Methods for detecting organisms and enzymatic reactions using raman spectroscopy

• US 20080274489A1
• Filed: 04/16/2008
• Published: 11/06/2008
• Est. Priority Date: 10/17/2005
• Status: Active Grant

• Current Assignee: Sword Diagnostics, Inc.
• Original Assignee: Sword Diagnostics, Inc.
• Inventors: Kundu, Samar Kumar, Siegel, Neal Arthur, Ginsburgh, Charles Lester
• Granted Patent: US 7,947,437 B2
• Time in Patent Office: Days
• US Class Current: 435/28
• CPC Class Codes: C12Q 1/28 involving peroxidase
• CPC Class Codes: G01N 21/65 Raman scattering
• CPC Class Codes: G01N 33/56916 Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
• CPC Class Codes: G01N 33/56938 Staphylococcus
• CPC Class Codes: G01N 33/56983 Viruses
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Abstract

The present disclosure provides systems for the rapid and sensitive detection of organisms and molecules in samples. Reactants that produce Raman-active products are used in combination with Raman light scattering. The present disclosure can also be used to measure enzyme-kinetics.

Claims

1. A method for detecting the activity of at least one enzyme in a sample comprising:

a) preparing a mixture comprising the sample and;

i. optionally at least one aromatic compound;

ii. at least one amine-containing compound; and

iii. at least one electron-donating compound;

b) incubating the mixture to form at least one Raman-active product; and

c) detecting the at least one Raman-active product with Raman spectroscopy.

2. The method of claim 1, wherein the at least one amine-containing compound is chosen from 4-aminoantipyrene and 5-aminosalicyclic acid.

3. The method of claim 1, wherein the at least one aromatic compound is chosen from 2-hydroxybenzyl alcohol, 4-chloro-3,5-dimethylphenol, 2-naphthol, 4-hydroxy-4-biphenyl-carboxylic acid, 5,7-dichloro-8-hydroxyquinoline, 4-chloro-1-naphthol, phenol, and 4,5 dihydroxy-naphthalene-2,7-disulfonic acid.

4. The method of claim 1, wherein the at least one amine-containing compound comprises:

5. The method of claim 1, wherein the at least one amine-containing compound comprises:

6. The method of claim 1, wherein the at least one amine-containing compound comprises:

7. The method of claim 1, wherein the amine-containing compound comprises:

8. The method of claim 1, wherein the at least one aromatic compound comprises:

9. The method of claim 1, wherein the at least one aromatic compound comprises:

10. The method of claim 1, wherein the at least one aromatic compound comprises:

11. The method of claim 1, wherein the at least one aromatic compound comprises:

12. The method of claim 1, wherein the at least one electron-donating compound is chosen from an organic hydrogen peroxide, urea hydrogen peroxide, and hydrogen peroxide (H₂O₂).

13. The method of claim 1, wherein the at least one enzyme is a peroxidase.

14. The method of claim 1, wherein the at least one aromatic compound is 2-hydroxybenzyl alcohol, the at least one amine containing compound is 5-aminosalicyclic acid, the at least one electron-donating compound is urea hydrogen peroxide, and the at least one enzyme is a peroxidase.

15. The method of claim 1, wherein step b) comprises incubating the mixture in the presence of a base.

16. The method of claim 1, wherein the Raman spectroscopy is resonance Raman Spectroscopy.

31. The method of claim 22, wherein the at least one aromatic compound is the compound of claim 10.

32. The method of claim 22, wherein the at least one aromatic compound is the compound of claim 11.

46. The kit of claim 43, wherein the at least one amine-containing compound is the compound of claim 4.

52. The kit of claim 43, wherein the at least one aromatic compound is the compound of claim 10.

53. The kit of claim 43, wherein the at least one aromatic compound is the compound of claim 11.

17. A method for detecting the activity of at least one enzyme in a sample comprising:

a) preparing a mixture comprising the sample, 5-aminosalicyclic acid, and a hydrogen peroxide;

b) incubating the mixture to form at least one Raman-active product; and

c) detecting the at least one Raman-active product with Raman spectroscopy.

18. The method of claim 17, wherein the mixture further comprises biotin.

19. The method of claim 17, wherein the hydrogen peroxide is urea hydrogen peroxide, and the enzyme is a peroxidase.

20. A method for detecting the activity of at least one enzyme in a sample comprising:

a) preparing a mixture comprising the sample, an aromatic amine chosen from o-phenylenediamine, m-phenylenediamine, and p-phenylenediamine, and a hydrogen peroxide chosen from an aromatic hydrogen peroxide, urea hydrogen peroxide, and hydrogen peroxide (H₂O₂);

b) incubating the mixture to form at least one Raman-active product; and

c) detecting the at least one Raman-active product with Raman spectroscopy.

21. The method of claim 20, wherein the aromatic amine is o-phenylenediamine and the hydrogen peroxide is urea hydrogen peroxide.

