JPS62237348A - Production of enzyme sensor - Google Patents

Abstract

PURPOSE:To effectively produce a sensor by forming two sets of combined electrodes of anode electrode and cathode electrodes consisting of thin metallic films on the same insulating substrate and forming a deactivated enzyme immobilized film thereto. CONSTITUTION:A positive type photoresist 3 is patterned on the substrate 1 in such a manner that the electrode parts are formed within the range of an exposed substrate surface 2. A thin chromium film 4 and thin gold or platinum film 5 are successively formed on the substrate 1 by a vacuum deposition method. The entire pat is thereafter immersed in a resist stripping soln. to remove the resist. The thin film 5 remaining on the exposed surface is sued as the electrode surface. The three electrodes to be used as the anode electrode and cathode electrode consisting of the thin metallic film are thus formed in the state of commonly using one cathode electrode at the prescribed point on the substrate 1. The enzyme immobilized film is installed to at least one electrode to be used as the anode electrode. The sensor is produced in the above-mentioned manner.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a method for manufacturing an enzyme sensor.

More specifically, the present invention relates to a method for manufacturing an enzyme sensor that can form a highly reliable hydrogen peroxide electrode on an insulating substrate.

[Conventional technology]

Recently, various biosensors that utilize biological reactions such as enzyme reactions and immune reactions have been developed, and especially in the clinical field, there is a growing demand for smaller, higher performance, and lower priced sensors. Enzyme sensors are a type of biosensor. For example, glucose sensors that utilize the catalytic action of glucose oxidase are used to measure glucose concentrations in blood and urine, and are practically used in clinical tests for diabetic patients. important as such.

However, when these enzyme sensors are put into practical use,
In most commercially available products, the electrode and the enzyme-immobilized membrane are not integrated, which hinders miniaturization and cost reduction through mass production.

[Problem that the invention seeks to solve]

In view of these problems with commercially available enzyme sensors, research has recently progressed to immobilize enzymes directly on electrodes, and as a result, considerably smaller versions have been developed. However, if a large number of sensors are to be formed on the same substrate at the same time and mass production is possible, for example, the connecting part with the external electrode is exposed and the enzyme is immobilized only on the detection part. , it is necessary to immobilize the enzyme only on the necessary portions of the electrode substrate.

In order to solve these problems, the present inventors found that it is effective to form an enzyme-immobilized film using a photocrosslinked polymer, and the anode electrode consists of a metal thin film formed on an insulating substrate. We have previously proposed an enzyme sensor in which an enzyme-immobilized membrane immobilized with a photo-crosslinked polymer is installed on at least the former cathode electrode (Japanese Patent Application No. 103/1989, No. 699).

Enzyme sensors configured in this way can simultaneously form a large number of microscopic sensors on the same substrate, making mass production possible. When constructing a hydrogen electrode, it has been found that there is a problem in that this electrode is sensitive to reducing interfering substances other than hydrogen peroxide.

Normally, as a countermeasure to this problem, a hydrogen peroxide selectively permeable membrane is installed between the enzyme-immobilized membrane and the electrode surface, but in the case of the enzyme sensor mentioned above, Since the enzyme is directly immobilized, it is impossible to install a single selective permeation system.

Therefore, the inventors of the present invention searched for a new solution to this problem and, as a result of various studies, decided to combine two sets on the same board. 1
! We have previously discovered that the above problem can be effectively solved by forming electrodes and forming an enzyme-immobilized membrane immobilized on at least the anode electrode for one set of the electrodes (patent application 60-171.850).

The enzyme sensor proposed here has at least one set of two combination electrodes of an anode and a cathode made of a metal membrane formed on the same insulating substrate, one of which is immobilized on the anode. It is made by installing an enzyme-immobilized membrane. Immobilization of the enzyme-immobilized membrane onto a set of combination electrodes is generally performed using a photocrosslinked polymer, and the formation of the enzyme-immobilized membrane immobilized with the photocrosslinked polymer is performed in the same manner as the previous proposal. , by applying the photoringraph method to an aqueous mixture of a photocrosslinkable polymer and an enzyme.

In addition, another set of combination electrodes without an enzyme-immobilized membrane is used with at least the anode electrode covered with a photocrosslinked polymer film, but the formation of the photocrosslinked polymer film in this case also This is carried out by applying a photolithography method to an aqueous solution containing a crosslinkable polymer.

In these two sets of combined electrodes, an anode electrode and a cathode electrode may be formed for each set.
It is also possible to use several cathode electrodes and switch them for each combination of electrodes.

