IP Library › Granted Patent US 50,754

Granted Patent E1

US 50,754 · App. 17/861,032 · Granted Jan 20, 2026

Compositions and method for multiplex biomarker profiling

Inventors: Xiaohu Gao (Shoreline, WA); Pavel Zrazhevskiy (Redmond, WA)

Assignee: The University of Washington through its Center for Commercialization

C12Q1/6841 C12Q1/6804 G01N33/587 G01N33/588 G01N2458/10

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Quick Facts

Patent No.: US 50,754
App. No.: 17/861,032
Granted: Jan 20, 2026
Kind: E1

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Abstract

Provided herein are compositions and methods for identifying or quantitating one or more analytes in sample. The composition can comprise an affinity molecule reversibly conjugated to a label moiety via a double-stranded nucleic acid linker or via an adaptor molecule. The affinity molecule and the label moiety can be linked to different strands of the double-stranded nucleic acid linker. Compositions can be used in any biological assays for detection, identification and/or quantification of target molecules or analytes, including multiplex staining for molecular profiling of individual cells or cellular populations. For example, the compositions can be adapted for use in immunofluorescence, fluorescence in situ hybridization, immunohistochemistry, western blot, and the like.

Claims (171)

1 . A composition comprising a plurality of affinity molecules, wherein:

(i) each member of the plurality binds a target, wherein each affinity molecule is conjugated via hybridized first and second nucleic acid strands to a label moiety, wherein detectable properties of the label moieties are distinguishable from one another, wherein the targets are distinguishable from one another, wherein the hybridized first nucleic acid strands are distinguishable from one another, wherein members of the plurality of affinity molecules are distinguishable from one another, wherein the affinity molecule is linked to the first nucleic acid strand via an adaptor molecule or via a linker selected from the group consisting of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl-6-hydrazino-nicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH 2 groups triazole bond from “click” reaction), a phosphodiester linkage, a phosphorothioate linkage, or a combination thereof, and wherein the label moiety is linked to the second nucleic acid strand via an adaptor molecule or via a linker selected from the group consisting of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl-6-hydrazino-nicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH 2 groups triazole bond from “click” reaction a phosphodiester linkage a phosphorothioate linkage, or a combination thereof; or

(ii) each member of the plurality binding a target, wherein each affinity molecule is conjugated to a first single strand nucleic acid molecule that is specifically hybridized to a second single strand nucleic acid molecule, wherein the second single strand nucleic acid molecule is conjugated to a label moiety, such that each affinity molecule is conjugated via hybridized first and second nucleic acid strands to a label moiety, wherein detectable properties of the label moieties are distinguishable from one another, wherein the targets are distinguishable from one another, wherein the first single strand nucleic acid molecules are distinguishable from one another, wherein members of the plurality of affinity molecules are distinguishable from one another, and wherein the affinity molecule is linked to the first single strand nucleic acid molecule via an adaptor molecule or via a linker selected from the group consisting of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl-6-hydrazino-nicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH 2 groups, triazole bond (from “click” reaction), a phosphodiester linkage, a phosphorothioate linkage, or a combination thereof, and wherein the label is linked to the second single strand nucleic acid molecule via an adaptor molecule or via a linker selected from the group consisting of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl-6-hydrazino-nicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH 2 groups triazole bond from “click” reaction a phosphodiester linkage, a phosphorothioate linkage, or a combination thereof.

2 . A composition comprising a solid support comprising a sample for analysis for the presence of analytes, and a plurality of affinity molecules, each member of the plurality of affinity molecules conjugated to a first single-strand nucleic acid, wherein each member of the plurality of affinity molecules is bound to a different analyte in the sample, and wherein each first single-strand nucleic acid has a different nucleotide sequence, wherein members of the plurality of affinity molecules are distinguishable from one another, and wherein the affinity molecule is linked to the first single-strand nucleic acid via an adaptor molecule or via a linker selected from the group consisting of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl-6-hydrazino-nicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH 2 groups, triazole bond (from “click” reaction), a phosphodiester linkage, a phosphorothioate linkage, or a combination thereof.

