METHOD FOR DETECTING MULTIPLE PREDETERMINED COMPOUNDS IN A SAMPLE

• US 20160041179A1
• Filed: 03/14/2014
• Published: 02/11/2016
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: JU, Jingyue, KUMAR, Shiv, Russo, James J., TAO, Chuanjuan, KALACHIKOV, Sergey
• Granted Patent: US 10,732,183 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C08G 65/48 Polymers modified by chemical after-treatment
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68
• CPC Class Codes: G01N 33/6818 Sequencing of polypeptides

Abstract

This invention provides methods for detecting the presence of a plurality of predetermined compounds in a sample using a plurality of tag moieties and at least one nanopore. This invention also provides methods for determining the quantity of each of a plurality of predetermined compounds in a sample using a plurality of tag moieties and at least one nanopore. This invention further provides methods for detecting interaction of at least two predetermined compounds using at least one tag moiety and at least one nanopore.

Claims

1. A method for detecting the presence of a plurality of predetermined compounds in a sample which comprises:

(a) contacting the sample with a plurality of detectable tag moieties, wherein each detectable tag moiety comprises at least one detectable component, under conditions permitting the tag moieties to attach to each of the predetermined compounds present in the sample to form detectably tagged predetermined compounds, wherein each tag moiety uniquely attaches to one of the predetermined compounds present in the sample and is distinguishable from any tag moiety attached to any other predetermined compound present in the sample;

(b) treating the sample from step (a) to form an electrolyte solution containing the detectably tagged predetermined compounds;

(c) contacting the electrolyte solution from step (b) with at least one nanopore, wherein said at least one nanopore comprises a means for binding each of the detectably tagged predetermined compounds present in the electrolyte solution, under conditions such that the detectably tagged predetermined compounds bind to said at least one nanopore;

(d) treating the detectably tagged predetermined compounds bound to said at least one nanopore so as to separate the tag moieties from the predetermined compounds; and

(e) detecting said at least one detectable component of each of the tag moieties so released as the tag moieties pass through said at least one nanopore so as to thereby detecting the presence of the plurality of predetermined compounds in the sample.

5. The method of any one of claims 1-4, further comprising a washing step to remove any detectably tagged predetermined compound which is not bound to said at least one nanopore prior to step (d).

6. The method of any one of claims 1-5, wherein the predetermined compound is a protein.

7. The method of claim 6, wherein the protein is an antibody.

8. The method of any one of claims 1-5, wherein the predetermined compound is non-proteinaceous.

9. The method of any one of claims 1-8, wherein at least one of said tag moieties comprises more than one detectable component.

10. The method of any one of claims 1-9, wherein each detectable component is independently detectable.

11. The method of any one of claims 1-10, wherein each of said at least one detectable component of said tag moieties is selected from the group consisting of ethylene glycol, an amino acid, a carbohydrate, a peptide, a dye, a fluorescent compound, a chemilluminiscent compound, a mononucleotide, a dinucleotide, a trinucleotide, a tetranucleotide, a pentanucleotide, a hexanucleotide, a polynucleotide, a nucleotide monophopshate, a nucleotide diphosphate, a nucleotide polyphosphate, an aliphatic acid, an aromatic acid, an unsubstituted alcohol or thiol, an alcohol or a thiol substituted with one or more halogens, a cyano group, a nitro group, an alkyl group, an alkenyl group, an alkynyl group, and an azido group.

12. The method of any one of claims 1-11, wherein said at least one detectable component of said tag moieties comprises a multiplicity of ethylene glycol units.

13. The method of any one of claims 1-12, wherein said multiplicity of ethylene glycol units comprises 16, 20, 24, or 36 ethylene glycol units.

14. The method of any one of claims 1-13, wherein at least one of said tag moieties attaches to the predetermined compound via a cleavable linker.

15. The method of claim 14, wherein the cleavable linker is a photocleavable linker.

16. The method of claim 14, wherein the cleavable linker is a chemically cleavable linker.

17. The method of any one of claims 1-16, wherein at least one of the predetermined compounds present in the sample is a protein and at least one of said tag moieties attaches to the carboxy or amino terminus of said protein.

18. The method of any one of claims 1-17, wherein at least one of the predetermined compounds present in the sample is a protein and at least one of said tag moieties attaches to a lysine, an arginine, or a cysteine residue of said protein.

19. The method of any one of claims 1-18, wherein said at least one nanopore is a biological nanopore, a modified biological nanopore, or a synthetic nanopore.

20. The method of claim 19, wherein said at least one nanopore is proteinaceous.

21. The method of any one of claims 1-20, wherein said at least one nanopore is an alpha hemolysin (α

-hemolysin).

