JPH0623719B2 - Method for manufacturing enzyme sensor - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing an enzyme sensor. More specifically, it relates to a method for producing an enzyme sensor capable of forming a highly reliable hydrogen peroxide electrode on an insulating substrate.

[Conventional technology]

Recently, various biosensors utilizing biological reactions such as enzyme reactions and immunoreactions have been developed, and particularly in the clinical field, smaller, higher performance and lower cost sensors have been demanded. An enzyme sensor is one of such biosensors.For example, a glucose sensor that utilizes the catalytic action of glucose oxidase is used to measure the glucose concentration in blood or urine, and is practically used for clinical examinations in diabetic patients. As important as.

However, it has been put to practical use with such enzyme sensors,
Most of the commercially available products do not have an integrated structure of the electrode and the enzyme-immobilized membrane, which hinders achievement of downsizing and cost reduction due to mass production.

[Problems to be solved by the invention]

In view of such problems of the commercially available enzyme sensor, studies have recently been conducted to immobilize the enzyme directly on the electrode, and accordingly, a considerably miniaturized one has been developed. However, when multiple sensors are simultaneously formed on the same substrate to enable mass production, for example, the connection 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 part on the electrode substrate.

In order to solve these problems, the present inventors have found that it is effective to form an enzyme-immobilized film using a photocrosslinking polymer, and an anode electrode made of a metal thin film formed on an insulating substrate. Also, an enzyme sensor in which an enzyme-immobilized membrane immobilized with a photocrosslinking polymer is installed on at least the former electrode of the cathode electrode is first proposed (Japanese Patent Publication No. 5-39432).

The enzyme sensor thus configured can form a large number of minute sensors at the same time on the same substrate,
It enables mass production, but when one formed combination electrode constitutes a hydrogen peroxide electrode, there is a problem that this electrode is also sensitive to reductive interfering substances other than hydrogen peroxide. It was found.

Usually, as a countermeasure for such a problem, a method of installing a hydrogen peroxide selective permeable membrane between the enzyme-immobilized membrane and the electrode surface is taken, but in the case of the above enzyme sensor, it is directly attached on the electrode surface. Since the enzyme is immobilized, the permselective membrane cannot be installed.

Therefore, as a result of various investigations for a new solution to this problem, the present inventors formed two sets of combination electrodes on the same substrate, and fixed one set at least on the anode electrode. It was previously found that the above problems can be effectively solved by forming a modified enzyme-immobilized membrane.
(Japanese Patent Publication No. 5-39433).

The enzyme sensor proposed here is at least one of two sets of combined electrodes of an anode electrode and a cathode electrode made of a metal thin film formed on the same insulating substrate, which is at least immobilized on the anode electrode. A chemical film is installed. Immobilization of the enzyme-immobilized membrane on one set of combination electrodes
Generally, a photocrosslinking polymer is used, and the enzyme-immobilized membrane immobilized by the photocrosslinking polymer is formed by a photolithographic method on an aqueous mixture of the photocrosslinking polymer and the enzyme, as in the previous proposal. It is done by applying.

Further, the other set of combination electrodes not provided with the enzyme-immobilized membrane is used by covering at least the anode electrode with a photocrosslinking polymer film, but in this case, the formation of the photocrosslinking polymer film is also used. It is carried out by applying a photolithographic method to an aqueous solution containing a crosslinkable polymer.

In these two sets of combination electrodes, an anode electrode and a cathode electrode may be formed for each set, but the cathode electrode is commonly used for the two sets of combination electrodes.
It is also possible to use one cathode electrode and switch it for each combination electrode.

When manufacturing an enzyme sensor, first, a glass plate, a hard resin plate such as vinyl chloride resin or polyimide resin, SiO ₂ , Si
Two sets of combination electrodes of an anode electrode and a cathode electrode are formed on a flat insulating substrate such as a silicon wafer having a surface coated with an insulating coating such as ₃ N ₄ . The electrodes are formed by using a metal material such as gold, platinum (for the anode electrode) or silver, gold, platinum (for the cathode electrode) or the like, a lift-off method as shown in FIG. Photoetching method for etching and patterning the thin metal film that has been removed, mask evaporation method for depositing an electrode-forming substance on the substrate by overlaying a mask with an electrode-shaped window on the substrate, and using the electrode-forming substance as a conductive material It can be carried out by a screen printing method of printing a conductive paint in the shape of an electrode, or a plating method of performing electroless plating of an electrode forming material instead of vapor deposition in the photo etching method or the mask vapor deposition method.

