FLUORESCENT COMPOUNDS
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Abstract
The present invention relates to fluorescent dyes in general. The present invention provides a wide range of fluorescent dyes and kits containing the same, which are applicable for labeling a variety of biomolecules, cells and microorganisms. The present invention also provides various methods of using the fluorescent dyes for research and development, forensic identification, environmental studies, diagnosis, prognosis, and/or treatment of disease conditions.
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Citations
113 Claims
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1. A compound of Formula I:
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2. The compound of claim 1, wherein the fluorophore is a xanthene dye, a coumarin dye, a pyrene dye or a cyanine dye.
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3. The compound of claim 1, wherein the fluorophore is a coumarin dye.
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4. The compound of claim 1, wherein the fluorophore is a pyrene dye.
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5. The compound of claim 1, wherein the fluorophore is a rhodamine dye.
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6. The compound of claim 1, wherein the water-soluble polymer is a polyalkylene oxide.
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7. The compound of claim 1, wherein the water-soluble polymer is a polyethylene oxide.
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8. The compound of claim 1, wherein the water-soluble polymer is a carbohydrate.
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9. The compound of claim 1, wherein the water-soluble polymer is a polypeptide.
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10. The compound of claim 1, wherein the water-soluble polymer has a molecular weight of greater than about 300 Da.
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11. The compound of claim 1, wherein the water-soluble polymer has a molecular weight of greater than about 800 Da.
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12. The compound of claim 1, wherein the water-soluble polymer has a molecular weight ranging from about 800 Da to about 3000 Da.
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13. The compound of claim 1, wherein the reactive group forms a covalent bond with an amino, a sulfhydryl or a hydroxy nucleophile.
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14. The compound of claim 1, wherein the reactive group is an isothiocyanate, an isocyanate, a monochlorotriazine, a dichlorotriazine, a halogen-substituted pyridine, a halogen-substituted diazine, a phosphoramidite, a maleimide, an aziridine, a sulfonyl halide, an acid halide, a hydroxysuccinimidyl ester, a hydroxysulfosuccinimidyl ester, a tetrafluorophenol ester, an imido ester, a hydrazine, an azidonitrophenyl, an azide, an alkyne, a 3-(2-pyridyl dithio)-propionamide, a glyoxal or an aldehyde.
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15. The compound of claim 1, wherein a maximal fluorescence excitation wavelength of the compound ranges from about 350 to about 1200 nm.
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16. The compound of claim 1, wherein a maximal fluorescence emission wavelength of the compound ranges from about 360 to 1250 nm.
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17. The compound of claim 1, wherein the fluorophore is a coumarin of the formula:
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18. The compound of claim 17, wherein
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19. The compound of claim 17, wherein the fluorophore is substituted by one or more sulfonate groups.
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20. The compound of claim 1, wherein the fluorophore is a compound of the formula:
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21. The compound of claim 20, wherein m is 0 and
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22. The compound of claim 21, wherein R1, comprises a water soluble polymer group and R2 comprises a reactive group.
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23. The compound of claim 22, wherein R1, comprises a polyethylene glycol.
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24. The compound of claim 22, wherein R2 comprises an N-hydroxysuccinimide group.
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25. A compound having a maximal fluorescence excitation wavelength, wherein the compound has a structure of Formula II:
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F—
Y═
Ψ
Formula IIwherein; F is a moiety having the structure;
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26. The compound of claim 25, wherein when at least two adjacent R1, and/or two adjacent R2 are present, the two adjacent R1, and/or the two adjacent R2 are combinable to form a 6-membered ring which is unsubstituted or substituted by one or more (R)p-(L)q-.
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27. The compound of claim 26 wherein when the two adjacent R1, and/or the two adjacent R2 are combinable to form a 6-membered ring, the ring so formed is aromatic.
