JPH01129155A - Enzyme electrode - Google Patents

Abstract

PURPOSE:To realize an enzyme electrode possible in mass production and capable of being reduced in cost, by forming a reference electrode and an acting electrode on insulating substrates separate each other to integrally bond and assemble both substrates. CONSTITUTION:A reference electrode 3 is formed to the surface of the first insulating substrate 2 and covered with an insulating protective film 4 so as to leave the reaction surface 3a and connection surface thereof. The second substrate 12 detachable to the engaging groove 2a of the first insulating substrate 2 is prepared and an acting electrode 13 is formed on the surface of said insulating substrate 12. This acting electrode 3 is covered with an insulating protective film 14 leaving the reaction surface and connection surface thereof and the aforementioned reaction surface is covered with an immobilized enzyme film 19. By this method, since the reference electrode part and the acting electrode part can be prepared separately, mass production, cost reduction and the enhancement of capacity can be achieved.

Description

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

(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 the 73 specific chemical substances of the enzyme contained in the test liquid.

As a conventional enzyme electrode, the one shown in FIG. 12 is known. 51 is a working electrode made of platinum in the shape of a round rod (for example, 0.7 mm in diameter), and 52 is a working electrode in the shape of a cylinder (for example, 2 m in inner diameter).
m, outer diameter 6sw*) is a control electrode made of silver [first
2(a) and FIG. 12(b)] 0 working electrode 51,
Lead wires 53 and 53 are respectively connected to the reference electrode 52 by appropriate means such as soldering.The working electrode 51 and the reference electrode 52 are connected to a support member 5 made of an insulator.
4 and arranged coaxially.

Working electrode 51. The control electrode 52 is housed in a case 55 and sealed with epoxy resin 56. This epoxy resin 5
6 is raised from the case opening 55a and completely covers the working electrode 51 and the control electrode 52 in the enclosed stage. This epoxy resin 56, together with the case 55, is ground and polished into a spherical surface, and reaction surfaces 51a and 52a are formed on the working electrode 51 and the reference electrode 52, respectively.

The diameter of the working electrode 51 and the inner and outer diameters of the reference electrode 52 are determined so that the reaction surfaces 51a and 52a have a predetermined area ratio. This state, ie, the state before the immobilized enzyme membrane is attached, is called the base electrode 57.

An immobilized enzyme membrane 58 is attached to the base electrode 57, and the reaction surfaces 51a and 52a are in close contact with the immobilized enzyme 1158. The immobilized enzyme membrane 5B is a polymer membrane in which an enzyme, such as glucose oxidase, is immobilized. This immobilized enzyme membrane 58 is fixed at its peripheral portion to a membrane holder 60 by an O-ring 59. And, this membrane holder 60 is
The immobilized enzyme F! is fixed to the outer peripheral surface of the case 55 with screws (not shown). 58 is installed.

(c) Problems to be Solved by the Invention The conventional enzyme electrode described above has the following problems.

i: The conventional enzyme electrodes described above are produced one by one manually, making mass production difficult.

11: The above manual work is a series of detailed operations, and
It requires a high level of skill and reduces the yield of enzyme electrode production.

iii:! The loss of material, especially the loss of electrode material, is large in the 1-meter manufacturing process, and the manual work increases processing costs, increasing manufacturing costs.

1v: The immobilized enzyme 1158 is attached to the electrode reaction surfaces 51a, 5
Since it covers not only the case 2a but also the end face of the case 55, the immobilized enzyme W158 becomes large (for example, 16 mm in diameter), and the final cost of the enzyme electrode increases.

V: Since grinding and polishing are applied to the formation of the electrode reaction surfaces 51a and 52a, cracks and gaps occur in the epoxy resin 56 around the reaction surfaces 51a and 52B.

