JPH069698A - Immobilization of protein - Google Patents

Abstract

PURPOSE:To strongly immobilize a protein on the surface of a metal without losing its activity. CONSTITUTION:A reactive single molecular film 5 having functional groups is formed on the surface of a metal such as a gate electrode 4, and the functional groups are chemically treated to impart hydroxyl groups, etc., to the functional groups. A protein 11 is reacted with and immobilized on the chemically treated part.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for immobilizing proteins.

More specifically, it relates to a method for immobilizing an arbitrary protein on a metal surface via a monomolecular film.

[0003]

2. Description of the Related Art Up to now, there have been almost no known examples of protein immobilization, and as a closer example, a method of immobilizing an enzyme on the surface of a glass electrode or a gate electrode of a field effect transistor (FET) is used. A method in which an enzyme is immobilized on is known. However, the conventional method has a problem as a protein immobilization method, and a highly reliable method has not been obtained.

That is, the conventional enzyme immobilization method is FET
The method of directly applying the enzyme to the electrode or the glass electrode, mixing the enzyme with the resin and applying it, applying the one fixed on the resin particles, or fixing the enzyme through the organic film was used. .

[0005]

However, in the method of direct coating, there is a problem in durability because the enzyme is desorbed during the measurement, and there is a problem in that the protein is mixed into the resin for coating. The method of applying the one immobilized on the plate had low activity, and the method of immobilizing the protein through the organic film also had a problem in durability.

[0006]

In view of the drawbacks of the conventional method described above, the present invention provides a method for reactively immobilizing a protein on a metal surface such as on a FET gate electrode through a reactive monolayer. Is. That is, an active group (eg, —SiCl ₃ etc.) that has a reactive group (eg, CH ₂ ═CH—, CH≡C— group, etc.) at one end and that reacts with an oxide film on the metal surface at the other end and is adsorbed (eg, —SiCl ₃ etc.). ) Or an Langmuir-Blodgett method using an organic molecule having a reactive group at one end and a hydrophilic group (for example, —COOH) at the other end (hereinafter (Hereinafter referred to as LB method), a reactive monolayer is formed, and then the reactive group is chemically treated to add an -OH group. Further, the cyanobromide method or the aldehyde method is used to coat the monolayer surface with a protein. through a process of converting a group reactive to an amino group (-NH ₂₎ an amino group of the protein is to provide a method of reacting fixed.

[0007]

By using the present invention, the protein is immobilized on the metal surface by the reaction between the amino group, which is abundant in the molecule, and the monolayer, so that the protein has high activity and is firmly immobilized. Become.

[0008]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described below with reference to the drawings. As shown in FIG. 1, a resist 3 is coated on the substrate 2 on which the FET 1 is formed in advance, and only the gate electrode 4 is exposed and developed to form an opening (FIG. 1).

Next, a silane surfactant such as CH ₂ ═CH— (CH ₂ ) n-is provided in the opening by a chemisorption method.
SiCl ₃ (n is an integer of 10 to 20 is good, and CH ₂
= CH- may be CH≡C-) (FIG. 2).

At this time, oxygen on the metal surface selectively reacts with -SiCl _{3 on the} surface of the gate electrode,

[0011]

[Chemical 1]

A monomolecular film 5 of is formed. For example, 2.
80% at a concentration of 0 × 10 ^{-3 to} 5.0 × 10 ^-2 Mol / l
When immersed in an activator solution dissolved in n-hexane, 12% carbon tetrachloride, 8% chloroform solution for 2 to 3 minutes, at the metal interface.

[0013]

[Chemical 2]

The bond 6 of is formed. And at this time,
The vinyl group 7 of the silane surfactant is lined up on the surface of the electrode 4 to form the monomolecular film 5 (FIG. 3).

Next, the resist pattern 3'shown in FIG.
After removing the solution, the substrate on which the monomolecular film 5 was formed was dipped in a THF solution containing 1 mol / l diborane at room temperature, and further dipped in an aqueous solution of NaOH 0.1 Mol / l 30% H ₂ O _{2 to} remove the monomer. A hydroxyl group (—OH) 8 is added to the vinyl group or the acetylene group on the surface of the molecular film 5 (FIG. 4).

