METHOD FOR SEPARATION AND IDENTIFICATION OF BIOMOLECULES USING UNCONVENTIONAL GEL ELECTROPHORESIS AND DETECTION OF SINGLE NANOPARTICLE PROBES

• US 20090277791A1
• Filed: 10/23/2007
• Published: 11/12/2009
• Est. Priority Date: 10/23/2006
• Status: Abandoned Application

• Current Assignee: Oregon Health & Science University
• Original Assignee: Oregon Health & Science University
• Inventors: Liu, Hong Yan, Vu, Tania Q.
• Application Number:
  US12/446,736
• Publication Number:
  US 20090277791A1
• Time in Patent Office: Days
• US Class Current: 204/461
• CPC Class Codes: G01N 33/5432 Liposomes or microcapsules
• CPC Class Codes: G01N 33/559 through a gel, e.g. Ouchterlony technique

Abstract

A method for identifying and separating of target biomolecules is disclosed. The method involves contacting a sample with a nanoparticle probe that is capable of specifically binding a target biomolecule. The complex formed by the nanoparticle target biomolecule is separated by electrophoresis using a non-conventional gel composed of a synthetically cross-linked polymer and a gel stabilizer. The nanoparticle probe:target biomolecule complex is detected, thereby identifying the target biomolecule. In certain examples, the nanoparticle probe emits a signal to identify its location. Particular examples of nanoparticle probes are semiconductor nanocrystals, such as quantum dots, which fluoresce at a particular wavelength. Also disclosed is a method of substantially sealing and/or increasing the transparency of a membrane. Such a method includes applying a film to a membrane. Sealing the membrane allows the detection of single nanoparticles.

Claims

1. A method for detecting a target biomolecule of interest in a sample comprising:

labeling the target biomolecule of interest in the sample with a nanoparticle probe comprising a detectable nanoparticle;

electrophoresing the sample comprising the labeled target biomolecule of interest in a composite gel to separate the labeled target biomolecule of interest from other biomolecules in the sample, wherein the composite gel comprises a cross-linked polymer and a polysaccharide gel stabilizer and is capable of separating the labeled target biomolecule of interest from other biomolecules in the sample; and

detecting the nanoparticle, whereby detection of the nanoparticle detects the target biomolecule of interest in the electrophoresed sample.

2. The method of claim 1, wherein labeling the target biomolecule comprises labeling the target biomolecule with a nanoparticle probe having a defined physical characteristic that separates the labeled target biomolecule during electrophoresis, and wherein the labeled target biomolecule is separated by the physical characteristic of the nanoparticle probe.

3. The method of claim 2, wherein the physical characteristic of the nanoparticle probe is the size of the nanoparticle probe and the labeled target biomolecule is separated by the size of the nanoparticle probe.

4. The method of claim 2, wherein the physical characteristic of the nanoparticle probe is the mass of the nanoparticle probe and the labeled target biomolecule is separated by the mass of the nanoparticle probe.

5. The method of claim 2, wherein the physical characteristic of the nanoparticle probe is the charge of the nanoparticle probe and the labeled target biomolecule is separated by the charge of the nanoparticle probe.

6. The method of claim 1, wherein the detectable nanoparticle comprises a semiconductor nanocrystal that emits a detectable electromagnetic signal that identifies the target biomolecule of interest by a characteristic emission spectrum of the semiconductor nanocrystal.

