Reagents for directed biomarker signal amplification

• US 10,288,620 B2
• Filed: 10/31/2017
• Issued: 05/14/2019
• Est. Priority Date: 01/19/2010
• Status: Active Grant

• Current Assignee: Sirigen Ii Limited (Becton, Dickinson & Co)
• Original Assignee: Sirigen Ii Limited (Becton, Dickinson & Co)
• Inventors: Gaylord, Brent S., Bartholomew, Glenn P., Baldocchi, Russell A., Hong, Janice W., Huisman, William H., Liang, Yongchao, Nguyen, Trung, Tran, Lan T., Wheeler, Jean M., Palmer, Adrian Charles Vernon, Uckert, Frank Peter
• Primary Examiner(s): Truong, Duc
• Application Number:
  US15/799,927
• Publication Number:
  US 20180106812A1
• Time in Patent Office: 560 Days
• Field of Search: 528371
• 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 polymer-biomolecule conjugate comprising:

a water soluble conjugated polymer; and

a biomolecule covalently linked to the water soluble conjugated polymer;

wherein the water soluble conjugated polymer is of the formula;

2. The polymer-biomolecule conjugate according to claim 1, wherein the biomolecule is a sensor biomolecule capable of interacting with a target molecule.

3. The polymer-biomolecule conjugate according to claim 1, wherein the biomolecule is a protein, a peptide, an affinity ligand, an antibody, an antibody fragment, a sugar, a lipid, a nucleic acid, an aptamer, avidin, streptavidin, neutravidin, avidinDN or avidinD.

4. The polymer-biomolecule conjugate according to claim 3, wherein the biomolecule is an antibody or antibody fragment.

5. The polymer-biomolecule conjugate according to claim 1, wherein MU is selected from optionally substituted benzothiadiazole, optionally substituted benzoxidazole, optionally substituted benzoselenadiazole, optionally substituted benzotellurodiazole, optionally substituted naphthoselenadiazole, optionally substituted 4,7-di(thien-2-yl)-2,1,3-benzothiadiazole, squaraine dyes, optionally substituted quinoxaline, optionally substituted perylene, optionally substituted perylene diimide, optionally substituted diketopyrrolopyrrole, thienopyrazine low bandgap commercial dyes, olefin and cyano-substituted olefins and isomers thereof.

6. The polymer-biomolecule conjugate according to claim 5, wherein MU is selected from the group consisting of a′

-k′

having the structures;

7. The polymer-biomolecule conjugate according to claim 6, wherein MU is selected from one of the following:

8. The polymer-biomolecule conjugate according to claim 1, wherein MU is a phenyl.

9. The polymer-biomolecule conjugate according to claim 8, wherein MU is

10. The method 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.

11. The polymer-biomolecule conjugate according to claim 1, wherein each R comprises an ethylene glycol oligomer.

12. The polymer-biomolecule conjugate according to claim 11, wherein each R is a non-ionic side group comprising mPEG5, mPEG8, mPEG11 or mPEG24.

13. The polymer-biomolecule conjugate according to claim 11, wherein each R is independently selected from (CH₂)_x′

(OCH₂CH₂)_y′

OCH₃ wherein x′

is independently an integer from 0-20 and y′

is independently an integer from 0 to 50, and a benzyl substituted with one or more groups selected from halogen, hydroxyl, C₁-C₁₂ alkoxy and (OCH₂CH₂)_zOCH₃ wherein z is independently an integer from 0 to 50.

14. The polymer-biomolecule conjugate according to claim 11, wherein each R is (CH₂)_x′

(OCH₂CH₂)_y′

OCH₃ wherein x′

is independently an integer from 0-20 and y′

is independently an integer from 0 to 50.

15. The polymer-biomolecule conjugate according to claim 1, wherein each R is a dendrimer of PAMAM, PEA, PEHAM, PPI, tri-branched benzoate, or glycerol with a generation of 1 to 4 and optional terminal substitutions selected from ( - - - - - )CH₂CH₂O)_jCH₃ and ( - - - - - )OCH₂CH₂)_jCH₃ where j is an integer from 0 to 25 and the dotted lines ( - - - - - ) are each independently selected from any one or a combination of, C₁-C₁₂ alkyl, C₁-C₁₂ alkoxy, C₂-C₁₂ alkene, amido, amino, aryl, (CH₂)_r(OCH₂CH₂)_s(CH₂)_r where each r is independently an integer from 0-20, s is independently an integer from 0 to 50, carbamate, carboxylate, C₃-C₁₂ cycloalkyl, imido, phenoxy and C₄-C₁₈(hetero)aryl group.

16. The method according to claim 1, wherein wherein L₁ and/or L₂ are present and have the structure:

17. The polymer-biomolecule conjugate according to claim 1, wherein L₁ and/or L₂ are present and have the structure:

18. The polymer-biomolecule conjugate according to claim 1, wherein L₁ and/or L₂ is conjugated to a signaling chromophore.

19. The polymer-biomolecule conjugate according to claim 1, wherein G₁ or G₂ comprise the covalently linked biomolecule.

20. The polymer-biomolecule conjugate according to claim 15, wherein at least one of G₁ and G₂ has the structure: