Quantum dot biolabeling and immunomagnetic separation for detection of contaminants

• US 20080135490A1
• Filed: 01/09/2006
• Published: 06/12/2008
• Est. Priority Date: 01/07/2005
• Status: Abandoned Application

• Current Assignee: The Board of Trustees of the University of Arkansas (University of Arkansas System)
• Original Assignee: The Board of Trustees of the University of Arkansas (University of Arkansas System)
• Inventors: Yang, Liju, Li, Yanbin, Su, Xiao-Li
• Application Number:
  US11/328,020
• Publication Number:
  US 20080135490A1
• Time in Patent Office: Days
• US Class Current: 210/695
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: G01N 33/588 with semiconductor nanocrystal label, e.g. quantum dots
• CPC Class Codes: G01N 35/0098 involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation magnetic particles used in immunoassays G01N33/54326; magnetic separation in general B03C

Abstract

Methods are provided for detecting, separating, isolating and quantifying contaminants in starting materials by separating the contaminant from the starting material using a bead coupled to an affinity moiety and quantum dot-labeling the contaminant. The contaminant is detected by the characteristic emission spectrum of the quantum dot. Also, competitive binding methods are provided wherein the starting material and a control material are contacted with a quantum dot coupled to an affinity moiety capable of binding the contaminant and a competitor complex. A decrease in the intensity of the characteristic emission spectrum of the quantum dot associated with the competitor complex from the starting material as compared to that of the control material is indicative of the presence of the contaminant in the starting material.

Claims

1. A method of detecting a contaminant in a starting material, comprising:

a) immunomagnetically separating the contaminant,

b) quantum dot-immunolabeling the contaminant, and

c) detecting and analyzing a characteristic emission spectrum of the quantum dot.

2. The method of claim 1, wherein step (a) comprises contacting the starting material with a magnetic bead coupled to a first antibody capable of binding the contaminant to form a bead-antibody-contaminant complex, and magnetically separating the complex from the starting material.

3. The method of claim 1, wherein step (b) comprises contacting the contaminant with a quantum dot coupled to a second antibody capable of binding the contaminant to form a quantum dot-antibody-contaminant complex.

4. The method of claim 1, wherein step (b) comprises quantum dot labeling the complex of step (a).

5. The method of claim 1, wherein step (a) and step (b) can be carried out simultaneously.

6. The method of claim 1, wherein step (b) can precede step (a).

7. The method of claim 1, wherein the starting material is selected from the group consisting of a food product, an environmental sample and a clinical sample.

8. The method of claim 1, wherein the contaminant is selected from the group consisting of a prokaryote, a eukaryote, a virus, and a polypeptide.

9. A method of detecting a contaminant in a starting material, the method comprising:

a) contacting the starting material with a bead coupled to a first affinity moiety capable of binding the contaminant to form a target, the target comprising the bead coupled to the contaminant;

b) separating the target from the starting material;

c) contacting the target with a quantum dot coupled to a second affinity moiety capable of binding the contaminant of the target of step (a) to form a labeled target, the labeled target comprising the target coupled to the quantum dot, the quantum dot having a characteristic emission spectrum; and

d) detecting the characteristic emission spectrum of the quantum dot in the labeled target of step (c), wherein the presence of the characteristic emission spectrum is indicative of the detection of the contaminant.

10. The method of claim 9, wherein more than one contaminant is simultaneously detected by a plurality of quantum dots, each of the plurality having a distinct characteristic emission spectrum and coupled to a distinct affinity moiety capable of binding a distinct contaminant to form more than one labeled target.

11. The method of claim 9, wherein the starting material is selected from the group consisting of a food product, an environmental sample and a clinical sample.

12. The method of claim 11, wherein the food is selected from the group consisting of a fruit, a vegetable, a raw food, a ready-to-eat food, a beef product, a poultry product, a sea food product, and a dairy product.

13. The method of claim 12, wherein the poultry product is selected from the group consisting of a chicken carcass, a chicken carcass wash water, a deboned chicken, a ground poultry meat sample, and a poultry patty.