In the embodiment shown in FIG. 1, the lift-off method is used. First, the positive photoresist 3 is patterned so that the electrode portion is formed within the area of the exposed surface 2 of the substrate on a cleaned flat insulating substrate, for example, the glass plate 1 (step a). Then, a chromium thin film 4 (thickness: about 500Å) and a gold or platinum thin film 5 (thickness: about 0.2 μm) are sequentially formed on this substrate by a vacuum evaporation method. Here, the chrome thin film is
It is provided in order to enhance the adhesion between the gold or platinum thin film forming the electrode and the glass plate substrate (step b). afterwards,
The whole was immersed in a resist stripping solution such as acetone to remove the resist, and the vapor-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 one cathode electrode is commonly used at a predetermined position on the insulating substrate and three electrodes serving as an anode electrode and a cathode electrode made of a metal thin film are formed, only a few of them are formed. In both cases, the enzyme-immobilized membrane is installed on one electrode that serves as an anode electrode, and the installation is performed using a photolithographic method, for example, as shown in FIG.

First, the electrode surface 5 on the insulating substrate 2 formed as shown in FIG. 1 is uniformly coated 6 with an aqueous mixture of a photocrosslinkable polymer and an enzyme by spin coating, spraying or the like. (Step d). As the photocrosslinkable polymer, a water-soluble polymer is generally used for dispersing it as an aqueous mixture together with an aqueous enzyme solution, and for example, a stilbazolium group as a photocrosslinkable group in the molecule, a photosensitive group such as a diazo group, Polyvinyl alcohol having a stilbazolium group is preferably used as an aqueous solution. The aqueous mixture is the photocrosslinkable polymer aqueous solution (concentration 8.5 to 12% by weight)
An enzyme aqueous solution prepared by dissolving 3 to 72 mg of enzyme in 0.8 ml of distilled water is added to 1 g, and the mixture is stirred for about several minutes and mixed to be used for coating.

When the coating liquid is coated on the electrode surface on the insulating substrate and it is naturally dried, it is covered with a photomask 7 having a negative or positive image and irradiated with ultraviolet rays to photocrosslink the photocrosslinkable polymer. The cross-linked portion is removed with pure water to form the enzyme-immobilized membrane 8 fixed with the photo-crosslinked polymer on the photo-crosslinked portion (step e). This was irradiated again with ultraviolet rays and then dried. The photomask used here is
Only one set of the anode electrode 11 of the two sets of combination electrodes,
A film having an image on which the enzyme-immobilized film 8 is formed is used.

Next, in order to equalize the sensitivity characteristics of the two sets of combination electrodes with respect to the reducing interfering substance, the anode electrode 12 of the other set of combination electrodes without the enzyme-immobilized membrane was treated by the same method as in FIG. A photolithographic method was applied to an aqueous solution containing a photocrosslinkable polymer, and the photocrosslinkable polymer film 9 was coated thereon, and this was used as a reference electrode (step f).

This is irradiated with ultraviolet rays again, dried, and divided into each element, and the lead wires 10, 1 are attached to the electrode exposed surfaces 5, 5 ', 5'.
Install 0'and 10 '. One embodiment of the enzyme sensor thus produced is shown in FIG. 3 as a plan view. In addition, FIGS. 4 to 5 show plan views of other embodiments of the enzyme sensor. In the embodiment of FIG. 4, two cathode electrodes 13, 13 'are extended from the electrode surface 5'. In this embodiment, the electrode distance between the anode and the cathode is shortened, and the combined electrodes are covered with the enzyme-immobilized film 8 and the photocrosslinking polymer film 9, respectively, and in the embodiment of FIG. By setting the area of the cathode electrode 13 wider than the areas of the electrodes 11 and 12, the cathode current to the anode is stabilized, and by increasing the facing width between both electrodes while keeping the distance between the electrodes constant, The response characteristics are improved.

