JPS6214276B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Application of the Invention) The present invention relates to a method for partially deactivating an immobilized enzyme membrane. This paper relates to a method for preparing an immobilized enzyme electrode in which an enzyme is immobilized on a polarographic electrode using a polarographic electrode. This provides a simple method for adjusting enzyme electrodes.

(Prior Art) A typical example of an enzyme electrode using an ISFET is one in which an enzyme membrane is formed in the gate portion, as in the invention described in Japanese Patent Application Laid-Open No. 53-149397 by Shimada et al.

In the invention described in the above-mentioned publication, a minute enzyme electrode can be realized using an electrode, but a reference electrode is required to provide a reference potential of the solution during use. Currently, a small and stable reference electrode is not in practical use, and therefore, the use of a small enzyme electrode alone is limited.

One of the methods used to compensate for this
One method is to use the two ISFETs described above and use one as a modified comparison electrode. For example, JP-A-55-
In Publication No. 12480, Yano et al. propose a reference electrode in which the gate portion of an ISFET is covered with a diene polymer. The reference electrode using this method uses a polydiene layer to block the Si ₃ N ₄ layer, which has hydrogen ion selectivity.
It is possible to have an output that eliminates the hydrogen ion concentration dependence of the ISFET output, and it is thought that there is a certain effect when using the differential output of the normal ISFET and this reference electrode as a PH meter. However, it is not suitable as a reference electrode for enzyme electrodes for the following reasons.

In other words, as a transducer for enzymatic reactions.
When using ISFET, if there is a PH change in the solution other than the PH change resulting from the action of the immobilized enzyme on the substrate, it will be indistinguishable from a change in substrate concentration, resulting in an error. In addition, in order to stabilize the solution PH, if the measurement is performed in a buffer with high ionic strength, the PH of the solution itself can be measured.
Errors caused by changes are greatly improved, but on the other hand, PH changes near the ion-sensitive membrane that occur as a result of enzyme reactions are also inhibited by the buffer, making it difficult to detect them as PH changes.

This method was developed to improve the above drawbacks and to measure samples with low buffer effects such as blood, by preparing two ISFETs, one with an immobilized enzyme membrane, and the other with a differential output as an untreated PH sensor. A method was devised to take the In this case, since the two ISFETs are differentially connected, it is sufficient to use a reference electrode that provides a relative potential, and a material such as Au.Pt that is easy to mold and can be miniaturized or formed on a semiconductor surface can be used.

Examples of such composite enzyme electrodes include those that have a glucose oxidase immobilized membrane on the gate part on the back side of the epoxy laminate, and those that have a urease immobilized membrane (such as a differential type enzyme electrode with a membrane immobilized on the immobilized enzyme membrane). A total of three electrodes were hardened with epoxy resin, one with no treatment (hereinafter referred to as the "active electrode"), and one untreated (hereinafter the differential type used for comparison is simply referred to as the "inactive electrode"). A model with platinum wire on the surface has been announced. This method, which is described by Hanasato et al. in the Proceedings of the 44th Japan Society of Applied Physics, P606, provides the concept of a multi-sensor that has many ion-sensing sections on one chip, and at the same time The ion sensor has the following deficiencies.
In other words, by attaching a plurality of electrodes on an epoxy substrate or the like, it is difficult to achieve a certain level of integration. In addition, this product is made by molding bulk silicon.
In the case of ISFET, the enzyme membrane is formed after each chip is separated because it involves three-dimensional processing, but even if a chip with multiple ion-sensing parts is supplied, the enzyme membrane will still be attached. It is difficult to apply the coating to non-stick surfaces when using minute sensors, making it impossible to supply large quantities industrially and making quality control difficult.

(Objective of the Invention) An object of the present invention is to provide a method for efficiently forming an immobilized enzyme membrane and a deactivated immobilized enzyme membrane in a minute area. The present invention provides a method for easily dividing ion sensing sections in a chip having a plurality of ion sensing sections formed on an insulating substrate such as sapphire into active electrodes and inactive electrodes.

(Structure of the Invention) The present invention is a method for partially inactivating an immobilized enzyme membrane, which is characterized by irradiating a predetermined portion of the immobilized enzyme membrane with electromagnetic waves to inactivate the enzyme in this portion.

(Detailed explanation of the configuration) The circuits included in one chip of the wafer on which the enzyme membrane is formed are at least two ion-sensitive parts,
ISFET is a typical example, but it is also possible to use polaro electrodes, semiconductor gas sensors, etc. The mask is defined by the type of light source, but if ultraviolet rays are used as the light source, it may be one normally used in semiconductor manufacturing in which the portions of the quartz glass surface that are not exposed to light are patterned with chrome or the like.