If liquid enters these cracks or gaps when using an enzyme electrode, noise may be generated or measurement accuracy may be reduced.

vi: The enzyme electrode output is determined by the area of the electrode reaction surfaces 51a and 52a, particularly the working electrode reaction surface 51a. However, in the conventional enzyme electrode, since the reaction surfaces 51a and 52a are formed manually, the reaction surfaces 51a and 52a are formed between the individual enzyme electrodes.
The areas of the electrodes 52a are different, resulting in variations in electrode output.

vfi: The immobilized enzyme membrane 58 is attached to the membrane holder 60 by an O-ring 59, but there is a risk that the immobilized enzyme membrane 58 will break during this attachment, making handling difficult.

vi:r! When attaching the holder 60 to the case, the degree of adhesion of the immobilized enzyme membrane 58 to the electrode reaction surfaces 51a and 52a is
It differs each time it is worn, and the electrode output fluctuates.

Only one item can be measured with one iX enzyme electrode, and when measuring multiple items, enzyme electrodes are required for the number of items.

This invention has been made in view of the above, and aims to provide an enzyme electrode that enables higher performance, mass production, and lower cost.

(d) Means for Solving the Problems In order to solve the above problems, the enzyme electrode of Mini's invention includes a reference electrode made of a thin metal film on the surface of a first tIA recording base, and a reaction surface of this reference electrode. an insulating film is formed to cover the reference electrode, leaving a connection surface and a contact surface; one or more insulating bases are removably provided on the first insulating base; Each surface of the insulating base is provided with a working electrode made of a gold rIA thin film, an insulating protective film covering the working electrode with the reaction surface and connection surface of the working electrode remaining, and the working electrode. The reaction surface is formed by integrally forming an immobilized enzyme membrane with *ni+.

(E) Function: The enzyme electrode of the present invention has a reference electrode and a working electrode formed on the surfaces of the first and second insulating bases, respectively.
A large number of electrodes are formed all at once on the surface of a large insulating substrate, and this insulating substrate is divided into individual electrodes to form an insulating base, making mass production possible.

The reference electrode and the working electrode are made of metal thin films, and the metal II film can be formed by sputtering, vacuum evaporation,
Well-known automated thin film formation techniques such as printing can be applied. Therefore, it is possible to reduce loss of electrode material. Furthermore, the formation of the insulating protective layer can be automated by applying photolithography technology, and together with the automation of electrode formation, it becomes possible to reduce the manufacturing cost of the enzyme electrode.

In addition, the immobilized enzyme membrane only needs to cover the working electrode, making it possible to reduce the cost of the enzyme electrode.

On the other hand, since the enzyme electrode of the present invention does not involve grinding or polishing during the manufacturing process, no cracks or gaps are generated in the insulating base, and noise and deterioration in measurement accuracy due to this are prevented.

Further, the area of the electrode reaction surface can be determined accurately by the photolithography technique, and variations in output between individual enzyme electrodes can be eliminated.

Furthermore, since the immobilized enzyme membrane is integrally provided on the reaction surface of the working electrode, there is no need to remove the immobilized enzyme membrane, making it easier to handle and speeding up the reaction on the immobilized enzyme membrane and the reaction surface of the working electrode. . Furthermore, the degree of adhesion of the immobilized enzyme membrane to the reaction surface of the working electrode is always constant, eliminating fluctuations in electrode output.

In addition, since the first insulating base and the second insulating base are detachable, only the second box board on which the working electrode is provided can be replaced, reducing running costs. . Furthermore, since two or more second insulating bases can be provided on one first insulating base, if an immobilized enzyme film containing a different enzyme is formed on each of these second insulating bases,
A single enzyme electrode can measure multiple items.

(F) Example (Example 1) A first example of the present invention will be described below based on FIGS. 1 to 7.

The enzyme electrode 1 of this embodiment is of a type that is applied to measuring the concentration of glucose contained in blood, etc., and detects hydrogen peroxide produced as a result of an enzyme reaction.

FIG. 1 is an external perspective view of an example enzyme electrode 1, in which 2 is a reference electrode substrate (first insulating base) and 12 is a working electrode substrate (second insulating base).

FIG. 2 shows an external perspective view of the working electrode substrate 12 before forming the immobilized enzymes n. This working electrode 75Fl
12 is made of, for example, alumina ceramic;
It has a shape of 8MX 13wa SW and a length of 0.4 tm.

