Reagents for directed biomarker signal amplification

• US 9,547,008 B2
• Filed: 08/07/2015
• Issued: 01/17/2017
• 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: Hong, Janice W., Liang, Yongchao, Baldocchi, Russell A., Huisman, William H., Bartholomew, Glenn P., Uckert, Frank Peter, Gaylord, Brent S., Palmer, Adrian Charles Vernon, Wheeler, Jean M., Tran, Lan T., Nguyen, Trung
• Primary Examiner(s): Truong, Duc
• Application Number:
  US14/821,386
• Publication Number:
  US 20160025735A1
• Time in Patent Office: 529 Days
• Field of Search: 525/541, 525/539, 525/542, 525/50
• 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. A water soluble conjugated polymer having the structure of Formula (Ia):

2. The water soluble conjugated polymer according to claim 1, wherein the at least 1 functional group is conjugated to a signaling chromophore.

3. The water soluble conjugated polymer according to claim 2, wherein the signaling chromophore is selected from the group consisting of fluorescent dyes, semiconductor nanocrystals, lanthanide chelates and fluorescent proteins.

4. The water soluble conjugated polymer according to claim 1, wherein each R is a non-ionic side group capable of imparting solubility in water in excess of 10 mg/mL.

5. The water soluble conjugated polymer according to claim 1, wherein each R is independently (CH₂)_x(OCH₂CH₂)_yOCH₃ wherein each x is independently an integer from 0-20 and each y is independently an integer from 0 to 50, or a benzyl substituted with at least one (OCH₂CH₂)_zOCH₃ group wherein each z is independently an integer from 0 to 50.

6. The water soluble conjugated polymer according to claim 5, wherein each R is a benzyl substituted with at least two (OCH₂CH₂)₁₀OCH₃ groups.

7. The water soluble conjugated polymer according to claim 5, wherein each R is (CH₂)₃(OCH₂CH₂)₁₁OCH₃.

8. The water soluble conjugated polymer according to claim 1, wherein MU is selected from optionally substituted 2,1,3-benzothiadiazole, benzoxidazole, benzoselenadiazole, benzotellurodiazole, naphthoselenadiazole, 4,7-di(thien-2-yl)-2,1,3-benzothiadiazole, squaraine dyes, quinoxalines, perylene, perylene diimides, diketopyrrolopyrrole, thienopyrazine low bandgap commercial dyes, olefins, and cyano-substituted olefins and isomers thereof.

9. The water soluble conjugated polymer according to claim 8, wherein MU is selected from the group consisting of a′

- k′

having the structure;

10. The water soluble conjugated polymer according to claim 9, wherein MU is selected from one of the following:

11. The water soluble conjugated polymer according to claim 1, wherein MU is selected from one of the following:

12. The water soluble conjugated polymer according to claim 1, wherein L₁ and/or L₂ are present.

13. The water soluble conjugated polymer according to claim 12, wherein linkers L₁ or L₂ have the structure:

14. The water soluble conjugated polymer according to claim 12, wherein linkers L₁ or L₂ have the structure:

15. The water soluble conjugated polymer according to claim 14, wherein at least one R₂₅ comprises an amine conjugation site.

16. The water soluble conjugated polymer of claim 15, wherein linkers L₁ or L₂ have the structure:

17. The water soluble conjugated polymer according to claim 14, wherein at least one R₂₅ comprises a carboxylic acid sidechain.

18. The water soluble conjugated polymer according to claim 14, wherein at least one R²⁵ is conjugated to a signaling chromophore.

19. The water soluble conjugated polymer according to claim 1, wherein at least one of G₁ and G₂ comprises a functional conjugation site.

20. The water soluble conjugated polymer according to claim 19, wherein at least one of G₁ and G₂ has the structure:

21. The water soluble conjugated polymer according to claim 19, wherein at least one of G₁ and G₂ is selected from the group consisting of capping units 1-31 having the structures:

22. The water soluble conjugated polymer according to claim 19, wherein at least one of G₁ and G₂ is a terminal carboxylic acid capping unit.

23. The water soluble conjugated polymer according to claim 22, wherein at least one of G₁ and G₂ is selected from one of the following structures:

24. The water soluble conjugated polymer according to claim 19, wherein at least one of G₁ and G₂ comprises a terminal amine capping unit.

25. The water soluble conjugated polymer according to claim 1, wherein the polymer has the structure of formula:

26. The water soluble conjugated polymer according to claim 1, wherein the polymer has the structure of formula:

27. The water soluble conjugated polymer according to claim 1, wherein the polymer is conjugated to a signaling chromophore.

28. The water soluble conjugated polymer according to claim 18, wherein the conjugated polymer is conjugated to both an acceptor dye and a biomolecule.

29. The water soluble conjugated polymer according to claim 26, wherein the conjugated polymer has multiple dye attachments positioned internally.

30. The water soluble conjugated polymer according to claim 26, wherein the biomolecule is conjugated at a terminus of the conjugated polymer.

31. The water soluble conjugated polymer according to claim 26, wherein the conjugated polymer is conjugated to an amine group on a protein.

32. The water soluble conjugated polymer according to claim 26, wherein the biomolecule is an antibody.

33. A polymer-dye-biomolecule conjugate comprising:

a conjugated polymer according to claim 1;

an organic dye; and

a sensor biomolecule conjugated to the at least one functional group within G1 or G2;

wherein the conjugated polymer is an energy transfer donor and the dye is an acceptor.

34. The polymer-dye-biomolecule conjugate according to claim 33, wherein the sensor biomolecule is a protein, peptide, affinity ligand, antibody, antibody fragment, sugar, lipid, nucleic acid or an aptamer.

35. The polymer-dye-biomolecule conjugate according to claim 33, wherein the sensor biomolecule is an antibody.

36. The polymer-dye-biomolecule conjugate according to claim 33, wherein the organic dye is a fluorophore.