Electrochemical detection of nucleic acid hybridization

• US 20100000881A1
• Filed: 11/01/2004
• Published: 01/07/2010
• Est. Priority Date: 10/30/2003
• Status: Abandoned Application

• Current Assignee: North Carolina State University (University of North Carolina System)
• Original Assignee: North Carolina State University (University of North Carolina System)
• Inventors: Feldheim, Daniel, Franzen, Stefan
• Application Number:
  US10/978,678
• Publication Number:
  US 20100000881A1
• Time in Patent Office: Days
• US Class Current: 205/780.5
• 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

Abstract

A nucleic acid hybridization detection assay is carried out at an electrode. A solid electrode, such as an indium tin oxide electrode, is modified by capture probes comprising single-stranded oligonucleotides immobilized to the surface of the electrode. In some embodiments using sandwich assay methodology, the capture probes hybridize complementary target nucleic acid sequences, which in turn are bound to detection probes comprising nanoparticle-oligonucleotide conjugates comprising target-complementary oligonucleotides. In some embodiments, detection probes comprise nanoparticles attached to molecules comprising one partner of a ligand-binding pair (e.g., streptavidin), while target sequences comprise the other partner of the ligand-binding pair (e.g., biotin). When the assay is carried out in the presence of a redox mediator, redox reactions catalyzed, and/or facilitated and/or enhanced by the presence of nanoparticles generate electrons that are transferred to the electrode, resulting in a detectable electrical signal.

Claims

1. A method of detecting a target nucleic acid, comprising:

providing a hybridization complex comprising (a) a capture probe that is attached to an electrode surface directly or via an attachment linker;

(b) a target nucleic acid that is hybridized to the capture probe; and

(c) at least one nanoparticle attached to the target nucleic acid;

removing unhybridized nucleic acid;

contacting the electrode with a redox solution comprising a redox mediator and an electrolyte, such that the complex is in contact with the redox solution; and

detecting a non-photogenerated electrical signal in the electrode, whereby detection of an increased non-photogenerated electrical signal relative to a signal that would be detected in the absence of said target nucleic acid or said nanoparticle indicates the presence or amount of target nucleic acid hybridized to the electrode.

2. The method of claim 1, wherein providing the hybridization complex comprises:

hybridizing a target nucleic acid to at least one capture probe to form a capture probe-target nucleic acid complex; and

hybridizing a detection probe to the capture probe-target nucleic acid complex to form the hybridization complex, wherein the detection probe comprises the nanoparticle.

3. The method of claim 1, wherein the target nucleic acid comprises RNA.

4. The method of claim 1, wherein the target nucleic acid comprises cDNA.

5. The method of claim 1, wherein the target nucleic acid is present in a biological sample.

6. The method of claim 1, wherein the electrode comprises a conducting material comprising one or more of metals and metal oxides.

7. The method according to claim 1, wherein the electrode comprises indium tin oxide.

8. The method according to claim 1, wherein the electrode is formed on a non-conducting solid substrate.

9. The method according to claim 1, wherein the nanoparticle comprises one or more of the group consisting of metals and metal oxides.

10. The method according to claim 9, wherein the nanoparticle comprises a metal comprising one or more of gold, silver, platinum and palladium.

11. The method according to claim 1, wherein the nanoparticle comprises gold.

12. The method according to claim 1, wherein the nanoparticle comprises silver.

13. The method according to claim 1, wherein the nanoparticle has a diameter from about 10 to about 20 nanometers.

15. The method according to claim 1, where the nanoparticle is attached to the target nucleic acid by one of the group consisting of a binding pair and complementary nucleic acids.

16. The method according to claim 1, where the nanoparticle is attached to the target nucleic acid by one of the group consisting of primer extension and ligation of a nanoparticle-labeled nucleic acid.

17. The method of claim 1, wherein the complex comprises a detection probe.

18. The method of claim 17, wherein the detection probe is attached to the target nucleic acid before, during, or after the target nucleic acid hybridizes to the capture probe.

19. The method of claim 1, comprising the sequential steps of hybridizing the target nucleic acid to the capture probe;

and then reacting the hybrid with a detection probe.

20. The method according to claim 2, wherein the detection probe further comprises an oligonucleotide attached to the nanoparticle.

21. The method according to claim 20, wherein the capture probe is complementary to a first target domain of the target nucleic acid, and the oligonucleotide of the detection probe is complementary to a second target domain of the target nucleic acid.

22. The method according to claim 2, wherein the detection probe further comprises one partner of a ligand-binding pair, and the target nucleic acid comprises the other partner of a ligand-binding pair.

23. The method according to claim 22, wherein one partner of the ligand-binding pair is streptavidin, and the other partner of the ligand binding pair is biotin.

24. The method according to claim 22, wherein the target nucleic acid comprises biotin.

25. The method according to claim 24, wherein the biotin has been incorporated into the target nucleic acid during nucleic acid amplification.

26. The method according to claim 22, wherein the detection probe comprises streptavidin.

27. The method according to claim 1, wherein the redox mediator comprises EDTA.

28. The method according to claim 1, wherein the redox mediator comprises ferrocene.

29. The method according to claim 1, wherein the electrical signal is electrical current, and the detecting step is carried out by cyclic voltammetry.

30. The method according to claim 1, wherein the detecting step is carried out by chronoamperometry.

31. The method according to claim 1, wherein a plurality of different capture probes is attached to the electrode in an array.

32. The method according to claim 1, wherein the redox solution further comprises a sacrificial electron donor.

33. The method according to claim 32, wherein the sacrificial electron donor comprises EDTA.

34. The method according to claim 1, wherein the target nucleic acid is selected from the group consisting of an mRNA sequence derived from a biological sample and a cDNA sequence derived from a biological sample.

35. The method according to claim 34, wherein an indication of hybridization complex formation is indicative of gene expression or a gene expression level.

36. The method according to claim 1, wherein the capture probe comprises a nucleic acid from a gene of interest.

37. The method according to claim 1, wherein a redox reaction catalyzed by the nanoparticle generates electron transfer to the electrode, resulting in a detectable electrical signal in the electrode.

38. The method according to claim 1, wherein:

the nanoparticle comprises platinum;

the redox solution comprises water;

the nanoparticle oxidizes the water, wherein the oxidation generates electrons; and

the electrons are transferred to the electrode, resulting in a detectable electrical signal in the electrode.

39. The method of claim 1, wherein the nanoparticle is free of a photochemically active moiety.

14. (canceled)

40. A method of detecting a target nucleic acid, comprising:

providing a hybridization complex comprising (a) a capture probe that is attached to an electrode surface directly or via an attachment linker;

(b) a target nucleic acid that is hybridized to the capture probe; and

(c) at least one nanoparticle attached to the target nucleic acid;

removing unhybridized nucleic acid;

contacting the electrode with a redox solution comprising a redox mediator and an electrolyte, such that the complex is in contact with the redox solution; and

detecting an electrical signal in the electrode, whereby detection of an increased electrical signal relative to a signal that would be detected in the absence of said target nucleic acid or said nanoparticle indicates the presence or amount of target nucleic acid hybridized to the electrode, wherein the method is free of exposing a photoelectrochemically active moiety to a laser.