Then, it is immersed in an aqueous solution of periodic acid and the OH groups on the surface are oxidized into aldehyde groups 9 according to the following formula (1) (FIG. 5).

[0017]

[Chemical 3]

Further, a protein having a specific activity is subjected to an addition reaction according to the following formula (2) and immobilized.

[0019]

[Chemical 4]

Therefore, the protein 10 is firmly fixed on the gate electrode 4 as a protein thin film 11 by a chemical reaction through the monomolecular film 5 at the time of selection (FIGS. 6 and 7).

Finally, the biosensor is completed by dicing the substrate and assembling the electrodes and the like.

Although the aldehyde method has been shown in the above examples, protein immobilization using the cyanobromide method represented by the following formula (3) can be carried out in the same manner.

[0023]

[Chemical 5]

Further, an example in which the chemisorption method is used for forming the monomolecular film is shown. CH ₂ ═CH— (CH ₂ ) nCOOH
It has been confirmed that a monomolecular film can be formed on the gate electrode even by the Langsuer-Blodgett (LB) method using CH≡C- (CH ₂ ) nCOOH or the like.

Furthermore, in the above example, the monolayer is selectively formed only on the gate electrode using the resist,
Since vinyl groups and acetylene groups are sensitive to energy rays to form polymers, after forming a monomolecular film on the entire surface,
After leaving only the gate electrode, it is exposed to energy rays 12 to partially deactivate (deactivate) vinyl groups and acetylene groups.
It is also possible to add an -OH group to the remaining vinyl group 14 (or acetylene group) after the reaction is performed (Fig. 8).

It will be apparent that the molecular device can be constructed by applying the method of the present invention, that is, the self-alignment mechanism of the protein to the LB membrane.

[0027]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, a protein is immobilized via a monolayer selectively and firmly without losing its activity. Therefore, a highly sensitive and highly reliable biosensor can be provided.

Further, since the manufacturing process is simple, there is also an effect that the cost can be greatly reduced.

[Brief description of drawings]

FIG. 1 is a sectional view of a part of a manufacturing process of a biosensor according to an embodiment of the present invention.

FIG. 2 is a sectional view of a part of the manufacturing process of the embodiment.

FIG. 3 is an enlarged view of a part of the manufacturing process of the embodiment.

FIG. 4 is an enlarged view of a part of the manufacturing process of the embodiment.

FIG. 5 is an enlarged view of a part of the manufacturing process of the embodiment.

FIG. 6 is an enlarged view of a part of the manufacturing process of the embodiment.

FIG. 7 is a sectional view of a part of the manufacturing process for the embodiment.

FIG. 8 is an enlarged view of a part of the manufacturing process of the embodiment.

[Explanation of symbols]

 1 FET 2 substrate 4 gate electrode 5 monomolecular film 11 protein film

Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location G01N 27/327 // H01L 29/78

Claims (5)

[Claims] 1. A method for immobilizing a protein, which comprises the steps of forming a monomolecular film having a sensitive group on a metal surface, chemically treating the sensitive group, and reacting and immobilizing the protein on the chemically treated portion. 2. The method for immobilizing a protein according to claim 1, wherein a chemisorption method is used in the step of forming a monomolecular film. 3. The method for immobilizing a protein according to claim 2, wherein a linear hydrocarbon molecule having a vinyl group or an acetylene group at one end and a chlorosilane group at the other end is used as a raw material for the monomolecular film. 4. The method for immobilizing a protein according to claim 2, wherein the Langure-Blodgett method is used in the step of forming a monomolecular film. 5. In the step of chemically treating, a film whose surface is a vinyl group or an acetylene group is used.
The method for immobilizing a protein according to claim 1, wherein the protein is immobilized by the cyanobromide method or the aldehyde method after once adding a hydroxyl group.