JPS6366454A - Enzyme sensor and manufacture thereof - Google Patents

Abstract

PURPOSE:To obtain an enzyme sensor with a high response speed excellent in the sensitivity, by providing an enzyme immobilize film 3-20mum thick on an ion sensitive film of an ion sensitive (IS) FET. CONSTITUTION:An insulating material 4 is placed on a P-type silicon substrate 1 and scraped off thin at a portion thereof 4 on a gate 5. An ion sensitive film 2 is deposited on the insulating material 4 by the reaction sputtering and then an enzyme immobilized film 3 with the thickness of 3-20mu is provided on the sensitive film 2 by the lift off using a photoresist. Thus, the gate 5 and the sensitive film 2 are connected through the insulation material 4 scraped off thin to allow the transmission of an interface potential of the sensitive film 2 to the gate 5. Furthermore, the immobilized film 3 with the thickness of about 3-20mum is provided on the sensitive film 2 and at a position separated from the gate 5, thereby enabling the production of an enzyme sensor excellent in the response speed with a high sensitivity.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an ISFET [Ion Sensitive Field Effect Transistor CIon 5ensi]
tive Field EJ'Tect Trar+5
istor)], especially ISFE
This invention relates to an enzyme sensor in which an enzyme-immobilized membrane is directly deposited on an ion-sensitive membrane of T.

[Prior Art] In recent years, along with advances in semiconductor technology and IC technology using this technology, sensors for measuring the concentration of hydrogen ions and sodium ions using ISFETs have been developed by P. Bergverd, Matsuo, K. D. Yisc. [
IE,E Transactions (iE,E,
Trans,) BFM-19°342 (1972);
Same, BEM-21, 485 (1974)] I 5FET
Generally consists of a substrate, a barrier film, and an ion-sensitive film, and the ion-sensitive film is connected to a gate portion. Then, when the ISFET is immersed in the test liquid, the surface potential of the ion-sensitive membrane changes depending on the ion concentration, and this potential change is measured, for example, as a current change between the source and drain.
The ion concentration can be determined by referring to the results with the standard solution.

By immobilizing an enzyme that decomposes the analyte to be measured and generates protons on the ion-sensitive membrane of the above-mentioned ISFET, it becomes an enzyme sensor. Conventionally used as a method for immobilizing enzymes on the ion-sensitive membrane. In this method, 3-aminoprobilt I mylythoxysilane (APTE), which is a type of silane coupling agent, is mixed in a distilled water bath, and the ISFET is immersed in this mixed solution to react. This introduces amino groups into the ion-sensitive membrane. Next, a fixed membrane stock solution containing geltaraldehyde as a main component is applied onto the ion-sensitive membrane using a dip coating method or the like.

As a result, the amino group and the aldehyde group form a -CH=N- type bond. Furthermore, after introducing aldehyde groups into the fixed membrane by immersing it in a dilute gel tar aldehyde solution, an enzyme (e.g., urease) solution is applied, and urease is applied to the fixed membrane by a reaction between the amino groups of urease and the aldehyde groups on the surface of the fixed membrane. There is a way to fix it on top. (“Biosensor” edited by Shu Suzuki, Kodansha Entific) [Problems to be solved by the invention] However, the enzyme sensor obtained by the above method has the problems of slow response speed and poor sensitivity. Was. The present inventor discovered that the cause of this problem lies in the enzyme-immobilized membrane fixed on the ion-sensitive membrane, leading to the present invention.

An object of the present invention is to provide an enzyme sensor with high response speed and excellent sensitivity by improving the enzyme membrane, and a method for producing the same.

[Means for Solving the Interference Point] What solves the above problem is an enzyme sensor in which an enzyme-immobilized membrane with a thickness of 3 to 20 μm is provided directly on the ion-sensitive membrane of the ISFET.

