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

• US 6,127,127 A
• Filed: 04/22/1999
• Issued: 10/03/2000
• Est. Priority Date: 06/27/1995
• Status: Expired due to Term

• Current Assignee: Board of Trustees of The University of Illinois, Xanthon, Inc. (Nokia Corporation)
• Original Assignee: Xanthon, Inc. (Nokia Corporation), University of North Carolina At Chapel Hill (University of North Carolina System)
• Inventors: Eckhardt, Allen E., Thorp, H. Holden, Napier, Mary E., Thomas, Robert S., Mikulecky, Jill C.
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): Chakrabarti, Arun K.
• Application Number:
  US09/296,929
• Time in Patent Office: 530 Days
• Field of Search: 435/6, 435/7, 324/715, 536/23.1
• 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 1/6825 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

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 to 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. A method of determining the presence of a label-bearing target in a sample, comprising:

(a) contacting a non-conductive self-assembled monolayer on an electrode having a conductive working surface, said monolayer comprising phosphonate molecules having at the minimum at least one phosphonate group and at least on R₁ group 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, with a sample suspected of containing a label-bearing target which is capable of being oxidized in an oxidation-reduction reaction, so that the immobilized member of the binding pair and the target, if present, form a target complex on the monolayer;

(b) contacting the monolayer and the target complex, if present, with a transition metal complex that oxidizes the label-bearing target in an oxidation-reduction reaction between the transition metal complex and the label-bearing target from which label-bearing target there is electron transfer to the transition metal complex, resulting in regeneration of the reduced form of the transition metal complex as part of a catalytic cycle;

(c) detecting the oxidation-reduction reaction; and

(d) determining the presence or absence of the target from the detected oxidation-reduction reaction.

2. The method of claim 1, wherein the transition metal complex is Ru(bpy)₃²⁺ and the detected oxidation-reduction reaction is guanine oxidation.

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

4. The method of claim 3, wherein R₂ comprises (CH₂)₁₁.

5. The method of claim 1, wherein the phosphonate molecules comprise a carboxy-alkyl phosphonate.

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

7. The method of claim 1, wherein the label-bearing target is selected from the group consisting of nucleic acids, proteins and carbohydrates.

8. The method of claim 1, wherein the conductive working surface comprises an ITO surface.

9. The method of claim 1, wherein the label-bearing target is a nucleic acid containing guanine and the immobilized member of the binding pair is an oligonucleotide probe hybridizable with said target to form a hybridized target complex.

10. The method of claim 9, further comprising amplifying the target nucleic acid to produce an amplified nucleic acid solution prior to contacting the self-assembled monolayer with the target.

11. The method of claim 10, wherein the amplification is carried out by a method selected from the group consisting of polymerase chain reaction, strand displacement amplification, ligase chain reaction, and nucleic acid sequence-based amplification.

12. The method of claim 1, wherein the member of the binding pair comprises an oligonucleotide probe.

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

14. The method of claim 13, wherein the protein-binding substance comprises a protein.

15. The method of claim 1, wherein the sample is selected from the group consisting of:

synthetic or natural oligonucleotides, surgical specimens, specimens used for medical diagnostics, specimens used for genetic testing, environmental specimens, cell culture specimens, food specimens, dental specimens and veterinary specimens.

16. The method of claim 1 wherein prior to step (a), the R₁ group was covalently bound to a member of a binding pair and the resulting phosphonate molecules were then applied to the electrode.

17. A method of determining the presence of a label-bearing target in a sample, comprising:

(a) contacting an electrode having a conductive working surface with phosphonate molecules having at the minimum at least one phosphonate group and at least one R₁ group, wherein the R₁ group is either covalently bound to a member of a binding pair or capable of being covalently bound to a member of a binding pair, to form a non-conductive self-assembled monolayer on said electrode, 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;

(b) binding the R₁ group to the member of the binding pair if not already so bound by activating the R₁ group with a coupling agent and contacting the activated R₁ group with a member of a binding pair capable of binding to a target to immobilize the member of the binding pair;

(c) contacting the self-assembled monolayer having the member of the binding pair immobilized thereon with a sample suspected of containing a label-bearing target which is capable of being oxidized in an oxidation-reduction reaction, so that the immobilized member of the binding pair and the target form a target complex on the monolayer;

(d) contacting the monolayer and the target complex, if present, with a transition metal complex capable of oxidizing the label-bearing target in an oxidation-reduction reaction;

(e) detecting the oxidation-reduction reaction; and

(f) determining the presence or absence of the target from the detected oxidation-reduction reaction.

