Functionalized nanocrystals and their use in detection systems

• US 6,114,038 A
• Filed: 08/11/1999
• Issued: 09/05/2000
• Est. Priority Date: 11/10/1998
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: BioCrystal, Ltd.
• Inventors: Barbera-Guillem, Emilio, Castro, Stephanie L.
• Primary Examiner(s): Nutter, Nathan M.
• Application Number:
  US09/372,729
• Time in Patent Office: 391 Days
• Field of Search: 428/402.24, 428/404, 257/65, 257/614, 257/642, 424/9.1, 424/9.32, 424/9.36, 424/9.42, 424/9.6, 427/213.3, 427/214, 427/215, 427/220
• US Class Current: 428/402.24
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07F 7/02 Silicon compounds
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: Y10T 428/2989 Microcapsule with solid core [includes liposome]
• CPC Class Codes: Y10T 428/2993 Silicic or refractory material containing [e.g., tungsten oxide, glass, cement, etc.]

Abstract

Provided are compositions comprising water-soluble, functionalized nanocrystals. The water-soluble functionalized nanocrystals comprise quantum dots capped with a layer of a capping compound, and further comprise, by operably linking and in a successive manner, one or more additional compounds. The one or more additional compounds is comprised of at least a diaminocarboxylic acid which is operatively linked to the capping compound, and may further comprise an amino acid, an affinity ligand, or a combination thereof. Also provided are methods of using the functionalized nanocrystals having an affinity ligand to detect the presence or absence of a target substrate in a sample. The method comprises contacting the functionalized nanocrystals with the sample so that complexes are formed between the functionalized nanocrystals and substrate, if the substrate is present; exposing the complexes in the detection system to a wavelength of light suitable for exciting the functionalized nanocrystals of the complexes to emit a luminescence peak; and detecting the emitted luminescence peak.

Claims

1. A water-soluble, functionalized nanocrystal comprising:

a quantum dot having a core and a shell;

a capping compound operably linked to the quantum dot, wherein the capping compound comprises a compound having the formula HS(CH₂)_n X, wherein X is a carboxylate; and

a diaminocarboxylic acid which is operably linked to the capping compound.

2. The water-soluble, functionalized nanocrystal according to claim 1, wherein the core comprises CdX, and wherein X is Se, Te or S.

3. The water-soluble, functionalized nanocrystal according to claim 1, wherein the shell comprises YZ, and wherein Y is Cd or Zn, and Z is S, or Se.

4. The water-soluble, functionalized nanocrystal according to claim 1, wherein the capping compound is selected from the group consisting of a mercaptocarboxylic acid, a mercaptofunctionalized amine, aminoethanethiol-HCl, homocysteine, and 1-amino-2-methyl-2-propanethiol-HCl.

5. The water-soluble, functionalized nanocrystal according to claim 1, wherein the diaminocarboxylic acid forms a layer over the capping compound.

6. The water-soluble, functionalized nanocrystal according to claim 1, wherein an d the diaminocarboxylic acid is selected from the group consisting of lysine, asparagine, glutamine, arginine, citrulline, ornithine, 5-hydroxylysine, djenkolic acid, β

-cyanoalanine, 3,4-diaminobenzoic acid, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2,5-diaminopentanoic acid, and 2,6-diaminopimelic acid.

7. The water-soluble, functionalized nanocrystal according to claim 1, further comprising an affinity ligand which is operably linked to the diaminocarboxylic acid.

8. The water-soluble, functionalized nanocrystal according to claim 7, wherein the affinity ligand forms a layer over the diaminocarboxylic acid.

9. The water-soluble, functionalized nanocrystal according to claim 7, wherein the affinity ligand is selected from the group consisting of a lectin, a monoclonal antibody, a peptide, an aptamer, a nucleic acid molecule, avidin, streptavidin, and an avidin derivative.

36. A method of using the water-soluble, functionalized nanocrystal according to claim 7 in a detection system, the method comprising the steps of:

(a) contacting the functionalized nanocrystals with a sample being analyzed for the presence or absence of a substrate for which the affinity ligand has binding specificity, wherein if the substrate is present in the sample, formed are complexes comprising the functionalized nanocrystals bound to the substrate;

(b) exposing the complexes, if formed, in the detection system to a wavelength of light suitable for exciting the functionalized nanocrystals of the complexes to emit a luminescence peak; and

(c) detecting the luminescence peak emitted by the complexes, if present, by a detection means for detecting the luminescence peak;

wherein the detection of a luminescence peak is indicative of the presence of the substrate.

37. The method according to claim 36, wherein the presence of the substrate is detected, and further comprises quantitating the amount of substrate present by measuring the intensity of the luminescence peak emitted.

38. The method according to claim 36, wherein the detection system is selected from the group consisting of an affinity assay, fluorescent staining, flow cytometry, nucleic acid sequencing, nucleic acid hybridization, nucleic acid synthesis or amplification, identification verification, and molecular sorting.