7. The method of claim 1, wherein the polysaccharide is hydroxyethyl cellulose, hydroxypropylmethyl cellulose, methyl cellulose, agarose, hydroxyethyl agarose, galactomannan, dextran or a combination thereof, and wherein the cross-linked polymer comprises the free radical polymerization reaction product of a mixture comprising at least one monomer and at least one cross-linker sufficient to cross-link the monomer, wherein the monomer comprises acrylamide, N-methylacrylamide, N,N-dimethylacrylamide, N-(hydroxymethyl)acrylamide, diacetonacrylamide, N-hydroxypropylacrylamide N-hydroxypropylacrylamide, N-acryloyl-tris(hydroxymethyl)aminomethane, N-acryloyl-1-amino-1-deoxy-D-galactitol, or a combination thereof, and the cross-linker comprises N,N′

-methylenebisacrylamide, N,N′

-propylenebisacrylamide, diacrylamide dimethylether, 1,2-diacrylamide ethyleneglycol, ethylenureabisacrylamide, ethylene diacrylate, N,N′

-diallyltartardiamide, N,N′

-bisacrylylcystamine, N,N′

-1,2-dihydroxyethylene-bisacrylamide, N,N-bisacrylyl cystamine, trisacryloyl-hexahydrotriazine, dihydroxyethylene-bis-acrylamide, piperazine-di-acrylamide, or a combination thereof.

8. The method of claim 1, wherein the composite gel comprises agarose and cross-linked acrylamide

9. The method of claim 8, wherein the composite gel comprises:

from about 0.1% agarose to about 3.0% agarose; and

from about 0.1% cross-linked acrylamide to about 7% cross-linked acrylamide.

10. The method of claim 9, wherein the composite gel comprises:

from about 0.5% agarose to about 1.5% agarose; and

from about 0.5% cross-linked acrylamide to about 3% cross-linked acrylamide.

11. The method of claim 1, wherein the target biomolecule of interest comprises a polypeptide or a nucleic acid molecule.

12. The method of claim 1, wherein the nanoparticle probe further comprises a specific binding agent that specifically binds the target biomolecule of interest.

13. The method of claim 12, wherein the specific binding agent is an antibody, a ligand, an aptamer, or a peptide.

14. The method of claim 12, wherein the detectable nanoparticle is conjugated directly to the specific binding agent.

15. The method of claim 12, wherein the probe further comprises a linker linking the detectable nanoparticle and the specific binding agent.

16. The method of claim 15, wherein the linker is streptavidin, avidin, biotin or a combination thereof.

17. The method of claim 1, further comprising transferring the target biomolecule of interest to a membrane.

18. The method of claim 17, wherein the membrane is a polyvinylidene fluoride membrane.

19. The method of claim 17, further comprising sealing the membrane to substantially inhibit contact of the membrane with the atmosphere.

20. The method of claim 19, wherein sealing the membrane increases the light transmissibility of the membrane.

21. The method of claim 20, wherein increasing the light transmissibility of the membrane comprises rendering the membrane at least translucent.

22. The method of claim 21, wherein increasing the light transmissibility of the membrane comprises rendering the membrane substantially transparent.

23. The method of claim 19, wherein sealing the membrane comprises coating a portion of the membrane with a film.

24. The method of claim 23, wherein the film is non-fluorescent.

25. The method of claim 23, wherein the film comprises a silica based polymer, monomer, or a combination thereof.

26. The method of claim 25, wherein the silica based polymer or monomer comprises a siloxane, a silicone, a silane, a specialty silane, a silane coupling agent, a polysilane, a silathiane, a stannoxane, a silazane, a polymethylsilsesquioxane, a polyvinylsilsequioxane, a polysilazane, a polysiloxane, a polysilsesquioxane, a polyphosphazine, a polyborosilane, a polycarbosilazane, a variant, a derivative or a combination thereof.

27. The method of claim 25, wherein the film comprises a polydimethylsiloxane.

28. The method of claim 17, further comprising identifying the target biomolecule of interest on the membrane, comprising:

contacting the membrane with a detectable probe capable of specifically binding to the target biomolecule of interest; and

detecting the detectable probe, wherein detecting the detectable probe identifies the target biomolecule of interest on the membrane.

29. The method of claim 28, wherein the detectable probe comprises a specific binding agent that specifically binds the target biomolecule of interest.

