JPS63134946A - Enzyme electrode - Google Patents

Abstract

PURPOSE:To manufacture an electrode at a low cost by a mass production and to improve its performance by covering an induction part having a metallic foil which is stuck by a conductive adhesive agent onto an induction part of an electrode with a fixed enzyme film, and covering other part with an insulating protective film. CONSTITUTION:On the surface 8a of an insulating substrate 8 having flexibility, two or more electrodes 4, 5 are provided which are constituted of conductive paste and are composed of induction parts 4a, 5a and connecting parts 4c, 5c, respectively. A metallic foil 6 is stuck onto the induction part 4a of the electrode 4 by a conductive adhesive agent, other part than the induction parts 4a, 5a and the connecting parts 4c, 5c is covered with an insulating protective film 7 and also the induction part 4a to which the metallic foil 6 is stuck is covered with a fixed enzyme film 15. Therefore, since the electrodes 4, 5 consist of conductive paste, its shape can be easily automatized and formed by printing, etc.

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.

Conventional enzyme electrodes are manufactured one by one by hand, as shown in FIG. 41 is a working electrode made of platinum in the shape of a round rod, and 42 is a control electrode made of cylindrical silver. A lead wire 43 is connected to the working electrode 41 and the control electrode 42.
．． 43 are connected to each other by means such as soldering. The working electrode 41 and the reference electrode 42 are connected to the support member 44
They are disposed coaxially within the cylindrical case 45 via the cylindrical case 45. An epoxy resin 46 is sealed in the space inside the case 45, and the working electrode 41 and the reference electrode 42 are fixed and insulated.

The end surface 45a of the case 45 is ground into a spherical surface using a cutting tool, and then polished. The end face of the working electrode 41 and the end face of the control electrode 42 are exposed on this end face 45a, and serve as sensing portions 41a and 42a, respectively.

Furthermore, an immobilized enzyme membrane 47 is provided on the case end surface 45a.
is attached, and the sensitive parts 41a and 42a are brought into close contact with this immobilized enzyme membrane 47. The immobilized enzyme membrane 47 is a polymer membrane in which an enzyme such as glucose oxidase is immobilized, and a peripheral portion 47 a is fixed to the outer surface of the case 45 by an O-ring 48 .

(c) Problems to be Solved by the Invention The above-mentioned conventional enzyme electrodes have been produced one by one manually in the manufacturing process, making mass production difficult. In addition, this manual work is a series of delicate operations, and there is a large loss of material, especially electrode material.
There was an inconvenience that the yield during production of enzyme electrodes decreased.

Furthermore, there were disadvantages such as high processing costs and high manufacturing costs.

Incidentally, this processing cost accounts for 60 to 80% of the manufacturing cost.

On the other hand, the conventional enzyme electrode described above has the following disadvantages when used. First, the sensitive surface 41 by grinding and polishing
During the formation of 42a and 42a, cracks and gaps are generated in the epoxy resin 46 around the working electrode 41. This is epoxy resin 4
6. This is due to the difference in hardness between the working electrode 41 and the control electrode 42. When using an enzyme electrode, if water or other liquid enters these cracks or gaps, noise is generated and measurement accuracy is reduced. In addition, the output of the enzyme electrode is
It is determined by the area of the sensitive surfaces 41a and 42a.

However, since the sensitive surfaces 41a and 42a are formed manually, the areas of the sensitive surfaces 41a and 42a are not constant between the enzyme electrodes, and the output varies (this is an inconvenience.Furthermore, the immobilized enzyme There is an inconvenience that a change in output occurs due to improper attachment of the membrane 47.

In addition, the enzyme electrode is cylindrical and has no degree of freedom in shape.
There are disadvantages such as restrictions on the design of clinical testing equipment using this enzyme electrode, which makes it difficult to use.

This 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 low in cost, and has excellent performance and ease of use.

