JPS5967452A - Biosensor - Google Patents

Abstract

PURPOSE:To obtain a biosensor excellent in the economy which enables the replacement of an enzyme immobilization film alone when the enzyme activity lowers by interposing a porous high polymer film having an enzyme immobilized between a preelectrolyzing porous metal electrode and a porous electrode for detecting H2O2. CONSTITUTION:In an immobilized enzyme electrode as biosensor for detecting and analyzing a biological substance, a film in which, for example, glucose oxidase 11 is immobilized to a polycarbonate porous film 10 or the like is provided between a corrosion resistant porous metal electrode made of stainless steel, Pt or the like as preelectrolyzing electrode 8 for removing disturbing substance in an object to be inspected and a porous metal electrode 9 for detecting H2O2 at Pt screen or stainless screen plated with Pt. The enzyme immobilized electrode is loaded in such a manner that the electrode 9 is positioned inside a holder 13 and a reference electrode 20 and an opposite pole 19 of the electrode 9 are provided in the holder 13 while a lead wire 14 of the electrode 8 and an opposite pole 17 thereof 8 are provided to obtain a biosensor. Thus, a sensor is obtained which facilitates the conversion of the film 10 with an excellent respoonse and a greater mechanical strength.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to improvements in biosensors using immobilized enzyme electrodes.

Identification of Conventional Structures and Their Problems Various types of so-called biosensors have been proposed for the purpose of detecting and analyzing biological substances using enzymatic reactions. Among these, a sensor using an immobilized enzyme electrode is one that is attracting attention due to its high accuracy and rapid response. Taking the glucose sensor as an example, a typical sensor using an immobilized enzyme, Clari et al.'s glucosenza, in which glucose oxidase (hereinafter referred to as GOD) was immobilized on an enzyme detection electrode, has been developed. Recently, an improved type has been proposed, as shown in FIG. 1, which is composed of a porous thin film having a platinum layer and a porous thin film having a platinum layer and immobilized GOD.

In FIG. 1, reference numeral 1 denotes an electrode for pre-electrolysis to remove interfering substances from the test liquid, and has a platinum layer 3 provided on one side of a porous thin film 20. 4 is an electrode for detecting hydrogen peroxide, which has a platinum layer 6 on one side of a porous thin film 6, and a G layer on the other side and the pores.
OD7 is fixed. Glucose in the test liquid is oxidized by this GOD, and the concentration of glucose is detected from the oxidation current value obtained by electrolytically oxidizing H2O2 generated at that time in the platinum layer 6.

The porous thin film used here is very thin in order to improve the diffusion of the test liquid, so it must be handled with care.
Providing a porous platinum layer also has the disadvantage that if the amount is too small, electron conductivity cannot be obtained, and if the amount is too large, the porous thin film becomes clogged and does not function properly. Furthermore, since the enzyme is fixed on the platinum layer, improvements in economical efficiency are also desired, such as the fact that the entire expensive platinum layer must be disposed of when the enzyme activity decreases and it becomes unusable.

Purpose of the Invention The present invention solves the above-mentioned conventional inconveniences, improves the characteristics of a sensor, makes it easy to handle in terms of strength, and is also economical because only the immobilized enzyme membrane can be replaced. The purpose is to provide excellent sensors.

Components of the Invention The biosensor of the present invention comprises a porous metal electrode for pre-electrolysis, a porous metal electrode capable of detecting hydrogen peroxide, and a porous polymer membrane on which an enzyme is immobilized and interposed between the two electrodes. It is composed of.

Description of examples FIG. 2 shows an example of the configuration of the biosensor of the present invention.

In FIG. 2, reference numeral 8 denotes a porous metal electrode made of stainless steel, nickel, platinum, etc., which has corrosion resistance, and is made of, for example, a wire mesh, a sintered plate, etc., and is used for pre-electrolysis to remove interfering substances. Reference numeral 9 denotes an electrode for measuring the oxidation current of hydrogen peroxide, and a platinum wire mesh or the like is mainly used.

In order to reduce costs, platinum may be electrodeposited or vapor-deposited on a perforated stainless steel plate or net as a catalyst. Reference numeral 10 denotes a thin polymer film sandwiched between electrodes 8 and 9, and enzyme 11 is immobilized on the surface thereof, including in the pores.

The immobilized enzyme is a so-called oxidoreductase in which hydrogen peroxide is produced in a reaction system, but it can also be immobilized together with other enzymes as a complex enzyme. The porous polymer membrane used is cellulose or polycarbonate. Regarding the location of enzyme immobilization, in order to increase the reaction efficiency, it is also possible to adopt a configuration in which the side of the porous polymer membrane in contact with electrode 9 has more enzyme immobilized, and the part without immobilized enzyme is placed on the electrode 8 side. good.

