measuring electrode and a counter electrode made of a material mainly composed of carbon is provided on an insulating substrate, and the surface of at least the measuring electrode of the electrode system is coated in advance with an organic polymer having a crosslinked structure, and further coated with an oxidoreductase and a counter electrode. A biosensor characterized in that a porous body supporting an electron acceptor and the insulating substrate are integrated.