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28. The compound of claim 25 wherein when Ψ
- is Formula 1, and the maximal fluorescence excitation wavelength of the compound is equal to or greater than 660 nm, or Ψ
is other than Formula 1, then R5 and R6 are independently (R)p (L)q-, or R5 and R6 are combinable to form a cyclic moiety which is unsubstituted or substituted by one or more (R)p-(L)q-.
- is Formula 1, and the maximal fluorescence excitation wavelength of the compound is equal to or greater than 660 nm, or Ψ
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29. The compound of claim 25, wherein Y is:
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30. The compound of claim 25, wherein at least one R of R1 and R2 is a charged moiety.
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31. The compound of claim 25, wherein at least one R of R1 and R2 comprises a sulfonate group or a phosphonate group.
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32. The compound of claim 25, wherein each R of R1 and R2 comprises a sulfonate group or a phosphonate group.
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33. The compound of claim 25, wherein X2 and X3 are independently
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34. The compound of claim 25, wherein X2 and X3 are independently
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35. The compound of claim 25, wherein the water-soluble polymer is a polyalkylene oxide.
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36. The compound of claim 25, wherein the water-soluble polymer is a polyethylene oxide.
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37. The compound of claim 25, wherein the water-soluble polymer has a molecular weight of greater than about 300.
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38. The compound of claim 25, wherein the water-soluble polymer has a molecular weight of greater than about 800.
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39. The compound of claim 25, wherein the water-soluble polymer has a molecular weight ranging from about 800 to about 3000.
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40. The compound of claim 26, wherein two adjacent (R1)a and the atoms in ring A to which it is attached are combined to form a carbocyclic ring.
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41. The compound of claim 40, wherein the carbocyclic ring is aromatic.
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42. The compound of claim 26, wherein two adjacent (R2)b and the atoms in ring B to which it is attached are combined to form a carbocyclic ring.
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43. The compound of claim 42, wherein the carbocyclic ring is aromatic.
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44. The compound of claim 25, wherein the compound has the formula:
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45. The compound of claim 25, wherein the compound has the formula:
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46. The compound of claim 25, wherein the compound has the formula:
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47. The compound of claim 25, wherein the compound has the formula:
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48. The compound of claim 25, wherein the compound has the formula:
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49. The compound of claim 25, wherein the compound has the formula:
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50. The compound of claim 25, wherein the maximal fluorescence excitation wavelength of the compound ranges from about 350 to about 1200 nm.
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51. The compound of claim 25, wherein a maximal fluorescence emission wavelength of the compound ranges from about 360 to about 1250 nm.
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52. The compound of claim 25, wherein the water-soluble polymer is a polyalkylene oxide.
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53. The compound of claim 25, wherein the water-soluble polymer is a polyethylene oxide.
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54. The compound of claim 25, wherein the water-soluble polymer is a carbohydrate.
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55. The compound of claim 25, wherein the water-soluble polymer is a polypeptide.
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56. The compound of claim 25, wherein the water-soluble polymer has a molecular weight of greater than 300.
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57. The compound of claim 25, wherein the water-soluble polymer has a molecular weight of greater than 800.
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58. The compound of claim 25, wherein the compound is
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59. A substituted cyanine dye comprising a reactive group and one or more water soluble polymer groups, wherein the cyanine dye has a maximal fluorescence excitation wavelength of equal to or greater than about 660 nm.
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60. A substituted cyanine dye comprising a reactive group and one or more water soluble polymer groups, wherein the cyanine dye has an absorption maximal wavelength of equal to or greater than about 660 nm.
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61. The substituted cyanine dye of claim 59 or 60 wherein the dye is substituted by a non-spiro moiety.
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62. A binding agent labeled with a substituted cyanine dye of claim 59 or 60, wherein the binding agent binds selectively to a target polypeptide to form a complex, and wherein formation of the complex yields a signal to noise ratio of fluorescence that is at least 100.
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63. The binding agent of claim 62 wherein the substituted cyanine dye is substituted with a non-spiro substituent.