An example of a combination of a substrate detectable by this enzyme sensor and an enzyme as a catalyst which reacts to this substrate is as follows, in which case both electrodes act as hydrogen peroxide electrodes. The substance to be detected enzyme glucose glucose oxidase galactose galactose oxidase L- amino L- amino Okidaze ethanol alcohol oxidase phospholipid phospholipase choline choline oxidase glycerol glycerol kinase (two enzymes L-alpha-glycero-3-simultaneously fix the phosphoric acid) oxidase When such an enzyme sensor is used as a glucose sensor, it acts as follows. First, a glucose sensor is immersed in a buffer solution containing no glucose, and a voltage of 0.8 V is applied to the electrode surface in the case of a gold electrode, for example. When glucose is added to this, the enzyme to which the enzyme is immobilized is At the hydrogen peroxide electrode side, glucose diffuses into the glucose oxidase enzyme-immobilized membrane and reacts as follows by the catalytic action of the immobilized enzyme.

Hydrogen peroxide generated by 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 glucose concentration flows.

H ₂ O ₂ → 2H ⁻ ＋ O ₂ + 2e ⁻ At this time, since the enzyme is not immobilized on the reference electrode, the current due to the enzyme reaction does not flow, so the differential output between the enzyme-immobilized side and the reference side is Even if it 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 by both combination electrodes regardless of the presence or absence of immobilized enzyme. By detecting the differential output of the current flowing through both electrodes, the current value due to the interfering substance is canceled out, and only the current value due to the enzyme reaction can be detected.For example, in the case of L-ascorbic acid It was confirmed that, when the concentration was within the range of 0.5 to 4 mg / dl, the electric current due to the interfering substances generated at both electrodes could be reduced to 3% or less.

However, when the enzyme-immobilized film and the photocrosslinking polymer film on the reference side are observed with an optical microscope, the photocrosslinking polymer film forms a transparent and homogeneous film, while the enzyme-immobilized film is immobilized. It was found that the film was whiter and innumerable minute irregularities were formed on the surface.

Therefore, the two membranes were made as equivalent as possible, and as a result of further investigation to find a means for more accurately detecting the differential output, immobilization enzyme was also immobilized on the membrane on the reference side. It has been found that the method of buffing is extremely effective (JP-A-62-75346).

In this enzyme sensor, two sets of combination electrodes of an anode electrode and a cathode electrode made of a metal thin film are formed on the same insulating substrate, and one set has at least an enzyme-immobilized film immobilized on the anode electrode, In each set, at least an inactivated enzyme-immobilized membrane immobilized on the anode electrode is installed.

The production of such an enzyme sensor is carried out according to the method described above with reference to FIGS. 1 and 2 of the drawings, in which the aqueous solution of the photocrosslinkable polymer-containing enzyme used in step (d) is used in an oven depending on the type of enzyme. Except that an aqueous solution in which the enzyme is inactivated is used in step (f) by heating at about 80 to 100 ° C for about 5 to 10 minutes,
The same method is used. And, for example, the third to fifth
In the embodiment shown in the figure, an enzyme sensor having an enzyme-immobilized membrane immobilized on the code 8 side and an inactivated enzyme-immobilized membrane immobilized on the code 9 side can be obtained.

In the production of the enzyme sensor thus performed, the formation of the inactivated enzyme-immobilized film to be immobilized on at least the anode electrode of another set of combination electrodes is carried out by using the photocrosslinkable polymer-containing enzyme aqueous solution as described above. It is carried out by heat treatment and use as an aqueous solution in which the enzyme is inactivated.

The prepared enzyme sensor immobilizes the deactivating enzyme on the reference side as well, so that the membrane quality of the reference side electrode is made equal to that of the enzyme side electrode. Are made equal to each other to further improve the differential elimination characteristic of the interfering substance signal. Specifically, in the measurement of the differential elimination characteristics of the interfering substance signal using L-ascorbic acid, when the concentration was within the range of 1 to 100 mg / dl, the current caused by the interfering substance at both electrodes was 2%. % Compared to the previously proposed concentration range of 0.5% to 4 mg / dl within the range of 3% or less and the current value, further improving the differential elimination characteristics of interfering substance signals. To achieve. Also,
Such characteristics are effectively exhibited not only when the interfering substance is L-ascorbic acid but also when it is uric acid or the like.

Thus, although this enzyme sensor achieves its intended purpose in terms of effect, two photolithographic methods are required to form the enzyme-immobilized membrane and the inactivated enzyme-immobilized membrane separately. There is a waste in production in that the enzyme must be applied and the same amount of enzyme as that required for forming the enzyme-immobilized membrane must be used also when forming the inactivated enzyme-immobilized membrane.

The present invention is intended to provide a method for producing an enzyme sensor that eliminates such waste.

