Enzymatic electrochemical detection assay using protective monolayer and device therefor

• US 20060160100A1
• Filed: 01/19/2005
• Published: 07/20/2006
• Est. Priority Date: 01/19/2005
• Status: Abandoned Application

• Current Assignee: Agency For Science Technology and Research
• Original Assignee: Agency For Science Technology and Research, National University of Singapore
• Inventors: Le, Van Dong, Xie, Fang, Gao, Zhiqiang, Gopalakrishnakone, Ponnampalam
• Application Number:
  US11/038,289
• Publication Number:
  US 20060160100A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: G01N 33/5438 Electrodes

Abstract

There is provided an electrochemical assay method for detecting a target molecule, for example a protein, in a sample, which involves the use of a protective monolayer and a redox polymer to form a bilayer immobilized on an electrode. The monolayer protects the electrode from non-specific adherence of reagents, particular proteins, to the electrode while simultaneously providing a surface that can be functionalized to immobilize a capture molecule and that can interact with the redox polymer.

Claims

1. A method of electrochemically detecting a target molecule in a sample, comprising:

coating an electrode with a monolayer capable of immobilizing a capture molecule thereon and of transferring electrons there across, thereby inhibiting non-specific binding of protein molecules at the electrode;

immobilizing the capture molecule on the monolayer;

adding a sample containing a target molecule to be captured by the capture molecule;

adding a detection molecule that specifically binds one of the capture molecule or the target molecule, wherein the detection molecule is labelled with an enzyme capable of oxidizing or reducing a substrate;

adding a redox polymer that interacts with the monolayer and that together with the monolayer forms a conductive path from the enzyme to the electrode;

adding the substrate to be oxidized or reduced by the enzyme; and

detecting current flow at the electrode.

2. The method of claim 1, wherein the detection molecule specifically binds to the target molecule and the detection molecule comprises an antibody.

3. The method of claim 1, wherein the detection molecule specifically binds to the capture molecule and the detection molecule comprises a competitor of the target molecule.

4. The method of claim 1 wherein the redox polymer has a charged group which interacts electrostatically with the monolayer through an oppositely charged group on the monolayer.

5. The method of claim 4 further comprising rinsing the electrode after each of said coating, said immobilizing, said adding the sample, said adding the detection molecule and said adding the redox polymer.

6. The method of claim 5 wherein the sample is a crude cell lysate, a partially purified cell lysate, a tissue culture medium containing secreted proteins, blood, serum, cerebrospinal fluid, saliva or urine.

7. The method of claim 6 wherein the capture molecule comprises a protein, an antibody, a monoclonal antibody, an antibody fragment, a receptor, a receptor fragment, a ligand, an inhibitor, a small molecule, a nucleic acid, a hormone or a non-cleavable substrate analogue.

8. The method of claim 7 wherein the target molecule comprises a protein, a peptide, a receptor, a receptor fragment, a nucleic acid, a ligand, an inhibitor, a small molecule, a hormone or a non-cleavable substrate analogue.

9. The method of claim 8 wherein the monolayer comprises mercaptoundecanoic acid or mercaptohexadecanoic acid.

10. The method of claim 9 wherein in the enzyme comprises an oxidoreductase, glucose oxidase, horse radish peroxidase, glucose-6-phosphate-dehydrogenase, catalase, peroxidase, microperoxidase, alkaline phosphatase, β

-galactosidase, urease, β

-lactamase, lactate oxidase or laccase.

11. The method of claim 10 wherein the substrate comprises glucose, hydrogen peroxide, glucose-6-phosphate, phenylphosphate, p-aminophenylphosphate, p-aminophenyl-β

-galactoside, urea or benzyl penicillin.

12. The method of claim 11 wherein the electrode comprises carbon, a metal, a metal oxide or a conductive polymeric material.

