Immobilized enzyme film, protein immobilized film and process for forming the same

• US 5,356,757 A
• Filed: 11/06/1992
• Issued: 10/18/1994
• Est. Priority Date: 11/08/1991
• Status: Expired due to Fees

• Current Assignee: NEC Corporation
• Original Assignee: NEC Corporation
• Inventors: Shionoya, Kiseko, Saito, Atsushi
• Primary Examiner(s): Kight, III, John
• Assistant Examiner(s): Mosley, T.
• Application Number:
  US07/972,560
• Time in Patent Office: 711 Days
• Field of Search: 430/315, 435/288
• US Class Current: 430/315
• CPC Class Codes: C12Q 1/003 Functionalisation
• CPC Class Codes: G01N 27/4145 specially adapted for biomolecules, e.g. gate electrode with immobilised receptors

Abstract

Disclosed are an immobilized enzyme film, characterized in that said film is formed using an enzyme solution prepared by adding 1 to 3 parts by weight of a 50 to 100 wt % water-soluble crosslinking agent having at least two epoxy groups in the molecule and 1 to 3 parts by weight of a 1 to 2 wt % glutaraldehyde to 1 to 3 parts by weight of a 10 to 50 wt % aqueous protein solution containing an enzyme, and a process for forming the same on an ion-sensitive field effect transistor. The thus formed immobilized enzyme film is of an uniform thickness and stable by virtue of its increased hydrophilicity and higher elasticity, whereby deactivation of the enzyme to be caused by the shrinkage of the film can be prevented.

Claims

1. An immobilized enzyme film, characterized in that said film is formed using an enzyme solution prepared by adding 1 to 3 parts by weight of a 50 to 100 wt % water-soluble crosslinking agent having at least two epoxy groups in the molecule wherein said water-soluble crosslinking agent is ethylene polyethylene glycol diglycidyl ether and 1 to 3 parts by weight of a 1 to 2 wt % glutaraldehyde to 1 to 3 parts by weight of a 10 to 50 wt % aqueous protein solution containing an enzyme.

9. The immobilized enzyme film of claim 1 wherein the enzyme is a urease.

2. A process for forming an immobilized enzyme film, which comprises:

a step of mixing 1 to 3 parts by weight of a 10 to 50 wt % aqueous protein solution containing an enzyme with 1 to 3 parts by weight of a 50 to 100 wt % water-soluble crosslinking agent having at least two epoxy groups in the molecule wherein said water-soluble crosslinking agent is ethylene polyethylene glycol diglycidyl ether followed by vigorous stirring of the resulting mixture with ice cooling;

a step of adding 1 to 3 parts by weight of a 1 to 2 wt % glutaraldehyde to the resulting mixture with continuous ice cooling, followed by vigorous stirring; and

a step of applying the resulting mixture by spin coating to a wafer treated with a silane coupling agent.

10. The process of claim 2 wherein the enzyme is a urease.

3. A process for forming an immobilized enzyme film, which comprises:

(a) a step of applying a photoresist soluble in an organic solvent onto a semiconductor wafer on which an ion-sensitive field effect transistor has been formed, followed by removal of the photoresist at a predetermined portion on the surface of the ion-sensitive field effect transistor where an immobilized enzyme film is to be formed;

(b) a step of applying a silane coupling agent by spin coating to treat the predetermined portion on the surface of the ion-sensitive field effect transistor with the silane coupling agent;

(c) a step of applying an aqueous protein solution containing an enzyme and a crosslinking agent onto the thus treated surface of the semiconductor wafer by spin coating; and

(d) a step of treating the resulting semiconductor wafer with an organic solvent in which the photoresist is soluble to dissolve the photoresist and lift off the immobilized enzyme film present on the portions other than the predetermined portion on the surface of the ion-sensitive field effect transistor;

wherein the above spin coating step is repeated at predetermined intervals necessary for the crosslinking agent to complete the crosslinking reaction, whereby to form an immobilized enzyme film having a desired thickness at the predetermined portion on the surface of the ion-sensitive field effect transistor;

the aqueous protein solution containing an enzyme and a crosslinking agent consisting at least of 1 to 4 parts by weight of a solution containing 1 to 20 % by weight of an enzyme and 10 to 50 % by weight of a protein, 1 to 2 parts by weight of a solution containing 50 to 100 % by weight of a water-soluble crosslinking agent having at least two epoxy groups in the molecule wherein said water-soluble crosslinking agent is ethylene polyethylene glycol diglycidyl ether and 1 to 2 parts by weight of a solution containing 1 to 2 % by weight of glutaraldehyde.

4. The process for forming an immobilized enzyme film according to claim 3, wherein the step of treating the predetermined portion on the surface of the ion-sensitive field effect transistor with a silane coupling agent is followed by a step of applying a water-soluble crosslinking agent having at least two epoxy groups in the molecule wherein said water-soluble crosslinking agent is ethylene polyethylene glycol diglycidyl ether onto the surface of the semiconductor wafer by spin coating.

5. The process for forming an immobilized enzyme film or a protein immobilized film according to claim 3, wherein said silane coupling agent is triethoxyvinylsilane, ethoxydimethylvinylsilane, allyltriethoxysilane or 3-aminopropylethoxysilane.

11. The process of claim 3 wherein the enzyme is a urease.

6. A process for forming a protein immobilized film, which comprises:

(a) a step of applying a photoresist soluble in an organic solvent onto a semiconductor wafer on which an ion-sensitive field effect transistor has been formed, followed by removal of the photoresist at a predetermined portion on the surface of the ion-sensitive field effect transistor where a protein immobilized film is to be formed;

(b) a step of applying a silane coupling agent by spin coating to treat the predetermined portion on the surface of the ion-sensitive field effect transistor with the silane coupling agent;

(c) a step of applying an aqueous protein solution containing a crosslinking agent onto the thus treated surface of the semiconductor wafer by spin coating; and

(d) a step of treating the resulting semiconductor wafer with an organic solvent in which the photoresist is soluble to dissolve the photoresist and lift off the protein immobilized film present on the portions other than the predetermined portion on the surface of the ion-sensitive field effect transistor;

wherein the above spin coating step is repeated at predetermined intervals necessary for the crosslinking agent to complete the crosslinking reaction, whereby to form a protein immobilized film having a desired thickness at the predetermined portion on the surface of the ion-sensitive field effect transistor;

the aqueous protein solution containing an enzyme and a crosslinking agent being a solution consisting at least of 1 to 4 parts by weight of a solution containing 10 to 50 % by weight of an aqueous protein solution, 1 to 2 parts by weight of a solution containing 50 to 100 % by weight of a water-soluble crosslinking agent having at least two epoxy groups in the molecule wherein said water-soluble crosslinking agent is ethylene polyethylene glycol diglycidyl ether and 1 to 2 parts by weight of a solution containing 1 to 2 % by weight of glutaraldehyde.

7. The process for forming a protein immobilized film according to claim 6, wherein the step of treating the predetermined portion on the surface of the ion-sensitive field effect transistor with a silane coupling agent is followed by a step of applying a water-soluble crosslinking agent having at least two epoxy groups in the molecule wherein said water-soluble crosslinking agent is ethylene polyethylene glycol diglycidyl ether onto the surface of the semiconductor wafer by spin coating.

8. The process for forming an immobilized enzyme film or a protein immobilized film according to claim 6, wherein said silane coupling agent is triethoxyvinylsilane, ethoxydimethylvinylsilane, allyltriethoxysilane or 3-aminopropylethoxysilane.