(d) Means for Solving the Problems As a means for solving the above-mentioned disadvantages, the enzyme electrode of the present invention has a flexible insulator as shown in FIG. 1(a) corresponding to the embodiment. The surface 8a of the substrate 8 is made of conductive paste, and has sensing parts 4a, 5a and connecting parts 4, respectively.
Two or more electrodes 4.5 comprising C%5C are provided, and a metal foil 6 is adhered to the sensitive part 4a of at least one of the electrodes 4 using a conductive adhesive, and each of the electrodes is The parts other than the sensitive parts 4a, 5a and the connecting part 4C15C are covered with an insulating protective film 7, and the sensitive part 4a, to which the metal foil 6 is adhered, is covered with an immobilized enzyme film 15. be.

(e) Function The enzyme electrode of the present invention has a working electrode 4 and a reference electrode 5 on a visible insulating substrate 8, and it is possible to manufacture a plurality of enzyme electrodes at once on a large flexible substrate. It becomes possible. Furthermore, since the electrodes 4.5 are made of conductive paste, their shapes can be easily and automatically formed by printing or the like. Additionally, material usage and losses are reduced. The above three effects make it possible to mass produce enzyme electrodes, reduce costs, and improve yield in the manufacturing process.

On the other hand, in the enzyme electrode of the present invention, since grinding and polishing of the electrodes 4.5 is not necessary, noise caused by this is prevented. Furthermore, since the positions and shapes of the electrodes 4.5 and the insulating protective film 7 are determined with high precision, the areas of the sensitive parts 4a and 5a are made uniform, and variations in output between the enzyme electrodes are eliminated. Furthermore, the immobilized enzyme membrane 15 can be integrally provided on the surface of the insulating substrate 8, eliminating fluctuations in output caused by improper attachment of the conventional immobilized enzyme membrane. In addition, absolutely 8M board 8
Since it has flexibility, the enzyme electrode can be deformed and attached to fit the space in which it will be used, making it easy to use.

(f) Example An example of this invention is shown in FIG. 1(a), FIG. 1(b),
The following description will be made based on FIG. 2 fal to FIG. 2 (phantom), FIG. 3 (a) to FIG. 3 (fl), FIG. 4, FIG. 5 (a), and FIG. 5 (b).

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 with different manufacturing steps.

FIG. 2 (al and FIG. 3 fa) shows a flexible substrate 2 made of polycarbonate. This flexible substrate 2 is later cut to form the insulating substrate 8 of the enzyme electrode 1, and for example, a 12×20 cm one is used. Note that as the flexible substrate, a material made of fluororesin (trade name: Kabuton) can also be used, and the size is not limited to the above-mentioned one.

A pattern 3 of silver paste (conductive paste) is directly printed on the surface 2a of the flexible substrate 2 [Fig.
b) and Figure 3 (see bl).This silver paste pattern 3 includes working electrodes 4,..., 4, reference electrodes 5,...
, 5 are included. The working electrode 4 has a rectangular sensing section 4
a, a strip-shaped wiring section 4b, and a rectangular connection section 4C. For example, the size of each part is 2X for the sensitive part 4a.
2mm, the width of the wiring part 4b is 0.5mm, and the width of the connection part 4C is 2mm.
It is said to be X7mm.

On the other hand, the control electrode 5 is also composed of a sensitive part 5a, a wiring part 5b, and a connecting part 5c. It has the same shape and size as the portion 4b and the connecting portion 4C.

Next, as shown in FIG. 2fc) and FIG. 3(C), a platinum foil (metallic foil) 6 is adhered onto the working electrode sensitive portion 4a using a conductive adhesive. The thickness of the platinum foil 6 is 5 μm.

FIG. 2(d) and FIG. 3(d) show an insulating protective film 7 made of photosensitive polyimide on the surface 2 of the flexible substrate.
. , 7 is shown. This insulating protective film 7 covers the working electrode wiring section 4b and the reference electrode wiring section 5b, and provides insulation protection for these.

