Monolayer and electrode for detecting a label-bearing target and method of use thereof

• US 6,387,625 B1
• Filed: 06/19/2000
• Issued: 05/14/2002
• Est. Priority Date: 06/27/1995
• Status: Expired due to Term

• Current Assignee: Xanthon, Inc. (Nokia Corporation), University of North Carolina At Chapel Hill (University of North Carolina System)
• Original Assignee: Xanthon, Inc. (Nokia Corporation), University of North Carolina At Chapel Hill (University of North Carolina System)
• Inventors: Thomas, Robert S., Mikulecky, Jill C., Eckhardt, Allen E., Thorp, H. Holden, Napier, Mary E.
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): Chakrabarti, Arun K.
• Application Number:
  US09/596,607
• Time in Patent Office: 694 Days
• Field of Search: 204/153.12, 324/715, 435/6, 435/288, 435/291, 435/810, 435/817, 436/94, 436/501, 536/23.1, 536/24.3
• US Class Current: 435/6.11
• 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 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

An electrode for detecting interactions between members of a binding pair, which electrode has been modified by formation of a non-conductive self-assembled monolayer, and a method of detecting biomolecules, such as nucleic acids or other targets, including receptors, ligands, antigens or antibodies, utilizing such an electrode. When contacted with a target nucleic acid, an oligonucleotide probe coupled to the self-assembled monolayer reacts with the target nucleic acid form a hybridized nucleic acid on the modified electrode surface. The hybridized nucleic acid is reacted with a transition metal complex capable of oxidizing a preselected base in the hybridized nucleic acid in an oxidation-reduction reaction, the oxidation-reduction reaction is detected, and the presence or absence of the nucleic acid is determined from the detected oxidation-reduction reaction.

Claims

1. An electrode, comprising:

(a) a substrate having a conductive working surface thereon; and

(b) a non-conductive self-assembled monolayer on said conductive working surface, said monolayer comprising phosphonate molecules having at the minimum at least one phosphonate group and at least one R₁ group, wherein the R₁ group is covalently bound to a member of a binding pair, and through which monolayer a transition metal complex can freely move from reactants immobilized on the monolayer to the conductive working surface to transfer electrons to the conductive working surface;

and wherein the conductive working surface comprises an ITO surface.

2. The electrode of claim 1, wherein an organic spacer group, R₂, is located between the phosphonate group and the R₁ group.

3. The electrode of claim 2, wherein R₂ comprises (CH₂)₁₁.

4. The electrode of claim 1, wherein the phosphonate molecules comprise a carboxy-alkyl phosphonate.

5. The electrode of claim 4, wherein the carboxy-alkyl phosphonate is 11-carboxyundecane phosphonic acid.

6. The electrode of claim 1, wherein the R₁ group was coupled to the member of the binding pair prior to formation of the self-assembled monolayer.

7. The electrode of claim 1, wherein the member of the binding pair comprises an oligonucleotide probe.

8. The electrode of claim 1, wherein the member of the binding pair comprises a protein-binding substance.

9. The electrode of claim 8, wherein the protein-binding substance comprises a protein.

10. The electrode of claim 1, wherein the R₁ group has been activated with a coupling agent.

11. The electrode of claim 10, wherein the coupling agent comprises a carbodiimide.

12. The electrode of claim 1, wherein the substrate is selected from the group consisting of metallic substrates and non-metallic substrates.

13. A method of preparing a self-assembled monolayer on an electrode, comprising:

(a) providing a substrate having a conductive working surface;

(b) providing phosphonate molecules having at the minimum at least one phosphonate group and at least one R₁ group, wherein the R₁ group is capable of covalent bonding with a member of a binding pair, wherein said self-assembled monolayer is nonconductive and a transition metal complex can freely move through the monolayer from reactants immobilized on the monolayer to the conductive working surface to transfer electrons to the conductive working surface; and

(c) contacting the substrate with the phosphonate molecules to form a self-assembled monolayer;

and wherein the conductive working surface comprises an ITO surface.

14. The method of claim 13, wherein an organic spacer group, R₂, is located between the phosphonate group and the R₁ group.

15. The method of claim 14, wherein R₂ comprises (CH₂)₁₁.

16. The method of claim 13, wherein the phosphonate molecules comprise a carboxy-alkyl phosphonate.

17. The method of claim 16, wherein the carboxy-alkyl phosphonate is 11-carboxyundecane phosphonic acid.

18. The method of claim 13, wherein the R₁ group is coupled to the member of the binding pair prior to formation of the self-assembled monolayer.

19. The method of claim 13, wherein the member of the binding pair comprises an oligonucleotide probe.

20. The method of claim 13, wherein the member of the binding pair comprises a protein-binding substance.

21. The method of claim 20, wherein the protein-binding substance comprises a protein.

22. The method of claim 13, further comprising activating the R₁ group with a coupling agent.

23. The method of claim 22, wherein the coupling agent comprises a carbodiimide.