22. The method of any one of claims 1-18, wherein said at least one nanopore is a solid-state nanopore.

23. The method of claim 22, wherein the solid-state nanopore comprises graphene.

24. The method of any one of claims 1-23, wherein said at least one nanopore is in a membrane.

25. The method of any one of claims 1-24, wherein said at least one nanopore is part of an array of nanopores.

26. The method of claim 25, wherein each nanopore in said array comprises identical means for binding the detectably tagged predetermined compounds.

27. The method of claim 25, wherein each nanopore in said array comprises different means for binding the detectably tagged predetermined compounds.

28. The method of any one of claims 1-27, wherein the means for binding the detectably tagged predetermined compounds is a protein.

29. The method of claim 28, wherein the protein is an antibody.

30. The method of any one of claim 1-27, wherein the means for binding the detectably tagged predetermined compounds is non-proteinaceous.

31. The method of any one of claims 1-30, wherein each tag moiety is distinguishable from any other tag moiety based on blockade signature of said tag moiety detectable with said at least one nanopore.

32. The method of claim 31, wherein the blockade signature is result of a change in current amplitude of said at least one nanopore.

33. The method of claim 31, wherein the blockade signature is result of a change in conductance of said at least one nanopore.

34. The method of any one of claims 1-33, wherein said at least one nanopore further comprising a means for ejecting said tag moiety from the nanopore.

70. The method of any one of claims 1-68, wherein the means for ejecting said tag moiety from the nanopore consisting of a means to adjust electric field of said nanopore.

71. A detectable tag moiety for use in the method of any one of claims 1-70 which comprises a protein reactive group, at least one detectable component, and a linker connecting the protein reactive group and the at least one detectable component.

72. The tag moiety of claim 71, wherein the linker is a cleavable linker.

73. The tag moiety of claim 72, wherein the cleavable linker is a photocleavable linker.

74. The tag moiety of claim 72, wherein the cleavable linker is a chemically cleavable linker.

75. The tag moiety of any one of claims 71-74, wherein the at one detectable component is selected from the group consisting of ethylene glycol, an amino acid, a carbohydrate, a peptide, a dye, a fluorescent compound, a chemilluminiscent compound, a mononucleotide, a dinucleotide, a trinucleotide, a tetranucleotide, a pentanucleotide, a hexanucleotide, a polynucleotide, a nucleotide monophopshate, a nucleotide diphosphate, a nucleotide polyphosphate, an aliphatic acid, an aromatic acid, an unsubstituted alcohol or thiol, an alcohol or a thiol substituted with one or more halogens, a cyano group, a nitro group, an alkyl group, an alkenyl group, an alkynyl group, and an azido group.

2. A method for detecting the presence of a plurality of predetermined compounds in a sample which comprises:

(a) contacting the sample with a plurality of detectable tag moieties, wherein each detectable tag moiety comprises at least one detectable component, under conditions permitting the tag moieties to attach to each of the predetermined compounds present in the sample to form detectably tagged predetermined compounds, wherein each tag moiety uniquely attaches to one of the predetermined compounds present in the sample and is distinguishable from any tag moiety attached to any other predetermined compound present in the sample;

(b) treating the sample from step (a) to form an electrolyte solution containing the detectably tagged predetermined compounds;

(c) contacting the electrolyte solution from step (b) with at least one nanopore, wherein said at least one nanopore comprises a means for binding each of the detectably tagged predetermined compounds present in the electrolyte solution, under conditions such that the detectably tagged predetermined compounds bind to said at least one nanopore, and wherein each of the tag moieties of said bound detectably tagged predetermined compounds localizes within said at least one nanopore;

(d) detecting said at least one detectable component of each of the tag moieties with said at least one nanopore so as to thereby detecting the presence of the plurality of predetermined compounds in the sample.

3. A method for determining the quantity of each of a plurality of predetermined compounds in a sample which comprises:

(a) contacting the sample with a plurality of detectable tag moieties, wherein each detectable tag moiety comprises at least one detectable component, under conditions permitting the tag moieties to attach to each of the predetermined compounds present in the sample to form detectably tagged predetermined compounds, wherein each tag moiety uniquely attaches to one of the predetermined compounds present in the sample and is distinguishable from any tag moiety attached to any other predetermined compound present in the sample;

(b) treating the sample from step (a) to form an electrolyte solution containing the detectably tagged predetermined compounds;

(c) contacting the electrolyte solution from step (b) with at least one nanopore, wherein said at least one nanopore comprises a means for binding each of the detectably tagged predetermined compounds present in the electrolyte solution, under conditions such that the detectably tagged predetermined compounds bind to said at least one nanopore;

(d) treating the detectably tagged predetermined compounds bound to said at least one nanopore so as to separate the tag moieties from the predetermined compounds;

(e) determining the amount of said at least one detectable component of each of the tag moieties so released as the tag moieties pass through said at least one nanopore; and

(f) determining, from the amounts of said detectable components determined in step (e), the quantity of each of the plurality of predetermined compounds present in the sample.