3 . The composition of claim 2 , wherein the composition further comprises a plurality of second single strand nucleic acid molecules, each conjugated to a label moiety, wherein each second single strand nucleic acid molecule specifically hybridizes to a first single strand nucleic acid molecule conjugated to a member of the plurality of affinity molecules, such that at least a subset of the plurality of affinity molecules bound to the analytes in the sample is specifically associated with a plurality of label moieties, wherein detectable properties of the label moieties are distinguishable from one another, wherein members of the plurality of second single stand nucleic acid molecules are distinguishable from one another, and wherein the label moiety is linked to the second single strand nucleic acid molecule via an adaptor molecule or via a linker selected from the group consisting of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl-6-hydrazino-nicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH 2 groups, triazole bond (from “click” reaction), a phosphodiester linkage, a phosphorothioate linkage, or a combination thereof.

4 . A method of analyzing a sample for a plurality of analytes, the method comprising, in order:

(a) contacting the sample with a plurality of affinity molecules under conditions that permit specific analyte binding by the affinity molecules, wherein each affinity molecule specifically binds a different member of the plurality of analytes, and wherein each affinity molecule is conjugated to a first single strand nucleic acid, such that members of the plurality of affinity molecules become bound to members of the plurality of analytes present in the sample, wherein the first single strand nucleic acids are distinguishable from each other, wherein members of the plurality of affinity molecules are distinguishable from one another, and wherein the affinity molecule is linked to the first single strand nucleic acid via an adaptor molecule or via a linker selected from the group consisting of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl-6-hydrazino-nicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH 2 groups triazole bond from “click” reaction), a phosphodiester linkage, a phosphorothioate linkage, or a combination thereof;

(b) contacting the sample, under conditions that permit specific nucleic acid hybridization, with a first set of second single strand nucleic acid molecules, each conjugated to a label moiety, wherein each second single strand nucleic acid molecule specifically hybridizes to a first single strand nucleic acid molecule conjugated to a member of the plurality of affinity molecules, such that at least a subset of the plurality of affinity molecules bound to members of the plurality of analytes in the sample becomes specifically associated with a plurality of label moieties, wherein detectable properties of the label moieties are distinguishable from one another, wherein members of the first set of second single strand nucleic acid molecules are distinguishable from one another, and wherein the label moiety is linked to the second single strand nucleic acid molecule via an adaptor molecule or via a linker selected from the group consisting of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl-6-hydrazino-nicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH, groups, triazole bond (from “click” reaction), a phosphodiester linkage, a phosphorothioate linkage, or a combination thereof; and

(c) detecting signal from label moieties associated with affinity molecules bound to the sample, thereby detecting the presence or amount of at least a subset of the plurality of analytes.

5 . The method of claim 4 , further comprising the steps of:

(d) erasing the signal from the label moieties conjugated to the first set of second single strand nucleic acid molecules;

(e) contacting the sample, under conditions that permit specific nucleic acid hybridization, with a second set of second single strand nucleic acid molecules, each conjugated to a label, wherein each second single strand nucleic acid molecule specifically hybridizes to a first single strand nucleic acid molecule conjugated to a member of the plurality of affinity molecules, such that a subset of the plurality of affinity molecules bound to members of the plurality of analytes in the sample becomes specifically associated with a plurality of label moieties, wherein members of the second set of second single strand nucleic acid molecules are distinguishable from one another and from members of the first subset of single strand nucleic acid molecules of step (b), and wherein the label moiety is linked to the second single strand nucleic acid molecule via an adaptor molecule or via a linker selected from the group consisting of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl-6-hydrazino-nicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH 2 groups, triazole bond (from “click” reaction), a phosphodiester linkage, a phosphorothioate linkage, or a combination thereof;

(f) detecting signal from label moieties associated with affinity molecules bound to the sample, thereby detecting the presence or amount of the subset of the plurality of analytes; and

(g) optionally repeating steps (d)-(f) with a further set of second single strand nucleic acid molecules.