10. A water-soluble, functionalized nanocrystal comprising:

a quantum dot having a core and a shell;

a capping compound operably linked to the quantum dot, wherein the capping compound comprises a compound having the formula HS(CH₂)_n X, wherein X is a carboxylate;

a diaminocarboxylic acid which is operably linked to the capping compound; and

an amino acid which is operably linked to the diaminocarboxylic acid.

11. The water-soluble, functionalized nanocrystal according to claim 10, wherein the core comprises CdX, and wherein X is Se, Te or S.

12. The water-soluble, functionalized nanocrystal according to claim 10, wherein the shell comprises YZ, and wherein Y is Cd or Zn, and Z is S, or Se.

13. The water-soluble, functionalized nanocrystal according to claim 10, wherein the capping compound is selected from the group consisting of a mercaptocarboxylic acid, a mercaptofunctionalized amine, aminoethanethiol-HCl, homocysteine, and 1-amino-2-methyl-2-propanethiol-HCl.

14. The water-soluble, functionalized nanocrystal according to claim 10, wherein the amino acid forms a layer over the diaminocarboxylic acid.

15. The water-soluble, functionalized nanocrystal according to claim 10, wherein the amino acid is selected from the group consisting of a monocarboxylic acid and a diaminocarboxylic acid.

16. The water-soluble, functionalized nanocrystal according to claim 15, wherein the amino acid comprises a diaminocarboxylic acid, and the diaminocarboxylic acid is selected from the group consisting of lysine, asparagine, glutamine, arginine, citrulline, ornithine, 5-hydroxylysine, djenkolic acid, β

-cyanoalanine, 3,4-diaminobenzoic acid, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2,5-diaminopentanoic acid, and 2,6-diaminopimelic acid.

17. The water-soluble, functionalized nanocrystal according to claim 15, wherein the amino acid comprises a monoaminocarboxylic acid, and the monoaminocarboxylic acid is selected from the group consisting of glycine, serine, threonine, cysteine, β

-alanine, homoserine, γ

-aminobutyric acid, and homocysteine.

18. The water-soluble, functionalized nanocrystal according to claim 10, further comprising an affinity ligand which is operably linked to the amino acid.

19. The water-soluble, functionalized nanocrystal according to claim 18, wherein the affinity ligand forms a layer over the diaminocarboxylic acid.

20. The water-soluble, functionalized nanocrystal according to claim 18, wherein the affinity ligand is selected from the group consisting of a lectin, a monoclonal antibody, a peptide, an aptamer, a nucleic acid molecule, avidin, streptavidin, and an avidin derivative.

39. A method of using the water-soluble, functionalized nanocrystal according to claim 18 in a detection system, the method comprising the steps of:

(a) contacting the functionalized nanocrystals with a sample being analyzed for the presence or absence of a substrate for which the affinity ligand has binding specificity, wherein if the substrate is present in the sample, formed are complexes comprising the functionalized nanocrystals bound to the substrate;

(b) exposing the complexes, if formed, in the detection system to a wavelength of light suitable for exciting the functionalized nanocrystals of the complexes to emit a luminescence peak; and

(c) detecting the luminescence peak emitted by the complexes, if present, by a detection means for detecting the luminescence peak;

wherein the detection of a luminescence peak is indicative of the presence of the substrate.

40. The method according to claim 39, wherein the presence of the substrate is detected, further comprising quantitating the amount of substrate present by measuring the intensity of the luminescence peak emitted.

41. The method according to claim 39, wherein the detection system is selected from the group consisting of an affinity assay, fluorescent staining, flow cytometry, nucleic acid sequencing, nucleic acid hybridization, nucleic acid synthesis or amplification, identification verification, and molecular sorting.

21. A water-soluble, functionalized nanocrystal comprising:

(a) a quantum dot having a core wherein the core comprises CdX, and wherein X is Se, Te or S;

(b) a shell, wherein the shell comprises YZ, and wherein Y is Cd or Zn, and Z is S, or Se;

(c) a capping compound operably linked to the quantum dot, wherein the capping compound comprises a compound having the formula HS(CH₂)_n X, and wherein X is a carboxylate; and

(d) a diaminocarboxylic acid which is operably linked to the capping compound.

22. The water-soluble, functionalized nanocrystal according to claim 21, wherein the capping compound is selected from the group consisting of a mercaptocarboxylic acid, a mercaptofunctionalized amine, aminoethanethiol-HCl, homocysteine, and 1-amino-2-methyl-2-propanethiol-HCl.

23. The water-soluble, functionalized nanocrystal according to claim 21, wherein the diaminocarboxylic acid forms a layer over the capping compound.

