ELECTROCHEMICAL LABELS DERIVED FROM SIDEROPHORES

• US 20090246763A1
• Filed: 03/31/2008
• Published: 10/01/2009
• Est. Priority Date: 03/31/2008
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Eason, Robert G.
• Granted Patent: US 8,492,158 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 15/26 Acyclic or carbocyclic radicals, substituted by hetero rings
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label

Abstract

Disclosed are tri-nuclear metal complexes and salts thereof, such as tri-nuclear osmium or ruthenium complexes or salts thereof, suitable for use as electrochemical labels. Also disclosed are oligonucleotide probes with an attached electrochemical label, methods of nucleic acid amplification, methods of sequencing, and kits for nucleic acid amplification and sequencing having oligonucleotide probes including an electrochemical label. The electrochemical labels are synthesized from siderophores.

Claims

1. A tri-nuclear metal complex comprising structure (I), structure, (II), structure (III) or structure (IV), or a salt thereof:

2. The tri-nuclear metal complex as set forth in claim 1, wherein A is Os.

3. The tri-nuclear metal complex as set forth in claim 1, wherein the complex has an E_1/2 value of from about −

200 to about 500 mV vs. Ag/AgCl.

4. The tri-nuclear metal complex as set forth in claim 1, wherein the tri-nuclear complex is conjugated to an oligonucleotide.

5. The tri-nuclear metal complex as set forth in claim 1, wherein the counterion is selected from the group consisting of PF₆⁻

, TFA⁻

, OAc⁻

, Cl⁻

and Br⁻

.

6. The tri-nuclear metal complex as set forth in claim 1, wherein the tri-nuclear metal complex has the structure (I′

), or a salt thereof;

7. The tri-nuclear metal complex as set forth in claim 1, wherein the tri-nuclear metal complex has the structure (II′

), or a salt thereof;

8. The tri-nuclear metal complex as set forth in claim 1, wherein the tri-nuclear metal complex has the structure (III′

), or a salt thereof;

9. The tri-nuclear metal complex as set forth in claim 1, wherein the tri-nuclear metal complex has the structure (IV′

), or a salt thereof;

10. A probe comprising an oligonucleotide labeled with an electrochemical label, the probe comprising structure (V), structure (VI), structure (VII) or structure (VIII), or a salt thereof:

11. The probe as set forth in claim 10, wherein A is Os.

12. The probe as set forth in claim 10, wherein the electrochemical label has an E_1/2 value of from about −

200 to about 500 mV vs. Ag/AgCl.

13. The probe as set forth in claim 10, wherein the counterion is selected from the group consisting of PF₆⁻

, TFA⁻

, OAc⁻

, Cl⁻

and Br⁻

.

14. The probe as set forth in claim 10, wherein the probe is cleavable.

15. A nucleic acid amplification method, the method comprising:

providing a probe comprising an oligonucleotide labeled with an electrochemical label;

displacing the electrochemical label from the oligonucleotide probe during amplification; and

detecting the displaced electrochemical label;

wherein the probe comprises structure (V), structure (VI), structure (VII), or structure (VIII), or a salt thereof;

16. The method of claim 15, wherein the detecting is done electrochemically.

17. The method of claim 15, wherein the detecting is done by electrochemiluminescence.

18. The method of claim 15, wherein the detecting is done by fluorescence.

19. A kit for polynucleotide amplification, the kit comprising:

a first primer and a second primer;

a polymerase; and

a probe comprising an oligonucleotide labeled with an electrochemical label, the probe comprising structure (V), structure (VI), structure (VII), or structure (VIII), or a salt thereof;

20. The kit as set forth in claim 19, wherein the probe is cleavable.

21. A method of preparing a tri-nuclear metal complex, the method comprising:

reacting a siderophore with a bipyridinyl osmium or ruthenium carbonato complex or salt thereof.

22. The method as set forth in claim 21, wherein the tri-nuclear metal complex is an tri-nuclear osmium (II) complex and the bipyrimidal osmium complex is bis(2,2′

-bipyridinyl)carbonatoosmium(II).

23. The method as set forth in claim 21, wherein the siderophore is selected from the group consisting of desferrioxamine B, desferrichrome A, desferrirhodin, and salmochelin 2.