Immobolization of biomolecules by enhanced electrophoretic precipitation

• US 5,166,063 A
• Filed: 06/29/1990
• Issued: 11/24/1992
• Est. Priority Date: 06/29/1990
• Status: Expired due to Term

• Current Assignee: Disetronic Licensing Ag
• Original Assignee: Eli Lilly and Company
• Inventors: Johnson, Kirk W.
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): CHAN, KWONG
• Application Number:
  US07/546,101
• Time in Patent Office: 879 Days
• Field of Search: 435/288, 435/291, 435/817, 435/173, 204/403, 204/14.1, 204/180.2, 204/15, 204/181.1, 205/83, 205/80
• US Class Current: 435/173.2
• CPC Class Codes: C12Q 1/003 Functionalisation
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode

Abstract

The present invention provides a method for immobilizing biomolecules on a conductive substrate to produce a biosensor.

Claims

1. A method of preparing a biosensor electrode having a biomolecular component comprising

preparing a solution of at least one species of biomolecule intended for deposition buffered range sufficient to prevent denaturation of the biomolecules, and to a pH such that all species of biomolecules intended for deposition have the same net electric charge sign due to their respective isoelectric points,

immersing said biosensor electrode and a counter electrode in the solution,

applying a variable potential of less than 1 volt across the biosensor electrode and counter electrode, with the potential varying in a manner selected to provide a substantially constant electric current between the electrodes sufficient to cause migration of the biomolecules intended for deposition to said biosensor electrode and accumulation thereon.

2. The method of claim 1 further comprising the step maintaining the substantially constant electric current between the electrodes for a time sufficient to allow accumulation on the biosensor electrode of a film of biomolecules having an average film thickness between 1.0 to 10 micrometers.

3. The method of claim 2 further comprising the step of introducing the biosensor electrode and accumulated deposition into a solution of a crosslinking agent present in an amount and for sufficient time to crosslink the biomolecules together to form a water-insoluble mass on the biosensor electrode.

4. The method of claim 1 wherein the preparation step includes preparation of a solution containing a protein to be deposited.

5. The method of claim 4 wherein the protein is an enzyme.

6. The method of claim 4 wherein the protein is an antibody.

7. A method of preparing a biosensor electrode having a proteinaceous component comprising

preparing a solution of at least one species of protein intended for deposition buffered sufficient to prevent denaturation of the protein

immersing said biosensor electrode and a counter electrode in the solution,

applying a variable potential of less than 1 volt across the biosensor electrode and counter electrode, with the potential varying with time in a manner selected to provide a substantially constant electric current between the electrodes sufficient to cause migration of the biomolecules intended for deposition to said biosensor electrode and accumulation thereon, and

crosslinking said deposited accumulation on the biosensor electrode with a crosslinking agent to form a water-insoluble mass on the biosensor electrode.

8. A method of preparing an enzymatic biosensor comprising

preparing a solution of glucose oxidase intended for deposition on a biosensor electrode,

immersing said biosensor electrode and a counter electrode in the solution,

applying a variable potential of less than 1 volt across the biosensor electrode and counter electrode, with the potential varying with time in a manner selected to provide a substantially constant electric current between the electrodes sufficient to cause migration of the glucose oxidase intended for deposition to said biosensor electrode and accumulation of a film less than 10 micrometers thereon.