JPH0921778A - Biosensor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To make it possible to rapidly measure the substrate concentration of even trace amount of a liquid to be inspected and to reduce the size. SOLUTION: An operating electrode 13 is provided on one surface of an open-cell porous board 12 in which an enzyme is immobilized, and a counter electrodes 14 are provided on the other surface, and an opening 18 is formed on the electrode 14 side. A board 15 and an insulating film 16 are sequentially formed on the outer surface of the electrodes 14. An insulating film 17 is formed on the entire one side surfaces of the electrode 13 and the board 12. A liquid to be inspected introduced from the opening 18 rapidly permeates between the electrodes 13 and 14 via the board 12 to be able to measure the substrate concentration.

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 measuring the concentration of various liquid components by using immobilized enzymes. Pertain.

[0002]

2. Description of the Related Art Conventionally, a biosensor using an immobilized enzyme is shown in FIG. In this biosensor, a working electrode 2 is provided on one surface of an insulating substrate 1 and a counter electrode 3 is provided on the other surface. The immobilized enzyme membrane 4 is attached to the surface of the working electrode 2. 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. 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 amperometric measurement. You can do it.

[0003]

However, in the above-mentioned conventional biosensor, since the working electrode 2 and the counter electrode 3 are separated by the insulating substrate 1, the biosensor is immersed in the liquid to be inspected. It is necessary to fill the space between the electrodes with the test liquid, which requires a considerable amount of the test liquid. As described above, the conventional biosensor has a problem that it is impossible to measure a trace amount of the test liquid. An object of the present invention is to achieve miniaturization and to enable rapid measurement of substrate concentration even with a small amount of test liquid.

[0004]

According to the first aspect of the present invention,
A continuous porous layer in which an enzyme or an enzyme and a mediator are immobilized is provided on the inner surface of one electrode of a pair of electrodes facing each other, and a test liquid that causes a reaction with the enzyme on the other electrode side. Is characterized in that an opening is formed in the continuous porous layer. In the invention according to claim 2, between the other electrode and the continuous porous layer,
It is characterized in that a gap is formed which communicates with the opening and into which the test liquid is introduced. According to a third aspect of the present invention, an insulating layer is provided on opposite outer surfaces of the pair of electrodes,
The opening is characterized by penetrating the insulating layer. The invention according to claim 4 is characterized in that the pair of electrodes and the continuous porous layer have flexibility, and the insulating layer has flexibility and flexibility.

According to the first aspect of the invention, the test liquid can be conducted through the opening formed on the other electrode side. The test liquid conducted from the opening permeates into the continuous pores in the continuous porous layer. Since the enzyme is immobilized in the continuous porous layer, the test liquid is surely subjected to the catalytic action of the enzyme to cause the enzymatic reaction. Further, the test liquid introduced from the opening can flow an electric current between the electrodes depending on the substrate concentration if the test liquid is in an amount that fills the voids except the porous layer between the pair of electrodes facing each other. It will be possible. By adjusting the diameter of the continuous pores, it is possible to prevent a polymer such as a protein, which is an interfering substance, from reaching one electrode (working electrode). In the invention according to claim 2, between the other electrode and the continuous porous layer,
Since the gap that communicates with the opening and into which the test liquid is introduced is formed, the test liquid can be promptly interposed between the electrodes. According to the third aspect of the invention, since the insulating layers are provided on the opposing outer surfaces of the pair of electrodes, it is possible to prevent the electrodes from being electrically influenced from the outside. Further, since the opening is provided with the insulating layer interposed therebetween, the liquid to be inspected can easily conduct to the porous layer from the outside of the insulating layer. In the invention according to claim 4, since the insulating layer has flexibility and flexibility and the pair of electrodes and the continuous porous layer have flexibility, for example, when the sensor is inserted under the eyelid. It has a function of preventing the human body from being damaged when the sensor is inserted into the mouth or the mouth.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, details of a biosensor according to the present invention will be described based on embodiments shown in the drawings. (First Embodiment) FIGS. 1A and 1B show a first embodiment of the present invention. As shown in FIG. 1 (A), reference numeral 11 in the figure is a biosensor. In this biosensor 11, for example, one surface of a continuous porous substrate 12 on which glucose oxidase which is a glucose oxidase is immobilized. A working electrode 13 is provided, and the counter electrode 1 is provided on the other surface.
4 are provided. In addition, this continuous porous substrate 12
Is formed of, for example, a fluororesin such as polytetrafluoroethylene having a continuous porous structure. Further, in order to immobilize glucose oxidase on the surface inside the continuous porous substrate 12, glucose oxidase can be immobilized on an organic film by a known method such as a crosslinking method or an encapsulation method. In this case, since the organic film does not block the continuous pores of the continuous porous substrate 12, for example, a method of attaching an organic film containing glucose oxidase to the inner surface of the continuous pore while supplying air to the continuous porous substrate 12 Can be used.

