AC methods for the detection of nucleic acids

• US 20030148328A1
• Filed: 09/11/2002
• Published: 08/07/2003
• Est. Priority Date: 11/05/1996
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Jon Faiz Kayyem, Oʼconnor, Stephen D.
• Inventors: Kayyem, Jon Faiz, OʼConnor, Stephen D.
• Granted Patent: US 7,056,669 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors

Abstract

The invention relates to nucleic acids covalently coupled to electrodes via conductive oligomers. More particularly, the invention is directed to the site-selective modification of nucleic acids with electron transfer moieties and electrodes to produce a new class of biomaterials, and to methods of making and using them.

Claims

1. A method for detecting the presence of a target sequence in a nucleic acid sample, comprising

a) applying a first input signal comprising an AC component and a non-zero DC component to a hybridization complex, said hybridization complex comprising at least a target sequence and a first probe single stranded nucleic acid, said hybridization complex being covalently attached to:

i) a first electron transfer moiety comprising an electrode; and

ii) a second electron transfer moiety; and

b) detecting the presence of said hybridization complex by receiving an output signal characteristic of electron transfer through said hybridization complex.

6. A method according to claims 1, 2, 3 or 4, wherein said hybridization complex comprises

a) a single stranded nucleic acid covalently attached to:

1) a first electron transfer moiety comprising an electrode; and

2) a second electron transfer moiety; and

b) a target sequence hybridized to said single stranded nucleic acid.

7. A method according to claims 1, 2, 3 or 4 wherein said hybridization complex comprises:

a) a single stranded nucleic acid covalently attached via a conductive oligomer to a first electron transfer moiety comprising an electrode; and

b) a target sequence hybridized to said single stranded nucleic acid; and

i) a second electron transfer moiety.

8. A method according to claims 1, 2, 3 or 4 wherein said output signal comprises a current.

9. A method according to claims 1, 2, 3 or 4 wherein said output signal comprises a voltage.

10. A method according to claims 1, 2, 3 or 4 wherein said output signal comprises a phase shift between said input and said output signals.

11. A method according to claims 1, 2, 3 or 4 wherein said output signal comprises a change in the faradaic impedance between the electron transfer moieties as a result of the formation of the hybridization complex.

12. A method according to claims 1, 2, 3 or 4 further comprising adding said target sequence to said single stranded nucleic acid to form said hybridization complex.

13. A method according to claims 1, 3 or 4 further comprising applying input signal at a plurality of frequencies.

14. A method according to claims 1, 2 or 4 further comprising applying signal at a plurality of DC voltages.

15. A method according to claims 1, 2, 3 or 4 wherein said single-stranded nucleic acid is covalently attached to said electrode via a spacer.

16. A method according to claims 1, 2, 3 or 4 wherein said single-stranded nucleic acid is a peptide nucleic acid.

17. A method according to claim 16, wherein said spacer is a conductive polymer.

18. A method according to claim 16, wherein said conductive polymer has the formula:

19. A method according to claim 16, wherein said electrode further comprises a monolayer.

2. A method for detecting the presence of a target sequence in a nucleic acid sample, comprising

a) applying a first input signal comprising an AC component at a first frequency and a non-zero DC component to a hybridization complex, said hybridization complex comprising at least a target sequence and a first probe single stranded nucleic acid, said hybridization complex being covalently attached to:

i) a first electron transfer moiety comprising an electrode; and

ii) a second electron transfer moiety;

b) applying a second input signal comprising an AC component at least a second frequency and a non-zero DC component to said hybridization complex; and

c) detecting the presence of said hybridization complex by receiving an output signal characteristic of electron transfer through said hybridization complex.

3. A method for detecting the presence of a target sequence in a nucleic acid sample, comprising

a) applying a first input signal comprising an AC component and a first non-zero DC component to a hybridization complex, said hybridization complex comprising at least a target sequence and a first probe single stranded nucleic acid, said hybridization complex being covalently attached to:

i) a first electron transfer moiety comprising an electrode; and

ii) a second electron transfer moiety;

b) applying a second input signal comprising said AC component and at least a second non-zero DC component to said hybridization complex; and

c) detecting the presence of said hybridization complex by receiving an output signal characteristic of electron transfer through said hybridization complex.

4. A method for detecting the presence of a target sequence in a nucleic acid sample, comprising

a) applying a first input signal comprising an AC component at a first voltage amplitude

to a hybridization complex, said hybridization complex comprising at least a target sequence and a first probe single stranded nucleic acid, said hybridization complex being covalently attached to:

i) a first electron transfer moiety comprising an electrode; and

ii) a second electron transfer moiety;

b) applying a second input signal comprising said AC component at a second voltage amplitude to said hybridization complex; and

c) detecting the presence of said hybridization complex by receiving an output signal characteristic of electron transfer through said hybridization complex.

5. A method according to claim 4 wherein said input signals further comprise a non-zero DC component.

20. An apparatus for the detection of target nucleic acids in a test sample, comprising:

a) a test chamber comprising a first and a second measuring electrode, wherein said first measuring electrode comprises a covalently attached conductive oligomer covalently attached to a single stranded nucleic acid; and

b) an AC/DC voltage source electrically connected to said test chamber.

22. An apparatus according to claim 20 or 21, further comprising:

d) a processor coupled to said electrodes.

23. An apparatus according to claim 20 or 21, wherein said AC voltage source is capable of delivering frequencies from between about 1 Hz to about 100 kHz.

24. An apparatus according to claim 23, wherein said single stranded nucleic acids are covalently attached to said electrode via a spacer.

25. An apparatus according to claim 24, wherein said spacer is a conductive oligomer.

26. An apparatus according to claim 20 or 25, wherein said conductive oligomer has the formula:

21. An apparatus for the detection of target nucleic acids in a test sample, comprising:

a) a test chamber comprising a first and a second measuring electrode, wherein said first measuring electrode comprises a covalently attached single stranded nucleic acid comprising a covalently attached second electron transfer moiety; and

b) an AC/DC voltage source electrically connected to said test chamber.