4. A method for determining the quantity of each of a plurality of predetermined compounds in a sample which comprises:

(a) contacting the sample with a plurality of detectable tag moieties, wherein each detectable tag moiety comprises at least one detectable component, under conditions permitting the tag moieties to attach to each of the predetermined compounds present in the sample to form detectably tagged predetermined compounds, wherein each tag moiety uniquely attaches to one of the predetermined compounds present in the sample and is distinguishable from any tag moiety attached to any other predetermined compound present in the sample;

(b) treating the sample from step (a) to form an electrolyte solution containing the detectably tagged predetermined compounds;

(c) contacting the electrolyte solution from step (b) with at least one nanopore, wherein said at least one nanopore comprises a means for binding each of the detectably tagged predetermined compounds present in the electrolyte solution, under conditions such that the detectably tagged predetermined compounds bind to said at least one nanopore, and wherein each of the tag moieties of said bound detectably tagged predetermined compounds localizes within said at least one nanopore;

(d) determining the amount of said at least one detectable component of each of the tag moieties with said at least one nanopore; and

(e) determining, from the amounts of said detectable components determined in step (d), the quantity of each of the plurality of predetermined compounds present in the sample.

35. A method for detecting interaction of at least two predetermined compounds which comprises:

(a) contacting at least a first predetermined compound present in a sample with a detectable tag moiety, wherein the detectable tag moiety comprises at least one detectable component, under conditions permitting the tag moiety to attach to the predetermined compound present in the sample to form a detectably tagged predetermined compound;

(b) treating the sample from step (a) to form an electrolyte solution containing the detectably tagged predetermined compounds;

(c) contacting the electrolyte solution from step (b) with at least one nanopore, wherein at least a second predetermined compound is immobilized on said at least one nanopore, under conditions permitting the binding of the detectably tagged predetermined compound with the second predetermined compound;

(d) treating the detectably tagged predetermined compound bound to said at least one nanopore so as to separate the tag moiety from the predetermined compound; and

(e) detecting said at least one detectable component of the tag moiety so released as the tag moiety passes through said at least one nanopore so as to thereby detecting the interaction of the predetermined compounds.

37. The method of claim 35 or 36, further comprising a washing step to remove any detectably tagged predetermined compound which is not bound to the second predetermined compound prior to step (d).

38. The method of any one of claims 35-37, wherein at least one of the predetermined compounds is a protein.

39. The method of claim 38, wherein the protein is an antibody.

40. The method of any one of claims 35-37, wherein at least one of the predetermined compounds is non-proteinaceous.

41. The method of any one of claims 35-40, wherein said tag moiety comprises more than one detectable component.

42. The method of any one of claims 35-41, wherein each detectable component is independently detectable.

43. The method of any one of claims 35-42, wherein each of said at least one detectable component of said tag moiety is selected from the group consisting of ethylene glycol, an amino acid, a carbohydrate, a peptide, a dye, a fluorescent compound, a chemilluminiscent compound, a mononucleotide, a dinucleotide, a trinucleotide, a tetranucleotide, a pentanucleotide, a hexanucleotide, a polynucleotide, a nucleotide monophopshate, a nucleotide diphosphate, a nucleotide polyphosphate, an aliphatic acid, an aromatic acid, an unsubstituted alcohol or thiol, an alcohol or a thiol substituted with one or more halogens, a cyano group, a nitro group, an alkyl group, an alkenyl group, an alkynyl group, and an azido group.

44. The method of any one of claims 35-43, wherein said at least one detectable component of said tag moiety comprises a multiplicity of ethylene glycol units.

45. The method of any one of claims 35-44, wherein said multiplicity of ethylene glycol units comprises 16, 20, 24, or 36 ethylene glycol units.

46. The method of any one of claims 35-45, wherein said tag moiety attaches to the predetermined compound via a cleavable linker.

47. The method of claim 46, wherein the cleavable linker is a photocleavable linker.

48. The method of claim 46, wherein the cleavable linker is a chemically cleavable linker.

49. The method of any one of claims 35-48, wherein the predetermined compound present in the sample is a protein and said tag moiety attaches to the carboxy or amino terminus of said protein.