18. The method of claim 17, wherein the transition metal complex is Ru(bpy)₃² + and the detected oxidation-reduction reaction is guanine oxidation.

19. The method of claim 17, wherein an organic spacer group, R₂, is located between the phosphonate group and the R₁ group.

20. The method of claim 19, wherein R₂ comprises (CH₂)₁₁.

21. The method of claim 17, wherein the phosphonate molecules comprise a carboxy-alkyl phosphonate.

22. The method of claim 21, wherein the carboxy-alkyl phosphonate is 11-carboxyundecane phosphonic acid.

23. The method of claim 17, wherein the label-bearing target is selected from the group consisting of nucleic acids, proteins and carbohydrates.

24. The method of claim 17, wherein the conductive working surface comprises an ITO surface.

25. The method of claim 17, wherein the label-bearing target is a nucleic acid containing guanine and the immobilized member of the binding pair is an oligonucleotide probe hybridizable with said target to form a hybridized target complex.

26. The method of claim 25, further comprising amplifying the target nucleic acid to produce an amplified nucleic acid solution prior to contacting the self-assembled monolayer with the target.

27. The method of claim 26, wherein the amplification is carried out by a method selected from the group consisting of polymerase chain reaction, strand displacement amplification, ligase chain reaction, and nucleic acid sequence-based amplification.

28. The method of claim 17, wherein the member of the binding pair comprises an oligonucleotide probe.

29. The method of claim 17, wherein the member of the binding pair comprises a protein-binding substance.

30. The method of claim 29, wherein the protein-binding substance comprises a protein.

31. The method of claim 17, wherein the coupling agent comprises a carbodiimide.

32. The method of claim 17, wherein the sample is selected from the group consisting of:

synthetic or natural oligonucleotides, surgical specimens, specimens used for medical diagnostics, specimens used for genetic testing, environmental specimens, cell culture specimens, food specimens, dental specimens and veterinary specimens.

33. A method of determining the presence of a target nucleic acid in a sample, comprising:

(a) providing a non-conductive self-assembled monolayer on an electrode having a conductive working surface, said monolayer comprising phosphonate molecules having at the minimum at least one phosphonate group and at least on R₁ group covalently bound to an oligonucleotide probe, 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;

(b) contacting the self-assembled monolayer having the oligonucleotide probe immobilized thereon with a sample suspected of containing a target nucleic acid which is capable of being oxidized in an oxidation-reduction reaction so that the immobilized oligonucleotide probe and the target nucleic acid, if present, form a target complex on the monolayer;

(c) contacting the monolayer and the target complex, if present, with a transition metal complex that oxidizes the target nucleic acid in an oxidation-reduction reaction between the transition metal complex and the target nucleic acid, from which target nucleic acid there is electron transfer to the transition metal complex, resulting in regeneration of the reduced form of the transition metal complex as part of a catalytic cycle;

(d) detecting the oxidation-reduction reaction; and

(e) determining the presence or absence of the target nucleic acid from the detected oxidation-reduction reaction.

34. The method of claim 33, wherein the transition metal complex is Ru(bpy)₃²⁺ and guanine oxidation is detected.

35. The method of claim 33, wherein an organic spacer group, R₂, is located between the phosphonate group and the R₁ group.

36. The method of claim 35, wherein R₂ comprises (CH₂)₁₁.

37. The method of claim 33, wherein the phosphonate molecules comprise a carboxy-alkyl phosphonate.

38. The method of claim 37, wherein the carboxy-alkyl phosphonate is 11-carboxyundecane phosphonic acid.

39. The method of claim 33, wherein the conductive working surface comprises an ITO surface.

40. The method of claim 33, wherein the R₁ group is coupled to the member of the binding pair prior to formation of the self-assembled monolayer.

