Nanoparticle and microparticle based detection of cellular products
View Patent ↗Embodiments of the present invention relate to devices and methods for detecting cellular products using detection particles having product-specific detection reagents and having a characteristic spectral feature. In particular, devices and methods are provided for measuring secreted cellular products including cytokines. Detection substrates, include microwells having product-specific capture reagents thereon or comprising hydrophobic membranes are described having greater capability to detect products from individual cells in a mixture of heterogeneous cells. With the use of multiple detection particles, multiple cellular products can be detected in a single well. Additionally, using the inherent spectral properties of detection particles, no enzymatic reactions are needed to visualize a secreted product, thereby increasing the sensitivity, reproducibility and ease of use.
1. A method of detecting a cell product, comprising:
a) providing a reaction chamber comprising:
a cell culture medium and detection substrate having a capture reagent immobilized thereon that binds to said cell product to form a cell product-capture reagent complex;
b) adding a sample of cells to said reaction chamber;
said cells producing said cell product, said product binding to said capture reagent forming a cell product-capture reagent complex at a location on said substrate near said cell;
c) adding a detection reagent/detection particle complex to said reaction chamber, said detection particle having a characteristic spectrographic feature, said detection reagent/detection particle complex binding to said cell product-capture reagent complex producing a detection reagent/detection particle-cell product-capture reagent complex;
d) washing away detection reagent/detection particle complexes not bound to said product-capture reagent complex, and
e) detecting said detection reagent/detection particle-cell product-capture reagent complex visually or via image analysis as a spot on said detection substrate.
2. The method of claim 1 , wherein said detection reagent/detection particle complex comprises a detection reagent coupled to said detection particle by streptavidin/biotin binding.
3. The method of claim 1 , wherein said capture reagent is a cell product-specific ligand covalently attached to said substrate.
4. The method of claim 3 , wherein said cell product-specific ligand is an antibody or a cell product-specific receptor.
5. The method of claim 3 , wherein said detection substrate comprises a microwell.
6. The method of claim 1 , wherein said capture reagent is a surface, and said cell product becomes associated with said surface by way of physical interaction.
7. The method of claim 6 , wherein said physical interaction is hydrophobic interaction.
8. The method of claim 1 , wherein said cell product is an interferon.
9. The method of claim 8 , wherein said interferon is interferon gamma.
10. The method of claim 1 , wherein said cell product is an interleukin.
11. The method of claim 10 , wherein said interleukin is IL-5.
12. The method of claim 1 , wherein said cell is a human cell.
13. The method of claim 1 , wherein said detection particle is a nanoparticle having a lower limit of size of about 90 Å and an upper limit of about 500 Å.
14. The method of claim 1 , wherein said detection particle is a microparticle having a lower limit of size of about 0.05 μm; and an upper limit of size of about 5 μm.
15. The method of claim 1 , wherein said particle is made of a material selected from the group consisting of organic polymers, inorganic semiconductors and heavy metals.
16. The method of claim 15 , wherein said organic polymer is selected from the group consisting of polystyrene, polyacrylamide, polyacrolein and polycarbonate.
17. The method of claim 15 , wherein said inorganic semiconductor is selected from the group consisting of silicon dioxide-containing semiconductors and cadmium selenide (CdSe).
18. The method of claim 15 , wherein said heavy metal is selected from the group consisting of gold, silver, platinum, palladium, and lead.
19. The method of claim 1 , wherein said detection particle exhibits a spectral property selected from the group consisting of absorption, fluorescence and phosphoresence.
20. The method of claim 19 , wherein said detection particle exhibits a spectral feature having a lower limit of wavelength in the ultraviolet portion of the spectrum and an upper limit in the infrared portion of the spectrum.
21. A method for determining alloreactivity, comprising:
a) providing a reaction chamber comprising: a cell culture medium and detection substrate having a capture reagent immobilized thereon that binds to said cell product to form a cell product-capture reagent complex;
b) adding a first cell to said reaction chamber;
c) adding a second cell to said reaction chamber, wherein one of said cells stimulates said other of said cells to produce a cellular product indicative of alloreactivity, said cellular product binding to said capture reagent forming a cell product-capture reagent complex on said substrate at a location near at least one of said cells;
d) adding a detection reagent/detection particle complex to said reaction chamber, said detection particle having a characteristic spectrographic feature, said detection reagent/detection particle complex binding to said cell product-capture reagent complex forming a detection reagent/detection particle cell product-capture reagent complex;
e) washing away detection reagent/detection particle complexes not bound to said product-capture reagent complex, and
f) detecting said detection reagent/detection particle-cell product-capture reagent complex visually or via image analysis as a spot on said detection substrate, said product being indicative of alloreactivity.
22. The method of claim 21 , wherein said detection reagent/detection particle complex comprises a detection reagent coupled to said detection particle by streptavidin/biotin binding.