With the above configuration, the sensor of the present invention has higher electron conductivity than conventional sensors because both electrodes 8 and 9 are made of metal, and has excellent responsiveness and sensitivity. It is stable and has a long lifespan with less deterioration compared to conventional enzymes immobilized on platinum layers. Of course, this polymer membrane with immobilized enzyme can be replaced if it deteriorates.

Next, a specific example of a glucose sensor using GOD will be described as one of the biosensors.

The electrode 8 is made of stainless steel and has a thickness of 80 μm.
A 20oO mesh net was used. As the electrode 9, a stainless steel net similar to that described above was used, in which platinum was deposited by sputtering to increase the reactivity with hydrogen peroxide. As the polymer membrane serving as the enzyme carrier, a polycarbonate porous membrane with a pore diameter of 2000 μm, a membrane thickness of 2 μm, and a pore density of 3×10 8 pores/cyl was used. 10 μl/ml of a 100 mo/ml aqueous solution of GOD as an enzyme was added to this membrane.
The membrane was vacuum dried at 4°C,
Next, a crosslinking reaction was carried out at 26°C for 1 hour in glutapraldehyde vapor, which is a bifunctional crosslinking reagent, for immobilization.

After the reaction was completed, it was thoroughly washed with water.

Figure 3 schematically shows the electrode holder and electrode system equipped with the enzyme-immobilized electrode described above. Reference numeral 12 in the figure represents the above-mentioned electrode, which is mounted so that the electrode 9 side is inside the holder 13. 14 is attached to the outer tube 15 of the holder,
It is a lead in contact with the porous metal electrode 8 for pre-electrolysis, and 1
6 is the lead of the opposite pole 17. A lead 18 is attached to the holder and is in contact with the porous metal electrode 9 having hydrogen peroxide detection capability. 19 is a counter electrode, 20 is a reference electrode for keeping the voltage applied to electrode 9 constant, and AqC
12/Ag electrode was used. Inside the electrode holder, the pH is 5.6.
phosphate buffer 21.

A comparative test of the response characteristics to glucose was conducted between the sensor A according to the present invention described above and the conventional sensor B, which uses a membrane electrode as shown in FIG. The potential of the electrode capable of detecting hydrogen peroxide was set to +0.6V with respect to the reference electrode 20, and the voltage applied between the electrode for pre-electrolysis and the counter electrode 170 was +0.55V.

FIG. 4 shows the response current to glucose concentration. From this, it can be seen that sensor A has higher sensitivity than sensor B because the slope is steeper, and sensor A also has better linearity in the high concentration region. Furthermore, regarding the repetition life, while A showed almost no deterioration after 300 repetitions, B showed a 20% deterioration in sensitivity (response current).

As mentioned above, this result was obtained by using a porous metal electrode with excellent electronic conductivity to create an electrochemical system with almost no electrical resistance, and in the meantime immobilizing the enzyme that controls the enzyme reaction. This is thought to be due to the sandwiching of the polymer membrane. Of course, the mechanical strength has also been improved, and the polymer membrane can be easily replaced, making it easier to handle and economically advantageous.

The above two effects are not limited to glucose oxidase, but are useful for all so-called redox enzymes such as alcohol oxidase and xanthine oxidase.

Effects of the Invention As described above, according to the present invention, it is possible to obtain a biosensor that not only has excellent properties but also is easy to handle and economically advantageous.

[Brief explanation of drawings]

Figure 1 is a cross-sectional schematic diagram showing the configuration of the electrodes of a conventional sensor.
FIG. 2 is a schematic cross-sectional view showing an example of the structure of the electrode of the sensor of the present invention, FIG. 3 is a vertical cross-sectional view of a cell using the same electrode, and FIG. 4 is a comparison of the response characteristics of the electrodes. 8... Electrode for pre-electrolysis, 9... Electrode for detecting hydrogen peroxide, 10... Polymer membrane, 11... Immobilized enzyme. Name of agent: Patent attorney Toshio Nakao and 1 other person No. 1
Figure 2 Figure 3 f'7 1z

Claims (1)

[Claims] A biosensor comprising a porous metal electrode for pre-electrolysis, a porous metal electrode capable of detecting hydrogen peroxide, and a porous polymer membrane on which an enzyme is immobilized and interposed between the two electrodes.