24. The water-soluble, functionalized nanocrystal according to claim 21, wherein and the diaminocarboxylic acid is selected from the group consisting of lysine, asparagine, glutamine, arginine, citrulline, ornithine, 5-hydroxylysine, djenkolic acid, β

-cyanoalanine, 3,4-diaminobenzoic acid, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2,5-diaminopentanoic acid, and 2,6-diaminopimelic acid.

25. The water-soluble, functionalized nanocrystal according to claim 21, further comprising an affinity ligand which is operably linked to the diaminocarboxylic acid.

26. The water-soluble, functionalized nanocrystal according to claim 25, wherein the affinity ligand forms a layer over the diaminocarboxylic acid.

27. The water-soluble, functionalized nanocrystal according to claim 25, wherein the affinity ligand is selected from the group consisting of a lectin, a monoclonal antibody, a peptide, an aptamer, a nucleic acid molecule, avidin, streptavidin, and an avidin derivative.

28. The water-soluble, functionalized nanocrystal according to claim 21, further comprising an amino acid which is operably linked to the diaminocarboxylic acid.

29. The water-soluble, functionalized nanocrystal according to claim 28, wherein the amino acid forms a layer over the diaminocarboxylic acid.

30. The water-soluble, functionalized nanocrystal according to claim 28, wherein the amino acid is selected from the group consisting of a monocarboxylic acid and a diaminocarboxylic acid.

31. The water-soluble, functionalized nanocrystal according to claim 30, wherein the amino acid comprises a diaminocarboxylic acid, and the diaminocarboxylic acid is selected from the group consisting of lysine, asparagine, glutamine, arginine, citrulline, ornithine, 5-hydroxylysine, djenkolic acid, β

-cyanoalanine, 3,4-diaminobenzoic acid, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2,5-diaminopentanoic acid, and 2,6-diaminopimelic acid.

32. The water-soluble, functionalized nanocrystal according to claim 30, wherein the amino acid comprises a monoaminocarboxylic acid, and the monoaminocarboxylic acid is selected from the group consisting of glycine, serine, threonine, cysteine, β

-alanine, homoserine, γ

-aminobutyric acid, and homocysteine.

33. The water-soluble, functionalized nanocrystal according to claim 28, further comprising an affinity ligand which is operably linked to the amino acid.

34. The water-soluble, functionalized nanocrystal according to claim 33, wherein the affinity ligand forms a layer over the amino acid.

35. The water-soluble, functionalized nanocrystal according to claim 33, wherein the affinity ligand is selected from the group consisting of a lectin, a monoclonal antibody, a peptide, an aptamer, a nucleic acid molecule, avidin, streptavidin, and an avidin derivative.

42. A method of using the water-soluble, functionalized nanocrystal according to claim 25 in a detection system, the method comprising the steps of:

(a) contacting the functionalized nanocrystals with a sample being analyzed for the presence or absence of a substrate for which the affinity ligand has binding specificity, wherein if the substrate is present in the sample, formed are complexes comprising the functionalized nanocrystals bound to the substrate;

(b) exposing the complexes, if formed, in the detection system to a wavelength of light suitable for exciting the functionalized nanocrystals of the complexes to emit a luminescence peak; and

(c) detecting the luminescence peak emitted by the complexes, if present, by a detection means for detecting the luminescence peak;

wherein the detection of a luminescence peak is indicative of the presence of the substrate.

43. The method according to claim 42, wherein the presence of the substrate is detected, further comprising quantitating the amount of substrate present by measuring the intensity of the luminescence peak emitted.

44. The method according to claim 42, wherein the detection system is selected from the group consisting of an affinity assay, fluorescent staining, flow cytometry, nucleic acid sequencing, nucleic acid hybridization, nucleic acid synthesis or amplification, identification verification, and molecular sorting.

45. A method of using the water-soluble, functionalized nanocrystal according to claim 33 in a detection system, the method comprising the steps of:

(a) contacting the functionalized nanocrystals with a sample being analyzed for the presence or absence of a substrate for which the affinity ligand has binding specificity, wherein if the substrate is present in the sample, formed are complexes comprising the functionalized nanocrystals bound to the substrate;

(b) exposing the complexes, if formed, in the detection system to a wavelength of light suitable for exciting the functionalized nanocrystals of the complexes to emit a luminescence peak; and

(c) detecting the luminescence peak emitted by the complexes, if present, by a detection means for detecting the luminescence peak;

wherein the detection of a luminescence peak is indicative of the presence of the substrate.

46. The method according to claim 45, wherein the presence of the substrate is detected, further comprising quantitating the amount of substrate present by measuring the intensity of the luminescence peak emitted.

47. The method according to claim 45, wherein the detection system is selected from the group consisting of an affinity assay, fluorescent staining, flow cytometry, nucleic acid sequencing, nucleic acid hybridization, nucleic acid synthesis or amplification, identification verification, and molecular sorting.