14. The method of claim 11, wherein the environmental sample is selected from the group consisting of a water sample, an air sample and a soil sample.

15. The method of claim 11, wherein the clinical sample is selected from the group consisting of a urine sample, a blood sample, a fecal sample, a swab from a skin surface, a swab from an organ surface and a tissue sample.

16. The method of claim 9, wherein the contaminant is selected from the group consisting of a prokaryote, a eukaryote, a virus, and a polypeptide.

17. The method of claim 16, wherein the prokaryote is Escherichia coli, Escherichia coli O157:

H7, Salmonella spp., and Listeria monocytogenes.

18. The method of claim 16, wherein the polypeptide is selected from the group consisting of a toxin and a prion.

19. The method of claim 9, wherein the bead is selected from the group consisting of a microbead, a magnetic microbead, a nanoparticle and a magnetic nanoparticle.

20. The method of claim 9, wherein the target is separated from the starting material by subjecting the starting material to a magnetic field.

21. The method of claim 9, wherein the first affinity moiety and the second affinity moiety comprise an antibody having affinity for the contaminant.

22. The method of claim 21, wherein the antibody is coupled directly to the quantum dot and to the bead.

23. The method of claim 21, wherein the antibody is coupled to the quantum dot and to the bead by a linker.

24. The method of claim 23, wherein the linker is selected from the group consisting of Protein A, Protein G, an Fc receptor, and an anti-Fc antibody.

25. The method of claim 23, wherein the linker comprises a bridging complex selected from the group consisting of:

biotin-streptavidin, biotin-avidin, Protein A-IgG, Protein G-IgG, and IgG-anti-IgG.

26. The method of claim 9, wherein the characteristic emission spectrum of the quantum dot is detected by fluorescence microscopy.

27. The method of claim 9, wherein the characteristic emission spectrum of the quantum dot is detected by spectroscopy.

28. The method of claim 9, wherein the target is separated from the starting material by centrifugation.

29. The method of claim 9, wherein the target is separated from the starting material by filtration.

30. The method of claim 9, wherein the first affinity moiety and the second affinity moiety are the same or different.

31. The method of claim 9, further comprising:

e) isolating the contaminant from the labeled target.

32. The method of claim 9, further comprising:

e) subjecting the labeled target and the separated contaminant to antibiotic resistance testing.

33. The method of claim 9, further comprising:

e) subjecting the labeled target to microbiologic, immunologic, or nucleic acid based assays.

34. A method of quantifying contaminants in a starting material, the method comprising:

a) contacting the starting material with a bead coupled to a first affinity moiety capable of binding the contaminant to form a target, the target comprising the bead coupled to the contaminant;

b) separating the target from the starting material;

c) contacting the target with a quantum dot coupled to a second affinity moiety capable of binding to the contaminant to form a labeled target, the labeled target comprising the target coupled to the quantum dot, the quantum dot having a characteristic emission spectrum;

d) detecting the intensity of the characteristic emission spectrum of the quantum dot in the labeled target of step (c); and

e) determining the quantity of the contaminant in the starting material by correlating the emission spectrum intensity of step (d) with the emission spectrum intensity of a labeled target containing a known number of contaminants.

35. The method of claim 34, wherein the starting material is selected from the group consisting of a food product, an environmental sample and a clinical sample.

36. The method of claim 34, wherein the contaminant is selected from the group consisting of a prokaryote, a eukaryote, a virus, and a polypeptide.

37. The method of claim 34, wherein the bead is selected from the group consisting of a microbead, a magnetic microbead, a nanoparticle and a magnetic nanoparticle.

38. The method of claim 34, wherein the target is separated from the starting material by subjecting the starting material to a magnetic field.

39. A method of separating a contaminant from a starting material, the method comprising:

a) contacting the starting material with a composition comprising a magnetic bead coupled to a first affinity moiety capable of binding the contaminant to form a target, the target comprising the magnetic bead coupled to the contaminant;

b) contacting the target with a quantum dot coupled to a second affinity moiety capable of binding the contaminant of the target of step (a) to form a labeled target, the labeled target comprising the target coupled to the quantum dot, the quantum dot having a characteristic emission spectrum;

c) separating the labeled target from the starting material; and

d) detecting the characteristic emission spectrum of the quantum dot in the labeled target of step (c), wherein the presence of the characteristic emission spectrum is indicative of the detection of the contaminant.