13. The method of claim 12 wherein the electrode comprises carbon paste, carbon fiber, graphite, glassy carbon, gold, silver, copper, platinum, palladium, indium tin oxide, poly(3,4-ethylenedioxythiophene) (PEDOT) or polyaniline.

14. The method of claim 13 wherein the redox polymer comprises quinone, ferrocene, osmium (4,4′

-dimethyl-2,2′

-bipyridine)₂, tetrathiafulvalene, an Ru complex, a Co complex, an Fe complex, or an Rh complex as a redox centre.

15. The method of claim 14 wherein the redox polymer comprises polyvinylpyridine, polysiloxane, polypyrrole, polyquinone or polyvinylpyridine-co-acrylamide.

16. A method of providing an electrochemical cell for detecting a target molecule in a sample, comprising:

coating an electrode with a monolayer capable of immobilizing a capture molecule thereon and of transferring electrons there across, thereby inhibiting non-specific binding of protein molecules at the electrode; and

immobilizing the capture molecule thereon.

17. A device for performing an electrochemical assay, comprising:

a monolayer formed on a surface of an electrode, said monolayer capable of immobilizing a capture molecule thereon and of transferring electrons there across, thereby inhibiting non-specific binding of protein molecules at the electrode; and

a capture molecule immobilized on the monolayer.

18. The device of claim 17 further comprising:

a target molecule captured by the capture molecule;

a detection molecule specifically bound to one of the capture molecule or the target molecule, wherein the detection molecule is labelled with an enzyme capable of oxidizing or reducing a substrate; and

a redox polymer forming an interaction with the monolayer and that together with the monolayer forms a conductive path from the enzyme to the electrode.

19. The device of claim 18 wherein the detection molecule is specifically bound by the target molecule and the detection molecule comprises an antibody.

20. The device of claim 18 wherein the detection molecule is specifically bound by the capture molecule and the detection molecule comprises a competitor of the target molecule.

21. The device of claim 18 wherein the interaction formed between the redox polymer and the monolayer is an electrostatic interaction.

22. The device of claim 21 wherein the capture molecule comprises a protein, an antibody, a monoclonal antibody, an antibody fragment, a receptor, a receptor fragment, a ligand, an inhibitor, a small molecule, a nucleic acid, a hormone or a non-cleavable substrate analogue.

23. The device of claim 22 wherein the target molecule comprises a protein, a peptide, a receptor, a receptor fragment, a nucleic acid, a ligand, an inhibitor, a small molecule, a hormone or a non-cleavable substrate analogue.

24. The device of claim 23 wherein the monolayer comprises mercaptoundecanoic acid or mercaptohexadecanoic acid.

25. The device of claim 24 wherein in the enzyme comprises an oxidoreductase, glucose oxidase, horse radish peroxidase, glucose-6-phosphate-dehydrogenase, catalase, peroxidase, microperoxidase, alkaline phosphatase, β

-galactosidase, urease, β

-lactamase, lactate oxidase or laccase.

26. The device of claim 25 wherein the substrate comprises glucose, hydrogen peroxide, glucose-6-phosphate, phenylphosphate, p-aminophenylphosphate, p-aminophenyl-β

-galactoside, urea or benzyl penicillin.

27. The device of claim 26 wherein the electrode comprises carbon, a metal, a metal oxide or a conductive polymeric material.

28. The device of claim 27 wherein the electrode comprises carbon paste, carbon fiber, graphite, glassy carbon, gold, silver, copper, platinum, palladium, indium tin oxide, poly(3,4-ethylenedioxythiophene) (PEDOT) or polyaniline.

29. The device of claim 28 wherein the redox polymer comprises quinone, ferrocene, osmium (4,4′

-dimethyl-2,2′

-bipyridine)₂,tetrathiafulvalene, an Ru complex, a Co complex, an Fe complex, or an Rh complex as a redox centre.

30. The device of claim 29 wherein the redox polymer comprises polyvinylpyridine, polysiloxane, polypyrrole, polyquinone or polyvinylpyridine-co-acrylamide.