[Means for solving problems]

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

Forming the enzyme-immobilized film on at least the anode electrode of the two sets of combination electrodes uses the photomask having an image in step (e) in the method described above with reference to FIGS. Without irradiation, or by using a photomask having an image pattern in which two sets of anode electrodes are exposed, and irradiating with ultraviolet rays. The ultraviolet light used for this ultraviolet irradiation is of a relatively long wavelength, such as that emitted from a mercury lamp,
When this is irradiated for about 30 seconds, the photocrosslinking polymer is photocrosslinked, but the effect on the enzyme is hardly observed.

After that, only the enzyme-immobilized film on the combination electrode serving as the reference electrode is irradiated with ultraviolet rays using a photomask to inactivate the enzyme once immobilized. The ultraviolet irradiation conditions in this case are generally as follows.

Light source: UV lamp for short wavelength Wavelength: Approx. 2000 to 3000Å Output: Approx. 10 to 20 W / cm ² Distance: Approx. 1 to 5 cm Time: Approx. 1 to 5 minutes [Effect of the invention] The following effects are obtained by the method of the present invention. Is played.

(1) The photolithography method for forming the enzyme-immobilized film and the inactivated enzyme-immobilized film on the electrode of the enzyme sensor is applied once.

(2) In this regard, since the film thickness and film quality of both of these immobilization films are the same, the differential removal characteristic with respect to the interfering substance is improved.

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

〔Example〕

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

Examples of photocrosslinkable polyvinyl alcohol (photocrosslinkable stilbazolium group content 1.4 mol%, saponification degree 88%, polymerization degree 1400)
0.4 ml of distilled water in which 30 mg of glucose oxidase enzyme is dissolved is added to 0.5 g of a 11.7% by weight aqueous solution, and the mixture is stirred and mixed for about several minutes to prepare a coating solution. This coating solution is spin-coated on two sets of hydrogen peroxide electrodes formed on a glass plate under the conditions of 4000 rpm and 20 seconds. After the coating liquid has dried naturally, cover the anode electrode part of each of the two sets of hydrogen peroxide electrodes with a photomask that has a negative image so that it can be irradiated with ultraviolet light, and perform ultraviolet irradiation (output 250 W) for 15 seconds, and then It was developed by washing with pure water, and again irradiated with ultraviolet rays to be dried.

Next, cover with a quartz photomask having a negative image such that only the anode electrode portion of the reference electrode is irradiated with ultraviolet rays, using a sterilizing ultraviolet lamp (wavelength 2537Å), irradiation distance 2 cm, irradiation time 2 minutes UV irradiation was carried out under the conditions described above to deactivate the enzyme immobilized on the immobilization film on the anode electrode.

[Brief description of drawings]

FIG. 1 is a sectional view sequentially showing a process of forming electrodes on an insulating substrate. FIG. 2 shows the previously proposed method, in which an enzyme-immobilized membrane and a photocrosslinking polymer immobilized by a photocrosslinking polymer on two anode electrodes formed on an insulating substrate, respectively. It is sectional drawing which showed the process of installing a membrane | membrane (or inactivated enzyme immobilization membrane) one by one, and FIG. 3 is a top view of one aspect of the enzyme sensor produced in this way. 4 to 5 are plan views of other embodiments of the enzyme sensor. (Explanation of symbols) 1 ... Insulating substrate 2 ... Exposed substrate 5 ... Electrode 6 ... Enzyme aqueous solution containing photo-crosslinkable polymer 7 ... Photomask with image 8 ... Enzyme-immobilized film 9 ... Light Crosslinked polymer membrane (or inactivated enzyme immobilization membrane) 11 …… Enzyme immobilization membrane side anode electrode 12 …… Reference side anode electrode 13 …… Cathode electrode

Claims (4)

[Claims] 1. A combination of an anode electrode and a cathode electrode composed of a metal thin film is formed on the same insulating substrate, and an enzyme immobilization film is immobilized on at least the anode electrode in each combination electrode, and then the reference side is formed. A method for producing an enzyme sensor, comprising irradiating an oxygen-immobilized film on an electrode forming an 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 by a photocrosslinking polymer. 3. The method for producing an enzyme sensor according to claim 1, wherein one cathode electrode is commonly used for two sets of combination electrodes. 4. The method for producing an enzyme sensor according to claim 1, wherein the combination electrode constitutes a hydrogen peroxide electrode.