AC methods for the detection of nucleic acids

• US 6,232,062 B1
• Filed: 08/14/1997
• Issued: 05/15/2001
• Est. Priority Date: 03/07/1997
• Status: Expired due to Term

• Current Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Original Assignee: Clinical Micro Sensors Inc. (Roche Holding AG)
• Inventors: Kayyem, Jon Faiz, OʼConnor, Stephen D.
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US08/911,589
• Time in Patent Office: 1,370 Days
• Field of Search: 435/6, 536/23.1, 536/24.3, 935/76, 935/77, 935/78, 436/501, 422/50, 422/68.1
• US Class Current: 435/6.11
• 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 target sequence by detecting the presence of 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.

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 spacer to a first electron transfer moiety comprising an electrode;

b) a target sequence; and

c) a second electron transfer moiety.

8. A method according to claims 1, 2, 3 or 4 wherein said detecting is accomplished by receiving an output signal comprising a current.

9. A method according to claims 1, 2, 3 or 4 wherein said detecting is accomplished by receiving an output signal comprising a voltage.

10. A method according to claims 1, 2, 3 or 4 wherein said detecting is accomplished by receiving an output signal comprising a phase shift between said input and said output signals.

11. A method according to claims 1, 2, 3 or 4 wherein said detecting is accomplished by receiving an output signal comprising 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 nucleic acid analog.

17. A method according to claim 15, wherein said spacer is a conductive oligomer.

18. A method according to claim 1, 2, 3 or 4 wherein said electrode further comprises a passivation agent monolayer.

19. A method according to claim 1, 2, 3 or 4 wherein said spacer is a conductive oligomer.

20. A method according to claim 1, 2, 3 or 4 wherein said spacer is an insulator.

21. A method according to claim 18 wherein said monolayer comprises insulators.

22. A method according to claim 18 wherein said monolayer comprises conductive oligomers.

23. A method according to claim 18 wherein said monolayer comprises a mixture of conductive oligomers and insulators.

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

a) a first single stranded nucleic acid covalently attached via a spacer to a first electron transfer moiety comprising an elecrode;

b) a target sequence; and

c) a second single stranded nucleic acid comprising a covalently attached second electron transfer moiety.

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

a) a single stranded nucleic acid covalently shed via a spacer to a first electron transfer moiety comprising an electrode; and

b) a target sequence comprising a covalently attached second electron transfer moiety.

27. A method according to claim 1, 2 or 3 further comprising applying input signal at a plurality of amplitudes.

29. A method according to claim 21 wherein said insulators comprise alkyl oligomers.

30. A method according to claim 22, wherein said conductive oligomers have the formula:

wherein

Y is an aromatic group;

n is an integer from 1 to 50;

g is either 1 or zero;

e is an integer from zero to 10; and

m is zero or 1;

wherein when g is 1, B-D is a conjugated bond; and

wherein when g is zero, e is 1 and D is preferably carbonyl, or a heteroatom moiety,

wherein the heteroatom is selected from oxygen, sulfur, nitrogen, silicon or phosphorus.

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 a second frequency and a non-zero DC component to said hybridization complex; and

c) detecting the presence of said target sequence by detecting the presence of 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 target sequence by detecting the presence of 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 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 said AC component at a second voltage amplitude and a non-zero DC component to said hybridization complex; and

c) detecting the presence of said target sequence by detecting the presence of said hybridization complex.

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

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

a) applying an 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, wherein said electrode comprises a passivation agent monolayer; and

ii) a second electron transfer moiety; and

b) detecting the presence of said target sequence by detecting the presence of said hybridization complex.

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

a) applying an input signal comprising an AC component and a non-zero DC component to a hybridization complex, said hybridization complex comprising;

i) a first single stranded nucleic acid covalently attached via a spacer to an electrode, said electrode comprising a passivation agent monolayer;

ii) a target sequence; and

iii) a second single stranded nucleic acid comprising a covalenty attached second electron transfer moiety; and

b) detecting the presence of said target sequence by detecting the presence of said hybridization complex.