A working electrode 13 is formed on the surface 12a of the working electrode substrate 12. The working electrode 13 is a platinum thin film formed by sputtering, for example, III
It is said to have a shape of IIxlOIIIIIIl and a thickness of 1,500 people.

Further, on the working electrode substrate surface 12a, an insulating film made of photosensitive polyimide resin is provided. ! A film 14 is formed. Maintains insulation! ! ! Windows 14a and 114b are formed in the membrane 14, each exposing a part of the working electrode 13, and forming a reaction surface 13a (for example, 0.2 mm x 0.2 am) and a connecting surface 1.
3b (for example, 0.8sm x 2m).

This working electrode substrate 12 is manufactured through the following steps. First, a large alumina ceramic VL (not shown) is prepared, and a plurality of working electrodes are collectively formed on the surface of this alumina ceramic plate.Next, a photosensitive polyimide film is formed on the surface of the alumina ceramic plate. The photosensitive polyimide film is exposed to light using a photomask, developed and rinsed to maintain its insulation properties. ! One film. The alumina ceramic plate is then divided into individual working electrodes.

A lead wire 6b is connected to the connection surface 13b of the working electrode substrate 12.
One end of the is connected by suitable means such as soldering. Epoxy resin 15 is applied on the connection surface 13b to protect the connection portion between the connection surface 13b and the lead wire 6b.

Note that the lead wire 6b is provided with a connector 7a.

An immobilized enzyme film 19 is formed on the working electrode substrate 12 (see FIG. 3). First, the working electrode substrate 12 is immersed in a 3% acetylcellulose solution (solvent composition: acetone:cyclohexanone-4=1). The mixture is air-dried to form a first acetylcellulose II'J16 (so-called dip coating).

Reaction surface 1 on the surface of this first acetyl cellulose membrane 16
Enzyme solution 17 is dropped onto the portion located above 3a and air-dried. Enzyme solution 17 is 0.1non phosphate buffer (pH 6,0) 100μj! Add 2 mg of glucose oxidase (C;00) to the solution and the same phosphate buffer! 0.5% gluculaldehyde solution 10
0.1 liquid were mixed in equal amounts.

Furthermore, a second acetylcellulose membrane 18 is formed,
Immobilized enzyme 1 (J19 is completed. This second acetylcellulose membrane 18 is made by coating the working electrode substrate 12 with a 2% acetylcellulose solution (the solvent is acetone:ethanol-4
:1) Formed by dipping inside.

FIG. 4 is a perspective view of the control electrode substrate 2 seen from below;
The figure shows a cross section of the control electrode substrate 2. The control electrode substrate 2 is also made of alumina ceramic, for example, 2 mg
*The shape is 15mm x 0.4cm thick (not including the guard part).

A guard portion 2b is provided on the upper surface 2a of the control electrode substrate 2. This guard portion 2b supports and fixes the working electrode substrate 12. On the lower surface 2c of the reference electrode substrate, there is a reference electrode 3 and an insulating layer 1 covering the reference electrode 3! A film 4 is formed.

This control electrode 3 is a thin silver film formed by sputtering or vacuum deposition, for example, IXI 1, 15 mm thick.
00 people.

The insulating protective film 4 is also made of photosensitive polyimide resin and has window portions 4a and 4b. Window part 4a
has a size of, for example, 0.4×4 squares, and a part of the reference electrode 3 is exposed to form the reaction surface 3a. On the other hand, the window portion 4b
is similar to the previous window portion 14b, and exposes another part of the reference electrode 3 to serve as the connection surface 3b. Like the working electrode substrate 12, this control electrode substrate 2 is also made by forming a control electrode and an insulating protective film all at once on a large alumina ceramic plate, and then dividing the plate into parts.

A lead wire 6a is connected to the connection surface 3b, and the connection between the two is protected by the epoxy resin 5.

The lead wire 6a constitutes the core wire of the cable 6 together with the lead wire 6C, and a connector 7b is provided at the tip of the lead wire 6C.

As shown in FIG. 1, the working electrode substrate 12 is inserted into the guard portion 2b of the reference electrode substrate 2, and the connectors 7a and 7b are connected.