An insulating substrate 15 and an insulating film 16 are sequentially formed on the outer side of the counter electrode 14. Also,
An insulating film 17 that covers the working electrode 13 and the continuous porous substrate 12 is also formed outside the working electrode 13. In addition, it is desirable that the substrate 15 and the insulating film 16 described above are made of a material having good wettability with respect to the test liquid. As shown in the plan view of FIG. 1 (B), the working electrode 13 has a narrow narrow portion 13A and a circular detection portion 13B having a predetermined area. Further, the counter electrode 14 is formed over the entire other surface of the continuous porous substrate 12 except for the opening 18 described later. Then, the counter electrode 14, the substrate 15, and the insulating film 16 are formed with an opening 18 for exposing the continuous porous substrate 12 for introducing a liquid to be inspected. The opening 18
The position where is formed is set to a position adjacent to the detection portion 13B of the working electrode 13 provided on one surface of the continuous porous substrate 12 when seen in a plan view. A voltage applying circuit 21 and a current measuring circuit 22 are connected to the end edge portion of the narrow portion 13A of the working electrode 13 and the counter electrode 14 via wirings 19 and 20.

In this embodiment, the narrow portion 13 of the working electrode 13 is
Since the area where A and the counter electrode 14 face each other is small, the substrate concentration in the test liquid substantially interposed between the detection unit 13B and the counter electrode 14 is measured. When measuring the substrate concentration of the test liquid in this manner, the detection unit 13B and the counter electrode 14
, The liquid to be inspected is introduced from the opening 18 located at a position adjacent to the detection unit 13B in plan view, so that a very small amount of the liquid to be inspected reaches between the electrodes very quickly.
Further, in the biosensor of this embodiment, the electrodes are opposed to each other in a three-dimensional structure, and the distance between the electrodes can be made extremely short, so that the device can be downsized. By the way, in the continuous pores of the continuous porous substrate 12,
Since the test liquid is held by the capillary phenomenon, the test liquid can be reliably interposed between the electrodes. For this reason,
The glucose concentration in the liquid to be detected can be measured with the liquid to be detected being interposed between the electrodes. Furthermore, in this embodiment, since the enzyme is immobilized on the surface of the continuous pores of the continuous porous substrate 12, the contact area between the enzyme and the test liquid is substantially increased, and the enzyme reaction is efficiently performed. Therefore, the measurement efficiency can be improved. In addition, the continuous porous substrate 12
If the diameter of the continuous pores is set to a diameter that does not allow passage of macromolecules such as proteins, the macromolecules that become interfering substances during measurement of the substrate concentration are prevented from reaching the working electrode 13 and fixed. It becomes possible to efficiently measure, for example, hydrogen peroxide generated by the converted enzyme.

Using the biosensor 11 of this embodiment,
In order to measure the concentration of glucose in a test liquid containing glucose, such as blood, urine, saliva, and tear fluid, first, a small amount of the test liquid is dropped into the opening 18. And
When the test liquid permeates into the continuous pores of the continuous porous substrate 12, glucose in the test liquid is oxidized by the glucose oxidase immobilized in the continuous pores, while the glucose oxidase itself is reduced to a reduced form. Becomes
At this time, if oxygen exists in the liquid to be inspected or the liquid existing together with the liquid to be inspected, the oxygen becomes 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 such an oxidation reaction of glucose. At this time,
When a predetermined voltage is applied between the working electrode 13 and the counter electrode 14 by the voltage application circuit 21, the generated hydrogen peroxide is electrolytically oxidized, whereby the peroxide between the working electrode 13 and the counter electrode 14 is oxidized. An oxidation current of hydrogen flows. This oxidation current is
It can be measured by the current measuring circuit 22. The magnitude of this oxidation 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, the glucose concentration in the test liquid can be determined by measuring the magnitude of the oxidation current. That is, the time change of the electric current at this time is a known function depending on the glucose concentration which is the substrate in the test liquid. Therefore, the substrate concentration can be measured by detecting the change in current.