The light source may be light of a wavelength that deactivates the enzyme without passing through the light shielding part of the mask, but in order to precisely align the mask and semiconductor wafer, a mask aligner that uses a low-pressure mercury lamp or Xe-Hg lamp as the light source is recommended. It is more advantageous to use

However, in general, the ISFET gate section is typically 10
Since it has a square size of μm to 100 μm, it is a large pattern among semiconductors, and it is not necessarily necessary to use a mask aligner. All you have to do is irradiate it. In this case as well, it is desirable to use an ultraviolet lamp such as a low-pressure mercury lamp or a Xe-Hg lamp as the light source.

The light source used for deactivation is not limited to ultraviolet light, and X-ray deactivation using an X-ray shielding mask, infrared deactivation using an infrared mask, etc. can also be realized as an extension of the present invention.

Note that the present invention will be explained using drawings.

FIG. 2 is a plan view of an ISFET chip used in the present invention in Japanese Patent Application No. 56-076959. From the region to the drain region 23, source region 24, and ground region 25
is extended to the other end, the drain region 23 is covered with the drain electrode 26, and the source region 24 and the ground region 25 are short-circuited to each other with the source electrode 27.
It has two ISFETs.

By deactivating the sensor portion of one electrode shown here and not deactivating the other electrode, a differential type sensor can be obtained.

In this case, the area surrounded by the dotted line shown in 21 corresponds to the area where no light is applied and deactivation is prevented.

FIG. 1 is a cross-sectional view taken along line a-a' in FIG. 2, showing how an ISFET having an enzyme-immobilized membrane is exposed to ultraviolet light through a photomask.

Here, 1 is a sapphire substrate, 2 is a p-type silicon gate layer, 3 is an n-type silicon source layer, 4 is an n-type silicon drain layer, 5 is a p-type ground layer, 6 is a thermal oxide film, 7 is an ion photosensitive film, 8 is a membrane on which enzymes are immobilized. The membrane may be, for example, a membrane based on cellulose triacetate with urease immobilized thereon as an enzyme. 9 is a quartz glass plate for a photomask, 10 is a chromium light-shielding pattern formed thereon, and 11 is parallel ultraviolet light used for enzyme deactivation.

The present invention has the effect of enabling the simple supply of differential enzyme sensors in large quantities. For example, ISFET,
At least two ion sensing parts such as polaro electrodes
In this method, an enzyme film is formed on a wafer on which a large number of chips, each having a number of chips or more, are formed, and the enzyme is irradiated with ultraviolet rays through a mask to deactivate the enzyme in the portion corresponding to the inert electrode. The present invention will be explained based on examples. Second
The figure shows a chip with two equivalent SOSFETs, 1 mm wide and 5 mm long, and about 250 chips can be made from a 2 inch diameter wafer. The wafer used here is a SOSFET that does not undergo a radical reaction that would deactivate the enzyme after cutting each chip individually.
It is preferable to have a circuit provided on an insulating substrate such as. When irradiating with ultraviolet rays, an immobilized enzyme film is formed on the entire surface of the wafer or at least on a portion that will be used as an active electrode or an inactive electrode in the future. It is best to limit the formation method to only the surface on which the circuit is formed, by pulling the enzyme-containing membrane with a spinner.
Alternatively, it is common to use a membrane capable of immobilizing enzymes, but it is also possible to form a membrane by dipping the wafer into the membrane and pulling it up. If the membrane allows enzyme immobilization, the enzyme is subsequently immobilized. The term "enzyme-immobilized membrane" as used herein has a broad meaning, including membranes directly immobilized on the semiconductor surface. The wafer having the immobilized enzyme film obtained as described above is irradiated with ultraviolet light or the like through a mask. If the light source is ultraviolet light, a normal mask patterned with chromium or the like on the surface of quartz glass may be used.The part that does not transmit light includes at least the part used as an active electrode, and the part that transmits light and is deactivated has at least an inactive mask. Must include the electrode side. As for the remaining part, it doesn't matter which one. Regarding deactivation, it is not necessary to completely deactivate it, as long as a signal can be taken out as a differential output. Generally
It is good if there is 50% or more deactivation.

Regarding the light source used here, in the case of ultraviolet rays, a low-pressure mercury lamp, a mercury-xenon lamp, etc. are standard, but any light source that includes a sufficient wavelength of 4000 Å or less may be used. The intensity and irradiation time are greatly affected by the thickness of the immobilized enzyme membrane, the enzyme used, the material of the membrane, etc., but the shorter the irradiation time, the less the enzyme electrode will be affected, so the irradiation time is 30 A strong light source that requires less than a minute is advantageous.
It is also desirable to use a filter that cuts out long wavelengths in advance to avoid the influence of heat from the light source.

For example, in the case of the low pressure mercury lamp in the example, 4 m
Good results were obtained by irradiating with an intensity of W/cm ² (wavelength 2500 Å) for 20 minutes.

After this, it is cut into pieces, and the surface film is removed in some cases at the terminal part, and then bonded with aluminum wire or the like to form a practical electrode. It can be used as a differential enzyme electrode.