Furthermore, it is preferable that the ISFET is an isolated gate type ISFET. Furthermore, it is preferred that the enzyme is urease. Furthermore, it is preferable that the ion-sensitive membrane is iridium oxide.

Also, what solves the above problems is ISFET
An enzyme-immobilized membrane with an enzyme immobilized thereon was coated on the I5PET ion-sensitive membrane by spin coating for 3 to 2 hours.
This is a method for manufacturing an enzyme sensor that is coated to a thickness of 0 μm.

Therefore, the enzyme sensor of the present invention will be explained using an example shown in FIG.

The enzyme sensor of the present invention is made of PE on a P-type silicon substrate l.
An ion-sensitive membrane 2 having an enzyme-immobilized membrane 3 at a position slightly apart from the PET is provided on an insulating material 4, and the FET has a drain D6 and a source S.
7 and gate) G5, and the gate is an ion-sensitive membrane 2.
is connected to. In the one shown in FIG. 1, a P-type silicon substrate is used, but the invention is not limited to this, and a sapphire substrate may be used. Because the Saf-Ia substrate is strong, the resulting enzyme sensor will also be stronger. Also, the first
In what is shown in the figure, a separated gate type ISFET is used, but the present invention is not limited to this, and an ion-sensitive film provided on the gate may be used. Preferably, it is a separated gate type. If it is a separate gate type I 5FET, only the ion-sensitive membrane part needs to be immersed in the test liquid, and there is no need to immerse the PET gate part and the source and drain components, and furthermore, these parts are shielded from light and the outside world. This is because the response can be stabilized because the

Note that an isolated gate type ISFET refers to a structure in which a substrate, a barrier film, and a gate portion are connected to a source, and in which an ion-sensitive film and a gate isolation are provided on the same substrate at a distance.

PET is formed on a P-type (or N-type) silicon substrate l by a conventional method. For example, by applying n-type (or p-type) silicon on a p-type (or n-type) silicon substrate and applying known methods such as photoresist method, discharge treatment method, thermal diffusion method, etc. to the portions that will become the source and drain, Further, they are combined to form an FET.

It is preferable that the PET be formed near the end of the P-type silicon substrate 1. In addition, the ion-sensitive membrane 2 is made of FE
It is preferable to provide it near the other end of the T apart from the gate. This is because the entire P-type silicon substrate can be used effectively and is advantageous in terms of insulation technology.

Then, the insulating material 4 is placed on the P-type silicon substrate l, the insulating material on the gate 5 is shaved thinly, and the ion-sensitive film 2 is formed on the thinly shaved insulating material and a part spaced apart from that part. has been done. As an insulating material, silicon oxide (S
in, ) is preferably used. Further, as the ion-sensitive film, iridium oxide (Ire-), palladium oxide (PdO, ), silicon nitride (Sj3Na), aluminum oxide (A+yo3), tantalum oxide (ratos), etc. can be used. Preferred are iridium oxide and palladium oxide because they have excellent pFI sensitivity.

Particularly preferred is iridium oxide. Iridium oxide is a less noisy ion-sensitive membrane and also acts as an electrical conductor.

The ion sensitive film 2 is formed by depositing the above material on the insulating material 4 on the P-type silicon substrate l using a reactive sputtering method or the like.

Then, F'ET formed on the P-type silicon substrate 1
As described above, the gate 5 and the ion-sensitive membrane 2 are connected through a thinly cut insulating material, so that the interface potential of the ion-sensitive membrane 2 can be transmitted to the gate 5.

Furthermore, an enzyme-immobilized membrane 3 with a thickness of 3 to 20 μm is provided above the ion-sensitive membrane 2 and at a position spaced apart from the gate 5. It has a high response speed and high sensitivity. The enzyme-immobilized membrane 3 contains an enzyme and has the function of supporting the enzyme on the ion-sensitive S membrane 2.