Next, in order to manufacture the biosensor 11 of this embodiment, first, the working electrode 13 is formed on one surface of the continuous porous substrate 12 by the vacuum deposition method, the magnet sputtering method, the screen printing method, and the electrolytic plating. Method, electroless plating method, or the like. Further, a substrate 15 made of an insulating resin and having the counter electrode 14 formed on one surface thereof is prepared.
4 and the other surface of the continuous porous substrate 12 are put together, and then the insulating film 16 and the insulating film 17 are covered by a known technique. In addition, a notch is previously formed in a portion corresponding to the opening 18 of the counter electrode 14, the substrate 15, and the insulating film 16.
In the above-described manufacturing method, the working electrode 13 and the continuous porous substrate 12, and the substrate 15 and the counter electrode 14 may be bonded with an adhesive, or the members may be bundled and finally held by a holding member. Of course, it may be fixed.

(Second Embodiment) FIG. 2 shows a second embodiment of the present invention. In the description of this embodiment, the same parts as those in the first embodiment will be designated by the same reference numerals and the description thereof will be omitted. In the present embodiment, as shown in FIG. 2B, the area occupied by the counter electrode 14 with respect to the continuous porous substrate 12 is reduced, and the detection portion 13B and the opening 18 of the working electrode 13 are formed.
The counter electrode 14 is the detection part 13 with the part between the bottom and the bottom as a boundary.
It is arranged on the side facing B. A narrow portion 14A for connection, which is the same member as the counter electrode 14, extends from one side edge of the counter electrode 14 along the side edge of the continuous porous substrate 12. The end of the narrow portion 14A is connected to the wiring 19. Further, in the region of the continuous porous substrate 12 where the counter electrode 14 is not in contact, the insulating layer 23 is formed with the same thickness as the counter electrode 14 except for the opening 18, as shown in FIG.

In the present embodiment, the liquid to be inspected introduced from the opening 18 passes through the continuous porous substrate 12 and the working electrode 13
It is similar to the first embodiment in that it is quickly guided between the detection unit and the counter electrode 14, but the area where the counter electrode 14 and the working electrode 13 face each other is equal to the area of the detection unit 13B of the working electrode 13. Due to the limitation, the electrode area for amperometric measurement associated with the enzymatic reaction can be precisely determined. Therefore, the substrate concentration can be measured with high accuracy.

(Third Embodiment) FIG. 3 shows a third embodiment of the present invention.
Is shown. In the description of this embodiment, the same parts as those in the first and second embodiments are designated by the same reference numerals and the description thereof will be omitted. 3A is a cross-sectional view of a main part of the biosensor of this embodiment, FIG. 3B is a plan view of the biosensor, and FIG. 3C is a cross-sectional view taken along the line AA of FIG. 3B. As shown in FIG. 3 (A), the biosensor of the present embodiment is provided such that the counter electrode 14 of the portion of the working electrode 13 facing the detecting portion 13B and the continuous porous substrate 12 face each other with a gap 24 therebetween. It is characterized by being.

In this embodiment, an insulating layer 23 as a spacer is interposed between the continuous porous substrate 12 and the counter electrode 14, and the counter electrode 14 faces the detecting portion 13B of the working electrode 13,
A gap 24 is formed between the continuous porous substrate 12. As shown in FIG. 3B, the insulating layer 23 has a substantially U-shape cut out from the center to one side edge in the longitudinal direction, and is formed on the counter electrode 14, the substrate 15, and the insulating film 16. The opening 18 and the detection portion 13B of the working electrode 13 are located in the notched region of the insulating layer 23 in plan view. In addition, the substrate 15, the counter electrode 14,
Continuous porous substrate 12, working electrode 13, insulating films 16, 17
Is made of a flexible material and has an insulating film 16,
Reference numeral 17 is made of a flexible resin material. Further, as shown in the figure, the corners of each member are rounded. Therefore, the biosensor of the present embodiment can be used without damaging the human body when it is inserted into the human body, for example, under the eyelid for inspection, or when inserted into the mouth for inspection. Further, since each member has flexibility, it can be used by being deformed in the mouth, for example.

Further, in this embodiment, the insulating layer 23
Between the counter electrode 14 and the working electrode 13 (narrow portion 13A) in which the
Since the oxidation current of hydrogen peroxide between the detection unit 13B of the working electrode 13 and the counter electrode 14 can be mainly measured, there is an advantage that highly accurate measurement can be performed. Further, since the liquid to be inspected quickly permeates into the gap 24 between the continuous porous substrate 12 and the counter electrode 14 by the capillary phenomenon, quick and reliable measurement can be performed.