50. The method of any one of claims 35-49, wherein the predetermined compound present in the sample is a protein and said tag moiety attaches to a lysine, an arginine, or a cysteine residue of said protein.

51. The method of any one of claims 35-50, wherein said at least one nanopore is a biological nanopore, a modified biological nanopore, or a synthetic nanopore.

52. The method of claim 51, wherein said at least one nanopore is proteinaceous.

53. The method of any one of claims 35-52, wherein said at least one nanopore is an alpha hemolysin (α

-hemolysin).

54. The method of any one of claims 35-50, wherein said at least one nanopore is a solid-state nanopore.

55. The method of claim 54, wherein the solid-state nanopore comprises graphene.

56. The method of any one of claims 35-55, wherein said at least one nanopore is in a membrane.

57. The method of any one of claims 35-56, wherein said at least one nanopore is part of an array of nanopores.

58. The method of claim 57, wherein each nanopore in said array comprises an identical immobilized second predetermined compound.

59. The method of claim 57, wherein each nanopore in said array comprises a different immobilized second predetermined compound.

60. The method of any one of claims 35-59, wherein the second predetermined compound is a protein.

61. The method of claim 60, wherein the protein is an antibody.

62. The method of any one of claim 35-59, wherein the second predetermined compound is non-proteinaceous.

63. The method of any one of claims 35-62, wherein said tag moiety produces a blockade signature detectable with said at least one nanopore.

64. The method of claim 63, wherein the blockade signature is result of a change in current amplitude of said at least one nanopore.

65. The method of claim 63, wherein the blockade signature is result of a change in conductance of said at least one nanopore.

66. The method of any one of claims 35-65, wherein said at least one nanopore further comprising a means for ejecting said tag moiety from the nanopore.

67. The method of any one of claims 35-66, wherein the sample comprises a plurality of predetermined compounds.

68. The method of claim 67, wherein in step (a) contacting the plurality of predetermined compounds present in the sample with a plurality of detectable tag moieties, under conditions permitting the tag moieties to attach to each of the predetermined compounds present in the sample to form detectably tagged predetermined compounds, wherein each tag moiety uniquely attaches to one of the predetermined compounds present in the sample and is distinguishable from any tag moiety attached to any other predetermined compound present in the sample.

69. The method of any one of claims 35-68, wherein said at least one nanopore comprises a plurality of predetermined compounds capable of binding to the plurality of detectably tagged predetermined compounds present in the sample.

36. A method for detecting interaction of at least two predetermined compounds which comprises:

(a) contacting at least a first predetermined compound present in a sample with a detectable tag moiety, wherein the detectable tag moiety comprises at least one detectable component, under conditions permitting the tag moiety to attach to the predetermined compound present in the sample to form a detectably tagged predetermined compound;

(b) treating the sample from step (a) to form an electrolyte solution containing the detectably tagged predetermined compound;

(c) contacting the electrolyte solution from step (b) with at least one nanopore, wherein at least a second predetermined compound is immobilized on said at least one nanopore, under conditions permitting the binding of the detectably tagged predetermined compound with the second predetermined compound, and wherein the tag moiety of said detectably tagged predetermined compound bound to the second predetermined compound localizes within said at least one nanopore;

(d) detecting said at least one detectable component of the tag moiety with said at least one nanopore so as to thereby detecting the interaction of the predetermined compounds.

76. A method for detecting the presence of a plurality of predetermined compounds in a plurality of samples, comprising:

(a) contacting each sample with a nanopore-detectable tag moiety, under conditions permitting the tag moiety to attach to the plurality of predetermined compounds present in the sample to form detectably tagged predetermined compounds for that sample, which are distinguishable from any nanopore-detectable tag moiety attached to similar predetermined compounds from any other sample;

(b) mixing the resulting tagged compounds from the plurality of samples from step (a) and treating them in such a way as to form an electrolyte solution containing the set of detectably and distinguishably tagged predetermined compounds;

(c) contacting the electrolyte solution from step (b) with at least one nanopore, wherein said at least one nanopore comprises a means for specific analyte moiety binding to each of the detectably tagged predetermined compounds in the electrolyte solution, under conditions such that the detectably tagged predetermined compounds bind to said at least one nanopore;

(d) treating the detectably tagged predetermined compounds bound to said at least one nanopore so as to separate the tag moieties from the predetermined compounds; and

(e) detecting said at least one detectable component of each of the tag moieties so released as the tag moieties pass through said at least one nanopore so as to thereby detect the presence of the plurality of predetermined compounds in the plurality of samples.