Method and system for sorting and separating particles
View Patent ↗The present invention relates to a method of particle separation comprising preparing a suspension of particles containing at least one recognizable target particle in a suspending solution, labeling the target particle with a conjugate marker, wherein the conjugate marker comprises at least one recognition unit for the recognizable target particle and at least one peroxidase enzyme, contacting a gelatin surface with the suspending solution, adding developer to the suspending solution in contact with the gelatin surface and in the presence of phenol to attach the target particle to the gelatin surface, and washing the gelatin surface to remove unattached particles. The method may also include detecting the presence of the target particle on the gelatin surface as well as detaching and recovering the attached particles after removal of the non-target particles.
1. A method of particle separation comprising:
preparing a suspension of particles containing at least one recognizable target particle in a suspending solution;
labeling said at least one recognizable target particle in said suspending solution with a conjugate marker, wherein said conjugate marker comprises at least one recognition unit specific for said recognizable target particle and at least one peroxidase enzyme;
contacting a gelatin surface with said suspending solution;
adding developer comprising oxidant to said suspending solution in contact with said gelatin surface in the presence of phenol to attach said recognizable target particle to said gelatin surface;
washing said gelatin surface to remove unattached particles.
2. The method of claim 1 wherein said target particle is organic or inorganic.
3. The method of claim 1 wherein said target particle is a cellular or subcellular particle.
4. The method of claim 1 wherein said target particle is circular, spherical, rectangular, or elliptical.
5. The method of claim 1 wherein said target particle is recognizable by characteristics of the particle itself.
6. The method of claim 1 wherein said target particle is recognizable through a specific marker attached to said target particle.
7. The method of claim 1 wherein said suspending solution is an aqueous solution.
8. The method of claim 7 wherein said suspending solution is buffered.
9. The method of claim 1 wherein said suspending solution further comprises phenol.
10. The method of claim 1 wherein said recognition unit is an antibody, antigen, or ligand.
11. The method of claim 1 wherein said recognition unit has an affinity binding constant to the target analyte of no less than 10 −6 M −1 .
12. The method of claim 1 wherein said recognition unit is a nucleic acid or nucleic acid ligand.
13. The method of claim 1 wherein said recognition unit is a protein scaffold or synthetic molecular moiety capable of recognizing a specific DNA sequence.
14. The method of claim 1 wherein said peroxidase is connected to the recognition unit covalently.
15. The method of claim 1 wherein said peroxidase is connected to the recognition unit non-covalently.
16. The method of claim 1 wherein said peroxidase is horseradish peroxidase (HRP).
17. The method of claim 1 wherein said gelatin is coated onto a support.
18. The method of claim 17 wherein said support is organic, inorganic or biological.
19. The method of claim 17 wherein said support is glass, quartz, plastics, metals, semiconductors, a porous membrane.
20. The method of claim 1 wherein said gelatin further contains hardener.
21. The method of claim 1 wherein said gelatin has a laydown of from 0.2 to 100 grams per square meter.
22. The method of claim 1 wherein said gelatin has a laydown of from 10 to 50 grams per square meter.
23. The method of claim 1 wherein said developer contains hydrogen peroxide.
24. The method of claim 1 wherein the concentration of said hydrogen peroxide in said developer is from 0.01% (w/v) to 10% (w/v).
25. The method of claim 1 wherein said developer is aqueous.
26. The method of claim 1 wherein said developer is buffered.
27. The method of claim 1 wherein said developer further contains phenol.
28. The method of claim 1 wherein said phenol is added prior to or simultaneously with said developer.
29. The method of claim 1 wherein said phenol is represented by the following general formula:
where R1, R2, R3, R4, R5 are independently a hydrogen, a substituted or unsubstituted linear or branched alkyl group of 1 to 10 carbon atoms, a substituted or unsubstituted aryl group of 6 to 14 carbon atoms, a substituted or unsubstituted cycloalkyl group of 5 to 14 carbon atoms, a substituted or unsubstituted heterocyclic group, a solubilizing group, or a halogen atom of fluoride, chloride, bromide, iodide.
30. The method of claim 29 wherein at least one of R1, R2, R3, R4, R5 is or contains a solubilizing substituent that is generally negatively charged.
31. The method of claim 1 wherein said phenol is represented by at least one of the following structures:
32. The method of claim 1 wherein said washing is aqueous.
33. The method of claim 1 further comprising:
detaching said attached target particle; and
recovering said detached target particle.
34. The method of claim 33 wherein said detaching occurs by treatment with a chemical which may detach said attached target particle from said gelatin surface.
35. The method of claim 33 wherein said detaching occurs by subjecting the attached target particle to a proteolytic enzyme.
36. The method of claim 35 wherein said proteolytic enzyme is trypsin, pepsin, or papain.
37. The method of claim 33 wherein said detaching occurs by selective dissolution said gelatin surface in order to release said attached target particle.
38. The method of claim 33 wherein said detaching occurs by sonication to release said attached particle.
39. The method of claim 33 wherein said recovering occurs by removal of said detached target particle from additional washing fluid.
40. The method of claim 33 wherein said recovering occurs by collection through a suction device under visual control.
41. The method of claim 40 wherein said suction device is a micro-pipette.
42. The method of claim 33 further comprising detecting said attached target particle prior to said detaching and said recovering.
43. The method of claim 1 further comprising detecting said attached target particle.
44. The method of claim 43 wherein said detecting is visual inspecting.
45. The method of claim 44 wherein said visual inspecting utilizes a microscope.
46. The method of claim 43 wherein said detecting is automatic imaging.
47. The method of claim 43 wherein said detecting is dark field illumination, epi-illumination, phase-contrast microscopy, differential interference contrast microscopy (DIC), polarization microscopy, multi-spectral or hyper-spectral microscopy.
48. The method of claim 43 wherein said detecting is full-frame image capture.