30. The method of claim 29, wherein the specific binding agent is an antibody, a ligand, an aptamer, or a peptide.

31. The method of claim 29, wherein the detectable probe comprises a label.

32. The method of claim 31, wherein the label comprises an electron-dense compound, an enzyme, a fluorochrome, a hapten, a radioisotope, or a nanoparticle.

33. The method of claim 18, further comprising identifying a second biomolecule of interest that interacts with the target biomolecule of interest, comprising:

contacting the membrane with a detectable probe capable of specifically binding to the second biomolecule of interest;

detecting the detectable probe; and

determining if the detectable probe co-localizes with the target biomolecule of interest, wherein co-localization of the detectable probe and the target biomolecule of interest indicates that that the second biomolecule of interest interacts with the target biomolecule of interest.

34. The method of claim 1, wherein the sample comprises a plurality of different biomolecules of interest, the method further comprising:

labeling each different biomolecule of interest with a different nanoparticle probe specific for each different biomolecule of interest, wherein the different nanoparticle probes comprise different detectable nanoparticles and each of the different detectable nanoparticle probes comprises a specific binding agent that specifically binds each different biomolecule of interest; and

detecting the different detectable nanoparticles, whereby detection of the different detectable nanoparticles detects the different biomolecules of interest in the electrophoresed sample.

35. The method of claim 34, wherein labeling each different biomolecule of interest comprises labeling each different biomolecule of interest with a nanoparticle probe having a defined physical characteristic different from the other nanoparticle probes that separate the different biomolecules of interest during electrophoresis, and wherein the different biomolecules of interest are separated by the physical characteristic of the nanoparticle probe.

36. The method of claim 35, wherein the physical characteristic of the nanoparticle probes is the size of the nanoparticle probes and the different biomolecules of interest are separated by the size of the nanoparticle probes.

37. The method of claim 35, wherein the physical characteristic of the nanoparticle probes is the mass of the nanoparticle probes and the different biomolecules of interest are separated by the mass of the nanoparticle probes.

38. The method of claim 35, wherein the physical characteristic of the nanoparticle probes is the charge of the nanoparticle probes and the different biomolecules of interest are separated by the charge of the nanoparticle probes.

39. The method of claim 34, wherein each of the different detectable nanoparticles comprises a different semiconductor nanocrystal that emits a characteristic detectable electromagnetic spectrum distinct from the other semiconductor nanocrystals.

40. The method of claim 39, wherein the characteristic emission spectra from the different semiconductor nanocrystals are light.

41. The method of claim 39, wherein the different biomolecules of interest are detected by detecting different wavelengths of light.

42. A kit for the detection of a target biomolecule of interest, the kit comprising

a nanoparticle probe for the target biomolecule of interest, comprising a detectable nanoparticle;

a composite gel comprises a cross-linked polymer and a gel stabilizer, wherein the gel stabilizer comprises a polysaccharide gel stabilizer and the cross-linked polymer comprises the free radical polymerization reaction product of a mixture comprising at least one monomer and at least one cross-linker sufficient to cross-link the monomer; and

instructions for labeling and electrophoresing the target biomolecule of interest.

43. The kit of claim 42, wherein the detectable nanoparticle comprises a semiconductor nanocrystal that emits a detectable electromagnetic signal that identifies the target biomolecule of interest by a characteristic emission spectrum of the semiconductor nanocrystal.