Further, the insulating protective film 7 is provided with a window portion 7a, through which the platinum foil on the working electrode sensitive portion 4a is exposed.

The insulating protective film 7 is formed by forming a photosensitive polyimide layer on the flexible substrate surface 2a, exposing the layer to light using a photomask (photolithography), and removing unnecessary portions. The shape of this insulating protective film 7 is determined with high precision using a photomask. Therefore, there is no variation in the exposed area of the exposed working electrode sensitive parts 4a, . . . , 4a and the area of the exposed reference electrode sensitive parts 5a, .

The ratio of the exposed area of the working electrode sensitive section 4a (platinum foil 6) to the area of the reference electrode sensitive section 5a is set to a predetermined value.

FIG. 2(e) shows a state in which the flexible substrate 2 is cut to form an insulating substrate 8. As shown in FIG. Lead wires 9.9 are soldered to the working electrode connection 4C and the reference electrode connection 5C. The electrode in this state is called the base electrode 11.

In FIG. 2 (fl and FIG. 3 (Ql), the soldering part of the lead wire 9 of the base electrode 11 is sealed with epoxy resin 10,
Furthermore, a state in which an acetylcellulose film 12 is formed on the insulating substrate surface 8a is shown. This acetylcellulose membrane 12 integrally covers the working electrode sensitive section 4a and the reference electrode sensitive section 5a.

To form the acetylcellulose film 12, the base electrode 1
1 was attached to a spinner (rotator 2, not shown), and a 5% acetyl cellulose solution (solvent composition: acetone:cyclohexanone = 3 = 1) was dropped onto the insulating substrate surface 8a. O
Rotate for 5 seconds at OOrpm.

Next, an enzyme solution 13 is placed on the acetylcellulose membrane 12.
is applied [see Figures 2 (g) and 3 (fl)].

This enzyme solution 13 is prepared by the following procedure.

First, glucose oxidase (COD) 2.0 mg
100μi of 0.1M phosphate buffer (PH6,0)
dissolve in 0 prepared in the same phosphate buffer,
Mix with 100 μl of 5% glutaraldehyde solution to obtain enzyme solution 13.

Finally, another acetylcellulose membrane 14 is formed on the acetylcellulose membrane 12 coated with the enzyme solution 13, and the enzyme electrode 1 is completed [see FIG. 1(a) and FIG. 1(bl)]. The acetylcellulose membrane 14 is for protecting the enzyme solution 13, and has a 2.5% acetylcellulose solution (solvent composition).

It is formed by applying a thin layer of acetone:ethanol=・1:1). The acetylcellulose membrane 12.14 and the enzyme solution 13 constitute an immobilized enzyme membrane 15.

Next, hydrogen peroxide (
The sensitivity characteristics of H20□) will be explained below with reference to FIG. Since the performance of the enzyme electrode 1 depends on the characteristics of the base electrode 11, it is important to confirm this.

FIG. 4 shows the respective H, O□ concentrations (0,
, 1, 3, 5 PPM) and the relationship between electrode voltage (V) and electrode output (μA). From FIG. 4, it is confirmed that the base electrode 1■ responds well to the H2O□ concentration. Further, it has been shown that a suitable value for the electrode voltage is 0.6 to 0.9V.

Next, the glucose detection characteristics of the enzyme electrode 1 of this example will be explained with reference to FIGS. 5(a) and 5(b).

FIG. 5 (al indicates the measurement system 18 used to measure the characteristics of the enzyme electrode 1. 19 is a constant temperature block;
0.1M phosphate buffer 2 adjusted to pH 7.0 inside
0 is stored. The enzyme electrode 1 is immersed in this phosphate buffer 20 . Moreover, this phosphate buffer 20 is
The mixture is stirred by a starter 19a built into the constant temperature block 19. 19b is the rotor of Stark 19a.

