System and method for electrochemical detection of biological compounds

• US 8,105,477 B2
• Filed: 10/22/2010
• Issued: 01/31/2012
• Est. Priority Date: 04/16/2003
• Status: Active Grant

• Current Assignee: HandyLab Incorporated (Becton, Dickinson & Co)
• Original Assignee: HandyLab Incorporated (Becton, Dickinson & Co)
• Inventors: Althaus, John S., Kyonghoon, Lee, Namasivayam, Vijay, Brahmasandra, Sundaresh N., Handique, Kalyan
• Primary Examiner(s): Barton, Jeffrey T
• Assistant Examiner(s): DIETERLE, JENNIFER M
• Application Number:
  US12/910,112
• Publication Number:
  US 20110039345A1
• Time in Patent Office: 466 Days
• Field of Search: 204400-40315, 204/600, 204/450, 205/777.5, 205/775, 205/787, 205/792, 205/793.5, 435/6, 435/6.11, 435/6.12, 435/7.1, 435/287.2, 436/94, 436/86, 436/2, 422/68.1, 422/82.01
• US Class Current: 205/792
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates to an electrochemical method for detecting a target polynucleotide. An electrode comprising an electrode surface is provided. The electrode surface includes at least one probe molecule reversibly immobilized with respect to the electrode surface. A first electrochemical signal indicative of an amount of probe molecule immobilized with respect to the electrode surface is obtained. The electrode surface is contacted with a liquid comprising the target polynucleotide. Upon the contacting step, at least some of the probe molecule immobilized with respect to the electrode surface dissociates therefrom. A second electrochemical signal indicative of an amount of probe molecule immobilized with respect to the electrode surface is obtained. The presence of the target polynucleotide is determined at least partially on the basis of the first and second electrochemical signals.

Claims

1. An electrochemical method for detecting a target polynucleotide, comprising:

obtaining a first electrochemical signal, a portion of the first electrochemical signal arising from a first amount of a probe molecule reversibly immobilized on an electrode and not intercalated with the target polynucleotide;

increasing or decreasing an amount of the target polynucleotide in fluid communication with the first amount of the probe molecule, wherein the probe molecule preferentially associates with the target polynucleotide over the electrode;

obtaining a second electrochemical signal, a portion of the second electrochemical signal arising from a second amount of the probe molecule reversibly immobilized on the electrode and not intercalated with the target polynucleotide; and

determining a difference between the first and second electrochemical signals.

2. The electrochemical method of claim 1, wherein the first and second electrochemical signals are obtained using an electrode and the second electrochemical signal is obtained without contacting the electrode with fresh probe molecule intermediate obtaining the first and second electrochemical signals.

3. The electrochemical method of claim 1, wherein the second electrochemical signal is substantially free of a portion arising from an oxidation or reduction of guanine residues, if present, of the target polynucleotide.

4. The electrochemical method of claim 1, wherein, upon increasing or decreasing an amount of target polynucleotide, a portion Δ

of the first amount of probe molecule intercalates with the target polynucleotide, the second electrochemical signal being different from the first electrochemical signal by an amount indicative of Δ

.

5. The electrochemical method of claim 4, wherein the first and second electrochemical signals are substantially free of an electrochemical signal from the intercalated probe molecule.

6. The electrochemical method of claim 1, wherein the probe molecule is substantially free of polynucleotides having a length of at least 8 bases.

7. The electrochemical method of claim 6, wherein the first and second electrochemical signals arise from an electrochemically active moiety of the probe molecule that is free of purines.

8. The electrochemical method of claim 7, wherein the probe molecule is selected from the group consisting of daunomycin, doxorubicin, methylene blue, toluidine blue O, azure A, azure B, azure C, and thionin.

9. The electrochemical method of claim 7, wherein the probe molecule comprises at least three 6 membered rings.

10. The electrochemical method of claim 7, wherein the probe molecule comprises at least two cyclic groups.

11. The electrochemical method of claim 10, wherein the probe molecule comprises an anthracycline, methylene blue, or derivative thereof.

12. The electrochemical method of claim 7, wherein the probe molecule comprises (a) a polynucleotide having a sequence sufficiently complementary to a sequence of the target polynucleotide to form a duplex therewith and (b) an electrochemically active moiety that is free of purines, the first and second electrochemical signals arising from the electrochemically active moiety.

13. The electrochemical method of claim 12, further comprising detecting a second target polynucleotide, the method comprising:

obtaining a third electrochemical signal, a portion of the third electrochemical signal arising from a first amount of a second probe molecule reversibly immobilized on an electrode and not intercalated with the target polynucleotide or the second target polynucleotide, the second probe molecule comprising (a) a polynucleotide having a sequence sufficiently complementary to a sequence of the second target polynucleotide to form a duplex therewith and different from the sequence of the polynucleotide of the probe molecule and (b) an electrochemically active moiety that is free of purines;

increasing or decreasing an amount of the second target polynucleotide in fluid communication with the first amount of the second probe molecule; and

obtaining a fourth electrochemical signal, a portion of the fourth electrochemical signal arising from a second amount of the second probe molecule reversibly immobilized on an electrode and not intercalated with either the target polynucleotide or the second target polynucleotide.

14. The electrochemical method of claim 13, wherein the second probe molecule is selected from the group consisting of daunomycin, doxorubicin, methylene blue, toluidine blue O, azure A, azure B, azure C, and thionin.

15. The electrochemical method of claim 13, wherein the third and fourth electrochemical signals are obtained using an electrode and the fourth electrochemical signal is obtained without contacting the electrode with fresh second probe molecule intermediate obtaining the third and fourth electrochemical signals.

16. The electrochemical method of claim 13, wherein the fourth electrochemical signal is substantially free of a portion arising from an oxidation or reduction of guanine residues, if present, of the second target polynucleotide.

17. The electrochemical method of claim 13, wherein the second probe molecule is substantially free of polynucleotides having a length of at least 8 bases.

18. The electrochemical method of claim 17, wherein the third and fourth electrochemical signals arise from an electrochemically active moiety of the second probe molecule that is free of purines.