Electrocatalytic nucleic acid hybridization detection

• US 20050084881A1
• Filed: 08/06/2004
• Published: 04/21/2005
• Est. Priority Date: 05/13/2003
• Status: Active Grant

• Current Assignee: Trustees of Boston College (Boston College)
• Original Assignee: Trustees of Boston College (Boston College)
• Inventors: Taft, Bradford, Kelley, Shana, Gasparac, Rahela, Lapierre-Devlin, Melissa
• Granted Patent: US 7,741,033 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: G01N 27/3278 involving nanosized elements, e.g. nanogaps or nanoparticles nanopores G01N33/48721; magnetic beads G01N27/745

Abstract

The detection of specific nucleic acid sequences using electrochemical readout would permit the rapid and inexpensive detection and identification of bacterial pathogens and the analysis of human genes. A new assay developed for this purpose is described that harnesses an electrocatalytic process to monitor nucleic acid hybridization. Furthermore, the new assay when used on nanoscale electrodes, provides ultrasensitive detection of nucleic acids.

Claims

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

(a) providing a device for detecting the presence of the target nucleic acid, said device comprising a nanoelectrode, wherein the nanoelectrode comprises at least one metallic nanowire embedded within a polycarbonate membrane and a nucleic acid probe attached to the metallic nanowire;

(b) contacting the nanoelectrode with the sample under a hybridization condition; and

(c) measuring an electrocatalytic signal generated by hybridization of the nucleic acid probe and the target nucleic acid in the sample, wherein an increase of the signal detected relative to a signal of a control sample comprising no target nucleic acid is indicative of the presence of the target nucleic acid in the sample.

2. The method of claim 1, wherein the metallic nanowire comprise gold.

3. The method of claim 1, wherein the nucleic acid probe comprises DNA.

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

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

6. The method of claim 1, wherein the nucleic acid probe has a sequence that is complementary to the target nucleic acid sequence.

8. The method of claim 1, wherein the first transition metal complex comprises a metal selected from the group consisting of cobalt, iron, molybdenum, osmium, ruthenium and rhenium, and wherein the second transition metal complex comprises a metal selected from the group consisting of iron, cobalt, molybdenum, iridium, osmium and rhenium.

9. The method of claim 8, wherein the first transition metal complex is a transition metal ammonium complex, and wherein the second transition metal complex is a transition metal cynate or chloride complex.

7. A method of detecting nucleic acid hybridization between a nucleic acid probe and a target nucleic acid in a sample, comprising:

(a) providing a nucleic acid probe immobilized on nanoelectrode ensembles;

(b) contacting under hybridization conditions the nanoelectrode ensembles and the immobilized nucleic acid probe to the sample, and a redox pair comprising a first transition metal complex and a second transition metal complex; and

(c) measuring a electroncatalytic signal generated by hybridization of the nucleic acid probe and the target nucleic acid in the sample, wherein an increase of the signal detected relative to a signal of a control sample comprising no target nucleic acid, indicates that the nucleic acid hybridization has occurred.

10. The method of claim 7, further comprising an additional step of contacting under hybridization conditions the nanoelectrode ensembles and the immobilized nucleic acid probe to a solution containing no sample, and a redox pair comprising a first transition metal complex and a second transition metal complex.

11. The method of claim 7, the nanoelectrode ensembles comprise a gold nanowire.

12. A method of detecting nucleic acid hybridization between a first nucleic acid and a second nucleic acid, comprising:

(a) providing the first nucleic acid immobilized on nanoelectrode ensembles;

(b) contacting under hybridization conditions the nanoelectrode ensembles and the immobilized first nucleic acid to a solution suspected of containing the second nucleic acid, and a redox pair comprising a first transition metal complex and a second transition metal complex; and

(c) measuring a electroncatalytic signal generated by hybridization of the first nucleic acid and the second nucleic acid, wherein an increase of the signal detected relative to a signal of a control sample comprising no second nucleic acid, indicates that the nucleic acid hybridization has occurred.

13. The method of claim 12, wherein the first transition metal complex comprises a metal selected from the group consisting of cobalt, iron, molybdenum, osmium, ruthenium and rhenium, and wherein the second transition metal complex comprises a metal selected from the group consisting of iron, cobalt, molybdenum, iridium, osmium and rhenium.

14. The method of claim 13, wherein the first transition metal complex is a transition metal ammonium complex, and wherein the second transition metal complex is a transition metal cynate or chloride complex.

15. The method of claim 12, further comprising an additional step of contacting under hybridization conditions the nanoelectrode ensembles and the immobilized nucleic acid probe to a solution containing no sample, and a redox pair comprising a first transition metal complex and a second transition metal complex.

16. The method of claim 12, wherein the nanoelctrode ensembles comprise a gold nanowire.

17. A method of detecting nucleic acid hybridization between a nucleic acid probe and a target nucleic acid in a sample, comprising:

(a) providing a nucleic acid probe immobilized on nanoelectrode ensembles;

(b) contacting under hybridization conditions the nanoelectrode ensembles and the immobilized nucleic acid probe to the sample, and a redox pair comprising a first transition metal complex and a redox-active probe comprising an ascorbic acid or tripropylamine; and

(c) measuring a electroncatalytic signal generated by hybridization of the nucleic acid probe and the target nucleic acid in the sample, wherein an increase of the signal detected relative to a signal of a control sample comprising no target nucleic acid, indicates that the nucleic acid hybridization has occurred.

18. The method of claim 17, wherein the first transition metal complex comprises a metal selected from the group consisting of cobalt, iron, molybdenum, osmium, ruthenium and rhenium.

19. The method of claim 18, wherein the first transition metal complex is a transition metal ammonium complex.

20. The method of claim 17, further comprising an additional step of contacting under hybridization conditions the nanoelectrode ensembles and the immobilized nucleic acid probe to a solution containing no sample, and a redox pair comprising a first transition metal complex and a redox-active probe comprising an ascorbic acid or tripropylamine.

21. The method of claim 17, the nanoelectrode ensembles comprise a gold nanowire.

22. A method of detecting nucleic acid hybridization between a first nucleic acid and a second nucleic acid, comprising:

(a) providing the first nucleic acid immobilized on nanoelectrode ensembles;

(b) contacting under hybridization conditions the nanoelectrode ensembles and the immobilized first nucleic acid to a solution suspected of containing the second nucleic acid, and a redox pair comprising a first transition metal complex and a redox-active probe comprising an ascorbic acid or tripropylamine; and

(c) measuring a electroncatalytic signal generated by hybridization of the first nucleic acid and the second nucleic acid, wherein an increase of the signal detected relative to a signal of a control sample comprising no second nucleic acid, indicates that the nucleic acid hybridization has occurred.

23. The method of claim 22, wherein the first transition metal complex comprises a metal selected from the group consisting of cobalt, iron, molybdenum, osmium, ruthenium and rhenium.

24. The method of claim 23, wherein the first transition metal complex is a transition metal ammonium complex.

25. The method of claim 22, further comprising an additional step of contacting under hybridization conditions the nanoelectrode ensembles and the immobilized nucleic acid probe to a solution containing no sample, and a redox pair comprising a first transition metal complex and a redox-active probe comprising an ascorbic acid or tripropylamine.

26. The method of claim 22, wherein the nanoelctrode ensembles comprise a gold nanowire.