6 . A method of analyzing a sample for a plurality of analytes, the method comprising, in order:

(a) contacting the sample with a first plurality of affinity molecules under conditions that permit specific analyte binding by the affinity molecules, wherein each affinity molecule specifically binds a different member of the plurality of analytes, wherein each affinity molecule is conjugated to a first single strand nucleic acid molecule that is specifically hybridized to a second single strand nucleic acid molecule, wherein the second single strand nucleic acid molecule is conjugated to a label moiety, such that each affinity molecule is conjugated via hybridized first and second nucleic acid strands to a label moiety and members of the plurality of different affinity molecules become bound to members of the plurality of analytes present in the sample, wherein detectable properties of the label moieties are distinguishable from one another, wherein the first single strand nucleic acids are distinguishable from each other, and wherein the affinity molecule is linked to the first single strand nucleic acid molecule via an adaptor molecule or via a linker selected from the group consisting of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl-6-hydrazino-nicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH 2 groups triazole bond from “click” reaction), a phosphodiester linkage, a phosphorothioate linkage, or a combination thereof, and wherein the label is linked to the second single strand nucleic acid molecule via an adaptor molecule or via a linker selected from the group consisting of succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) linker, sulfo-SMCC linker, succinimidyl-6-hydrazino-nicotinamide (S-HyNic) linker, N-succinimidyl-4-formylbenzamide (S-4FB) linker, bis-aryl hydrazone bond, an amide bond, two amide bonds on a spacer for cross-linking two —NH 2 groups, triazole bond (from “click” reaction), a phosphodiester linkage, a phosphorothioate linkage, or a combination thereof; and

(b) detecting signal from label moieties associated with first plurality of affinity molecules bound to the sample, thereby detecting the presence or amount of the plurality of analytes.

7 . The method of claim 6 , further comprising the steps of:

(c) erasing the signal from the label molecules conjugated to the first set of second single strand nucleic acid molecules;

(d) contacting the sample with a second plurality of affinity molecules under conditions that permit specific analyte binding by the affinity molecules wherein members of the second plurality of affinity molecules are distinguishable from one another and from members of the first plurality of affinity molecules of step (a);

(e) detecting signal from label moieties associated with second plurality of affinity molecules bound to the sample, thereby detecting the presence or amount of at least a subset of the plurality of analytes; and

(f) optionally repeating steps (c)-(e) with a further second set of the affinity molecules.

8. A method, comprising:

(a) introducing into a sample a plurality of probes, wherein:

each probe of the plurality of probes comprises an affinity moiety linked to at least one tag moiety through at least one phosphodiester moiety;

the affinity moiety comprises a first nucleic acid sequence that hybridizes to a target RNA sequence in the sample; and

each tag moiety comprises a second nucleic acid sequence;

(b) contacting the sample with at least one labeling agent comprising a third nucleic acid sequence linked to an optical label through a phosphodiester moiety, wherein the third nucleic acid sequence hybridizes to the second nucleic acid sequence;

(c) obtaining at least one image of the sample, the at least one image comprising one or more optical signals corresponding to at least one optical label of the at least one labeling agent;

(d) removing the at least one optical label from the sample; and

(e) repeating steps (b)-(d) with at least one additional labeling agent.

9. The method of claim 8 , wherein the first nucleic acid sequence is a DNA sequence.

10. The method of claim 8 , wherein the second nucleic acid sequence is a DNA sequence.

11. The method of claim 8 , wherein the first nucleic acid sequence comprises at least 10 nucleotides.

12. The method of claim 11 , wherein the first nucleic acid sequence comprises at least 20 nucleotides.

13. The method of claim 8 , wherein the second nucleic acid sequence comprises at least 10 nucleotides.

14. The method of claim 13 , wherein the second nucleic acid sequence comprises at least 20 nucleotides.

15. The method of claim 8 , wherein the affinity moiety and the at least one tag moiety form a contiguous nucleic acid sequence.

16. The method of claim 8 , wherein at least one additional moiety is interposed between the affinity moiety and the at least one tag moiety.

