JPS6232351A - Enzyme sensor - Google Patents

Abstract

PURPOSE:To detect the signal based on enzyme reaction by immobilizing enzyme to one set of two sets of hydrogen peroxide electrodes on the same substrate and detecting said signal by the differential output from both electrodes. CONSTITUTION:A positive type photoresist is patterned on a glass plate 1 and a thin chromium film 4 and a thin gold or platinum film 5 is formed on the substrate 1 by a vacuum deposition method. The substrate is then immersed in a resist stripping liquid to remove the resist so that the thin vapor deposited film 5 is used as an electrode surface. A coating liquid is coated on the substrate 1 and is covered by a photomask of such as image with which an enzyme immobilized film 8 is formed only on the anode electrode 11. The photocrosslinkable polymer is photocrosslinked to form the film 8. A photocrosslinkable film 9 is further coated to an anode electrode 12 on which the film 8 is not provided to form a reference electrode so that the sensitive characteristics of two sets of the electrodes 11, 12 for a reducing disturbance material are made equal. The enzyme is immobilized only to the electrode 11 of two sets of the hydrogen peroxide electrodes 11, 12 and the differential output is detected from both electrodes, by which the signal from the reducing disturbance material to be equally sensed by both electrodes is offset. Only the signal based on the enzyme reaction is thus detected.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an enzyme sensor. More specifically, the present invention relates to 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, many sensors are formed on the same substrate at the same time.

If mass production is to be possible, 1) Immobilize the enzyme only on the necessary parts of the electrode substrate, for example, expose the connection part with the external electrode and immobilize the enzyme only on the detection part. There is a need.

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 by photocrosslinking 1 is installed on at least the former electrode 1 of the electrodes 1 to 1. No. 699).

One embodiment of such an enzyme sensor is shown diagrammatically in FIG.
A pair of combined electrodes 22 and 22' made of metal thin films such as gold and platinum are formed on 21 with a chromium thin film interposed therebetween, and an enzyme-immobilized film immobilized with a photo-crosslinked polymer is formed on the detection part of these electrodes. 23 is installed to constitute an enzyme sensor. In this case, the formation of an enzyme-immobilized film immobilized with a photocrosslinkable polymer is performed by applying a photoringraph method to an aqueous mixture of a photocrosslinkable polymer and an enzyme.

Enzyme sensors configured in this way can simultaneously form many microscopic sensors on the same substrate.
Although mass production is possible, when one combined electrode formed constitutes a hydrogen peroxide electrode, there is a problem that this electrode becomes sensitive to reducing interfering substances other than hydrogen peroxide. A new thing has been discovered.

Normally, as a countermeasure to this problem, a hydrogen peroxide selectively permeable membrane is installed between the enzyme-immobilized membrane and the electrode surface. Because the enzyme is directly immobilized on the membrane, it is impossible to install a selectively permeable membrane.

Therefore, the present inventors have conducted various studies in search of a new solution to this problem, and as a result, formed two sets of combination electrodes on the same substrate, and for IMi among them, at least anode electrode 1. We have found that the above problems can be effectively solved by forming an enzyme-immobilized membrane in which humans are immobilized.

[Means for solving problems]

Therefore, the present invention relates to an enzyme sensor, and this enzyme sensor has at least two electrodes, one of which is a combination of an anode and a cathode formed on the same insulating substrate. An enzyme-immobilized membrane is installed. The enzyme-immobilized membranes are generally immobilized on the 14 sets of combination electrodes using a photo-crosslinked polymer, and the formation of the enzyme-immobilized membrane immobilized with the photo-crosslinked polymer is carried out in the same manner as the previous proposal. , by applying photolithographic methods to an aqueous mixture of photocrosslinkable polymers and enzymes.