The enzyme used in the present invention can be any enzyme that decomposes the analyte to generate protons, such as urease (for urea detection), glucose oxidase (for glucose detection), beninlinase (for penicillin detection), Examples include trypsin (for detecting peptides), lipase [for detecting fatty acids, such as phospholipase (for detecting acetylcholine)], and peptidase (for detecting threonine).

The method of providing the enzyme-immobilized membrane 3 on the ion-sensitive membrane 2 in the present invention is preferably carried out by a lift-off method using a photoresist. After coating, exposing the area other than the area where the enzyme-immobilized film will be formed, and developing, the photoresist of the area of the ion-sensitive membrane where the enzyme-immobilized film will be formed is removed. For photoresist,
Known products can be used, such as those obtained by adding N-methyl-P-formylstylpyridinium methosulfate to an aqueous solution of polyvinyl alcohol (Japanese Patent Application Laid-Open No. 56-57
61), polyethylene glycol methacrylate (to which benzoin ethyl ether is added as a sensitizer), polyvinyl alcohol (to which a diazide compound is mixed as a crosslinking agent), polyvinylpyrrolidone (to which a diazide compound is mixed as a crosslinking agent) and so on.

Then, a solution of a silane coupling agent (for example, 1 wt% 3-aminopropyltritrifluoride) is applied to the exposed portion of the ion-sensitive membrane (the portion where the enzyme-immobilized membrane is formed) and the ion-sensitive membrane where the hardened photoresist is formed. ethoxysilane solution) and heated (e.g. 110°C,
5 minutes) to introduce amino groups onto the surface, and then immersed in a glutaraldehyde solution of about 5 wt % (for example, 15 minutes), washed with water, and dried.

Buffer solution [e.g., PIPES-NaOH buffer (0.1M, pH 6.8), HEPES buffer (0.1M, pH 6.8),
1M, pl-! 7.5), Tris-hydrochloride buffer] as a solution (e.g., 300x9/
ml) and urease solution (e.g. 50x9/a+1)
and a glutaraldehyde solution (for example, 2 wt%) is spin-coated.

Spin coat is manufactured by Mikasa Co., Ltd., for example, 3
When the reaction is carried out at 25° C. for 10 minutes at 000 rpm, an enzyme-immobilized membrane of about 1 μm can be coated on the ion-sensitive membrane in one operation.

Therefore, an enzyme-immobilized membrane of any thickness can be formed. Furthermore, the thickness of the enzyme-immobilized membrane deposited in one operation can be changed by changing the viscosity of the enzyme solution. After spin-coating the enzyme solution, it is left to stand (for example, for 30 minutes or more), and then immersed in a solvent (for example, acetone) that dissolves the hardened photoresist but does not dissolve the enzyme-immobilized film, or preferably, applies ultrasound. ) Remove the photoresist. As a result, an enzyme-immobilized membrane is formed on a portion of the ion-sensitive membrane.

Further, in order to protect the FET, it is preferable to provide a protective film 13 in a portion other than the enzyme immobilized film 3 and the insulating material 4.

By using the above-mentioned conventional method, the thickness of the enzyme-immobilized membrane 3 can be accurately controlled, and a membrane having a thickness of 3 to 20 μm can be reliably and easily produced.

Then, it is preferable to provide a control electrode on the P-type silicon substrate l, which has the same configuration as the enzyme sensor described in 1-1 except that it does not have the enzyme immobilized I03. It also works to check drifts, etc. The reference electrode may be provided on a separate substrate, but it is better to provide it on the same substrate because it is easier to measure by simply immersing one sensor in the test liquid. 4. It is preferable that the PET to be formed is fairly homogeneous.It is also preferable to provide an immobilized membrane of an enzyme inactivated by heat or acid on the ion-sensitive membrane of the control electrode. This is preferable, because in this way it is possible to better understand the effects of drift and the like.