When producing an enzyme sensor, first a glass plate, a hard resin plate such as vinyl chloride resin or polyimide resin, or a SiO□
, Two combination electrode sets of an anode electrode and a cathode electrode are formed on a flat insulating substrate such as a silicon wafer having an insulating film such as Si□N4 formed on the surface. The electrodes are formed using metal materials such as gold, platinum (for anode electrodes) or silver, gold, platinum (for cathode electrodes), and are deposited on the substrate using a lift-off method as shown in Figure 1 of the drawings. The photo-etching method involves etching away and patterning the metal thin film that has been etched, the mask evaporation method involves placing a mask with an electrode-shaped window on the substrate, and depositing an electrode-forming material through the mask, and the electrode-forming material is a conductive material. This can be carried out by a screen printing method in which a conductive paint is printed in the shape of an electrode, or a plating method in which electroless plating of an electrode forming material is performed instead of vapor deposition in the above-mentioned photoetching method or mask vapor deposition method.

In the embodiment shown in FIG. 1, a lift-off method is used. First, an electrode portion is formed on a clean flat insulating substrate, for example a glass plate 1, within the exposed surface 2 of the substrate. , the positive photoresist 3 is patterned (step a). Next, a chromium thin film 4 (about 500 mm thick) and a gold or platinum thin film 5 are deposited on this substrate by vacuum evaporation.
(thickness approximately 0.2 μm) are sequentially formed. Here, the chromium thin film is provided to improve the adhesion between the gold or platinum thin film forming the electrode and the glass plate substrate (step b
).

Thereafter, the whole was immersed in a resist stripping solution such as acetone to remove the resist, and the deposited thin film 5 remaining on the exposed surface 2 of the substrate was used as an electrode surface (Step C).

In this way, if three electrodes are formed at a predetermined location on the insulating substrate in such a manner that one cathode electrode is used in common, the anode electrode and the cathode electrode made of the metal thin film are formed. An enzyme-immobilized membrane is installed on one electrode which serves as an anode electrode, and the installation is carried out using a photolithography method, for example, as shown in FIG. 2.

First, an insulating group F formed as shown in FIG.
An aqueous mixture of a photocrosslinkable polymer and an enzyme is uniformly coated 6 on the electrode surface 5 on i2 by a spin coating method, a spray method, etc. (step d).

As the photocrosslinkable polymer, a water-soluble polymer is generally used because it is dispersed as an aqueous mixture together with an aqueous enzyme solution. Polyvinyl alcohol having a stilbazolium group is used as an aqueous solution.

The aqueous mixture was the photocrosslinkable polymer aqueous solution (concentration 8.5
An enzyme aqueous solution prepared by dissolving 3 to 72 ml of enzyme in 0.8+nQ of distilled water was added to (~12% by weight) Ig.

The mixture is stirred and mixed for several minutes before being used for coating.

The coating liquid is coated on the electrode surface on the insulating substrate, and when it dries naturally, it is covered with a photomask 7 having a negative or positive image, and the photocrosslinkable polymer is photocrosslinked by irradiation with ultraviolet rays. The crosslinked portion is eluted with pure water (step 1) to form an enzyme-immobilized membrane 8 immobilized with a photocrosslinked polymer on the photocrosslinked portion (step e). This was irradiated with ultraviolet light again and then dried. The photomask used here is
Among the two sets of combined electrodes, only one set of anode electrodes 11 has an image such that the enzyme-immobilized film 8 is formed thereon.

Next, in order to equalize the sensitivity characteristics of the two sets of combination electrodes to reducing interfering substances, the anode electrode 12 of the other set of combination electrodes in which the enzyme-immobilized membrane was not installed was coated with the same method as shown in FIG. A photolithographic method was applied to an aqueous solution containing a photocrosslinkable polymer, and a photocrosslinkable polymer film 9 was coated thereon, and this was used as a reference electrode (step f).

This is irradiated with ultraviolet rays again and then dried, divided into each element, and the lead wires 10.
Attach 10' and 10'. One embodiment of the enzyme sensor produced in this manner is shown in FIG. 3 as a plan view. 4 and 5 show plan views of other embodiments of the enzyme sensor. In the embodiment of FIG. 4, two cathode electrodes 13 and 13' are extended from the electrode surface 5'. Let it form.

In addition to shortening the inter-electrode distance between each anode and cathode, each of these combined electrodes is covered with an enzyme immobilization film 8 and a photocrosslinked polymer film 9, and in the embodiment shown in FIG. 5, the cathode electrode 13 By setting the area of the electrodes 11 and 12 to be larger than the area of the electrodes 11 and 12, the cathode current to the anode is stabilized.

Furthermore, the response characteristics of the electrodes are improved by increasing the facing width between both electrodes while keeping the distance between the electrodes constant.

Examples of combinations of a substrate detectable by this enzyme sensor and an enzyme as a catalyst that reacts with this substrate are as follows, in which both electrodes act as hydrogen peroxide electrodes.

