JPS6390755A - Enzyme electrode - Google Patents

Abstract

PURPOSE:To permit mass production by providing electrodes consisting of a conductor film and insulating protective film for coating the parts exclusive of the sensitive parts and junctures of said electrodes on the two surfaces of an insulating base plate and providing an immobilized enzyme film for coating the sensitive parts of the electrodes formed on one surface. CONSTITUTION:The working electrodes 5,...5 are formed on the respective blocks 4 on the upper surface 2a of a insulating flat plate 2 and in succession thereof, the reference electrodes 6,...6 are formed on the lower surface 2b of the flat plate 2. The insulating protective films 7,...7 are then formed on the upper surface 2a to coat and insulate the entire surfaces except the two ends of the working electrodes 5. The insulating protective films 8,...8 are formed on the lower surface 2b as well to coat and insulate the entire surface except the two ends of the reference electrode 6. The plate 2 is then divided to individual blocks 4 to form insulating substrates 9,...9. An acetyl cellulose film 13 is formed on the upper surface 9a of the insulating substrate of the underlying electrode 12. An enzyme soln. 14 is dropped onto the film 13 and further a polycarbonate film 15 is imposed thereon, by which an enzyme electrode 1 is completed.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application This invention relates to an enzyme electrode suitable for mass production and high performance.

(b) Conventional technology Enzyme electrodes are immersed in a liquid to be tested, and through an enzymatic reaction,
This makes it possible to electrically measure the concentration of a specific chemical substance that is a substrate for the enzyme contained in the test liquid.

Conventionally, as an enzyme electrode, the one shown in FIG. 6 (el) is known. This conventional enzyme electrode, together with its manufacturing method, will be explained below with reference to FIG. 6 (al to 6 tel). do.

FIG. 6(a) shows a state in which lead wires 30 and 30 are connected to the working electrode 25 and the reference electrode 26.

The working electrode 25 is platinum shaped into a pin.

On the other hand, the control electrode 26 is made of silver and is shaped into a cylinder.

FIG. 6(b) shows a state in which the working electrode 25 and the control electrode 26 are housed in a cup-shaped case 29.

The control electrode 26 is fitted and stored through the opening 29a of the case 29. On the other hand, the working electrode 25 is the control electrode 26
The reference electrode 26 is supported in the hollow part 26b by a support member 27 while being insulated from the reference electrode 26.
and the working electrode 25 have a coaxial structure. The lead wire 30
．． 30 is pulled out downward from the window hole 29b at the bottom of the case 29.

FIG. 6(C) shows the inside of the case 29 made of epoxy resin 2.
8 shows the sealed state. This epoxy resin 28
further rises above the case opening 29a and completely covers the working electrode 25 and the reference electrode 26. It takes about 6 days for the epoxy resin 28 to cure at room temperature.

FIG. 6 (dl) is the upper surface of the device shown in FIG.
5 and the reference electrode 26, respectively, sensitive parts 25a and 26a.
This shows the state in which . In the sensitive parts 25a and 26a, the diameter of the working electrode 25 and the inner and outer diameters of the reference electrode 26 are determined so as to have a predetermined area ratio. The electrode in this state is called the base electrode 32.

FIG. 6 (81) shows the completed enzyme electrode 31 by attaching the immobilized enzyme membrane 36 to the base electrode 32 shown in FIG. 6(d).The immobilized enzyme membrane 36 is a polymer membrane. It is an immobilized enzyme that uses a specific chemical substance to be detected as a substrate, and is prepared separately from the base electrode 32.The immobilized enzyme film 36 is attached to the base electrode so as to be in close contact with the sensitive parts 25a and 26a. 32 and its peripheral edge 36a is fixed to the outer circumferential surface of the case 29 by an O-ring 37. A groove 29c is provided on the outer circumferential surface of the case 29 to fix the O-ring 37. .

(C) Problems to be Solved by the Invention The conventional enzyme electrode 31 described above is produced one by one manually in the manufacturing process, and has the disadvantage that mass production is difficult. In addition, this manual work involves a series of delicate operations, resulting in a large loss of materials, especially electrode materials, and has the disadvantage of lowering the yield during the production of enzyme electrodes. Furthermore, there are disadvantages in that processing costs are high and manufacturing costs are high. By the way, this processing cost is 6% of the manufacturing cost.
It accounted for 0 to 80%.

On the other hand, the conventional enzyme electrode described above has the following disadvantages when used.