Next, a measurement circuit used in the enzyme sensor of the present invention will be explained. The measurement circuit is as shown in Figure 2, which is
This includes the measurement circuit for the reference electrode described above. This measurement circuit is for directly measuring the change in interfacial potential between the test liquid and the ion-sensitive membrane. In addition, the response characteristics to the test solution were evaluated using an enzyme sensor with an enzyme-immobilized membrane and a control FET with no enzyme-immobilized membrane or an inactivated enzyme-immobilized membrane. Measured from the operating output with the electrode.

The measurement circuit will be briefly explained.

A constant drain current flows through the enzyme sensor 8 and the reference electrode 11 by a feedback circuit consisting of an operational amplifier, and a constant voltage is always applied between the source and drain. The potential of the solution is kept constant by the Ag/AgCl electrode, and changes in the interfacial potential between the solution and the ion-sensitive membrane caused by changes in the pH of the solution appear at the A and B terminals. These two outputs are input to the operational amplifier 9, and the difference between the two outputs is recorded on the recorder 10 as a time change.

Next, an example will be given and explained.

Examples 1 to 4 According to the following method, IsFE with the structure shown in ':jS1 figure
We created an enzyme sensor using T.

(1) Creation of FEAT Using a P-type silicon substrate, PET was formed on a part of the substrate by combining a photoresist method and a discharge treatment method.

(2) Creation of ISFET Using the structure in which the FET was formed, an insulating material (5iOx) was provided over the gate of the FET from a distance from the gate of the FET. Furthermore, the insulating material on the gate part of the FET was shaved thin. Then, an iridium oxide film, which will become an ion-sensitive film, is placed on the gate of the FET and on the insulating material at a distance from the gate.
It was formed using the reactive subabuta method in pure oxygen under a vacuum of 10-3 Torr.

The film thickness was 800 people.

Furthermore, electrodes were provided at the source and drain portions of the PET, respectively.

(3) Creation of enzyme sensor Then, a positive photoresist was spin-coated on the ion-sensitive membrane of FET, and the area other than the part (150 x 500 μm1) where the enzyme-immobilized membrane was formed was exposed and developed. The photoresist in the part of the sensitive membrane where the enzyme-immobilized membrane was to be formed was removed.

Next, a solution of silane coupling agent 1
A wt% 3-aminopropyltriethoxysilane solution was spin-coated, heated at 110°C for 5 minutes to introduce amino groups onto the surface, and then immersed in a 5wt% glutaraldehyde solution for 15 minutes, washed with water, Dry.

From above, add 5% to 300 xv/zQ bovine serum albumin solution [0, IM, pi-17,5 HEPES buffer].
An enzyme solution prepared by mixing a 0x9/toge urease solution and a 2 wt % glutaraldehyde solution was spin coated. Spin coating is made by Mikasa Co., Ltd., Spinner IH-D
By repeating this operation, the thickness of the urease immobilized film was 3 μm (Example 1), 5 μm (Example 2), and 10 μm.
20 μm (Example 4) and 20 μm (Example 4) were prepared.

After spin-coating the enzyme solution, the hardened photoresist is left for 30 minutes or more and removed by immersing it in acetone and applying ultrasound to remove the enzyme immobilization film and insulating material to protect the FET. We created a urea sensor with a protective film on the part.

By using the spin coating method, the thickness of the urease-immobilized film could be controlled with an accuracy of about 1 μm. Furthermore, the adhesion between the exposed surface of the ion-sensitive membrane and the immobilized membrane was good.

Comparative Example 1 A urea sensor with a urease-immobilized film thickness of I μm was prepared using the same method as in Example 1, except that spin coating was not repeated.

Comparative Example 2 A 0.2 M Tris-hydrochloride buffer (pH 8,5
) - 5 μQ of a solution of urease dissolved in a 15% bovine serum albumin solution was collected with a microsyringe, applied, and air-dried.
5 μe was added dropwise, and a urease-immobilized membrane was formed by a crosslinking reaction to produce a urea sensor. The thickness of the urease-immobilized membrane of this urea sensor was about 80 μm.