Next, the characteristics of the example enzyme electrode I will be explained.

First, the measurement system 41 used to measure the characteristics of the enzyme electrode 1 will be explained (see Fig. 6). 42 is a constant temperature block, inside which the pH is adjusted to 0°1 moI! , 10- phosphate buffer As is stored. The enzyme electrode 1 is immersed in this phosphate buffer solution 43. Further, this phosphate buffer solution 43 is stirred by a starter 42a built in the constant temperature block 42. 42b is a rotor of the starter 42a.

The lead wires 6a, 6c are connected to an electron meter 44, and a predetermined electrode voltage (+0.5V in this measurement) is applied. Recorder 45 is used for electron make 44.
is connected and the electrode output (current) is recorded.

A predetermined amount, for example 100 μ2, of glucose solution is dropped into the phosphate buffer 43 using a micropipette (not shown). This glucose (Gj!c) causes the following reaction within the immobilized enzyme membrane 19.

An increase in hydrogen peroxide (H, 0) accompanying this reaction appears in the electrode output as a change in oxidation current due to the working electrode reaction surface 13a.

Figure 7 shows several glucose concentrations (lIIg/d1)
This is a plot of the electrode output (nA) versus . In addition, the straight line shown in Figure 7 connects the plotted points to
This is the calibration curve obtained by connecting. Based on this calibration curve, the glucose concentration of any sample can be quantified.

In this example enzyme electrode 1, if the immobilized enzyme membrane 19 deteriorates, only the working electrode substrate 12 needs to be replaced, and running costs can be reduced. In addition, if a working electrode substrate on which different enzymes are immobilized is attached, other substances t
Mt can be detected.

<Embodiment 2> A second embodiment of the present invention will be described below based on FIGS. 8 to 11.

The enzyme electrode of this example is applied to simultaneous measurement of glucose concentration and uric acid concentration contained in blood or the like.

FIG. 8 is an exploded perspective view of the enzyme electrode 21 of this embodiment. This enzyme electrode 21 includes a control electrode substrate (first insulating base) 2 and two working electrode substrates (second insulating base) 32.
．． 329 is detachably provided.

The working electrode substrate 32 (32') is made of alumina ceramic and has a protrusion 32b (height 8-) on the lower surface.The working electrode substrate 32 (32') has the same working electrode 33 and insulating protection on the surface 32a of the working electrode substrate. A film 34 is formed (see Figure 10).
In addition, the lead wire 26b is connected to the working electrode connection surface (not shown), and the connection portion between the two is covered with the epoxy resin 3.
5. A connector 27a is provided at the other end of the lead wire 26b.

The working electrode substrate 32 contains the immobilized enzyme F! 39 is formed, and the working electrode reaction surface 33a is covered with the immobilized enzyme film 39. As in the first embodiment, this immobilized enzyme film 39 is composed of the 11th, 2nd acetylcellulose [36, 38 and the enzyme solution 37]. An enzyme solution 37 is placed on the zero working electrode substrate 32, which is
The same COD solution as before is used. On the other hand, a uricase solution is used as the enzyme solution 37'' for the working electrode substrate 32''. Preparation of uricase solution is performed by GO
Just change 02■ to uricase lO■, and the rest are the above C.
Same as OD solution.

1st. The second acetyl cellulose membranes 36 and 38 are formed by dip coating. At this time, a masking tape is attached to the lower surface of the working electrode substrate, and the masking tape is removed after the dip and coating are completed.

The reference electrode substrate 22 is an alumina ceramic plate having a convex cross-sectional shape as shown in FIG. The height is about 1.6 gua.

A control electrode 23 and an insulating protective film 24 are formed on the center surface 22a of the control electrode substrate 22, as in the first embodiment. 23a is the reaction surface of the reference electrode 23. Further, a lead wire 26a is connected to a connection surface (not shown) of the control electrode 23, and an epoxy resin 25 is applied thereto. Lead wire 26a
is one of the lead wires that make up the cable 26, and the other two 〇)')-F*ji! 26 c, 26 c c,
Connected to connector 27b.

