Precision fluorescently dyed particles and methods of making and using same

• US 20050260676A1
• Filed: 08/02/2005
• Published: 11/24/2005
• Est. Priority Date: 10/14/1997
• Status: Active Grant

• Current Assignee: Luminex Corporation (DiaSorin S.p.A.)
• Original Assignee: Luminex Corporation (DiaSorin S.p.A.)
• Inventors: Chandler, Van S., Chandler, Don J., Phipps, Stacie L., Reber, Janet J., Lambert, Beth A.
• Granted Patent: US 7,445,844 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: D06P 1/0012 Effecting dyeing to obtain luminescent or phosphorescent dyeings
• CPC Class Codes: D06P 1/922 hydrocarbons
• CPC Class Codes: D06P 1/924 Halogenated hydrocarbons
• CPC Class Codes: D06P 1/926 Non-halogenated hydrocarbons
• CPC Class Codes: D06P 1/928 Solvents other than hydrocarbons
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: Y10T 428/2982 Particulate matter [e.g., sphere, flake, etc.]
• CPC Class Codes: Y10T 428/2989 Microcapsule with solid core [includes liposome]
• CPC Class Codes: Y10T 428/2991 Coated
• CPC Class Codes: Y10T 428/2998 including synthetic resin or polymer

Abstract

An improved method of making a series of bead or microsphere or particle populations characterized by subtle variation in a proportion or ratio of at least two fluorescent dyes distributed within a single bead of each population is provided. These beads, when excited by a single excitation light source are capable of giving off several fluorescent signals simultaneously. A set containing as many as 64 distinct populations of multicolored, fluorescent beads is provided and when combined with analytical reagents bound to the surface of such beads is extremely useful for multiplexed analysis of a plurality of analytes in a single sample. Thus, methods of staining polymeric particles, the particles themselves, and methods of using such particles are claimed.

Claims

1-40. -40. (canceled)

41. A population of polymeric microspheres dyed with two or more fluorescent dyes, obtained by a method comprising:

treating polymeric microspheres by adding a polar organic solvent to an aqueous solution containing the polymeric microspheres to produce a mixture; and

adding at least two fluorescent dyes to the mixture such that the polymeric microspheres are stained with the at least two fluorescent dyes, wherein the at least two fluorescent dyes are selected such that on isolation and excitation of the polymeric microspheres, a distinct fluorescence signal is emitted from each dye, and wherein the distinct fluorescence signals have an intensity proportional to the amount of the dye in the polymeric microspheres.

42. The population of claim 41, wherein the mixture comprises about a 50% mixture of the aqueous solution and the polar organic solvent.

43. The population of claim 41, wherein the polar organic solvent is an aliphatic alcohol, methanol, ethanol, or propanol.

44. The population of claim 41, wherein the intensity exhibits a coefficient of variation among all of the polymeric microspheres that is no greater than about 15 percent.

45. The population of claim 41, wherein the intensity exhibits a coefficient of variation among all of the polymeric microspheres that is no greater than about 10 percent.

46. The population of claim 41, wherein the intensity exhibits a coefficient of variation among all of the polymeric microspheres that is no greater than about 8 percent.

47. The population of claim 41, wherein the at least two fluorescent dyes are hydrophobic.

48. The population of claim 41, wherein the at least two fluorescent dyes comprise squaric acid-based dyes.

49. The population of claim 48, wherein the squaric acid-based dyes are cyclobutenedione derivatives, symmetrical and unsymmetrical squaraines, substituted cephalosporin compounds, fluorinated squaraine compositions, alkylalkoxy squaraines, or squarylium compounds.

50. The population of claim 48, wherein the squaric acid-based dyes are a red fluorescent dye or an orange fluorescent dye.

51. The population of claim 50, wherein the red fluorescent dye comprises 1,3-bis[(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)methyl]-2,4-dihydroxycyclobutenediylium, bis(inner salt), and wherein the orange fluorescent dye comprises 2-(3,5-dimethylpyrrol-2-yl)-4-(3,5-dimethyl-2H-pyrrol-2-ylidene)-3-hydroxy-2-cyclobuten-1-one.

