Fluorescent methods and materials for directed biomarker signal amplification

• US 8,158,444 B2
• Filed: 10/08/2007
• Issued: 04/17/2012
• Est. Priority Date: 10/06/2006
• Status: Active Grant

• Current Assignee: Sirigen, Inc. (Becton, Dickinson & Co)
• Original Assignee: Sirigen, Inc. (Becton, Dickinson & Co)
• Inventors: Gaylord, Brent S., Hong, Janice W., Fu, Tsu-Ju, Sun, Cheng-Jun, Baldocchi, Russell
• Primary Examiner(s): Haq, Shafiqul
• Application Number:
  US11/868,870
• Publication Number:
  US 20080293164A1
• Time in Patent Office: 1,653 Days
• Field of Search: None
• US Class Current: 436/546
• CPC Class Codes: C08G 2261/3142 fluorene-based, e.g. fluorene, indenofluorene, or spirobifluorene
• CPC Class Codes: C08G 2261/3221 containing one or more nitrogen atoms as the only heteroatom, e.g. pyrrole, pyridine or triazole
• CPC Class Codes: C08G 2261/3241 containing one or more nitrogen atoms as the only heteroatom, e.g. carbazole
• CPC Class Codes: C08G 2261/3246 containing nitrogen and sulfur as heteroatoms
• CPC Class Codes: C08G 2261/522 fluorescent
• CPC Class Codes: C08G 2261/78 Complexation
• CPC Class Codes: C08G 2261/94 Applications in sensors, e.g. biosensors
• CPC Class Codes: C08G 61/02 Macromolecular compounds containing only carbon atoms in the main chain of the macromolecule, e.g. polyxylylenes
• CPC Class Codes: C08G 61/12 Macromolecular compounds containing atoms other than carbon in the main chain of the macromolecule
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: H10K 85/115 Polyfluorene; Derivatives thereof
• CPC Class Codes: Y10T 436/13 Tracers or tags

Abstract

Methods and compositions are provided that include a multichromophore and/or multichromophore complex for identifying a target biomolecule. A sensor biomolecule, for example, an antibody can be covalently linked to the multichromophore. Additionally, a signaling chromophore can be covalently linked to the multichromophore. The arrangement is such that the signaling chromophore is capable of receiving energy from the multichromophore upon excitation of the multichromophore. Since the sensor biomolecule is capable of interacting with the target biomolecule, the multichromophore and/or multichromophore complex can provide enhanced detection signals for a target biomolecule.

Claims

1. A multichromophore complex comprising:

a multichromophore covalently bound by a unique bioconjugation site pendant to at least one biomolecule selected from the group consisting of a sensor biomolecule, a bioconjugate and a target biomolecule wherein the multichromophore is used as a reporter or further comprises

a signaling chromophore covalently bound to the multichromophore or the biomolecule;

wherein the multichromophore comprises the structure

2. The multichromophore complex of claim 1, wherein the multichromophore comprises a single bioconjugation site.

3. The multichromophore complex of claim 1, wherein the signaling chromophore is covalently bound to the multichromophore or the biomolecule.

4. The multichromophore complex of claim 3, wherein the signaling chromophore is covalently bound to the multichromophore.

5. The multichromophore complex of claim 3, wherein the signaling chromophore is covalently bound to the biomolecule.

6. The multichromophore complex of claim 3, wherein the signaling chromophore is an organic dye.

7. The multichromophore complex of claim 1, wherein the multichromophore is a water-soluble conjugated polymer.

8. The multichromophore complex of claim 3, wherein both the signaling chromophore and the biomolecule are covalently linked to the multichromophore through a central linking site that covalently binds the multichromophore, the signaling chromophore and the biomolecule.

9. The multichromophore complex of claim 8, wherein the multichromophore comprises a single central linking site which covalently binds the multichromophore, the signaling chromophore, and the biomolecule.

10. The multichromophore complex of claim 1, wherein the conjugated polymer is a charge-neutral conjugated polymer.

11. The multichromophore complex of claim 1, wherein the bioconjugation sites are selected from the group consisting of maleimide, thiol, succimidylester (NHS ester), amine, azide chemistry, carboxylate, carboxy/EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride, Sulfo-SMCC (Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate), amine/BMPH (N-[β

-Maleimidopropionic acid]hydrazide·

TFA), and Sulfo-SBED Sulfosuccinimidyl[2-6-(biotinamido)-2-(p-azidobenzamido)-hexanoamidol-ethyl-1,3′

-dithiopropionate.