First, when the sensitive parts 25a and 26a are generated by grinding and polishing, cracks and gaps are generated in the epoxy resin around the working electrode 25. This is due to the difference in hardness between the epoxy resin 28, the working electrode 25, and the reference electrode 26. Enzyme electrode 3
When water or other liquids enter these cracks or gaps during use, noise is generated and measurement accuracy is reduced. In addition, the output of the enzyme electrode 31 is
Although it is determined by the area of the sensitive parts 25a and 26a,
Since a is generated manually, the sensitive parts 25a and 26
There was a problem that the area of a was not constant between the enzyme electrodes, and the output varied. Furthermore, there is a problem in that the output changes due to improper attachment of the immobilized enzyme membrane 36.

The present invention was made in view of the above-mentioned disadvantages, and an object of the present invention is to provide an enzyme electrode that can be mass-produced, is inexpensive, and has excellent performance.

(d) Means for Solving the Problems As a means for solving the above-mentioned disadvantages, the enzyme electrode of the present invention has an electrode made of a conductive film on at least two surfaces of the insulating base, and a sensitive electrode of the electrode. An insulating protective film is provided on at least one of the surfaces, and an insulating protective film is provided to cover the portions excluding the connecting portions and the connecting portions.

An immobilized enzyme membrane is provided to cover the area.

(e) Function In the enzyme electrode of the present invention, a plurality of enzyme electrodes can be manufactured all at once on one insulating flat plate. Also,
To form the electrode on this electrode plate, sputtering,
Vacuum deposition can be applied and electrode formation can be easily automated.

Furthermore, since the electrode is a conductive film formed on the surface of the insulating substrate, the amount of electrode material used and its loss during the process can be reduced. Of course, the usage and loss of other materials can also be reduced. The above three effects make it possible to mass produce enzyme electrodes, improve yields, and reduce costs.

On the other hand, the shapes of the electrodes and the insulating film can be determined with high precision by applying photomask technology to their formation. Therefore, at the same time, the area of the electrode sensitive portion is determined with high precision, and variations in output between enzyme electrodes are eliminated. In addition, the immobilized enzyme membrane is integrally formed on the surface of the insulating substrate, and unlike the conventional method, there is no output change due to whether or not the immobilized enzyme membrane is properly attached. Furthermore, since the process does not involve grinding or polishing the electrodes, there are no cracks or gaps that allow liquid to enter the surface of the insulating substrate, thereby preventing the generation of noise.

(f) Example An example of this invention is shown in FIGS. 1(a) to 1(h).
, FIGS. 2(a) to 2(g), and FIGS. 3 to 5.

The enzyme electrode 1 according to this embodiment is applied to detecting the concentration of glucose contained in blood or the like. Hereinafter, this enzyme electrode 1 will be explained below while following its manufacturing process.

FIG.
. . , 3 are formed and divided into individual sections 4, . . . , 4. The insulating flat plate 2, which is later divided to form an insulating substrate (insulating base) 9, is an alumina ceramic plate (alumina 96%) with an appropriate size, for example, 50 x 50 mm, thickness 0.5 mm. Use a rough surface instead of a mirror finish. The reason why the insulating flat plate 2 has a rough surface is that the working electrode 5 and the reference electrode 6, which will be described later,
This is to increase the effective area of the acetylcellulose film 13 and to prevent the acetylcellulose film 13 from peeling off from the insulating substrate 9.

The partition line 3 is a cut formed on the upper surface 2a of the insulating flat plate by laser processing, and its depth is approximately 172 mm thick than the thickness of the insulating flat plate 2 (see FIG. 2 Ta).

These division lines 3 divide the upper surface 2a of the insulating flat plate into divisions 4 of appropriate size (for example, 2×15 mm). Note that the material and size of the insulating flat plate 2 and the method of forming the partition lines 3 are not limited to those of this embodiment.

A working electrode 5 is formed in each section 4 of the upper surface 2a of the insulating flat plate [see FIG. 1 (bl and 2)].

The working electrode 5 is formed by sputtering, and is a band-shaped platinum thin film with one end wide (for example, size 1×11 II).
11, thickness 1500 people). A photoresist method is applied to form the working electrode 5, and the position and shape of the working electrode 5 are accurately determined using a photomask.

Subsequently, on the lower surface 2b of the insulating flat plate 2, a control electrode 6,
..., 6 is formed [Fig. 1 (C) and Fig. 2 (C)]
reference〕. The control electrodes 6 are silver thin films (for example, 1.5×1 mm in size) formed by vacuum deposition, and are formed at positions corresponding to the working electrodes 5 on the upper surface 2a of the insulating flat plate. Both the position and shape of this reference electrode 6 are accurately determined using a photomask.

As mentioned above, the reason why the working electrode 5 and the reference electrode 6 are separately formed on the upper surface 2a and the lower surface 2b of the insulating flat plate is to simplify the manufacturing process. Forming a pair of working and reference electrodes made of different metal thin films on one surface complicates the process and does not meet the purpose of the invention. However, if the working electrode and the reference electrode are made of the same type of metal reed membrane, this process will not be complicated, but the detection performance of the enzyme electrode will be lower than when the electrode materials are made of different metals.