52. The population of claim 41, wherein the polymeric microspheres comprise polystyrene, brominated polystyrene, polyacrylic acid, polyacrylonitrile, polyacrylamide, polyacrolein, polydimethylsiloxane, polybutadiene, polyisoprene, polyurethane, polyvinyl acetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene, polyglycidylmethacrylate, polymethylmethacrylate, or copolymers, blends, composites, or combination thereof.

53. The population of claim 41, wherein the polymeric microspheres comprise at least one analytical reactant bound covalently to functional groups present on a surface of the polymeric microspheres or passively adsorbed to the surface of the polymeric microspheres.

54. The population of claim 41, wherein the polymeric microspheres have a diameter between about 10 nm and 100 μ

m.

62. A set of distinct populations of polymeric microspheres dyed with at least two fluorescent dyes, wherein the set of distinct populations of polymeric microspheres is dyed using a method comprising:

treating separate populations of polymeric microspheres by adding a polar organic solvent to aqueous solutions containing the separate populations of polymeric microspheres to produce different mixtures;

preparing solutions of at least two fluorescent dyes, wherein each of the solutions has a different concentration of at least one of the at least two fluorescent dyes; and

adding said each of the solutions to one of the different mixtures to provide the set of distinct populations of polymeric microspheres, wherein each of the distinct populations of polymeric microspheres has a different fluorescence intensity from the at least one of the at least two fluorescent dyes.

63. The set of claim 62, wherein the different mixtures comprise about a 50% mixture of the aqueous solution and the polar organic solvent.

64. The set of claim 62, wherein the polar organic solvent is an aliphatic alcohol, methanol, ethanol, or propanol.

65. The set of claim 62, wherein the different fluorescence intensity of said each of the distinct populations exhibits a coefficient of variation of no greater than about 15 percent.

66. The set of claim 62, wherein the different fluorescence intensity of said each of the distinct populations exhibits a coefficient of variation of no greater than about 10 percent.

67. The set of claim 62, wherein the different fluorescence intensity of said each of the distinct populations exhibits a coefficient of variation of less than about 8 percent.

68. The set of claim 62, wherein the at least two fluorescent dyes are hydrophobic.

69. The set of claim 68, wherein the at least two fluorescent dyes comprise squaric acid-based dyes.

70. The set of claim 69, wherein the squaric acid-based dyes comprise cyclobutenedione derivatives, symmetrical and unsymmetrical squaraines, substituted cephalosporin compounds, fluorinated squaraine compositions, alkylalkoxy squaraines, or squarylium compounds.

71. The set of claim 69, wherein the squaric acid-based dyes comprise a red fluorescent dye or an orange fluorescent dye.

72. The set of claim 71, wherein the red fluorescent dye comprises 1,3-bis[(1,3-dihydro-1,3,3-trimethyl2H-indol-2-ylidene)methyl]-2,4-dihydroxycylobutenediylium, bis(inner salt), and wherein the orange fluorescent dye comprises 2(3,5-dimethylpyrrol-2-yl)-4-(3,5-dimethyl-2H-pyrrol-2-ylidene)-3-hydroxy-2-cyclobuten-1-one.

73. The set of claim 62, wherein the polymeric microspheres comprise polystyrene, brominated polystyrene, polyacrylic acid, polyacrylonitrile, polyacrylamide, polyacrolein, polydimethylsiloxane, polybutadiene, polyisoprene, polyurethane, polyvinylacetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene, polyglycidylmethacrylate, polymethylmethacrylate, or copolymers, blends, composites, or combinations thereof.

74. The set of claim 62, wherein the polymeric microspheres comprise at least one analytical reactant bound covalently to functional groups present on a surface of the polymeric microspheres or passively adsorbed to the surface of the polymeric microspheres.

75. The set of claim 62, wherein the polymeric microspheres have a diameter between about 10 nm and 100 μ

m.