44. The kit of claim 42, wherein the polysaccharide is hydroxyethyl cellulose, hydroxypropylmethyl cellulose, methyl cellulose, agarose, hydroxyethyl agarose, galactomannan, dextran or a combination thereof, the monomer comprises acrylamide, N-methylacrylamide, N,N-dimethylacrylamide, N-(hydroxymethyl)acrylamide, diacetonacrylamide, N-hydroxypropylacrylamide N-hydroxypropylacrylamide, N-acryloyl-tris(hydroxymethyl)aminomethane, N-acryloyl-1-amino-1-deoxy-D-galactitol, or a combination thereof, and the cross-linker comprises N,N′

-methylenebisacrylamide, N,N′

-propylenebisacrylamide, diacrylamide dimethylether, 1,2-diacrylamide ethyleneglycol, ethylenureabisacrylamide, ethylene diacrylate, N,N′

-diallyltartardiamide, N,N′

-bisacrylylcystamine, N,N′

-1,2-dihydroxyethylene-bisacrylamide, N,N-bisacrylyl cystamine, trisacryloyl-hexahydrotriazine, dihydroxyethylene-bis-acrylamide, piperazine-di-acrylamide, or a combination thereof.

45. The kit of claim 44, wherein the composite gel comprises agarose and cross-linked acrylamide.

46. The kit of claim 45, wherein the composite gel comprises:

from about 0.1% agarose to about 3.0% agarose; and

from about 0.1% cross-linked acrylamide to about 7% cross-linked acrylamide.

47. The kit of claim 46, wherein the composite gel comprises:

from about 0.5% agarose to about 1.5% agarose; and

from about 0.5% cross-linked acrylamide to about 3% cross-linked acrylamide.

48. The kit of claim 42, wherein the nanoparticle probe further comprises a specific binding agent that specifically binds the target biomolecule of interest.

49. The kit of claim 42, further comprising different nanoparticle probe specific for different biomolecules of interest, wherein the different nanoparticle probes comprise different detectable nanoparticles, wherein each of the different detectable nanoparticle probes comprises a specific binding agent for each different biomolecule of interest.

50. The kit of claim 42, further comprising a film for application to the membrane.

51. The kit of claim 50, wherein the film is non-fluorescent.

52. The kit of claim 50, wherein the film comprises a silica based polymer, monomer, or a combination thereof.

53. The kit of claim 52, wherein the silica based polymer or monomer comprises a siloxane, a silicone, a silane, a specialty silane, a silane coupling agent, a polysilane, a silathiane, a stannoxane, a silazane, a polymethylsilsesquioxane, a polyvinylsilsequioxane, a polysilazane, a polysiloxane, a polysilsesquioxane, a polyphosphazine, a polyborosilane, a polycarbosilazane, a variant, a derivative or a combination thereof.

54. The kit of claim 52, wherein the film comprises a polydimethylsiloxane.

55. The kit of claim 42, wherein the membrane is a polyvinylidene fluoride membrane.

56. A method of inhibiting photo-bleaching of a semiconductor nanocrystal on a membrane, comprising

providing a membrane containing at least one semiconductor nanocrystal;

and

applying a film to a portion of the membrane wherein the film substantially inhibits contact of the membrane with the atmosphere, thereby inhibiting photo-bleaching of the semiconductor nanocrystal on the membrane.

57. The method of claim 56, wherein applying the film to the membrane increases the light transmissibility of the membrane.

58. The method of claim 57, wherein increasing the light transmissibility of the membrane comprises rendering the membrane at least translucent.

59. The method of claim 58, wherein increasing the light transmissibility of the membrane comprises rendering the membrane substantially transparent.

60. The method of claim 56, wherein the film is non-fluorescent.

61. The method of claim 56, wherein the film comprises a silica based polymer, monomer, or a combination thereof.

62. The method of claim 61, wherein the polymer or monomer comprises a siloxane, a silicone, a silane, a specialty silane, a silane coupling agent, a polysilane, a silathiane, a stannoxane, a silazane, a polymethylsilsesquioxane, a polyvinylsilsequioxane, a polysilazane, a polysiloxane, a polysilsesquioxane, a polyphosphazine, a polyborosilane, a polycarbosilazane, a variant, a derivative or a combination thereof.

63. The method of claim 61, wherein the film comprises a polydimethylsiloxane.

64. The method of claim 56, wherein the membrane is a polyvinylidene fluoride membrane.