Next, an insulating protective film 7 is formed on the upper surface 2a of the insulating flat plate.
7 is formed [see Figure 1 (d) and Figure 2 (d)]
.

This insulating protective film 7 covers and insulates the entire surface of the working electrode 5 except for both ends thereof. One end and the other wide end of the working electrode 5 are exposed and serve as a sensing part 5a and a connecting part 5b, respectively.

The insulating protective film 7 is formed by applying a photosensitive resin (for example, photosensitive polyimide), exposing it to light using a photomask, and removing unnecessary portions. The position and shape of the insulating protective film 7 are accurately determined by a photomask. Therefore, since the position and shape of the working electrode 5 and the insulating protective film 7 are accurate, the working electrode sensitive part 5
The area of a will be determined with high accuracy.

Insulating protection 11 also on the lower surface 2b of the insulating flat plate! a...
, 8 are formed [see FIG. 1 (el) and FIG. 2 (e)].

This insulating protective film 8 covers and insulates the entire surface of the reference electrode 6 except for both ends thereof. Both ends of the control electrode 6 are exposed and serve as a sensing part 6a and a connecting part 6b, respectively. The formation of the insulating protective film 8 is the same as that of the insulating protective film 7, and the area of the sensitive portion 6a is determined with high precision. The area St of the sensitive part 6a makes a predetermined ratio (for example, S+:5z=1:20) with the area S1 of the working electrode sensitive part 5a.

The insulating flat plate 2 then separates the individual compartments 4 along the compartment lines 3.
The substrate is divided into insulating substrates 9, . . . , 9 [see FIG. 1(f) and FIG. 2(f)]. A lead wire 10 is connected to the working electrode connection portion 5b and the reference electrode connection portion 6b of the insulating substrate 9.
．． 10 are soldered [see also FIG.
Epoxy resins 11a and llb are applied to protect the solder portions of the lead wires 10 from insulation. The electrode in this state is called the base electrode 12.

A state in which an acetylcellulose film 13 is formed on the upper surface 9a of the insulating substrate of the base electrode 12 is shown in FIG. 1 if). This acetylcellulose membrane 13 completely covers the working electrode sensitive portion 5a. Formation of the acetyl cellulose membrane 13 is performed by the following procedure.

First, the base electrode 12 is mounted on a spinner (rotator) with the upper surface 9a of the insulating substrate facing upward. Next, 5% acetylcellulose solution (solvent composition, acetone: cyclohexanone =
3:1) onto the upper surface 9a of the insulating substrate, and
is rotated for 5 seconds at a rotation speed of 200 Qrpm to spread the acetylcellulose solution on the upper surface 9a of the insulating substrate.

An enzyme solution 14 is dripped onto the acetyl cellulose membrane 13 formed as described above, and a polycarbonate membrane 15 is further placed on it to complete the enzyme electrode 1 [Fig. 1(g)]
and FIG. 2 (see a). Preparation of the enzyme solution 14 is performed in the following procedure.

First, 20 mg of glucose oxidase (COD) was added to P
100 μl of 0.1M phosphate buffer prepared at H6.0
dissolve in Next, this is mixed with 100 μl of a 0.5% glutaraldehyde solution prepared with the same phosphate buffer to obtain enzyme solution 14.

The polycarbonate membrane 15 allows polymers such as proteins in the test liquid to be separated from the acetyl cellulose membrane 13 and the enzyme solution 14.
This is to prevent water from entering the interior. The acetylcellulose membrane 13, enzyme solution 14, and polycarbonate membrane 15 constitute an immobilized enzyme membrane 16.

Next, a test on the characteristics of the base electrode 12 (shown in FIG. 1(f), on which the acetylcellulose film 13 is formed) and its results will be explained below with reference to FIG. . Since the performance of the enzyme electrode 1 depends on the hydrogen peroxide (H20□) sensitivity characteristics of the base electrode 12, it is meaningful to confirm this.

FIG. 4 shows the respective HtOz concentrations (Oll, Oll,
Applied voltage (V) and electrode current (n
It shows the relationship with A). From this, the base electrode 12
It is confirmed that H20□ responds well to the H20□ concentration.

Further, it has been shown that a suitable value for the applied voltage is 0.4 to 0.7 .

Next, the glucose detection characteristics of the enzyme electrode 1 of this example will be explained below with reference to FIGS. 3 and 5.