Concave grooves 2 are formed on both sides 22b, 22b of the control electrode substrate 22.
2c is engraved. By inserting the protrusion 32b of the working electrode substrate 32 into this groove 22c, the working electrode substrate '32, 32'' is attached to the reference electrode substrate 22.

Then, the connector 27a127a is connected to the connector 27b.

The enzyme electrode 21 of this embodiment is completely similar to the previous embodiment in terms of glucose detection. On the other hand, the characteristics of uric acid detection are shown in FIG. FIG. 11 is obtained using the above-mentioned measurement system 41 (see FIG. 6), and plots the electrode output (n^) for several different uric acid concentrations (lRg/dl).

Uric acid causes the following reaction in the immobilized enzyme membrane 39.

An increase in hydrogen peroxide (Hz) accompanying the reaction of allantoin 10Hz Ot + COz2 appears as an electrode output.

Note that even if glucose and uric acid exist simultaneously in the test liquid, they will not interfere with each other and cause measurement errors.

Further, the number of working electrode substrates attached to the reference electrode substrate may be three or more (and the enzymes are not limited to glucose oxidase and uricase, 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 yield during manufacturing, and reducing costs.

In addition, the electrode output is stable, noise is reduced, highly accurate measurements can be made, and there is little variation in the output between individual enzyme electrodes.

Furthermore, there is no need to attach or detach the immobilized enzyme membrane, making it easier to handle, and when the immobilized enzymes 11 deteriorate, only the second substrate needs to be replaced, which has the advantage of reducing running costs. ing.

In addition, since two or more second substrates, each having immobilized enzyme films containing different enzymes, can be attached to the first substrate, it has the advantage of being able to measure multiple items with one enzyme electrode. ing.

[Brief explanation of the drawing]

FIG. 1 is an external perspective view of an enzyme electrode according to a first embodiment of the present invention, and FIG. 2 is a perspective view showing a working electrode substrate of the enzyme electrode in a state before an immobilized enzyme film is formed. Figure 3 shows
Fig. 1 - Cross-sectional view taken along line m-m of the same working electrode substrate, Fig. 4
The figure is a perspective view of the reference electrode substrate of the enzyme electrode viewed from below, FIG. 5 is a sectional view of the reference electrode substrate taken along the width v-v line in FIG. 4, and FIG. 6 is the characteristic of the enzyme electrode. FIG. 7 shows the measurement system used in the measurement, and FIG. 8 shows the relationship between the glucose concentration and electrode output of the same enzyme electrode. FIG. 8 shows the enzyme electrode according to the second embodiment of the present invention. Exploded perspective view of No. 9
The figure is a sectional view of the reference electrode substrate of the same enzyme electrode taken along the line XX in FIG. 8, FIG. 10 is a sectional view taken along the line XX of FIG. , a diagram showing the relationship between uric acid concentration and electrode output of the same enzyme electrode, Figure 12 (
FIG. 12(b) is a partially cutaway perspective view of a reference electrode and a working electrode of a conventional enzyme electrode, and FIG. 12(b) is a central vertical sectional view of the conventional enzyme electrode. !・21: Enzyme electrode, 2.22: Control electrode substrate, 3.2
3: Control electrode, 3a/23a: Control electrode reaction surface, 4/14/24/34: Insulating protective film, 12/32/3
2°: Working electrode substrate, 13.33: Working electrode, 13a.33a: Working electrode reaction surface, 19.39.39": Immobilized enzyme membrane. Patent applicant: Tateishi Electric Co., Ltd. Agent, Patent attorney: Shigeru Nakamura Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Glucose 1 degree (mg/dl) Figure 11 Uric acid concentration (mg/dl)

Claims (1)

[Claims] (1) Forming a control electrode made of a metal thin film on the surface of the first insulating base, and an insulating protective film covering the control electrode while leaving the reaction surface and connection surface of the control electrode; or 2
The above second insulating base is removably provided on the first insulating base, and on the surface of each of the second insulating bases,
A working electrode made of a metal thin film, an insulating protective film covering the working electrode, leaving the reactor and connection surface of the working electrode, and an immobilized enzyme film integrally covering the reaction surface of the working electrode. Enzyme electrode made by forming.