Test Example The output voltage and response speed of the urea sensors of Examples 1 to 4 and Comparative Example 1.2 were measured by the following method.

Measurement was carried out using 205M% pH 7.5 HEPS $
Urea concentration in 1 buffer solution: 10 B/dL I OOB/
dL 1000 m9/di, Ag/
Gate bias was adjusted using an AgCl electrode, and changes in gate surface potential (output voltage) and response speed at that time were measured.

Figure 3 shows the results regarding the output voltage,
The vertical axis is the output voltage, and the horizontal axis is the thickness of the enzyme-immobilized membrane.

Figure 4 shows the results regarding response speed.
The vertical axis is the response time, and the horizontal axis is the thickness of the enzyme-immobilized membrane.

FIG. 3 shows that as the enzyme-immobilized film thickness increases from 1 μm, the output voltage increases rapidly, and after reaching saturation at about 10 μm, it tends to decrease. Examples 1 to 4 in which the enzyme immobilization film thickness was 3 to 20 μm
It was found that the one with sufficient output voltage and high sensitivity.

FIG. 4 shows that the thinner the enzyme-immobilized membrane is, the faster the response speed is. In addition, the response speed was found to be dependent on the concentration of the substrate (urea). And enzyme immobilization film thickness 3-20
It was found that Examples 1 to 4 having a diameter of μm had sufficient response speed. In particular, it has been found that the enzyme-immobilized film has a thickness of 3 to 10 μm.

[Effects of the Invention] The enzyme sensor of the present invention has an enzyme-immobilized membrane with a thickness of 3 to 20 μm directly on the ion-sensitive membrane of the ISFET, so it has a fast response speed and sufficient sensitivity. Therefore, the intensity of the substance to be measured in the test liquid can be measured quickly and reliably.

In addition, if the I5FET is a separated gate type ISFET, it is only necessary to immerse only the ion-sensitive membrane part in the test liquid, and
It is not necessary to immerse the ET gate portion and the source and drain constituent portions, and furthermore, since these portions are shielded from light and the outside world, the response can be stabilized, which is preferable.

In addition, according to the method for manufacturing an enzyme sensor of the present invention, an ISFET is prepared, and an enzyme-immobilized film on which an enzyme is immobilized is coated on the ion-sensitive membrane of the ISFET to a thickness of 3 to 20 μm. For example, since the thickness of the enzyme-immobilized film formed on the ion-sensitive membrane can be accurately controlled, the enzyme sensor described above can be manufactured reliably and easily.

[Brief explanation of the drawing]

FIG. 1 is a drawing showing an example of the structure of the enzyme sensor of the present invention. FIG. 2 is a diagram showing a circuit diagram of the enzyme sensor measuring device of the present invention. FIG. 3 is a drawing showing the relationship between the enzyme immobilization film thickness and the output voltage. FIG. 4 is a drawing showing the relationship between enzyme immobilization film thickness and response time.

Claims (7)

[Claims] (1) Directly on the ion-sensitive membrane of the ISFET with a thickness of 3 to 2
An enzyme sensor characterized by having a 0 μm enzyme-immobilized suppuration. (2) The enzyme sensor according to claim 1, wherein the ISFET is a separated gate type ISFET. (3) Claim 1, wherein the enzyme is urease.
The enzyme sensor according to item 1 or 2. (4) The enzyme sensor according to any one of claims 1 to 3, wherein the ion-sensitive membrane is made of iridium oxide. (5) Production of an enzyme sensor characterized by creating an ISFET and depositing an enzyme-immobilized membrane on which an enzyme is immobilized to a thickness of 3 to 20 μm on the ion-sensitive membrane of the ISFET by spin coating. Method. (6) The enzyme sensor according to claim 5, wherein the ISFET is a separated gate type ISFET. (7) Claim 5, wherein the enzyme is urease.
The enzyme sensor according to item 6 or item 6.