Example 1 An embodiment of the present invention will be described below.

FIG. 2 shows an ISFET that constitutes the present invention. This is two sensors in one according to patent application No. 56-076959.
Approximately 250 chips can be collected per 2-inch wafer. The wafer used was an SOS substrate with silicon epitaxially grown on saphire. Regarding the formation of an enzyme-immobilized film on the surface, the wafer surface was treated with a silanizing agent ( _Y -aminopropyltriethoxysilane), and 1,8-diamino-4-aminomethyloctane, glutaraldehyde, and Apply the material containing triacetylcellulose evenly with a spinner, 40
The samples were used after being left at ℃ for one day and night. After washing this with pure water, it was immersed in water containing urease as an enzyme, and after washing with water, it was replaced with acetone and quickly dried to obtain an enzyme-immobilized wafer. The thickness of the enzyme membrane is approximately 10 μm, and the amount of immobilized enzyme is approximately 5×
It was 10 ^-2 U/ ^cm2 . Note that wafers prepared in the same manner were also used in Examples 2 and 3 described below. Exposure operations were carried out promptly to prevent deactivation of light other than ultraviolet light as much as possible.

A Xe-Hg lamp was used as a light source, and light transmitted through a filter that satisfactorily transmits 250 nm was used. The intensity at 250 nm was 4 mW/cm ² and the irradiation time was 20 minutes. After irradiation, the film at the lead wire attachment part was removed, connected to an external circuit, and the characteristics of various concentrations of urea were measured. The potential was taken as the value 1 minute after the electrode was immersed, the buffer was PH7, the temperature was 37°C in a phosphoric acid buffer. Figure 3 shows the relationship between urea concentration and differential output, and good results were obtained in the range of 10 mg/dl to 100 mg/dl.

As shown here, the electrodes adjusted using the method of the present invention succeeded in obtaining stable output as a differential output.

Example 2 Using an enzyme-immobilized wafer prepared in the same manner as in Example 1, enzyme inactivation was performed using X-rays. Aluminum Ka-rays were used as the X-ray light source. The intensity of the source was 1 mW/cm ² . The mask used was one in which gold was patterned approximately 10 μm thick on quartz glass. In this case, it was impossible to irradiate all the wafer surfaces with X-rays at the same time, so the irradiation was divided into several times. The irradiation time per location was 30 minutes.
One chip was extracted from the wafer thus exposed in the same manner as in Example 1 and the output was measured. Urea concentration and differential output showed a good correlation in the range of 10 mg/dl to 100 mg/dl.

Example 3 An enzyme-immobilized wafer prepared in the same manner as in Example 1 was prepared and enzyme deactivated using infrared rays. A 250W infrared lamp was used as a light source. The lamp used was an infrared moisture type TV-250D manufactured by Shimadzu Rika. 100 silicon wafers as masks
The material used was made thin to μm thickness and patterned with gold. The distance between the wafer and the infrared lamp is 10cm.
and exposed for 20 minutes. One chip was extracted from the wafer thus exposed in the same manner as in Example 1 and the output was measured. Urea concentration and differential output are 10mg/dl
A good correlation was shown in the range of ~100 mg/dl.

Conventionally, for example, in the example of Hanasato et al., a bare electrode was used as the inert electrode, but in this method, the inactive side also has no enzyme activity but has a film on the surface equivalent to the active electrode. , it has a more ideal form as a differential type.

(Effects of the Invention) As described above, it has been found that by using the enzyme deactivation method according to the present invention, a differential enzyme electrode using two ISFETs can be provided through a very simple process.

Furthermore, the present invention is not limited to this, and can be used in any method for deactivating a membrane having enzyme activity over its entire surface except for a certain portion.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing an embodiment of the present invention, FIG. 2 is a plan view of a semiconductor element used here, and FIG. 3 is a diagram showing an actually obtained differential output. In the figure, 1... Sapphire substrate, 2... P-type silicon gate layer, 3... N-type silicon source layer, 4
... N-type silicon drain layer, 5 ... P-type silicon ground layer, 6 ... Insulating film, 7 ... Ion-sensitive film, 8 ... Enzyme immobilization film, 9 ... Quartz glass for photomask, 10 ... Photomask Upper light shielding pattern, 11... Parallel ultraviolet light, 21... Portion where light is shielded by the mask, 22... Sensor section, 23... Drain region, 24... Source region, 25... Earth region, 26...Drain electrode, 27...Source electrode.

Claims (1)

[Scope of Claims] 1. A method for partially inactivating an immobilized enzyme membrane, which comprises irradiating a predetermined portion of the immobilized enzyme membrane with electromagnetic waves to inactivate the enzyme in this portion. 2. The method for partially inactivating an immobilized enzyme membrane according to claim 1, wherein the immobilized enzyme membrane is formed on an element having a plurality of ion-sensitive parts.