Biosensor including chemically modified enzymes

• US 5,543,326 A
• Filed: 03/04/1994
• Issued: 08/06/1996
• Est. Priority Date: 03/04/1994
• Status: Expired due to Fees

• Current Assignee: Adam Heller, Ioanis Katakis, Ling Ye
• Original Assignee: Adam Heller, Ioanis Katakis, Ling Ye
• Inventors: Ye, Ling, Katakis, Ioanis, Heller, Adam
• Primary Examiner(s): Beisner, William H.
• Application Number:
  US08/207,035
• Time in Patent Office: 886 Days
• Field of Search: 435/14, 435/25-28, 435/174, 435/176, 435/177, 435/180, 435/181, 435/188, 435/189, 435/190, 435/288, 435/291, 435/817, 204/153.12, 204/403
• US Class Current: 204/403.14
• CPC Class Codes: C12Q 1/001 Enzyme electrodes
• CPC Class Codes: Y10S 435/817 Enzyme or microbe electrode

Abstract

Oxidoreductases are chemically modified so as to change their net charge at a given pH. Through such modification the adsorption of the enzymes on surfaces, their retention in similarly or oppositely charged polymer membranes and their binding with similarly or oppositely charged macromolecules, particularly redox macromolecules, is controlled. The modified enzymes, of which modified recombinant glucose oxidase is an example, are useful in biosensors, e.g. for glucose; as marker enzymes of antigens or antibodies in immunosensing; and as labels of nucleotide sequences in probes, e.g. for DNA nucleotide sequences.

Claims

1. A biosensor comprising:

an electrode having a surface;

a polyionic polymer; and

a modified enzyme formed by increasing the number of amine groups covalently bound to a glycosylated redox enzyme such that the pI of the modified redox enzyme is increased by at least 2 pH units with respect to the glycosylated redox enzyme;

wherein the modified redox enzyme and the polyionic polymer are adsorbed or crosslinked on the surface of the electrode.

2. The biosensor of claim 1, wherein the polyionic polymer is an electron-relaying polymer.

3. The biosensor of claim 2, wherein the polyionic polymer is a polycationic polymer and the modified redox enzyme is a polycationic enzyme.

4. The biosensor of claim 1, wherein the polyionic polymer does not have redox properties and electron transfer is effected by a synthetic mediator adsorbed on the surface of the electrode.

5. The biosensor of claim 4, wherein the synthetic mediator is covalently linked to the modified redox enzyme.

6. The biosensor of claim 1, having a redox potential in the range of -0.05 to +0.3 V (SCE).

7. The biosensor of claim 1, wherein the pI of the modified redox enzyme is increased at least four pH units.

8. The biosensor of claim 1, wherein the pI of the modified redox enzyme is increased at least six pH units.

9. The biosensor of claim 1, wherein the polyionic polymer and the modified redox enzyme are adsorbed on the surface of the electrode by electrophoretic or physico/chemical deposition on the surface.

10. The biosensor of claim 1, wherein the polyionic polymer is polyanionic and the modified redox enzyme is polycationic.

11. The biosensor of claim 1, wherein the polyionic polymer is polycationic and the modified redox enzyme is polycationic.

12. A biosensor comprising:

an electrode having a surface;

a polyionic polymer; and

a modified redox enzyme formed by oxidation of a glycosylated redox enzyme to form an oxidized enzyme and reductive amination of the oxidized enzyme to form amines,

wherein the polyionic polymer and the modified redox enzyme are adsorbed or crosslinked onto the electrode.

13. A biosensor comprising:

an electrode having a surface;

a polyionic polymer; and

a modified redox enzyme formed by oxidation of oligosaccharide moieties of a glycosylated redox enzyme to form a polyaldehyde-enzyme, reaction of the polyaldehyde-enzyme with an excess of polyamines or amino-polyacids to form Schiff bases, and subsequent reduction of the Schiff bases to form stable, polyaminated or polycarboxylated redox enzymes, wherein the polyionic polymer and the modified redox enzyme are adsorbed or crosslinked on the surface of the electrode.

14. A biosensor comprising:

an electrode having a surface;

a polyionic redox polymer and;

a modified redox enzyme formed by oxidation of oligosaccharide moieties of a glycosylated redox enzyme to form a polyaldehyde-enzyme, reaction of the polyaldehyde-enzyme with an excess of polyamines to form Schiff bases, and subsequent reduction of the Schiff bases to form a stable, polyaminated redox enzyme,

wherein the polyionic redox polymer and the modified redox enzyme are coated on the surface of the electrode.

15. The biosensor of claim 14, wherein the polyionic redox polymer is poly(vinyl pyridine) derivitized with bis(bipyridine)osmium.