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64. The binding agent of claim 62 wherein the binding agent is a polypeptide.
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65. The binding agent of claim 63 wherein the polypeptide is an antibody.
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66. A compound of Formula III:
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67. The compound of claim 66 wherein at least one of R6, R7, R8 or R9 is combinable with a neighboring R1 or R2 and any intervening atoms in a ring to which the neighboring R1 or R2 is attached, to form a 5- or 6-membered ring which is unsubstituted or substituted by one or more (R)p (L)q-.
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68. The compound of claim 67 wherein when the at least one of R6, R7, R8 or R9 and a neighboring R1 or R2 are combinable to form a 5- or 6-membered ring, the ring is unsaturated.
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69. The compound of claim 67 wherein when the at least one of R6, R7, R8 or R9 and a neighboring R1 or R2 are combinable to form a 5- or 6-membered ring, the ring is saturated.
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70. The compound of claim 66, wherein X1 is ═
- NH2 .
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71. The compound of claim 66, wherein X1 is ═
- O.
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72. The compound of claim 66, wherein X2 is —
- OH.
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73. The compound of claim 66, wherein X2 is —
- NH2.
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74. The compound of claim 66, wherein a maximal fluorescence excitation wavelength of the compound ranges from about 450 to 750 nm.
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75. The compound of claim 66, wherein a maximal fluorescence emission wavelength of the compound ranges from about 470 to 800 nm.
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76. A compound of Formula V:
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77. A kit comprising:
i) the compound of claim 1, 25, 59, 60, 66, or 76;
ii) a buffer;
iii) materials or devices for purifying conjugation products; and
iv) instructions instructing the use of the compound.
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78. A biomolecule comprising a label having a structure of a Formula of claim 1, 25, 59, 60, 66, or 76, wherein the at least one reactive moiety of the Formula has undergone a reaction which attaches the label to the biomolecule.
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79. The biomolecule comprising a label of claim 78 wherein the biomolecule comprises a polynucleotide.
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80. The biomolecule comprising a label of claim 78 wherein the biomolecule comprises a polypeptide.
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81. The biomolecule of claim 80, wherein the polypeptide further comprises an antigen binding site.
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82. The biomolecule of claim 80, wherein the polypeptide is a whole immunoglobulin.
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83. The biomolecule of claim 80, wherein the polypeptide is a Fab fragment.
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84. An immunoglobin comprising a label having a structure of a Formula of claim 1, 25, 59, 60, 66, or 76 wherein the at least one reactive moiety of the Formula has undergone a reaction which attaches the label to the immunoglobin, wherein the immunoglobin is an antibody that binds specifically to an antigen on a cancer cell.
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85. The immunoglobin of claim 84 wherein the antibody binds to erb2.
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86. A method of preparing a labeled biomolecule comprising reacting a compound of claim 1, 25, 59, 60, 66, or 76 and a substrate biomolecule under conditions sufficient to effect crosslinking between the compound and the substrate biomolecule.
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87. The method of claim 86, wherein the substrate biomolecule is a polypeptide, a polynucleotide, a carbohydrate, a lipid or a combination thereof.
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88. The method of claim 87, wherein the substrate biomolecule is a polynucleotide.
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89. A method for labeling a cell within a population of cells whereby the cell is differentially labeled relative to neighboring cells within the population, the method comprising contacting the cell with a biomolecule of claim 78, wherein the biomolecule comprises a targeting moiety that binds to a binding partner that is indicative of the cell, and thereby differentially labeling the cell relative to neighboring cells within the population.
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90. The method of claim 89, further comprising the step of imaging the cell, the imaging step comprising:
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i) directing exciting wavelength to the cell; and ii) detecting emitted fluorescence from the cell.
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91. The method of claim 89, wherein the labeling takes place in vitro.
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92. The method of claim 89, wherein the labeling takes place in vivo.