22. A method for detecting at least one target in a sample comprising:

a) preparing a mixture comprising the at least one target;

b) incubating the mixture with at least one ligand specific for the at least one target, wherein the at least one ligand comprises an enzyme;

c) providing to the mixture;

i. optionally at least one aromatic compound;

ii. at least one amine-containing compound; and

iii. at least one electron-donating compound;

d) incubating the mixture to form at least one Raman-active product; and

e) detecting the at least one Raman-active product with Raman spectroscopy.

23. The method of claim 22, wherein the at least one amine-containing compound is chosen from 4-aminoantipyrene and 5-aminosalicyclic acid.

24. The method of claim 22, wherein the at least one aromatic compound is chosen from 2-hydroxybenzyl alcohol, 4-chloro-3,5-dimethylphenol, 2-naphthol, 4-hydroxy-4-biphenyl-carboxylic acid, 5,7-dichloro-8-hydroxyquinoline, 4-chloro-1-naphthol, phenol, and 4,5 dihydroxy-naphthalene-2,7-disulfonic acid.

25. The method of claim 22, wherein the at least one amine-containing compound is the compound of claim 4.

26. The method of claim 22, wherein the at least one amine-containing compound is the compound of claim 5.

27. The method of claim 22, wherein the at least one amine-containing compound is the compound of claim 6.

28. The method of claim 22, wherein the at least one amine-containing compound is the compound of claim 7.

29. The method of claim 22, wherein the at least one aromatic compound is the compound of claim 8.

30. The method of claim 22, wherein the at least one aromatic compound is the compound of claim 9.

33. The method of claim 22, wherein the at least one electron-donating compound is chosen from an organic hydrogen peroxide, urea hydrogen peroxide, and hydrogen peroxide (H₂O₂).

34. The method of claim 22, wherein the enzyme is a peroxidase.

35. The method of claim 22, wherein the at least one aromatic compound is 2-hydroxybenzyl alcohol, the amine-containing compound is 5-aminosalicyclic acid, the electron-donating compound is urea hydrogen peroxide, the at least one enzyme is a peroxidase, and the ligand is an antibody.

36. The method of claim 22, wherein step b) comprises incubating the mixture in the presence of a base.

37. The method of claim 22, wherein the Raman spectroscopy is resonance Raman Spectroscopy.

38. The method of claim 22, wherein the at least one target is an organism.

39. The method of claim 38, wherein the organism is chosen from E. coli, Listeria, Salmonella, Vibrio, Camphelbacter, Staphylococcus, HIV, Hepatitis, Adenovirus, Rhino virus, and Human papilloma virus.

40. The method of claim 22, wherein the target is chosen from a protein, amino acids, nucleic acids, nucleotides, carbohydrates, metabolites, hormones, and metabolic intermediates.

41. The method of claim 40, wherein the protein is chosen from IL-2, C-Reactive protein, Tumor Necrosis Factor Receptor II, and Human Cardiac Troponin I.

42. A method for detecting the activity of at least one enzyme in a sample comprising:

a) preparing a mixture comprising the sample and;

i. optionally at least one aromatic compound;

ii. at least one amine-containing compound; and

iii. at least one electron-donating compound;

b) incubating the mixture to form at least one charge transfer complex; and

c) detecting the at least one charge transfer complex with Raman spectroscopy.

43. A kit for detecting at least one enzyme activity comprising:

a) optionally, at least one aromatic compound;

b) at least one amine-containing compound;

c) at least one electron-donating compound; and

d) optionally, suitable buffers for the at least one enzyme.

44. The kit of claim 43, wherein the at least one amine-containing compound is chosen from 4-aminoantipyrene, 5-aminosalicyclic acid, and o-phenylenediamine, the at least one optional aromatic compound is chosen from 2-hydroxybenzyl alcohol, 4-chloro-3,5-dimethylphenol, 2-naphthol, 4-hydroxy-4-biphenyl-carboxylic acid, 5,7-dichloro-8-hydroxyquinoline, 4-chloro-1-naphthol, phenol, and 4,5 dihydroxy-naphthalene-2,7-disulfonic acid, and the at least one electron-donating compound is chosen from urea hydrogen peroxide and hydrogen peroxide.

45. The kit of claim 43, wherein the at least one aromatic compound is chosen from 2-hydroxybenzyl alcohol, 4-chloro-3,5-dimethylphenol, 2-naphthol, 4-hydroxy-4-biphenyl-carboxylic acid, 5,7-dichloro-8-hydroxyquinoline, 4-chloro-1-naphthol, phenol, and 4,5 dihydroxy-naphthalene-2,7-disulfonic acid.

47. The kit of claim 43, wherein the at least one amine-containing compound is the compound of claim 5.

48. The kit of claim 43, wherein the at least one amine-containing compound is the compound of claim 6.

49. The kit of claim 43, wherein the at least one amine-containing compound is the compound of claim 7.

50. The kit of claim 43, wherein the at least one aromatic compound is the compound of claim 8.

51. The kit of claim 43, wherein the at least one aromatic compound is the compound of claim 9.

54. The kit of claim 43, wherein the at least one aromatic compound is an aromatic amine chosen from o-phenylenediamine, p-phenylenediamine, and m-phenylenediamine.