Novel Reagents for Directed Biomarker Signal Amplification

• US 20160349267A1
• Filed: 08/17/2016
• Published: 12/01/2016
• 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: BARTHOLOMEW, Glenn P., GAYLORD, Brent S., 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
• Granted Patent: US 10,094,838 B2
• Time in Patent Office: Days
• 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

1-40. -40. (canceled)

41. A method of determining whether a target is present in a sample, the method comprising:

contacting the sample with a conjugated polymer complex comprising a sensor for the target conjugated to a water soluble conjugated polymer having the structure of Formula (Ia);

42. The method according to claim 41, wherein the at least 1 functional group is conjugated to a signaling chromophore.

43. The method according to claim 42, wherein the signaling chromophore is selected from the group consisting of fluorescent dyes, semiconductor nanocrystals, lanthanide chelates and fluorescent proteins.

44. The method according to claim 41, wherein each R is a non-ionic side group capable of imparting solubility in water in excess of 10 mg/mL.

45. The method according to claim 41, 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.

46. The method according to claim 45, wherein each R is a benzyl substituted with at least two (OCH₂CH₂)₁₀OCH₃ groups.

47. The method according to claim 46, wherein each R is (CH₂)₃(OCH₂CH₂)₁₁OCH₃.

48. The method according to claim 41, 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.

49. The method according to claim 48, wherein MU is selected from the group consisting of a′

-k′

having the structure;

50. The method according to claim 49, wherein MU is selected from one of the following:

51. The method according to claim 41, wherein MU is selected from one of the following:

52. The method according to claim 41, wherein L₁ and/or L₂ are present.

53. The method according to claim 52, wherein linkers L₁ or L₂ have the structure:

54. The method according to claim 52, wherein linkers L₁ or L₂ have the structure:

55. The method according to claim 54, wherein at least one R₂₅ comprises an amine conjugation site.

56. The method according to claim 54, wherein linkers L₁ or L₂ have the structure:

57. The method according to claim 54, wherein at least one R₂₅ comprises a carboxylic acid sidechain.

58. The method according to claim 54, wherein at least one R²⁵ is conjugated to a signaling chromophore.

59. The method according to claim 41, wherein at least one of G₁ and G₂ comprises a functional conjugation site.

60. The method according to claim 59, wherein at least one of G₁ and G₂ has the structure:

61. The method according to claim 60, wherein at least one of G₁ and G₂ is selected from the group consisting of capping units 1-31 having the structures:

62. The method according to claim 59, wherein at least one of G₁ and G₂ is a terminal carboxylic acid capping unit.

63. The method according to claim 62, wherein at least one of G₁ and G₂ is selected from one of the following structures:

64. The method according to claim 59, wherein at least one of G₁ and G₂ comprises a terminal amine capping unit.

65. The method according to claim 41, wherein the polymer has the structure of formula:

66. The method according to claim 41, wherein the polymer has the structure of formula:

67. The method according to claim 41, wherein the sensor is a protein, peptide, affinity ligand, antibody, antibody fragment, sugar, lipid, nucleic acid or an aptamer.

68. The method according to claim 41, wherein the sensor is an antibody.

69. The method according to claim 41, wherein the sensor is an avidin, streptavidin, neutravidin, avidinDN or an avidinD.

70. The method according to claim 41, wherein the target is a target protein of a cell.

71. The method according to claim 41, wherein the method is configured for immunohistochemistry or FISH.

72. The method according to claim 41, wherein the method is configured for intracellular staining.

73. The method according to claim 41, wherein the method is configured for flow cytometry.