17. The method of claim 8 , wherein the third nucleic acid comprises at least 10 nucleotides.

18. The method of claim 17 , wherein the third nucleic acid comprises at least 20 nucleotides.

19. The method of claim 8 , wherein the optical label comprises a fluorescent moiety.

20. The method of claim 8 , wherein the optical label comprises at least one fluorescent nucleotide.

21. The method of claim 8 , wherein the at least one tag moiety comprises multiple tag moieties linked to the affinity moiety.

22. The method of claim 21 , wherein the multiple tag moieties are the same.

23. The method of claim 8 , wherein:

the plurality of probes comprises multiple different types of probes;

each different type of probe comprises an affinity moiety comprising a first nucleic acid sequence that hybridizes to a different target RNA sequence relative to other types of probes; and

the at least one tag moiety of each different type of probe is different from the at least one tag moiety of each other type of probe of the multiple different types of probes.

24. The method of claim 23 , wherein the at least one tag moiety of one or more types of probes of the multiple different types of probes comprises multiple tag moieties.

25. The method of claim 24 , wherein the multiple tag moieties are the same.

26. The method of claim 23 , wherein a first type of probe of the multiple different types of probes comprises a different number of tag moieties relative to a second type of probe of the multiple different types of probes.

27. The method of claim 23 , wherein:

the at least one labeling agent comprises multiple different types of labeling agents; and

each different type of labeling agent comprises a third nucleic acid that is different from the third nucleic acid of other types of labeling agents among the multiple different types of labeling agents, and an optical label that is different from the optical label of other types of labeling agents in step (b).

28. The method of claim 27 , wherein the one or more optical signals comprise different optical signals corresponding to the optical labels of the multiple different types of labeling agents.

29. The method of claim 23 , wherein the plurality of probes comprises at least 25 different types of probes.

30. The method of claim 29 , wherein the plurality of probes comprises at least 100 different types of probes.

31. The method of claim 27 , wherein the multiple different types of labeling agents comprises at least 3 different types of labeling agents.

32. The method of claim 31 , wherein the multiple different types of labeling agents comprises at least 5 different types of labeling agents.

33. The method of claim 8 , wherein removing the at least one optical label from the sample comprises dehybridizing the third nucleic acid sequence from the second nucleic acid sequence.

34. The method of claim 8 , wherein removing the at least one optical label from the sample comprises erasing optical signals arising from the at least one optical label.

35. The method of claim 8 , further comprising detecting at least one target RNA sequence in the sample based on the one or more optical signals of the at least one obtained image.

36. The method of claim 23 , further comprising detecting multiple different target RNA sequences in the sample based on the one or more optical signals of the at least one obtained image.

37. The method of claim 23 , further comprising, by repeating steps (b)-(d), obtaining multiple images of the sample each comprising one or more optical signals associated with different types of probes, and detecting multiple different target RNA sequences in the sample based on the optical signals of the multiple images of the sample.

38. A method, comprising:

(a) introducing into a sample a plurality of probes, wherein the plurality of probes comprises:

a first set of probes, each of which comprises an affinity moiety comprising a first nucleic acid sequence that hybridizes to a target RNA sequence in the sample and is linked to at least one tag moiety through a first linker; and

a second set of probes, each of which comprises an affinity moiety that binds to a protein or peptide in the sample and is linked to at least one tag moiety through a second linker,

wherein each tag moiety comprises a second nucleic acid sequence; and

(b) contacting the sample with at least one labeling agent comprising a third nucleic acid sequence linked to an optical label through a third linker, wherein the third nucleic acid sequence hybridizes to the second nucleic acid sequence;

(c) obtaining at least one image of the sample, the at least one image comprising one or more optical signals corresponding to at least one optical label of the at least one labeling agent;

(d) removing the at least one optical label from the sample; and

(e) repeating steps (b)-(d) with at least one additional labeling agent,

wherein the first, second, and third linkers each independently comprise at least one member selected from the group consisting of a succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) moiety, a sulfo-SMCC moiety, a succinimidyl-6-hydrazino-nicotinamide (S-HyNic) moiety, a N-succinimidyl-4-formylbenzamide (S-4FB) moiety, a bis-aryl hydrazine bond, an amide bond, two amide bonds on a spacer for cross-linking two amino groups, a triazole bond, a phosphodiester moiety, or a phosphorothioate moiety.