41. The method of claim 33, further comprising amplifying the target nucleic acid to produce an amplified nucleic acid solution prior to contacting the self-assembled monolayer with the target.

42. The method of claim 41, wherein the amplification is carried out by a method selected from the group consisting of polymerase chain reaction, strand displacement amplification, ligase chain reaction, and nucleic acid sequence-based amplification.

43. The method of claim 33, wherein the sample is selected from the group consisting of:

synthetic or natural oligonucleotides, surgical specimens, specimens used for medical diagnostics, specimens used for genetic testing, environmental specimens, cell culture specimens, food specimens, dental specimens and veterinary specimens.

44. A method of determining the presence of a target protein in a sample, comprising:

(a) providing a nonconductive self-assembled monolayer on an electrode having a conductive working surface, said monolayer comprising phosphonate molecules having at the minimum at least one phosphonate group and at least on R₁ group covalently bound to a protein binding substance, 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;

(b) contacting the self-assembled monolayer having the protein-binding substance immobilized thereon with a sample suspected of containing a target protein;

(c) contacting the target protein bound to the monolayer, if present, with a second protein-binding substance which has bound to it a label capable of being oxidized in an oxidation-reduction reaction so that the protein-binding substance and the target protein, if present, form a target complex on the monolayer;

(d) contacting the monolayer and the target complex, if present, with a transition metal complex that oxidizes the label in an oxidation-reduction reaction between the transition metal complex and the label from which label there is electron transfer to the transition metal complex resulting in regeneration of the reduced form of the transition metal complex as part of a catalytic cycle;

(e) detecting the oxidation-reduction reaction; and

(f) determining the presence or absence of the target protein from the detected oxidation-reduction reaction.

45. The method of claim 44, wherein the label comprises an oligonucleotide.

46. The method of claim 44, wherein protein-binding substance is a protein.

47. The method of claim 44, wherein an organic spacer group, R₂, is located between the phosphonate group and the R₁ group.

48. The method of claim 47, wherein R₂ comprises (CH₂)₁₁.

49. The method of claim 44, wherein the phosphonate molecules comprise a carboxy-alkyl phosphonate.

50. The method of claim 49, wherein the carboxy-alkyl phosphonate is 11-carboxyundecane phosphonic acid.

51. The method of claim 44, wherein the conductive working surface comprises an ITO surface.

52. The method of claim 44, wherein the sample is selected from the group consisting of:

synthetic or natural oligonucleotides, surgical specimens, specimens used for medical diagnostics, specimens used for genetic testing, environmental specimens, cell culture specimens, food specimens, dental specimens and veterinary specimens.

53. A method of determining the presence of a target protein in a sample, comprising:

(a) providing a non-conductive self-assembled monolayer on an electrode having a conductive working surface, said monolayer comprising phosphonate molecules having at the minimum at least one phosphonate group and at least one R₁ group covalently bound to a protein-binding substance, 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;

(b) contacting the self-assembled monolayer having the protein-binding substance immobilized thereon with a sample suspected of containing a target protein which has bound to it a label capable of being oxidized in an oxidation-reduction reaction, so that the immobilized protein-binding substance and the target protein, if present, form a target complex on the monolayer;

(c) contacting the monolayer and the target complex, if present, with a transition metal complex capable of oxidizing the label in an oxidation-reduction reaction;

(d) detecting the oxidation-reduction reaction; and

(e) determining the presence or absence of the target protein from the detected oxidation-reduction reaction.

54. The method of claim 53, wherein the label comprises an oligonucleotide.

55. The method of claim 53, wherein the protein-binding substance comprises a protein.

56. The method of claim 53, wherein an organic spacer group, R₂, is located between the phosphonate group and the R₁ group.

57. The method of claim 56, wherein R₂ comprises (CH₂)₁₁.

58. The method of claim 53, wherein the phosphonate molecules comprise a carboxy-alkyl phosphonate.

59. The method of claim 58, wherein the carboxy-alkyl phosphonate is 11-carboxyundecane phosphonic acid.

60. The method of claim 53, wherein the conductive working surface comprises an ITO surface.