40. A kit for detecting a contaminant in a starting material, the kit comprising:

a) a bead capable of being coupled to a first affinity moiety;

b) the first affinity moiety capable of binding the contaminant;

c) a quantum dot capable of being coupled to a second affinity moiety, the quantum dot having a characteristic emission spectrum; and

d) the second affinity moiety capable of binding to the contaminant.

41. The kit of claim 40, wherein the first affinity moiety and the second affinity moiety comprise at least one antibody.

42. The kit of claim 41, wherein the at least one antibody is coupled to the quantum dot and to the bead by a linker.

43. The method of claim 42, wherein the linker is selected from the group consisting of Protein A, Protein G, an Fc receptor, and an anti-Fc antibody.

44. The method of claim 42, wherein the linker comprises a bridging complex selected from the group consisting of:

biotin-streptavidin, biotin-avidin, Protein A-IgG, Protein G-IgG, and IgG-anti-IgG.

45. The method of claim 40, wherein the bead is selected from the group consisting of a microbead, a magnetic microbead, a nanoparticle and a magnetic nanoparticle.

46. The kit of claim 40, further comprising:

e) a solution comprising a known concentration of the contaminant.

47. A method of detecting a contaminant in a starting material, the method comprising:

a) contacting the starting material containing a contaminant and a control material not containing the contaminant each with

     1) a quantum dot coupled to an affinity moiety, the affinity moiety capable of binding the contaminant and

     2) a competitor complex comprising a bead coupled to the contaminant;

b) separating the competitor complex from each of the starting material and the control material; and

c) detecting the characteristic emission spectrum of the quantum dot associated with the competitor complex from each of the starting material and the control material, wherein a decrease in the intensity of the characteristic emission spectrum of the competitor complex from the starting material as compared to the intensity of the characteristic emission spectrum of the competitor complex from the control material is indicative of the presence of the contaminant in the starting material.

48. The method of claim 47, wherein the starting material is selected from the group consisting of a food product, an environmental sample and a clinical sample.

49. The method of claim 47, wherein the contaminant is selected from the group consisting of a prokaryote, a eukaryote, a virus, a polypeptide and a chemical.

50. The method of claim 49, wherein the chemical is selected from the group consisting of a herbicide and a pesticide.

51. The method of claim 50, wherein the herbicide comprises 2-chloro-4-(ethylamine)-6-(isopropylamine)-s-triazine.

52. The method of claim 47, wherein the bead is selected from the group consisting of a microbead, a magnetic microbead, a nanoparticle and a magnetic nanoparticle.

53. The method of claim 47, wherein the target is separated from the starting material by subjecting the starting material to a magnetic field.

54. A method for quantifying a contaminant in a starting material, the method comprising:

a) contacting the starting material containing the contaminant, a control material not containing the contaminant and at least one control material containing a known amount of the contaminant each with

     1) a quantum dot coupled to an affinity moiety capable of binding the contaminant and

     2) a competitor complex comprising a bead coupled to the contaminant;

b) separating the competitor complex from each of the starting material, the control material not containing the contaminant and at least one control material containing a known amount of the contaminant;

c) detecting the characteristic emission spectrum of the quantum dot associated with the competitor complex from each of the starting material, the control material not containing the contaminant and at least one control material containing a known amount of the contaminant; and

d) comparing the intensity of the characteristic emission spectrum of the quantum dot-competitor complex from the starting material to the intensity of the characteristic emission spectrum of the quantum dot-competitor complex from the control material containing a known amount of the contaminant, wherein the intensity of the characteristic emission spectrum is indicative of the quantity of the contaminant in the starting material.

55. The method of claim 54, further comprising:

e) repeating steps (a) through (d) with a set of control samples containing various concentrations of the contaminant to form a dose curve; and

f) comparing the intensities of the quantum dot-competitor complex of the dose curve to the intensity obtained from the quantum dot-competitor complex from the starting material.