The lead wire 9 of the enzyme electrode 1 is connected to an electron meter 21, and a predetermined electrode voltage (0.7 V in this measurement) is applied. The electron meter 21 has a recorder 22
is connected, and the electrode output 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 (Gj!c) causes the following reaction within the immobilized enzyme membrane 15 of the enzyme electrode 1.

0D QIV, c+Q, --gluconic acid 10 Hz O2 These H, O□ are the working electrode sensitive part 4a and the reference electrode sensitive part 5a
is sensed, and an electrode output corresponding to the H20□ concentration is generated between both electrodes 4.5. Figure 5fb) shows the electrode output (nA) for several glucose concentrations (mg/d 1 ).
) is plotted. The curve shown in FIG. 5 (bl) is a calibration curve connecting the plotted points.

Based on this calibration curve, the glucose concentration in any sample, such as blood, can be quantified.

In addition, in the above example, the electrode is made of silver paste,
Although the metal foil is platinum foil, it is not limited to this and can be changed as appropriate.

Furthermore, the enzyme is not limited to glucose oxidase (eg, uricase, etc.), and can be changed as appropriate.

(G) As described in detail of the invention, the enzyme electrode of the present invention has the advantage of being able to be mass-produced, improving the manufacturing yield, and reducing the cost of the enzyme electrode (up to about 10% of the conventional cost). Exists. Further, it has the advantage that noise is reduced, output is stabilized, and variations in output between enzyme electrodes are eliminated.

Furthermore, since the enzyme electrode itself has flexibility, it has the advantage of being easier to use.

[Brief explanation of the drawing]

FIG. 1(a) is a plan view of an enzyme electrode according to an embodiment of the present invention, and FIG. 1('b) is a plan view of rb-1b in FIG. 1(a).
Enlarged cross-sectional view along the line, Fig. 2 (al, Fig. 2 (b),
Figure 2 (C), Figure 2 fdl, Figure 2 fQ), Figure 2 (
fl and FIG. 2(g) are diagrams explaining the manufacturing process of the enzyme electrode, FIG. 3(a) is an enlarged sectional view of the main part taken along the ma-ma line in FIG. bl is Fig. 2 fb
Figure 3 (C
1 is an enlarged sectional view of the main part taken along the line mc-mc in FIG. 2(C), and FIG. 3(d) is 111d-11 in FIG. 2(d).
The enlarged sectional view of the main part along the line 1d, Fig. 3je) is the
Fig. 3(f) is an enlarged sectional view taken along line IIIe-m e in Fig. 2, Fig. 4 is an enlarged sectional view taken along line f-mf in Fig. 2 fgl, and Fig. 4 is an enlarged sectional view taken on A diagram showing the sensitivity characteristics of the electrode, Figure 5 (al is a diagram showing the measurement system used to measure the characteristics of the enzyme electrode, Figure 5 (bl is a diagram showing the sensitivity characteristics of the enzyme electrode, The figure is a partially cutaway side view of a conventional enzyme electrode. 4: Working electrode, 5: Control electrode, 4a/5a: Sensing part, 4cm5c: Connection part, 6: Platinum foil, 7
: insulating protective film, 8: insulating substrate, 8a: surface, 15: immobilized enzyme membrane. Patent applicant Tateishi Electric Co., Ltd. Agent
Patent Attorney Shigeru Nakamura Figure 1 (σ) Figure 2 ((7) Figure 2 (e) Figure 2 (C7) Figure 4 (V) Figure 52 (o) Figure 5 ( b) qLucose pain (relaxation)

Claims (1)

[Claims] (1) 2 formed of conductive paste on the surface of a flexible insulating substrate, each comprising a sensing part and a connecting part.
The above electrodes are provided, a metal foil is adhered to the sensitive part of at least one of these electrodes using a conductive adhesive, and a portion other than the sensitive part and the connecting part of each of the electrodes is covered with an insulating protective film. An enzyme electrode characterized in that at least the sensitive part to which the metal foil is bonded is covered with an immobilized enzyme membrane.