12. The multichromophore complex of claim 1, wherein 1, 2, and 3 are independently a 7-conjugated unit selected from the group consisting of a, b, c, d, e, f, g, h, and i having the structure

13. The multichromophore complex of claim 1, wherein 1, 2, and 3 are independently a π

-conjugated unit selected from the group consisting of a, b, c, d, e, f, g, h, and i having the structure

14. The multichromophore complex of claim 13, wherein the linker (L) terminates with a bioconjugation site (A) selected from the group consisting of amine, maleimide, and carboxylate.

15. The multichromophore complex of claim 1, wherein the multichromophore further comprises the structure

16. The multichromophore complex of claim 1, wherein the multichromophore further comprises the structure

17. The multichromophore complex of claim 1, wherein the multichromophore further comprises the structure

18. The multichromophore complex of claim 1, wherein the multichromophore contains two or more unique bioconjugation sites.

19. The multichromophore complex of claim 1, wherein the multichromophore further comprises the structure

20. The multichromophore complex of claim 1, wherein the multichromophore further comprises the structure

21. The multichromophores of any one of claims 13, 14, 15, 16, and 17, further comprising a intermediary linker, which alters the pendant functionality on the multichromophore.

40. The multichromophore complex of claim 1, wherein the multichromophore comprises two bioconjugation sites.

41. The multichromophore complex of claim 10, wherein the charge-neutral conjugated polymer cannot be ionized.

22. A multichromophore complex for identifying a target biomolecule comprising:

(1) a multichromophore, wherein the multichromophore comprises the structure

23. The multichromophore complex of claim 22, wherein the multichromophore comprises a single bioconjugation site.

24. The multichromophore complex of claim 22, wherein the signaling chromophore is covalently bound to the multichromophore or to the sensor biomolecule.

25. The multichromophore complex of claim 24, wherein the signaling chromophore is covalently bound to the multichromophore.

26. The multichromophore complex of claim 24, wherein the signaling chromophore is covalently bound to the biomolecule.

27. The multichromophore complex of claim 24, wherein the signaling chromophore is an organic dye.

28. The multichromophore complex of claim 24, wherein both the signaling chromophore and the sensor biomolecule are covalently linked to the multichromophore through a plurality of linkers.

29. The multichromophore complex of claim 24, wherein both the signaling chromophore and the sensor biomolecule are covalently linked to the multichromophore through a central linking site that covalently binds the multichromophore, the signaling chromophore and the sensor biomolecule.

30. The multichromophore complex of claim 22, wherein the sensor biomolecule to multichromophore covalent linkage comprises a bioconjugation site selected from the group consisting of maleimide/thiol, succimidylester (NHS ester)/amine, azide chemistry, carboxy/EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide Hydrochloride)/amine, amine/Sulfo-SMCC (Sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate)/thiol, and amine/BMPH(N-[β

-Maleimidopropionic acid]hydrazide·

TFA)/thiol.

31. The multichromophore complex of claim 22, wherein the sensor biomolecule is selected from the group consisting of an avidin, streptavidin, neutravidin, avidinDN, and avidinD.

32. The multichromophore complex of claim 22, wherein the multichromophore is a water-soluble conjugated polymer.

33. The multichromophore complex of claim 22, further configured to bind to a complex selected from the group consisting of a biotin-labeled antibody, biotin-labeled protein, biotin-labeled nucleic acid and biotin-labeled target biomolecule.

34. The multichromophore complex of claim 22, wherein the sensor biomolecule is an antibody.

35. The multichromophore complex of claim 22, wherein the sensor biomolecule is an anti-digoxigenin antibody.

36. The multichromophore complex of claim 22, further configured to bind to a complex selected from the group consisting of digoxigenin-labeled antibody, digoxigenin-labeled protein, biotin-labeled nucleic acid or digoxigenin-labeled target biomolecule.

37. The multichromophore complex of claim 22, wherein the sensor biomolecule is a goat-anti-mouse antibody or a donkey-anti-mouse antibody.

38. The multichromophore complex of claim 22, wherein the sensor biomolecule is one member of a binding pair.

39. A multichromophore complex comprising:

a multichromophore covalently bound by a bioconjugation site pendant to at least one biomolecule selected from the group consisting of a sensor biomolecule, a bioconjugate and a target biomolecule wherein the multichromophore is used as a reporter or further comprises

a signaling chromophore covalently bound to the multichromophore or the biomolecule;

wherein the multichromophore comprises the structure