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 making an enzyme sensor, first, a glass plate, a hard resin plate such as vinyl chloride resin, polyimide resin, etc.
Two sets of combined electrodes of an anode electrode and a cathode electrode are formed on a flat insulating substrate such as a silicon wafer having an insulating film of N2, Si, N, etc. formed on its surface. The electrodes are formed using gold, platinum (for the anode electrode) or silver, gold, platinum (for the cathode electrode)
As shown in Figure 1 of the drawings, using metal materials such as A mask vapor deposition method in which the electrode forming material is deposited over a mask through a mask, a screen printing method in which a conductive paint using the electrode forming material as a conductive material is printed in the shape of the electrode, or an alternative to vapor deposition in the above photoetching method or mask vapor deposition method. This can be done by a plating method that performs electroless plating of electrode forming material.

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

Thereafter, the whole is immersed in a resist stripping solution such as acetone to remove the resists 1 to 3, and the deposited thin film 5 remaining on the exposed surface 2 of the substrate is used as an electrode surface in step C).

In this way, after forming an anode salt (anode salt) made of a metal thin film and three electrodes that will serve as the cathode electrode at a predetermined location on the insulating substrate in such a manner that one cathode electrode is commonly used, one of the three electrodes is formed. 1 which becomes at least the anode electrode of
An enzyme-immobilized membrane is installed on each electrode, and the installation is carried out using the photoringraph method, for example, as shown in FIG. 2.

First, an aqueous mixture of a photocrosslinkable polymer and an enzyme is uniformly coated 6 on the Ia electrode surface on the insulating substrate 2 formed as shown in FIG. 1 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 in which enzymes 3 to 72 Ifig were dissolved in 0.8 ml of distilled water was added to ~12% by weight,
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 to form an enzyme-immobilized membrane 8 in which the photocrosslinked portion is immobilized with a photocrosslinked polymer (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. The photoringraph method was applied to the aqueous solution containing the photocrosslinkable polymer, and the photocrosslinkable polymer film 9 was coated thereon, and this was used as a reference electrode (step f).

After irradiating the fabric with ultraviolet rays again and drying it, divide it into individual elements and connect the lead wires io, 5.5' and 5'' to the electrode exposed surfaces.
to', io''. One embodiment of the enzyme sensor produced in this way is shown in FIG. 3 as a plan view. In addition, other embodiments of the enzyme sensor are shown in FIGS. A plan view of - is shown in the embodiment of FIG.
Two cathode electrodes 13, 13' are formed by extending from the electrode surface -5', and the distance between each anode and cathode is shortened, and each of these combined electrodes is connected to the enzyme-immobilized membrane 8. and a photocrosslinked polymer film 9, and in the embodiment of FIG. 5, the cathode 11! By setting the area of the pole 13 to be larger than the area of the electrodes 11 and 12,
The response characteristics of the electrodes are improved by stabilizing the cathode current to the anode and increasing the facing width between the two 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.

1- East-person 1111 Glucose Glucose Oxidase Galactose
Galactose oxidase monoamino acid L-amino acid oxidase ethanol alcohol oxidase phospholipid phospholipase choline choline oxidase [Function] When such an enzyme sensor is used as a glucose sensor, it functions as follows. First, a glucose sensor is immersed in a buffer solution that does not contain glucose, and a voltage of O, SV is applied to the electrode surface in the case of a gold electrode, for example. 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.

Hz Ox → 20" + Oz + 2e - At this time, since no enzyme is immobilized on the reference side electrode, no current due to the enzyme reaction flows, so the differential output between the enzyme immobilized side and the reference side is detected. However, 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 resulting from the enzymatic reaction can be detected.

〔Effect of the invention〕

By immobilizing the enzyme on only one side of two sets of hydrogen peroxide electrodes made of thin metal films formed on the same substrate and detecting the differential output from both electrodes, both electrodes are equally sensitive. The enzyme sensor of the present invention can cancel out signals from reductive interfering substances that would otherwise cause oxidation, and detect only signals based on enzymatic reactions.

As a result, the hydrogen peroxide selectively permeable membrane that was previously required is no longer necessary, and a small, mass-producible enzyme sensor that fully utilizes the features of the enzyme immobilization method using photocrosslinked polymers can be obtained. It became so.