Figure 3 shows the measurement system 1 used to measure the characteristics of the enzyme electrode 1.
8 is shown. Reference numeral 19 denotes a constant temperature block, in which a 0.1M phosphate buffer 20 adjusted to pH 7.0 is stored. This phosphate buffer 20 contains an enzyme electrode 1.
is immersed. Further, this phosphate buffer solution 20 is stirred by a starter 19a built in the constant temperature block 19. 19b is the rotor of Stark 19a.

The lead wire 10 of the enzyme electrode 1 is connected to an electron meter 21
and a predetermined applied voltage (0.6V in this measurement)
is added. The electron meter 21 has a recorder 2
2 is connected, and the electrode output (current) of the enzyme electrode 1 is recorded.

A predetermined amount of glucose solution is dropped into the phosphate buffer 20 using a micropipette (not shown). This glucose (Glc) undergoes the following reaction within the immobilized enzyme membrane 16 of the enzyme electrode 1. This H, O□ causes the working electrode sensitive part 5a to become sensitive, and the HzOz? An electrode output corresponding to a degree is generated. In FIG. 5, the electrode output (n A) is plotted with open circles for several glucose concentrations c (mg/d 1 ). Moreover, the curve shown in FIG. 5 is a calibration curve that connects the plotted points. Based on this calibration curve, the glucose concentration in the blood can be determined.

In the above embodiment, glucose oxidase is immobilized as an enzyme on the immobilized enzyme membrane 16, but the enzyme is not limited to this and can be changed as appropriate.

Further, in the above embodiment, only the working electrode sensitive part 5a is covered with the immobilized enzyme membrane 16, but the reference electrode sensitive part 6a may also be covered with the immobilized enzyme membrane (the design can be changed as appropriate). be.

Furthermore, the number, shape, arrangement, etc. of the working electrodes and the control electrodes are not limited to those of the above embodiments, and the design can be changed as appropriate.

(G) Effects of the Invention The enzyme electrode of the present invention includes an electrode made of a conductive film on at least two surfaces of an insulating base, and an insulating protective film covering the part of the electrode excluding the sensitive part and the connecting part. and at least one of the surfaces is provided with an immobilized enzyme membrane covering the sensitive part of the electrode formed on this surface. Therefore, it has the advantage of making it possible to mass-produce enzyme electrodes, improving the yield in manufacturing, and reducing the cost of enzyme electrodes (less than 1,710 yen of the conventional cost). Further, it has the advantage that variations in output between enzyme electrodes are eliminated, the output of the enzyme electrode is stable, there is less noise, and measurement accuracy can be improved.

[Brief explanation of the drawing]

Figure 1(a), Figure 1(b), Figure 1(C), Figure 1(
d), FIG. 1(a), FIG. 1(f), FIG. 1(gl), and FIG. 1(h) are perspective views illustrating an enzyme electrode and its manufacturing process according to an embodiment of the present invention. , FIG. 2(a) is an enlarged sectional view of the main part taken along line ■a-Ila in FIG. 1+al,
FIG. 2(b) is an enlarged cross-sectional view of main parts taken along line nb-nb in FIG. 1(b), and FIG.
Fig. 2(d) is an enlarged cross-sectional view of the main part taken along the line l1d-IId in Fig. 1(d), and Fig. 2+81 is an enlarged sectional view of the main part taken along the line cml1c in Fig. 1 (line ne-11e in el). A sectional view of the main part in FIG. 2 ([1 is lf in FIG. 1(f)
A cross-sectional view taken along the f-lff line, FIG. 2(g) is a cross-sectional view taken along the ng-ng line in FIG. , 4th
The figure shows the characteristics of the base electrode that makes up the enzyme electrode.
Figure 5 is a diagram showing the characteristics of the enzyme electrode, Figure 6 (a),
6 [g (b), 6 (C), 6 (d) and 6
Figure (e) is a diagram illustrating a conventional enzyme electrode and its manufacturing method. 5: Working electrode, 5a: Sensing part, 5b: Connection part,
6: Control electrode, 6a: Sensing part, 6b
: Connection part, 7/8: Insulating protective film, 9: Insulating substrate, 9a
: Upper surface, 9b: Lower surface. Patent applicant Tateishi Electric Co., Ltd. Agent
Patent Attorney Shigeru Nakamura Nobumi 4 Figure 7' Leco Z U1 Seat C (mg/d)) Figure 6<a)

Claims (2)

[Claims] (1) At least two surfaces of the insulating base are provided with an electrode made of a conductive film, and an insulating protective film that covers the electrode except for the sensitive part and the connecting part, and at least one of the surfaces An enzyme electrode characterized in that it is provided with an immobilized enzyme membrane covering a sensitive part of the electrode formed on the surface of the enzyme electrode. (2) The enzyme electrode according to claim 1, wherein the surface of the insulating base is a rough surface.