Ill enzyme glucose glucose oxidase galactose galactose oxidase monoamino acid
L-amino acid oxidase ethanol alcohol oxidase phospholipid phospholipase choline choline oxidase glycerin
Glycerokinase When such an enzyme sensor is used as a glucose sensor, it acts in a first-order manner. First, the glucose sensor is immersed in a buffer solution that does not contain glucose, and a voltage of 1, for example, 0.8 V in the case of a gold electrode is applied to the electrode surface. When glucose is added to this, the enzyme is immobilized. Glucose diffuses into the glucose oxidase enzyme-immobilized membrane on the hydrogen peroxide electrode side, and reacts as follows due to the catalytic action of the immobilized enzyme.

Hydrogen peroxide generated as a result of this reaction is oxidized on the anode electrode as follows, and a current proportional to the amount of hydrogen peroxide generated, that is, a current proportional to the glucose concentration flows.

H20□→2H-+ 0. +2e- At this time, since no enzyme is immobilized on the reference side electrode, no current due to the enzyme reaction flows, so even if differential output between the enzyme immobilized side and the reference side is detected.

Only the current on the enzyme-immobilized side can be detected.

In this case, even if the sample solution contains a reducing interfering substance such as L-ascorbic acid, the interfering substance is oxidized equally at both combination electrodes regardless of the presence or absence of the immobilized enzyme. By detecting the differential output of the current flowing through these two electrodes, the current region caused by interfering substances is canceled out, and
Only the current value caused by the enzyme reaction can be detected,
For example, in the case of ascorbic acid, its concentration is 0.5~
It has been confirmed that within the range of 4/min, the current caused by interfering substances generated at both electrodes can be reduced to 3% or less.

However, the enzyme-immobilized membrane and the photocrosslinked polymer 1 serving as the reference side
When g is observed with an optical rIft microscope, the photocrosslinked polymer film forms a transparent and homogeneous film, whereas the enzyme-immobilized film is white with minute irregularities on its surface. It turns out that there are countless numbers of them.

Therefore, as a result of further investigation in search of a means to more accurately detect the differential output by making these two membrane bodies as similar as possible, we decided to immobilize the inactivated enzyme on the membrane body that would serve as the reference side. It has been found that the method of inducing the same effect is extremely effective (Japanese Patent Application No. 149-217-1988).

This enzyme sensor has two sets of combined electrodes consisting of an anode electrode and a cathode electrode made of metal thin films formed on the same insulating substrate, one set has at least an enzyme immobilized film immobilized on the anode electrode, and the other Each of the sets includes a deactivated enzyme-immobilized membrane immobilized on at least an anode electrode.

The production of such an enzyme sensor is carried out by using the method described above with reference to FIGS. 1 and 2, in which the photocrosslinkable polymer-containing enzyme aqueous solution used in step (d) is heated in an oven according to the type of enzyme. The same method is used except that an aqueous solution in which the enzyme is inactivated by heating at about 80 to 100° C. for about 5 to 10 minutes is used in step (f). For example, in the embodiments shown in FIGS. 3 to 5, an enzyme sensor is obtained in which an enzyme-immobilized membrane is immobilized on the 8 side and an inactivated enzyme-immobilized membrane is immobilized on the 9 side.

In the production of an enzyme sensor carried out in this way, the formation of the inactivated enzyme immobilized film to be immobilized on at least the anode electrode of the other set of combination electrodes involves the formation of an enzyme aqueous solution containing a photocrosslinkable polymer as described above. This is done by using an aqueous solution that has been heat-treated to deactivate the enzyme.

The fabricated enzyme sensor has an inactivated enzyme immobilized on the reference side as well, thereby making the membrane quality of the reference side electrode equal to that of the enzyme side electrode, thereby improving the response characteristics to interfering substances between the two sets of combined electrodes. are made equal, thereby further improving the differential rejection characteristics of interfering substance signals. Specifically, in measuring the differential rejection characteristics of interfering substance signals using L-ascorbic acid, the current caused by the interfering substance generated at both electrodes was %, compared to the concentration range and current value of 3% or less within the range of 0.5 to 4/m in the previous proposal, which further improves the differential rejection characteristics of the interference signal. Achieve further improvement. Moreover, such characteristics are not limited to cases where the interfering substance is L-ascorbic acid.

It is similarly effective in cases such as uric acid.

As described above, although this enzyme sensor is effective enough to achieve its intended purpose, two photolithographic methods were applied to separately form the enzyme-immobilized membrane and the inactivated enzyme-immobilized membrane. Moreover, the same amount of enzyme required for forming the enzyme-immobilized membrane must be used when forming the inactivated enzyme-immobilized membrane, which is wasteful in production.

The present invention aims to provide a method for manufacturing an enzyme sensor that eliminates such waste.

