JPH08285814A - Biosensor - Google Patents

Abstract

PURPOSE: To provide a small-sized biosensor which can measure a liquid specimen in a very small quantity. CONSTITUTION: A working electrode 14 and a counter electrode 16 are installed on opposite inside faces of an upper substrate 11 and a lower substrate 12 which are faced, and an immobilized enzyme film 15 is formed on the surface of the working electrode 14. A spacer 13 is interposed between the immobilized enzyme film 15 and the counter electrode 16 so as to be dispersed, the interval between both electrodes is made uniform, and both electrodes are held by a seal material. A drop introduction part 12a which is extended to a side part from the opposite part is formed on the lower substrate 12. By this constitution, both substrates can be formed to be of a stacked and compact structure as viewed from a plane. In addition, a liquid specimen 18 which has been soaked between both electrodes can be set to be a very small quantity, it is sufficient to drop the liquid onto the drop introduction part 12a, and a measurement can be performed surely and easily.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biosensor, and more particularly to an enzyme sensor used in clinical tests, water quality tests, etc., for detecting the concentration of various liquid components by using immobilized enzymes. Pertain.

[0002]

2. Description of the Related Art Conventionally, as one of biosensors using an immobilized enzyme, those shown in FIGS. 3A and 3B are known. FIG. 3A is a plan view.
3B is a cross-sectional view taken along the line ab of FIG. In this biosensor, a working electrode 2 and a counter electrode 3 are both provided on one surface of a substrate 1. The counter electrode 3 has a substantially U-shaped planar shape, and is formed at a predetermined distance from the three sides of the rectangular working electrode 2. Further, the surface of the working electrode 2 is coated with an immobilized enzyme membrane 4 on which, for example, glucose oxidase (glucose oxidase) is immobilized. As shown in FIG. 3 (B), the biosensor having such a configuration is prepared by dropping a test liquid (eg, blood, urine, etc.) 5 so as to straddle the working electrode 2 and the counter electrode 3. It becomes possible to measure. This biosensor reduces the oxygen consumed when glucose, which is a substrate (substance that changes by the action of an enzyme), is oxidized by the catalytic action of glucose oxidase, or the amount of oxygen generated at this time is reduced. By measuring the increase in hydrogen oxide amperometrically, the glucose concentration can be measured.

As another biosensor, one having a structure as shown in FIG. 4 is known. In this biosensor, the working electrode 7 is provided on one surface of the insulating substrate 6, and the counter electrode 8 is provided on the other surface. The surface of the working electrode 7 is coated with the immobilized enzyme film 9. The biosensor having such a structure is tested by immersing the sensor itself in the test liquid so that the test liquid exists between the electrodes. Also in this biosensor, the glucose concentration is measured by measuring the decrease in oxygen consumed when glucose is oxidized by the catalytic action of glucose oxidase or the increase in hydrogen peroxide produced at this time, by measuring the current. You can do it.

[0004]

However, in the former of the above-mentioned conventional biosensors, the substrate 1
Since the working electrode 4 and the counter electrode 3 are arranged so as not to overlap each other in plan view on one surface of the substrate 1, these electrodes are arranged on the substrate 1
The area occupied by one side surface was inevitably large. Therefore, it is difficult to reduce the size of the biosensor. On the other hand, in the latter case, since the working electrode 7 and the counter electrode 8 are formed so as to face each other with the insulating substrate 6 sandwiched therebetween, they can be overlapped with each other when seen in a plan view, so that the size can be reduced. However, since the electrodes are separated from each other by the insulating substrate 6, it is necessary to immerse the biosensor in the liquid to be inspected so that both electrodes come into contact with the liquid to be inspected. Is required. As described above, the latter method has a problem that it is impossible to measure a trace amount of the test liquid.

The problem to be solved by the present invention lies in what means should be taken to obtain a biosensor which can be downsized and which can measure a small amount of test liquid.

[0006]

According to the first aspect of the present invention,
An enzyme or an enzyme and a mediator are immobilized on the inner surface of one of the pair of electrodes facing each other, and the space between the pair of electrodes is held by a spacer.

