Methods of detecting nucleic acids using electrodes

• US 6,221,583 B1
• Filed: 07/24/1997
• Issued: 04/24/2001
• Est. Priority Date: 11/05/1996
• Status: Expired due to Term

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Clinical Micro Sensors Inc. (Roche Holding AG)
• Inventors: Kayyem, Jon Faiz, Meade, Thomas J., Yu, Changjun, Gozin, Michael, OʼConnor, Stephen D.
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/899,510
• Time in Patent Office: 1,370 Days
• Field of Search: 435/6, 435/810, 422/50, 422/68.1, 436/501, 935/77, 935/78
• 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 of detecting a target nucleic acid sequence in a nucleic acid sample comprising:

a) hybridizing a probe nucleic acid to said target sequence, if present, to form a hybridization complex, wherein said probe nucleic acid is covalently attached to a first electron transfer moiety comprising an electrode, wherein said probe nucleic acid is covalently attached to said electrode via a conductive oligomer, and wherein said hybridization complex comprises a second electron transfer moiety; and

b) detecting electron transfer between said electrode and said second electron transfer moiety, as an indicator of the presence or absence of said target sequence.

2. A method according to claim 1 wherein said conductive oligomer has 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 selected from acetylene, alkene, substituted alkene, amide, azo, esters, thioesters, —

CH═

N—

, —

CR═

N—

, —

N═

CH— and

—

N═

CR—

, —

SiH═

SiH—

, —

SiR═

SiH—

, —

SiR═

SiH—

, and —

SiR═

SiR—

, —

SiH═

CH—

, —

SiR═

CH—

, —

SiH═

CR—

, —

SiR═

CR—

, —

CH═

SiH—

, —

CR═

SiH—

, —

CH═

SiR—

, and —

CR═

SiR—

, wherein R is a substitution group; and

wherein when g is zero, e is 1 and D is carbonyl or a moiety comprising oxygen, sulfur, nitrogen or phosphorus.

3. A method according to claim 1 wherein said conductive oligomer has the formula:

wherein

n is an integer from 1 to 50;

m is 0 or 1;

C is carbon or substituted carbon when G is a single or double bond;

J is carbonyl or a heteroatom moeity, wherein the heteroatom is selected from the group consisting of nitrogen, silicon, phosphorus, sulfur; and

G is a bond selected from single, double or triple bonds, wherein when G is a single bond, two R groups are attached to each C, and when G is a double bond, one R group is attached to each C, wherein R is a substitution group.

4. A method according to claim 1 wherein said second electron transfer moiety is covalently attached to said probe nucleic acid.

5. A method according to claim 1 wherein said second electron transfer moiety is covalently attached to said target sequence.

6. A method according to claim 1 wherein said probe nucleic acid comprises a nucleic acid analog.

7. A method according to claim 1 wherein said covalent attachment of said conductive oligomer to said probe nucleic acid is to a base of said probe nucleic acid.

8. A method according to claim 1 wherein said probe nucleic acid comprises a ribose-phosphate backbone and said covalent attachment of said conductive oligomer to said probe nucleic acid is to a ribose of said probe nucleic acid.

9. A method according to claim 1 wherein said probe nucleic acid comprises a ribose-phosphate backbone and said covalent attachment of said conductive oligomer to said probe nucleic acid is to a phosphate of said probe nucleic acid.

10. A method according to claim 1 wherein said covalent attachment of said second electron transfer moiety to said probe nucleic acid is to a base of said probe nucleic acid.

11. A method according to claim 1 wherein said probe nucleic acid comprises a ribose-phosphate backbone and said covalent attachment of said second electron transfer moiety to said probe nucleic acid is to a ribose of said probe nucleic acid.

12. A method according to claim 1 wherein said probe nucleic acid comprises a ribose-phosphate backbone and said covalent attachment of said second electron transfer moiety to said probe nucleic acid is to a phosphate of said probe nucleic acid.

13. A method according to claim 1 wherein said electrode further comprises at least one passivation agent.

14. A method according to claim 1 wherein said electrode further comprises a monolayer of passivation agents.

15. A method according to claim 1 wherein said second electron transfer moiety comprises a transition metal complex.

16. A method according to claim 15 wherein said transition metal complex is a metallocene.

17. A method according to claim 16 wherein said metallocene is ferrocene.

18. A method according to claim 1 wherein said second electron transfer moiety comprises an organic electron transfer moiety.

19. A method according to claim 2 wherein Y is phenyl or substituted phenyl.

20. A method according to claim 2 or 19 wherein g is 1 and B—

D is acetylene.

21. A method according to claim 2 wherein n+m is four.

22. A method according to claim 2 wherein n+m is five.