Electrochemical detection of nucleic acid sequences

• US 6,391,558 B1
• Filed: 04/14/2000
• Issued: 05/21/2002
• Est. Priority Date: 03/18/1997
• Status: Expired due to Term

• Current Assignee: Magellan Diagnostics, Inc. (Meridian Bioscience, Inc.)
• Original Assignee: Andcare Incorporated
• Inventors: Henkens, Robert W., Wegner, Steven E., Stewart, Thomas N., Sundseth, Rebecca, Wojciechowski, Marek, Naser, Najih, OʼDaly, John P., Zhang, Honghua, Roe, R. Michael, Thompson, Deborah M.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/549,853
• Time in Patent Office: 767 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 422/68.1, 422/50, 422/62, 422/63, 422/67, 422/69, 422/82.01
• US Class Current: 435/6.11
• 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 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

An electrochemical detection system which specifically detects selected nucleic acid segments is described. The system utilizes biological probes such as nucleic acid or peptide nucleic acid probes which are complementary to and specifically hybridize with selected nucleic acid segments in order to generate a measurable current when an amperometric potential is applied. The electrochemical signal can be quantified.

Claims

1. A method of detecting a target molecule having at least one selected nucleic acid or protein segment, comprising:

(a) interacting a bioreporter molecule attached to a working electrode surface with the target molecule to form a complex;

(b) adding a reporter molecule that can be electrochemically oxidized or reduced upon interaction with said complex;

(c) applying a pulse of potential to the working electrode that electrochemically oxidizes or reduces the reporter molecule;

(d) repeating step (c); and

(e) measuring the current close to the end of step (c) wherein said current is indicative of the presence of the target molecule.

2. The method of claim 1 wherein the detecting is with an array of sensors, each sensor comprising a potentiostat, a working electrode with attached target molecule and a reference electrode comprised within a suitable circuit.

3. The method of claim 1 wherein step (e) is repeated and an average or summation signal is determined.

4. The method of claim 1 wherein each sensor of an array of sensors comprises a working electrode and a reference electrode comprised within a suitable circuit.

5. The method of claim 1 further comprising determining a concentration of said target molecule comprising relating a signal from a standard concentration of the target molecule to the signal determined from the target molecule in a sample.

6. The method of claim 1 wherein the intermittent pulse is applied for a period of from 0.1 millisecond to about 100 milliseconds.

7. The method of claim 1 wherein intermittent pulse separation time is from 1 millisecond to about 10 seconds.

13. The method of claim 1 or claim 8 wherein the nucleic acid target molecule is an oligonucleotide selected from the group consisting of DNA, RNA or a peptide nucleic acid.

14. The method of claim 1 or claim 8 wherein the protein target molecule is a protein, peptide or antigen.

15. The method of claim 1 or 8 for detecting a nucleic acid target molecule wherein the bioreporter molecule is complementary to a first selected region of the target nucleic acid segment attached to the surface of the working electrode and wherein the reporter molecule complementary to a second selected region of the target nucleic acid segment is labeled.

16. The method of claim 15 wherein the bioreporter molecule is attached to the working electrode by adsorption, crosslinking, covalent bonding, or charge-charge interaction.

17. The method of claim 16 wherein the bioreporter molecule is attached to the working electrode with avidin, streptavidin, protein G, protein A, neutravidin or antibody.

18. The method of claim 15 wherein the reporter molecule is labeled with fluorescein, digoxigenin, HRP or alkaline phosphatase.

19. The method of claim 15 further comprising:

(a) incubating the target molecule and the bioreporter molecule under conditions to form a primer extended molecule; and

(b) detecting the presence of the primer extended molecule.

20. The method of claim 19 wherein the primer extended molecule is labeled.

21. The method of claim 19 wherein a single primer is used.

22. The method of claim 19 wherein the bioreporter is extended before binding to the working electrode.

23. The method of claim 15 wherein the bioreporter has a blocked 3′

-end.

24. The method of claim 15 wherein the detector probe comprises a 3′

-hydroxyl group.

25. The method of claim 1 or claim 8 wherein the target molecule is a nucleic acid obtained from a DNA sequence comprising a deletion, insertion or single base alteration.

26. The method of claim 19 wherein the primer extended molecule is formed by enzyme catalysis.

27. The method of claim 26 wherein the enzyme is a DNA polymerase, an RNA polymerase or a reverse transcriptase.

8. A method of detecting a target molecule having at least one selected nucleic acid or protein segment, comprising:

(a) interacting a bioreporter molecule attached to a working electrode surface with the target molecule to form a complex;

(b) adding a reporter molecule that can be electrochemically reduced or oxidized upon interaction with said complex;

(c) applying a first potential to the working electrode at or close to the open circuit potential;

(d) measuring current close to the end of step (c);

(e) applying a second potential or potential pulse to the working electrode that electrochemically oxidizes or reduces the reporter molecule at or near the electrode surface;

(f) measuring a current close to the end of step (e); and

(g) determining a signal by subtracting the current measured in step (d) from the current measured in step (f);

wherein the signal indicates the presence of said target molecule.

9. The method of claim 8 wherein steps (c) through (g) are repeated and a signal is determined from individual signals determined in step (g) by averaging or summation.

10. The method of claim 8 further comprising determining a concentration of said target molecule comprising relating a signal from a standard concentration of the target molecule to the signal determined from the target molecule in a sample.

11. The method of claim 8 wherein the detecting is with an array of sensors, each sensor comprising a potentiostat, the working electrode with attached target molecule and a reference electrode comprised within a suitable circuit.

12. The method of claim 8 wherein the potential pulse separation time is from 1 millisecond to about 10 seconds.