The invention according to claim 2 is characterized in that a drop introducing portion for the liquid to be inspected extending in the lateral direction is formed on one electrode side of the pair of electrodes. The invention according to claim 3 is characterized in that the pair of electrodes are bonded to each other via a sealing material at least at a portion excluding a portion facing the drip introducing portion.

According to a fourth aspect of the present invention, there is provided a voltage application circuit for applying a voltage between the pair of electrodes, and a current detection circuit for detecting a current flowing between the electrodes. There is.

[0009]

According to the first aspect of the invention, since the spacer is interposed between the pair of electrodes facing each other, a predetermined space can be provided between the two electrodes. Since the test liquid can be injected, the measurement can be performed quickly. Further, if the distance between both electrodes is narrowed, even if a small amount of the liquid to be inspected can be reliably infiltrated between both electrodes by the capillary phenomenon and can be detected. in this way,
By making both electrodes face each other and narrowing the interval between them, the sensor main body can be downsized, and even a small amount of test liquid can be detected sufficiently and quickly. Further, by keeping the interval between both electrodes uniform, a uniform electric field can be formed over the entire electrodes even with a small amount of test liquid, and highly accurate quantitative analysis can be performed.

According to the second aspect of the present invention, when the liquid to be inspected is dripped into the drip introducing portion formed on one electrode side so as to extend in the vicinity of the side, the liquid to be inspected is dropped between both electrodes. Immediate penetration and contact with both electrodes. For this reason, it is not necessary to immerse the sensor itself in a large amount of the liquid to be inspected, and it is possible to perform measurement with a minute amount that can be dropped.

According to the third aspect of the present invention, since the spacers are scattered between the two electrodes, both substrates can be easily opposed to each other at a predetermined interval by using a sealing material.
At this time, since the portion other than the portion facing the drip introducing portion for dropping the liquid to be inspected is joined by the sealing material, the sealing material does not prevent the infiltration of the liquid to be inspected between the electrodes.

According to the fourth aspect of the invention, by providing a voltage applying circuit for applying a voltage between the electrodes and a current detecting circuit for detecting a current flowing between the electrodes, one electrode is provided. By the catalytic action of the enzyme immobilized on the substrate, for example, the decrease in oxygen consumed when the substrate is oxidized or the increase in hydrogen peroxide produced at this time is measured by current, and It becomes possible to measure the substrate concentration of. Oxygen is electrochemically reduced on the surface of one electrode, and hydrogen peroxide is electrochemically oxidized. Utilizing this property, for example, when the voltage application circuit is controlled so that the potential of one electrode becomes a predetermined potential, oxygen is electrochemically reduced, and current detection is performed in the state where there is no substance other than oxygen that reacts at the electrode. The current value detected by the circuit is proportional to the oxygen partial pressure in the test liquid. In this way, the consumption of oxygen is known, so that the concentration of the substrate oxidized by the enzyme can be measured. When hydrogen peroxide increases, the polarity of the potential to be set is opposite to that when oxygen decreases because the hydrogen peroxide is electrochemically oxidized, but the measurement principle is the same as when oxygen is observed. . When the enzyme and the mediator are immobilized on one of the electrodes, a predetermined potential is applied between the pair of electrodes by a voltage application circuit, and the electron transfer from the enzyme can be directly measured by the current detection circuit. . Thereby, the concentration of the substrate in the test liquid can be measured.

[0013]

Embodiments of the present invention will be described below in detail with reference to the drawings. 1 is a sectional explanatory view showing an embodiment of a biosensor according to the present invention, FIG. 2 (A) is a plan view of the biosensor, and FIG. 2 (B) is a sectional view taken along line cd of FIG. 1 (A). There is. In the figure, 10 indicates the biosensor of the present embodiment. The biosensor 10 includes an upper substrate 11 and a lower substrate 12.
And are opposed to each other, and electrodes are provided on the surfaces of the two substrates 11 and 12 facing each other. The lower substrate 12 has a width substantially equal to that of the upper substrate 11, and the length is set longer than that of the upper substrate 11. The both substrates 11 and 12 are arranged such that one end edges in the length direction are aligned with each other, and the lower substrate 12 extends closer to the lateral side on the other end edge side. The upper surface of the extending portion of the lower substrate 12 serves as a drop introducing portion 12a for the test liquid.