39. The method of claim 38 , wherein the first set of probes comprises multiple different types of probes, wherein for each different type of probe of the first set of probes:

the first nucleic acid sequence of the affinity moiety hybridizes to a different target RNA sequence relative to other types of probes of the first set of probes; and

the at least one tag moiety is different from the at least one tag moiety of each other type of probe of the first set of probes.

40. The method of claim 38 , wherein the second set of probes comprises multiple different types of probes, wherein for each different type of probe of the second set of probes:

the affinity moiety binds to a different protein or peptide relative to other types of probes of the second set of probes; and

the at least one tag moiety is different from the at least one tag moiety of each other type of probe of the second set of probes.

41. The method of claim 38 , wherein:

the first set of probes comprises multiple different types of probes and the second set of probes comprises multiple different types of probes;

for each different type of probe of the first set of probes, the first nucleic acid sequence of the affinity moiety hybridizes to a different target RNA sequence relative to other types of probes of the first set of probes; and

for each different type of probe of the second set of probes, the affinity moiety binds to a different protein or peptide relative to other types of probes of the second set of probes.

42. The method of claim 41 , wherein for each different type of probe of the first and second sets of probes, the at least one tag moiety is different from the at least one tag moiety of each other type of probe among the first and second sets of probes.

43. The method of claim 38 , wherein the affinity moiety of each probe of the second set of probes comprises an antibody or antibody fragment.

44. The method of claim 38 , wherein:

the at least one labeling agent comprises multiple different types of labeling agents; and

45. The method of claim 44 , wherein the third nucleic acid hybridizes to the at least one tag moiety of only one type of probe among the first set of probes.

46. The method of claim 44 , wherein the third nucleic acid hybridizes to the at least one tag moiety of only one type of probe among the second set of probes.

47. The method of claim 44 , wherein the third nucleic acid hybridizes to the at least one tag moiety of only one type of probe among the first and second sets of probes.

48. The method of claim 44 , wherein the one or more optical signals comprise different optical signals corresponding to the optical labels of the multiple different types of labeling agents.

49. The method of claim 44 , wherein the multiple different types of labeling agents comprises at least 3 different types of labeling agents.

50. The method of claim 49 , wherein the multiple different types of labeling agents comprises at least 5 different types of labeling agents.

51. The method of claim 38 , further comprising detecting at least one target RNA sequence in the sample based on the one or more optical signals of the at least one obtained image.

52. The method of claim 38 , further comprising detecting multiple different target RNA sequences in the sample based on the one or more optical signals of the at least one obtained image.

53. The method of claim 38 , further comprising detecting multiple different target RNA sequences and at least one protein or peptide in the sample based on the one or more optical signals of the at least one obtained image.

54. The method of claim 41 , further comprising, by repeating steps (b)-(d), obtaining multiple images of the sample each comprising one or more optical signals associated with the different types of probes of the first and second sets of probes, and detecting multiple different target RNA sequences and multiple different proteins or peptides in the sample based on the one or more optical signals of the multiple images of the sample.

55. The method of claim 38 , wherein the first nucleic acid sequence is a DNA sequence.

56. The method of claim 38 , wherein the second nucleic acid sequence is a DNA sequence.

57. The method of claim 38 , wherein the first nucleic acid sequence comprises at least 10 nucleotides.

58. The method of claim 38 , wherein the second nucleic acid sequence comprises at least 10 nucleotides.

59. The method of claim 38 , wherein the affinity moiety and the at least one tag moiety of the first set of probes form a contiguous nucleic acid sequence.

60. The method of claim 38 , wherein at least one additional moiety is interposed between the affinity moiety and the at least one tag moiety of the first set of probes.

61. The method of claim 38 , wherein at least one additional moiety is interposed between the affinity moiety and the at least one tag moiety of the second set of probes.