〔Example〕

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

Example Photocrosslinkable polyvinyl alcohol (photocrosslinkable stilbazolium group content 1.4 mol%, saponification degree 88%, polymerization degree 1)
400) in 0.5 g of an 11.7 wt% aqueous solution of 0.4 g of distilled water in which 30 mg of glucose oxidase enzyme was dissolved.
Add mQ and stir and mix for several minutes to prepare a coating liquid. This coating liquid was applied onto two sets of hydrogen peroxide electrodes formed on a glass plate at 4000 rpm.
, spin code for 20 seconds. After the coating solution dries naturally, cover it with a photomask with a negative image that allows only the anode electrode part of one set of the two sets of hydrogen peroxide electrodes to be irradiated with ultraviolet rays.
(output 250 W) for 15 seconds, then development was carried out by washing with pure water, and ultraviolet rays were irradiated again.

Next, a coating solution prepared by adding 0.4+s Q of distilled water to 0.5 g of a photocrosslinkable polyvinyl alcohol aqueous solution containing the same components as above was added to a coating solution without immobilized enzyme using the same method as above. was applied onto the anode electrode of a hydrogen peroxide electrode, and a photocrosslinked polymer film was coated thereon.

Regarding the glucose sensor obtained in this way,
When a glucose calibration curve showing the relationship between glucose concentration and output current value was measured at pH 7.0 and temperature 37°C, the results shown in Figure 6 were obtained, and the glucose concentration was 0.5.
A good linear relationship was observed within the range of ~100 mg/dQ.

In addition, L-ascorbic acid was used as a reducing interfering substance, and the differential removal characteristics of the interfering substance signal were evaluated at pH 7.0° and temperature 37° C.
When measured at ℃, the curve I (L
- shows the relationship between the ascorbic acid concentration and the current value flowing through the enzyme-immobilized side electrode) and curve II (1, - shows the relationship between the ascorbic acid concentration and the current value flowing through the reference side electrode). From the results of the difference curve 11 (which shows the relationship between the L-ascorbic acid concentration and the differential output value of the current flowing through these two electrodes), it was found that the interfering substance, 9 degrees 0.5 to 4
It was confirmed that within the range of mg/dQ, the current caused by harmful substances generated in one and both electrodes could be reduced to 3% or less.

[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 is a cross-sectional view showing the steps of sequentially installing an enzyme immobilization film and a photocrosslinked polymer film each immobilized with a photocrosslinked polymer on two anode electrodes formed on an insulating substrate. FIG. 3 is a plan view of one embodiment of the enzyme sensor produced in this manner. 4 and 5 are plan views of other embodiments of the enzyme sensor according to the present invention. FIG. 6 is a graph of a calibration curve of the enzyme sensor according to the present invention. FIG. 7 is a graph showing differential output characteristics in the presence of a reducing interfering substance. Also, the 8th
The figure is a plan view of the previously proposed 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 9...Photocrosslinked polymer membrane 11...Enzyme immobilization membrane side anode electrode 12...
... Reference side anode electrode 13 ... Cathode electrode

Claims (1)

[Scope of Claims] 1. One of 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.
An enzyme sensor comprising an enzyme-immobilized membrane fixed on at least an anode electrode. 2. The enzyme sensor according to claim 1, wherein the enzyme immobilization membrane is immobilized by a photocrosslinked polymer. 3. The enzyme-immobilized membrane immobilized with a photocrosslinkable polymer is formed by applying a photolithographic method to an aqueous mixture of a photocrosslinkable polymer and an enzyme, as described in claim 2. enzyme sensor. 4. The enzyme sensor according to claim 1, wherein at least the anode electrode is covered with a photocrosslinked polymer film in the other set of combination electrodes in which the enzyme immobilization membrane is not installed. 5. The enzyme sensor according to claim 4, wherein the photocrosslinkable polymer film is formed by applying a photolithography method to an aqueous solution containing the photocrosslinkable polymer. 6. The enzyme sensor according to claim 1, wherein one cathode electrode is used in common for two sets of combined electrodes. 7. The enzyme sensor according to claim 1, wherein the combined electrode constitutes a hydrogen peroxide electrode.