The electrodes provided on the inner surface of the upper substrate 11 facing each other are, for example, a vacuum vapor deposition method, a magnet sputtering method,
The working electrode 14 is formed by a known method such as a screen printing method, and the surface of the working electrode 14 has the immobilized enzyme membrane 1
5 are provided. The immobilized enzyme 15 is obtained by immobilizing glucose oxidase on a polymer membrane by a known method such as a crosslinking method or an encapsulation method. On the other hand, the electrode provided on the inner surface facing the lower substrate 12 is the working electrode 1 of the upper substrate 11.
The counter electrode 16 faces the counter electrode 4 and is formed in the same manner as the working electrode 14. Further, between the immobilized enzyme film 15 provided on the upper substrate 11 and the counter electrode 16 provided on the lower substrate 12, a plurality of, for example, electrically insulating resins are formed.
The spherical spacers 13 are dispersed and interposed. Therefore, a uniform gap is formed between the upper substrate 11 and the lower substrate 12. In addition, the immobilized enzyme membrane 15 and the counter electrode 16
The distance between and is set to such a distance that the liquid to be inspected dropped in the dropping introduction part 12a can be introduced between the immobilized enzyme membrane 15 and the counter electrode 16 by the capillary phenomenon. This distance can be determined by the diameter dimension of the spacer 13. The upper substrate 11 and the lower substrate 12 are shown in FIG.
As shown in (A) and (B), both sides are fixed to each other by the sealing material 17.

Further, as shown in FIG. 1, the voltage application circuit 2 is connected to the working electrode 14 and the counter electrode 16 via wirings 19 and 20.
1 and the current measuring circuit 22 are connected.

In the present embodiment, by adopting the above-mentioned structure, as shown in FIG.
When the test liquid 18 is dripped on, the test liquid 18 has a function of being promptly introduced between the immobilized enzyme membrane 15 and the counter electrode 16 by a capillary phenomenon. In this way, the working electrode 14
In a state where the liquid 18 to be inspected is interposed between the counter electrode 16 and the counter electrode 16,
It becomes possible to measure the glucose concentration.

That is, a small amount of the test liquid 18 containing glucose, such as blood, urine, saliva, etc., is dropped onto the dropping introduction part 12a of the lower substrate 12. Then, when the test liquid 18 permeates between the immobilized enzyme film 15 and the counter electrode 16, glucose in the test liquid 18 is oxidized by the glucose oxidase immobilized in the immobilized enzyme film 15, On the other hand, glucose oxidase itself is reduced to a reduced form.
At this time, if oxygen is present in the test liquid 18, the oxygen serves as an electron acceptor, and the glucose oxidase in the reduced form returns to the original oxidized form.

Further, hydrogen peroxide is produced at the same time as the glucose oxidation reaction. At this time, if a predetermined voltage is applied between the working electrode 14 and the counter electrode 16 by the voltage application circuit 21, the generated hydrogen peroxide is reduced, and as a result, the hydrogen peroxide is reduced between the working electrode 14 and the counter electrode 16. A reduction current of hydrogen peroxide flows. This reduction current can be measured by the current measuring circuit 22. The magnitude of this reduction current depends on the amount of hydrogen peroxide produced. Therefore, since the amount of hydrogen peroxide produced depends on the glucose concentration in the test liquid 18, the glucose concentration in the test liquid 18 can be determined by measuring the magnitude of the reduction current. . That is, the time change of the current at this time is a known function depending on the glucose concentration which is the substrate in the test liquid 18. Therefore, the substrate concentration can be measured by detecting the change in current.