[Means to solve the problem]

Therefore, the present invention relates to a method for manufacturing an enzyme sensor, in which two sets of combined electrodes of an anode electrode and a cathode electrode made of a gold R thin film are formed on the same insulating substrate, and in each combination electrode, at least an anode electrode is formed. After an enzyme-immobilized membrane is immobilized thereon, the enzyme-immobilized membrane on the electrode forming the reference side electrode is irradiated with ultraviolet rays to form an inactivated enzyme-immobilized membrane.

Forming an enzyme-immobilized mass on at least the anode electrode of the two sets of combined electrodes is performed by using a photomask having an image in step (e) in the method described above with reference to FIGS. 1 and 2 of the drawings. without using or 2
This is carried out by irradiating ultraviolet light using a photomask having an image pattern that exposes a set of anode electrodes. The ultraviolet light used for this ultraviolet irradiation has a relatively long wavelength, such as that emitted from a mercury lamp, and when irradiated with this for about 30 seconds, the photocrosslinkable polymer is photocrosslinked, but There is almost no effect on enzymes.

After that, only the enzyme-immobilized membrane on the combination electrode that will become the reference side electrode is irradiated with ultraviolet rays using a photomask etc.
Once immobilized, the enzyme is deactivated. The ultraviolet irradiation conditions in this case are generally as follows.

Light source: short wavelength ultraviolet lamp Wavelength: Approximately 2000-3000 people Approximately 10-20W/cd Distance: Approximately 1-50 Time: Approximately 1-5 minutes 〔Effect of the invention〕 The method of the present invention provides the following effects.

(1) A photolithographic method is applied at once to form an enzyme-immobilized membrane and an inactivated enzyme-immobilized membrane on the electrode of an enzyme sensor.

(2) In connection with this, since the film thickness and film quality of both of these immobilized films are the same, the differential removal characteristics for interfering substances are improved.

(3) The amount of enzyme used is only half that of the heat inactivation method.

〔Example〕

Next, the present invention will be explained with reference to examples.

Example 1 Photocrosslinkable polyvinyl alcohol (photocrosslinkable stilbazolium group content 1.4 mol%, saponification degree 88%, polymerization degree 1)
400) in which 30 mg of glucose oxidase enzyme was dissolved in 0.5 g of an 11.7% aqueous solution of distilled water.
4 (Add IIQ and stir and mix for several minutes to prepare a coating liquid. This coating liquid.

On top of two sets of hydrogen peroxide electrodes formed on a glass plate,
Spin code at 4000 rpm for 20 seconds. After the coating solution dries naturally, each of the two sets of hydrogen peroxide electrodes is covered with a photomask having a negative image that allows only the anode electrode portion to be irradiated with ultraviolet rays.
Ultraviolet irradiation (output 250 W) was performed for 15 seconds, and then 131 images were taken by washing with pure water, and ultraviolet ray irradiation was performed again to dry.

Next, it was covered with a quartz photomask having a negative image so that only the anode electrode part of the reference side electrode was irradiated with ultraviolet rays, and an irradiation distance of 2 c! a. UV irradiation was performed under conditions of irradiation time of 2 minutes to deactivate the enzyme immobilized on the immobilization film on the anode electrode.

[Brief explanation of drawings]

FIG. 1 is a cross-sectional view sequentially showing the steps of forming an electrode on an insulating substrate. Figure 2 shows the previously proposed method, in which an enzyme-immobilized film and a photo-crosslinked polymer are each immobilized with a photo-crosslinked polymer on two anode electrodes formed on an insulating substrate. FIG. 3 is a cross-sectional view sequentially showing the steps of installing a membrane (or inactivated enzyme i-immobilized membrane), and FIG. 3 is a plan view of one embodiment of the enzyme sensor produced in this way. 4-5 are plan views of other embodiments of the enzyme sensor. (Explanation of symbols) 1... Insulating substrate 2... Substrate exposed surface 5... Electrode 6... Enzyme aqueous solution containing photocrosslinkable polymer 7...
- Photomask 8 having an image...Enzyme immobilization membrane 11...Enzyme immobilization membrane side anode electrode 12...
...Reference side 7 node electrode l:3...Cathode electrode

Claims (1)

[Claims] 1. After forming two sets of combined electrodes consisting of an anode electrode and a cathode electrode made of metal thin films on the same insulating substrate, and immobilizing an enzyme-immobilized film on at least the anode electrode in each combined electrode. A method for producing an enzyme sensor, which comprises irradiating an enzyme-immobilized film on an electrode forming a reference side electrode with ultraviolet rays to form an inactivated enzyme-immobilized film. 2. The method for producing an enzyme sensor according to claim 1, wherein the immobilization is performed using a photocrosslinked polymer. 3. The method for manufacturing an enzyme sensor according to claim 1, wherein one cathode electrode is used in common for two sets of combined electrodes. 4. The method for manufacturing an enzyme sensor according to claim 1, wherein the combined electrode constitutes a hydrogen peroxide electrode.