23. The method of claim 21 , wherein said first cell and said second cell are derived from related human beings.
24. The method of claim 21 , wherein said first cell and said second cell are derived from unrelated human beings.
25. A method of detecting a plurality of cell products, comprising:
a) providing a reaction chamber comprising: a cell culture medium and detection substrate having a plurality of capture reagents immobilized thereon, where each of said reagents binds to at least one of said plurality of cell products;
b) adding a sample of cells to said reaction chamber;
said cells producing said plurality of cell products, wherein each of said cell products binds to at least one of said capture reagents forming a plurality of cell product-capture reagent complexes at locations on said substrate near to at least one of said cells;
c) adding a plurality of distinct detection reagent/detection particle complexes to said reaction chamber, each of said detection reagent/detection particle complexes binding to one type of said cell product-capture reagent complexes, thereby producing a plurality of detection reagent/detection particle-cell product-capture reagent complexes, each distinct type of detection reagent/detection particle complexes having a different spectrographic feature;
d)washing away detection reagent/detection particle complexes not bound to a cell product-capture reagent complex, and
e)detecting said plurality of detection reagent/detection particle-cell product-capture reagent complexes visually or via image analysis as a spot on said detection substrate.
26. The method of claim 25 , wherein each of said plurality of capture reagents comprise antibodies specific for one of said plurality of cell products.
27. The method of claim 25 , wherein said capture reagent comprises a surface to which said cell products become associated through physical interaction.
28. The method of claim 27 , wherein said physical interaction is hydrophobic interaction.
29. A spot assay kit for detecting the presence of a cell product, comprising:
a cell culture reaction chamber comprising:
a detection substrate having a capture reagent immobilized thereon that binds to said cell product forming a cell product-capture reagent complex;
a detection reagent/detection particle complex that binds to said cell product-capture reagent complex, said detection reagent/detection particle complex formed by a detection reagent bound to a detection particle via streptavidinlbiotin binding, said detection particle having a characteristic spectrographic feature;
a solution for mixing said detection reagent/detection particle complex; and
instructions for use that allow detection of the detection reagent/detection particle-cell product capture reagent complex visually or via image analysis as a spot on the detection substrate.
30. The kit of claim 29 , further comprising at least one culture medium suitable for growing cells.
31. The kit of claim 29 , wherein said capture reagent is a cell product-specific ligand covalently attached to said substrate.
32. The kit of claim 31 , wherein said cell product-specific ligand is an antibody or a cell product-specific receptor.
33. The kit of claim 31 , wherein said detection substrate comprises a microwell.
34. The kit of claim 29 wherein said capture reagent is a surface, and said cell product is adsorbed onto said surface by way of physical interaction.
35. The kit of claim 34 , wherein said physical interaction is hydrophobic interaction.
36. The kit of claim 29 , wherein said cell product is an interferon.
37. The kit of claim 36 , wherein said interferon is interferon gamma.
38. The kit of claim 29 , wherein said cell product is an interleukin.
39. The kit of claim 38 , wherein said interleukin is 1 L-5.
40. The kit of claim 29 , wherein said cell is a human cell.
41. The kit of claim 29 , wherein said detection particle is a nanoparticle having:
a lower limit of size of about 90 Å; and
an upper limit of about 500 Å.
42. The kit of claim 29 , wherein said detection particle is a microparticle having:
a lower limit of size of about 0.05 μm; and
an upper limit of size of about 5 μm.
43. The kit of claim 29 , wherein said particle is made of a material selected from the group consisting of organic polymers, inorganic semiconductors, and heavy metals.
44. The kit of claim 43 , wherein said organic polymer is selected from the group consisting of polystyrene, polyacrylamide, polyacrolein, and polycarbonate.
45. The kit of claim 43 , wherein said inorganic semiconductor is selected from the group consisting of silicon dioxide-containing semiconductors and cadmium selenide (CdSe).
46. The kit of claim 43 , wherein said heavy metal is selected from the group consisting of gold, silver, platinum, palladium, and lead.
47. The kit of claim 29 , wherein said detection particle exhibits a spectral property selected from the group consisting of absorption, fluorescence and phosphorescence.
48. The kit of claim 47 , wherein said detection particle exhibits a spectral feature having a lower limit of wavelength in the ultraviolet portion of the spectrum and an upper limit in the infrared portion of the spectrum.
49. A spot assay kit for detecting the presence of a plurality of cell products, comprising:
a reaction chamber comprising:
a cell culture medium and a detection substrate having a plurality of capture reagents immobilized thereon, where each of said plurality of capture reagents binds to one of said plurality of cell products forming a cell product-capture reagent complex;
a plurality of detection reagent/detection particle complexes, each of said complexes binding to one type of said cell product-capture reagent complex;
where each of said plurality of detection reagent/detection particle complexes binds to said detection particle via streptavidin/biotin binding; where each distinct type of detection particles has a characteristic spectrographic feature associated therewith sufficient to permit differential detection of each of said plurality of said detection particles;
a solution for mixing said detection reagent/detection particle complexes; and
instructions for use that allow detection of the detection reagent/detection particle-cell product capture reagent complexes visually or via image analysis as one or more spots on the detection substrate.
50. The kit of claim 49 , wherein each of said plurality of capture reagents comprise antibodies specific for one of said plurality of cell products.
51. The kit of claim 49 , wherein said capture reagents comprise a surface onto which said cell products become adsorbed through physical interaction.
52. The kit of claim 51 , wherein said physical interaction is hydrophobic interaction.