Method for sensitive detection of multiple biological analytes
View Patent ↗The present invention relates to a method of detecting biological analytes comprising suspending a target analyte in a suspending solution containing polymeric particles marked with a probe, wherein the probe has an affinity for said target analyte; adding recognition unit-peroxidase conjugate marker to the suspending solution; forming a complex of the target analyte, the polymeric particles marked with a probe, and the recognition unit-peroxidase conjugate marker; contacting a gelatin surface with the suspending solution; adding developer to the suspending solution in contact with the gelatin surface in the presence of phenol to attach the complex to the gelatin surface; washing the gelatin surface; and detecting the complex attached to the gelatin surface.
1. A method of detecting biological analytes comprising:
suspending a target analyte in a suspending solution containing polymeric particles with an attached probe, wherein said probe has specific affinity for said target analyte;
adding at least one recognition unit-peroxidase conjugate marker to said suspending solution, wherein said recognition unit associates with said target analyte;
forming a complex of said target analyte, said polymeric particles with an attached probe, said recognition unit-peroxidase conjugate marker;
contacting a gelatin surface with said suspending solution;
adding developer to said suspending solution in contact with said gelatin surface in the presence of phenol to attach said complex to said gelatin surface;
washing said gelatin surface; and
detecting said complex attached to said gelatin surface.
2. The method of claim 1 wherein said target analyte is a protein.
3. The method of claim 1 wherein said target analyte is bacteria.
4. The method of claim 1 wherein said suspending solution is an aqueous solution.
5. The method of claim 4 wherein said suspending solution is buffered.
6. The method of claim 1 wherein said suspending solution further comprises phenol.
7. The method of claim 1 wherein said polymeric particles are organic.
8. The method of claim 1 wherein said polymeric particles are inorganic.
9. The method of claim 1 wherein said polymeric particles have a distinct color, shape or size.
10. The method of claim 9 wherein said shape is curvilinear, spherical, donut shaped, elliptical, rectangular, cubic, or rod.
11. The method of claim 9 wherein said color is derived from mixing three dyes representing the primary colors R, G, B to create distinct “color addresses.”
12. The method of claim 11 wherein each of said distinct “color addresses” corresponds to a specific bioactive probe.
13. The method of claim 9 wherein said color is derived by covalently coupling one or more dyes to the surface of said polymeric particles.
14. The method of claim 9 wherein said color is derived from micro-encapsulated colorants and pigments incorporated into said polymeric particles.
15. The method of claim 1 wherein said polymeric particles are polystyrene, poly(methyl methacrylate) and poly(butyl acrylate).
16. The method of claim 1 wherein said polymeric particles have a mean diameter of from 1 to 100 microns.
17. The method of claim 1 wherein said polymeric particles have a mean diameter of from 2 to 30 microns.
18. The method of claim 1 wherein said polymeric particles have a mean diameter of front 3 to 20 microns.
19. The method of claim 1 wherein said polymeric particles are monodisperse wherein the coefficient of the particle size distribution (i.e. the standard deviation as a percentage of the mean) will be less than 20%.
20. The method of claim 1 wherein said polymeric particles are a plurality of particles types, wherein each type of particle is distinguishable from each other type of particle.
21. The method of claim 1 wherein said probe is a protein capture agent.
22. The method of claim 1 wherein said probe is an antibody.
23. The method of claim 1 wherein said probe has an affinity binding constant to said target analyte of greater than 10 6 M −1 .
24. The method of claim 1 wherein said recognition unit is an antibody.
25. 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 .
26. The method of claim 1 wherein said recognition unit is the same as said probe.
27. The method of claim 1 wherein said recognition unit is a nucleic acid.
28. The method of claim 1 wherein said peroxidase is connected to the recognition unit covalently.
29. The method of claim 1 wherein said peroxidase is connected to the recognition unit non-covalently.
30. The method of claim 1 wherein said peroxidase is horseradish peroxidase (HRP).
31. The method of claim 1 wherein said gelatin is coated onto a support.
32. The method of claim 31 wherein said support is organic, inorganic or biological.
33. The method of claim 31 wherein said support is glass, quartz, plastics, metals, semiconductors, a porous membrane.
34. The method of claim 1 wherein said gelatin further comprises hardener.
35. The method of claim 1 wherein said gelatin has a laydown of from 0.2 to 100 grams per square meter.
36. The method of claim 1 wherein said gelatin has a laydown of from 10 to 50 grams per square meter.
37. The method of claim 1 wherein said developer contains hydrogen peroxide.
38. 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).
39. The method of claim 1 wherein said developer is aqueous.
40. The method of claim 1 wherein said developer is buffered.
41. The method of claim 1 wherein said developer further comprises phenol.
42. The method of claim 1 wherein said phenol is added prior to or simultaneously with said developer.
43. 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 6to 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.
44. The method of claim 43 wherein at least one of R1, R2, R3, R4, R5 is or contains a solubilizing substituent that is generally negatively charged.
45. The method of claim 1 wherein said phenol is represented by at least one of the following structures:
46. The method of claim 1 wherein said washing is aqueous.
47. The method of claim 1 wherein said detecting is visual inspecting.
48. The method of claim 47 wherein said visual inspecting utilizes a microscope.
49. The method of claim 1 wherein said detecting is automatic imaging.
50. The method of claim 1 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.
51. The method of claim 1 wherein said detecting is full-frame image capture.