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93. An immunoglobulin labeled with a fluorescent compound comprising a polyalkylene oxide and a fluorophore that has an absorption maximal wavelength equal to or greater than 685 nm.
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94. An immunoglobulin labeled with a fluorescent compound comprising a polyalkylene oxide and a fluorophore that has an absorption maximal wavelength at or greater than 750 nm.
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95. The immunoglobulin of claim 93 or 94, wherein the immunoglobulin retains binding specificity to a target upon conjugation to the fluorescent compound.
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96. The immunoglobulin of claim 95, wherein the immunoglobin is an antibody that binds specifically to an antigen on a cancer cell.
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97. The immunoglobin of claim 96 wherein the antibody binds to erb2.
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98. The immunoglobulin of claim 93 or 94, wherein the fluorescent compound is a compound of claim 1, 25, 59, 60, or 66.
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99. A polypeptide labeled with a fluorescent compound, the polypeptide exhibiting a serum half-life no shorter than that of a corresponding polypeptide that lacks the fluorescent compound, wherein the fluorescent compound is a compound of claim 1, 25, 59, 60, 66, or 76.
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100. A method of labeling a polypeptide comprising:
- forming a complex that comprises the polypeptide and a binding agent, wherein the binding agent comprises a fluorescent label having a structure of formula of claim 1, 25, 59, 60, 66, or 76, wherein the at least one reactive moiety of the Formula has undergone a reaction which attaches the label to the binding agent.
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101. The method of claim 100 wherein the binding agent is an antibody.
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102. The method of claim 101 wherein the complex comprises (a) a primary antibody that binds to the polypeptide, and (b) the binding agent which functions as a secondary antibody exhibiting binding capability to the primary antibody.
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103. The method of claim 102, wherein the labeling occurs on a solid substrate.
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104. The method of claim 102 that labels a polypeptide intracellularly.
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105. The method of claim 102, wherein the complex yields a signal to noise ratio greater than about 100, wherein the signal to noise ratio is calculated by the formula:
(fluorescent signal from a complex comprising the polypeptide bound by a primary antibody which in turn is bound to the binding agent)/(fluorescent signal from a mixture of the polypeptide, an isotype control primary antibody and the binding agent).
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106. The method of claim 102, wherein the complex yields a signal to noise ratio greater than about 250, wherein the signal to noise ratio is calculated by the formula:
(fluorescent signal from a complex comprising the polypeptide bound by a primary antibody which in turn is bound to the binding agent)/(fluorescent signal from a mixture of the polypeptide, an isotype control primary antibody and the binding agent).
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107. The method of claim 102, wherein the complex yields a signal to noise ratio greater than about 270, wherein the signal to noise ratio is calculated by the formula:
(fluorescent signal from a complex comprising the polypeptide bound by a primary antibody which in turn is bound to the binding agent)/(fluorescent signal from a mixture of the polypeptide, an isotype control primary antibody and the binding agent).
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108. The method of claim 102, wherein the complex yields a total fluorescence signal at least 5% greater than that generated by a complex formed with the same primary antibody and with the same secondary antibody that has a comparable degree of labeling with a DyLight 680™
- dye.
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109. The method of claim 102 wherein the complex yields a total fluorescence signal at least 5% greater than that generated by a complex formed with the same primary antibody and with the same secondary antibody that has a comparable degree of labeling with a Cy5.5®
- dye.
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110. The method of claim 102 wherein the complex yields a total fluorescence signal at least 5% greater than that generated by a complex formed with the same primary antibody and with the same secondary antibody that has a comparable degree of labeling with an Alexa Fluor 680®
- dye.
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111. The method of claim 108, 109, or 110 wherein the label having a structure of a Formula of claim 25 is the compound having the structure:
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112. The method of claim 111 wherein each complex is excited at 635 nm or 633 nm.
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113. The method of claim 111 wherein each complex is present at identical protein concentrations.
Specification