62. The method of claim 38 , wherein the third nucleic acid comprises at least 10 nucleotides.

63. The method of claim 38 , wherein the optical label comprises a fluorescent moiety.

64. The method of claim 38 , wherein the optical label comprises at least one fluorescent nucleotide.

65. The method of claim 38 , wherein the at least one tag moiety of the first set of probes comprises multiple tag moieties linked to the affinity moiety.

66. The method of claim 65 , wherein the multiple tag moieties are the same.

67. The method of claim 38 , wherein the at least one tag moiety of the second set of probes comprises multiple tag moieties linked to the affinity moiety.

68. The method of claim 67 , wherein the multiple tag moieties are the same.

69. The method of claim 39 , wherein the plurality of probes comprises at least 25 different types of probes among the first set of probes.

70. The method of claim 40 , wherein the plurality of probes comprises at least 3 different types of probes among the second set of probes.

71. The method of claim 41 , wherein the plurality of probes comprises at least 25 different types of probes among the first and second sets of probes.

72. The method of claim 38 , wherein removing the at least one optical label from the sample comprises dehybridizing the third nucleic acid sequence from the second nucleic acid sequence.

73. The method of claim 38 , wherein removing the at least one optical label from the sample comprises erasing optical signals arising from the at least one optical label.

74. A kit, comprising:

a first composition comprising multiple different types of probes, wherein each different type of probe comprises:

a different affinity moiety comprising a first nucleic acid sequence that hybridizes to a different target RNA sequence relative to other types of probes among the multiple different types of probes; and

linked to the different affinity moiety through at least one phosphodiester moiety, at least one tag moiety comprising a second nucleic acid sequence that is different from the at least one tag moiety of the other types of probes among the multiple different types of probes; and

a second composition comprising multiple different types of labeling agents, wherein each different type of labeling agent comprises:

a different third nucleic acid sequence that hybridizes to only one of the second nucleic acid sequences of the different types of probes; and

linked to the third nucleic acid sequence through at least one phosphodiester moiety, an optical label.

75. The kit of claim 74 , wherein the first nucleic acid sequence is a DNA sequence.

76. The kit of claim 74 , wherein the second nucleic acid sequence is a DNA sequence.

77. The kit of claim 74 , wherein the first nucleic acid sequence comprises at least 10 nucleotides.

78. The kit of claim 74 , wherein the second nucleic acid sequence comprises at least 10 nucleotides.

79. The kit of claim 74 , wherein the different affinity moiety and the at least one tag moiety form a contiguous nucleic acid sequence.

80. The kit of claim 74 , wherein at least one additional moiety is interposed between the different affinity moiety and the at least one tag moiety.

81. The kit of claim 74 , wherein the third nucleic acid sequence comprises at least 10 nucleotides.

82. The kit of claim 74 , wherein the optical label comprises a fluorescent moiety.

83. The kit of claim 74 , wherein the optical label comprises at least one fluorescent nucleotide.

84. The kit of claim 74 , wherein the at least one tag moiety comprises multiple tag moieties linked to the different affinity moiety.

85. The kit of claim 84 , wherein the multiple tag moieties are the same.

86. The kit of claim 74 , wherein a first type of probe of the multiple different types of probes comprises a different number of tag moieties relative to a second type of probe of the multiple different types of probes.

87. The kit of claim 74 , wherein the first composition comprises at least 25 different types of probes.

88. The kit of claim 74 , wherein the second composition comprises at least 3 different types of labeling agents.

89. The kit of claim 88 , wherein the second composition comprises at least 5 different types of labeling agents.

90. A kit, comprising:

a first composition comprising multiple different types of first probes, wherein each different type of first probe comprises:

a different first affinity moiety comprising a first nucleic acid sequence that hybridizes to a different RNA target sequence relative to other types of first probes among the multiple different types of first probes; and

linked to the different first affinity moiety through a first linker, at least one tag moiety comprising a second nucleic acid sequence that is different from the at least one tag moiety of other types of first probes among the multiple different types of first probes;

a second composition comprising multiple different types of second probes, wherein each different type of second probe comprises:

a different second affinity moiety that binds to a different protein or peptide relative to other types of second probes among the multiple different types of second probes; and

linked to the different second affinity moiety through a second linker, at least one tag moiety comprising a third nucleic acid sequence that is different from the at least one tag moiety of other types of second probes among the multiple different types of second probes; and

a third composition comprising:

a first set of multiple different types of labeling agents, wherein each different type of labeling agent of the first set comprises:

a different fourth nucleic acid sequence that hybridizes to the second nucleic acid sequence of only one type of first probe; and

linked to the different fourth nucleic acid sequence through a third linker, an optical label,