76. The set of claim 62, wherein said each of the distinct populations exhibits an emission spectrum in a Fluorescence Bead Map that is unique to said each of the distinct populations.

77. The set of claim 62, comprising eight or more of the distinct populations of polymeric microspheres.

78. The set of claim 62, comprising sixteen or more of the distinct populations of polymeric microspheres.

79. The set of claim 62, comprising twenty-four or more of the distinct populations of polymeric microspheres.

80. The set of claim 62, comprising thirty-two or more of the distinct populations of polymeric microspheres.

81. The set of claim 62, comprising sixty-four or more of the distinct populations of polymeric microspheres.

82. The set of claim 81, wherein said each of the distinct populations exhibits an emission spectrum in a Fluorescence Bead Map that is unique to said each of the distinct populations, and wherein there is substantially no overlap between any of the sixty-four or more emission spectra associated with the sixty-four or more of the distinct populations of polymeric microspheres.

83. A population of polymeric microspheres dyed with two or more fluorochromes, obtained by a method comprising:

treating polymeric microspheres by adding a polar organic solvent to an aqueous solution containing the polymeric microspheres to produce a mixture; and

adding at least two fluorochromes to the mixture such that the polymeric microspheres are stained with the at least two fluorochromes, wherein the at least two fluorochromes are selected such that on isolation and excitation of the polymeric microspheres, a distinct fluorescence signal is emitted from each fluorochrome, and wherein the distinct fluorescence signals have an intensity proportional to the amount of the fluorochrome in the polymeric microspheres.

84. The population of claim 83, wherein the mixtures comprise about a 50% mixture of the aqueous solution and the polar organic solvent.

85. The population of claim 83, wherein the polar organic solvent is an aliphatic alcohol, methanol, ethanol, or propanol.

86. A set of distinct populations of polymeric microspheres dyed with at least two fluorochromes, wherein the set of distinct populations of polymeric microspheres is dyed using a method comprising:

treating separate populations of polymeric microspheres by adding a polar organic solvent to aqueous solutions containing the separate populations of polymeric microspheres to produce different mixtures;

preparing solutions of at least two fluorochromes, wherein each of the solutions has a different concentration of at least one of the at least two fluorochromes; and

adding said each of the solutions to one of the different mixtures to provide the set of distinct populations of polymeric microspheres, wherein each of the distinct populations of polymeric microspheres has a different fluorescence intensity from that at least one of the at least two fluorochromes.

87. The set of claim 86, wherein the different mixtures comprise about a 50% mixture of the aqueous solution and the polar organic solvent.

88. The set of claim 86, wherein the polar organic solvent is an aliphatic alcohol, methanol, ethanol, or propanol.

89. The set of claim 86, wherein the different fluorescence intensity of said each of the distinct populations exhibits a coefficient of variation of no greater than about 15 percent.

90. The set of claim 86, wherein the different fluorescence intensity of said each of the distinct populations exhibits a coefficient of variation of no greater than about 10 percent.

91. The set of claim 86, wherein the different fluorescence intensity of said each of the distinct populations exhibits a coefficient of variation of no greater than about 8 percent.

92. The set of claim 86, wherein at least one of the at least two fluorochromes is hydrophobic.

93. The set of claim 86, wherein the at least two fluorochromes are selected from fluorochromes that emit a red colored signal and fluorochromes that emit an orange colored signal.

94. The set of claim 86, wherein the polymeric microspheres comprise polystyrene, brominated polystyrene, polyacrylic acid, polyacrylonitrile, polyacrylamide, polyacrolein, polydimethylsiloxane, polybutadiene, polyisoprene, polyurethane, polyvinylacetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene, polyglycidylmethacrylate, polymethylmethacrylate, or copolymers, blends, composites, or combinations thereof.

95. The set of claim 86, wherein the polymeric microspheres comprise at least one analytical reactant bound covalently to functional groups present on a surface of the polymeric microspheres or passively adsorbed to the surface of the polymeric microspheres.

96. The set of claim 86, wherein the polymeric microspheres have a diameter between about 10 nm and 100 μ

m.