Although the embodiment has been described above, the present invention is not limited to this, and various design changes accompanying the gist of the configuration can be made. In the above example, the enzyme to be immobilized on the working electrode 14 was glucose oxidase, and the biosensor was provided for measurement of glucose concentration. However, the present invention is not limited to this.
By immobilizing an enzyme that oxidizes a substrate (measurement target) by its catalytic action on the working electrode and detecting hydrogen peroxide generated by oxidizing the substrate, it can be applied to a biosensor for measuring the substrate. . For example, if alcohol oxidase, which is an alcohol oxidase, is used as the immobilized enzyme, it can be used as a biosensor for measuring alcohol concentration. If cholesterol oxidase whose enzyme to be immobilized is cholesterol oxidase is used, it can be used as a biosensor for cholesterol measurement.

Further, in the above embodiment, only the enzyme was immobilized on the surface of the working electrode 14, but if a mediator coexists in addition to this, the enzyme that has oxidized the substrate and changed to the reduced form is the original oxidized form. When returning to, the mediator takes an electron from the enzyme and becomes a reduced mediator. Then, this reduced mediator gives electrons to the electrode by an electrode reaction, thereby returning to the original oxidized form. That is, if the substrate containing the enzyme and the mediator is present on the surface of the working electrode, the electrons move to the electrode via the enzyme and the mediator, and a current corresponding to the substrate concentration flows. Therefore, the substrate concentration can be measured by detecting this current.

Further, one of the working electrode 14 and the counter electrode 16 may be set as an anode electrode and the other as a cathode electrode according to a chemical reaction performed between a substrate and an enzyme or the like. Furthermore, in the above-described embodiment, the drop introducing portion 12a is provided on the lower substrate 12 side, but the counter electrode 16 may be longer than the working electrode 14 and the drop introducing portion may be formed on the counter electrode 16 itself.

Further, in the above embodiment, the spacers 13 made of an insulating resin are spherical particles, but may be made of an inorganic material such as glass as long as they are insulating. Further, it may be a cylindrical particle having a predetermined diameter dimension,
Other shapes may be used as long as the distance between the working electrode and the counter electrode can be kept uniform.

[0023]

As is apparent from the above description, according to the present invention, it is possible to arrange the electrodes facing each other in a plan view and to reduce the distance between the electrodes.
It is possible to downsize the biosensor and speed up the measurement. In particular, since the diameter of the spacer can be reduced to such an extent that the test liquid permeates between the electrodes due to the capillary phenomenon, the biosensor can be thinned. Also,
Since the liquid to be inspected only needs to enter between the narrow electrodes, there is an effect that the amount of the liquid to be inspected used for the measurement can be small.
Further, since the drip introducing portion is formed, the liquid to be inspected can be easily and surely supplied, and there is an effect of realizing an easy-to-use biosensor.

[Brief description of drawings]

FIG. 1 is an explanatory sectional view showing a biosensor according to an embodiment of the present invention.

FIG. 2A is a plan view of the biosensor of this embodiment,
FIG. 2B is a sectional view taken along line cd of FIG.

FIG. 3A is a plan view showing a conventional biosensor,
FIG. 3B is a cross-sectional view taken along the line ab of FIG.

FIG. 4 is a sectional view showing another conventional example.

[Explanation of symbols]

 10 Biosensor 13 Spacer 14 Working Electrode 15 Immobilized Enzyme Membrane 16 Counter Electrode 17 Sealing Material 18 Test Liquid 21 Voltage Application Circuit 22 Current Measurement Circuit

Claims (4)

[Claims] 1. An enzyme or an enzyme and a mediator are immobilized on the inner surfaces of one of the pair of electrodes facing each other, and the space between the pair of electrodes is held by a spacer. Characteristic biosensor. 2. The one electrode side of the pair of electrodes,
The biosensor according to claim 1, wherein a drop introducing portion for the liquid to be inspected is formed extending in the vicinity of the side. 3. The biosensor according to claim 2, wherein the pair of electrodes are bonded to each other via a sealing material at least at a portion excluding a portion facing the drip introducing portion. 4. A voltage applying circuit for applying a voltage between the pair of electrodes, and a current detecting circuit for detecting a current flowing between the electrodes, according to claim 1. Biosensor.