91. The kit of claim 90 , wherein the third composition comprises:

a second set of multiple different types of labeling agents, wherein each different type of labeling agent of the second set comprises:

a different fifth nucleic acid sequence that hybridizes to the third nucleic acid sequence of only one type of second probe; and

linked to the different fifth nucleic acid sequence through a fourth linker, an optical label,

wherein the fourth linker independently comprises at least one member selected from the group consisting of a succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) moiety, a sulfo-SMCC moiety, a succinimidyl-6-hydrazino-nicotinamide (S-HyNic) moiety, a N-succinimidyl-4-formylbenzamide (S-4FB) moiety, a bis-aryl hydrazine bond, an amide bond, two amide bonds on a spacer for cross-linking two amino groups, a triazole bond, a phosphodiester moiety, or a phosphorothioate moiety.

92. The kit of claim 90 , wherein the first nucleic acid sequence is a DNA sequence.

93. The kit of claim 90 , wherein the second nucleic acid sequence is a DNA sequence.

94. The kit of claim 90 , wherein the first nucleic acid sequence comprises at least 10 nucleotides, and wherein the second nucleic acid sequence comprises at least 10 nucleotides.

95. The kit of claim 90 , wherein the different first affinity moiety and the at least one tag moiety of each type of first probe form a contiguous nucleic acid sequence.

96. The kit of claim 90 , wherein at least one additional moiety is interposed between the different first affinity moiety and the at least one tag moiety of one or more different types of first probes.

97. The kit of claim 90 , wherein the at least one tag moiety of one or more of the different types of first probes comprises multiple tag moieties linked to the different first affinity moiety.

98. The kit of claim 97 , wherein the multiple tag moieties linked to the different first affinity moiety are the same.

99. The kit of claim 97 , wherein a first type of different first probe comprises a different number of tag moieties relative to a second type of different first probe.

100. The kit of claim 90 , wherein the third nucleic acid sequence comprises at least 10 nucleotides.

101. The kit of claim 90 , wherein each second affinity moiety comprises an antibody or antibody fragment.

102. The kit of claim 90 , wherein the optical label comprises a fluorescent moiety.

103. The kit of claim 90 , wherein the first composition comprises at least 25 different types of first probes.

104. The kit of claim 90 , wherein the second composition comprises at least 3 different types of second probes.

Assignments (2)

RELEASE OF SECURITY INTEREST Recorded Jul 11, 2025

From: MIDCAP FINANCIAL TRUST

To: AKOYA BIOSCIENCES, INC.

Reel/Frame 071893/0033 →

SECURITY INTEREST Recorded Jun 4, 2025

From: AKOYA BIOSCIENCES, INC.

To: MIDCAP FINANCIAL TRUST

Reel/Frame 071484/0056 →

Continuity (7)

Continuation 16840107 · Apr 3, 2020

Continuation 16022679 · Jun 28, 2018

Reissue 14577507 · Dec 19, 2014

Division 13455674 · Apr 25, 2012

Provisional Application 61478618 · Apr 25, 2011

Provisional Application 61478626 · Apr 25, 2011

Reissue 14577507 · Dec 19, 2014

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Jin et al., Molecular BioSystems, 6: 2325-2331 (2010). “Antibody-ProteinA conjugated quantum dots for multiplexed imaging of surface receptors in living cells.” [cited by applicant]

Kang et al., Small, 5:2519-2522 (2009). “Multiplex imaging of single tumor cells using quantum-dot-conjugated aptamers.” [cited by applicant]

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