Reagents for directed biomarker signal amplification

• US 9,139,869 B2
• Filed: 09/05/2013
• Issued: 09/22/2015
• Est. Priority Date: 01/19/2010
• Status: Active Grant

• Current Assignee: Sirigen Ii Limited (Becton, Dickinson & Co)
• Original Assignee: Sirigen, Inc. (Becton, Dickinson & Co)
• Inventors: Liang, Yongchao, Huisman, William H., Hong, Janice W., Baldocchi, Russell A., Bartholomew, Glenn P., Uckert, Frank Peter, Gaylord, Brent S., Palmer, Adrian Charles Vernon, Wheeler, Jean M., TRAN, Lan P., Nguyen, Trung
• Primary Examiner(s): Truong, Duc
• Application Number:
  US14/018,985
• Publication Number:
  US 20140206557A1
• Time in Patent Office: 747 Days
• Field of Search: 506/9, 436/501, 435/72, 435/61, 422/69
• US Class Current: 1/1
• CPC Class Codes: C07K 16/2812 against CD4
• CPC Class Codes: C07K 16/2815 against CD8
• CPC Class Codes: C08G 2261/1424 containing ether groups, including alkoxy
• CPC Class Codes: C08G 2261/143 Side-chains containing nitrogen
• CPC Class Codes: C08G 2261/1432 containing amide groups
• CPC Class Codes: C08G 2261/146 Side-chains containing halogens
• CPC Class Codes: C08G 2261/148 Side-chains having aromatic units
• CPC Class Codes: C08G 2261/164 comprising organic end groups
• CPC Class Codes: C08G 2261/1642 comprising reactive double bonds or triple bonds
• CPC Class Codes: C08G 2261/3142 fluorene-based, e.g. fluorene, indenofluorene, or spirobifluorene
• CPC Class Codes: C08G 2261/334 containing heteroatoms
• CPC Class Codes: C08G 2261/3422 conjugated, e.g. PPV-type
• CPC Class Codes: C08G 2261/411 Suzuki reactions
• 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 61/10 only aromatic carbon atoms, e.g. polyphenylenes
• CPC Class Codes: C09B 57/00 Other synthetic dyes of known constitution
• CPC Class Codes: C09B 68/41 Polymers attached to the pigment surface C09B68/444, C09B68/446 take precedence
• CPC Class Codes: C09B 69/00 Dyes not provided for by a single group of this subclass
• CPC Class Codes: C09D 165/00 Coating compositions based on macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain C09D107/00 - C09D157/00, C09D161/00 take precedence; Coating compositions based on derivatives of such polymers
• CPC Class Codes: C09K 11/06 : containing organic luminescent materials
• CPC Class Codes: C12Q 1/6818 : involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/682 : Signal amplification
• CPC Class Codes: G01N 33/58 : involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/582 : with fluorescent label
• CPC Class Codes: H10K 85/115 : Polyfluorene; Derivatives thereof
• CPC Class Codes: H10K 85/151 : Copolymers

Abstract

Described herein are methods, compositions and articles of manufacture involving neutral conjugated polymers including methods for synthesis of neutral conjugated water-soluble polymers with linkers along the polymer main chain structure and terminal end capping units. Such polymers may serve in the fabrication of novel optoelectronic devices and in the development of highly efficient biosensors. The invention further relates to the application of these polymers in assay methods.

Claims

1. An assay method for detecting a target biomolecule in a sample comprising:

providing a sample that is suspected of containing a target biomolecule;

providing a conjugated polymer complex comprising sensor biomolecule conjugated to a water soluble conjugated polymer having the structure of Formula (Ia);

2. The assay method of claim 1, wherein each R is independently (CH₂)_x(OCH₂CH₂)_yOCH₃ where each x is independently an integer from 0-20, each y is independently an integer from 0 to 50, or a benzyl optionally substituted with one or more halogen, hydroxyl, C₁-C₁₂ alkoxy, or (OCH₂CH₂)_zOCH₃ where each z is independently an integer from 0 to 50.

3. The assay method of claim 1, wherein the sensor biomolecule is a protein, peptide, affinity ligand, antibody, antibody fragment, sugar, lipid, nucleic acid or an aptamer.

4. The assay method of claim 1, wherein the sensor biomolecule is an antibody.

5. The assay method of claim 1, wherein the sensor biomolecule is an avidin, streptavidin, neutravidin, avidinDN or an avidinD.

6. The assay method of claim 1, wherein the sensor biomolecule is bound to a substrate.

7. The assay method of claim 1, wherein the target biomolecule is a target protein expressed on a cell surface.

8. The assay method of claim 1, wherein the method is configured as an sandwich immunoassay, protein array or microarray.

9. The assay method of claim 1, wherein the method is configured for immunohistochemistry or FISH.

10. The assay method of claim 1, wherein the method is configured for intracellular staining.

11. The assay method of claim 1, wherein the method is configured for flow cytometry.

12. The assay method of claim 1, wherein the method further comprises providing additional sensor biomolecules for detecting additional target biomolecules simultaneously.

13. The assay method of claim 1, wherein each R is (CH₂)₃(OCH₂CH₂)₁₁OCH₃.

14. The assay method of claim 1, wherein R is an non-ionic side group.

15. The assay method of claim 1, wherein optional linkers L₁ or L₂ are selected from the group consisting of a-j having the structures:

16. The assay method of claim 1, wherein optional linkers L₁ or L₂ are

17. The assay method of claim 1, wherein G₁ and G₂ each independently have the structure

18. The assay method of claim 1, wherein G₁ and G₂ are each independently selected from the group consisting of 1-33 having the structures:

19. The assay method of claim 1, wherein the polymer contains a single conjugation site at only one terminus of the polymer chain G₁ or G₂.

20. The assay method of claim 1, wherein G₁ and G₂ is

21. The assay method of claim 1, wherein MU is selected from the group consisting of—

a′

-k′

having the structure;

22. The assay method of claim 1, wherein MU is selected from the group consisting of

23. The assay method of claim 1, wherein the optional linkers L₁ or L₂ have the structure:

24. The assay method of claim 1, wherein optional linkers L₁ or L₂ are selected from the group consisting of

25. The assay method of claim 1, wherein the polymer has the structure of formula:

26. The assay method of claim 1, wherein the polymer has the structure of formula:

27. The assay method of claim 1, wherein the polymer has the structure of formula:

28. The assay method of claim 1, wherein the polymer is conjugated to a signaling chromophore selected from the group consisting of rhodamine, coumarin, xanthene, cyanine, polymethine, pyrene, dipyrromethene borondifluoride, napthalimide, phycobiliprotein, peridinum chlorophyll protein, conjugates thereof, and combinations thereof.

29. The assay method of claim 1, wherein the polymer is further conjugated to a secondary dye reporter.

30. The assay method of claim 1, wherein the polymer is >

95% pure.

31. The assay method of claim 1, wherein the polymer comprises a minimum number average molecular weight of greater than 40,000 g/mol and a water solubility of greater than 50 mg/mL in pure water or a phosphate buffered saline solution.

32. The assay method of claim 1, wherein a is from about 40% to 60%.

33. The assay method of claim 1, wherein b is from about 40% to 60%.

34. The assay method of claim 1, wherein the polymer has the structure:

35. The assay method of claim 1, wherein the polymer has the structure:

36. A flow cytometry system comprising:

a flow cytometer;

a sample that is suspected of containing a target biomolecule;

a sensor protein conjugated to a water soluble conjugated polymer having the structure of Formula (Ia);