Further, when the continuous porous substrate 12 and the counter electrode 14 are cleaned, the cleaning liquid can quickly penetrate into the gap 24 and the liquid does not remain in the opening 18, so that the cleaning can be easily carried out. Although the respective embodiments have been described above, the present invention is not limited to these, and various design changes associated with the gist of the configuration can be made.

Porous substrate 1 used in each of the above embodiments
2 is, for example, a hydrophilic Teflon membrane (pore diameter 0.2 μm
m) [trade name: Omnipore Membrane] may be impregnated with a mixed solution of glucose oxidase and bovine serum albumin, cross-linked with glutaraldehyde, and glucose oxidase may be immobilized on the surface and the inner surface of the Teflon membrane. However, it is not limited to this. Although glucose oxidase is used as the enzyme immobilized on the continuous porous substrate 12 in each of the above-described embodiments, the enzyme may be immobilized in the presence of a mediator. In this way, coexistence of a mediator oxidizes a substrate (glucose), and when the reduced enzyme, which has been reduced by itself, returns to the original oxidized form, the mediator takes an electron from the enzyme and becomes a reduced mediator. . Then, the reduced mediator gives electrons by the reaction that occurs between the electrodes, and thereby returns to the original oxidized mediator. That is, if the substrate is present in the porous substrate containing the enzyme and the mediator, 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.

When the enzyme and the mediator are coexisted and immobilized in the porous substrate in this manner, even if the amount of dissolved oxygen in the test liquid is extremely small, a current corresponding to the substrate concentration flows, so that the dissolved oxygen is dissolved. The test measurement can be performed without depending on the concentration. Further, an appropriate amount of buffer solution may be immersed in the opening to keep the pH in the solution constant.

In the above embodiment, the narrow portion of the working electrode overlaps the portion corresponding to the opening, but the invention is not limited to this, and the narrow portion may be formed to bypass the opening. Further, in the present embodiment, glucose oxidase was used to measure the glucose concentration as a substrate, but various enzymes or enzymes and mediators may be immobilized depending on the substrate to be measured.

In each of the above embodiments, the substrate 1
Although the configuration having 5 is adopted, it may of course be omitted.
Further, the shapes of the counter electrode 14 and the working electrode 13 can be various shapes other than the above-described embodiments, and the point is that the working electrode and the counter electrode sandwich a continuous porous substrate, or have a continuous porous structure. Any configuration may be used as long as it is provided with a gap from the substrate and has an opening for introducing the test liquid in the vicinity of the position where both electrodes face each other.

[0021]

As is apparent from the above description, according to the present invention, there is an effect that the substrate concentration can be promptly measured even if the test liquid is a small amount. Further, since the enzyme is immobilized in the continuous porous layer, there is an effect that the enzyme reaction can be efficiently carried out. Further, even if a small amount of the liquid to be inspected can be reliably interposed between the pair of electrodes, the distance between the electrodes can be short, and the device can be downsized. Further, by configuring each member with a material having flexibility or flexibility, it is possible to realize a biosensor that does not damage the human body.

[Brief description of drawings]

1A is a sectional view of Embodiment 1 of the present invention, FIG.
3 is a plan view of Embodiment 1. FIG.

2A is a cross-sectional view of Embodiment 2 of the present invention, FIG.
3 is a plan view of Embodiment 2. FIG.

3A is a sectional view of Embodiment 3 of the present invention, FIG.
Is a plan view of the third embodiment, and (C) is a cross-sectional view taken along line AA of (B).

FIG. 4 is a sectional view of a main part of a conventional example.

[Explanation of symbols]

 11 biosensor 12 continuous porous substrate 13 working electrode 14 counter electrode 16, 17 insulating film 18 opening

Claims (4)

[Claims] 1. A continuous porous layer having an enzyme or an enzyme and a mediator immobilized thereon is provided on the inner surface of one of a pair of electrodes facing each other, and the enzyme reacts with the enzyme on the other electrode side. A biosensor characterized in that an opening is formed so that a liquid to be inspected, which causes the above-mentioned, is conducted to the continuous porous layer. 2. A gap between the other electrode and the continuous porous layer, which is in communication with the opening and into which a liquid to be inspected is introduced is formed. Biosensor. 3. The biosensor according to claim 1, wherein an insulating layer is provided on opposite outer surfaces of the pair of electrodes, and the opening penetrates the insulating layer. 4. The pair of electrodes and the continuous porous layer have flexibility, and the insulating layer has flexibility and flexibility. The biosensor according to claim 2.