Claims
- 1. A method of detecting a reaction, comprising:
providing a first reagent mixture which comprises a first reagent having a fluorescent label; introducing a second reagent into the first reagent mixture to produce a second reagent mixture, the second reagent reacting with the first reagent to produce a fluorescently labeled product having a different charge than the first reagent; introducing a polyion into at least one of the first and second reagent mixtures; and comparing a fluorescent polarization level in the second reagent mixture relative to the first reagent mixture.
- 2. The method of claim 1, wherein the polyion associates with the product.
- 3. The method of claim 1, wherein the polyion associates with the first reagent.
- 4. The method of claim 1, wherein the polyion bears a charge that is opposite to a charge of the product.
- 5. The method of claim 1, wherein the polyion is a polycation.
- 6. The method of claim 5, wherein the polycation is a polyamino acid.
- 7. The method of claim 6, wherein the polyamino acid comprises a protein that associates with one of the fluorescent first reagent or product in a charge dependent manner.
- 8. The method of claim 6, wherein the polyamino acid is selected from polylysine, polyhistidine, polyarginine, or copolymers thereof.
- 9. The method of claim 1, wherein the first reagent comprises a substantially uncharged or positively charged nucleic acid analog, and the second reagent comprises a nucleic acid having a nucleotide sequence complementary to a sequence of the nucleic acid analog, and the product comprises a hybrid of the nucleic acid analog and the nucleic acid.
- 10. The method of claim 9, wherein the nucleic acid analog is selected from a peptide nucleic acid (PNA), a methyl phosphonate polymer and a cationic nucleic acid.
- 11. The method of claim 1, wherein the first reagent comprises a phosphorylatable compound, and the product comprises a phosphorylated first reagent.
- 12. The method of claim 11, wherein the second reagent comprises a kinase enzyme.
- 13. The method of claim 11, wherein the phosphorylatable compound comprises a peptide that comprises a phosphorylation site.
- 14. The method of claim 1, wherein the first reagent comprises a phosphorylated compound, and the product comprises a dephosphorylated first reagent.
- 15. The method of claim 14, wherein the second reagent comprises a phosphatase enzyme.
- 16. The method of claim 14, wherein the first reagent comprises a fluorescently labeled phosphorylated peptide.
- 17. The method of claim 1, wherein the first reagent comprises a first polypeptide sequence, and the product comprises a fragment of the polypeptide sequence, the fragment having a different charge than the first polypeptide sequence.
- 18. The method of claim 17, wherein the second reagent comprises a proteolytic reagent.
- 19. The method of claim 18, wherein the proteolytic reagent comprises a protease enzyme.
- 20. The method of claim 1, wherein the first reagent is transported along a first channel, and the second reagent is introduced into the first channel to mix with the first reagent.
- 21. The method of claim 20, wherein the first and second reagents are introduced into the first channel via second and third channels, respectively, the second and third channels having first and second ends, and intersecting the first channel at their second ends.
- 22. The method of claim 21, wherein the first ends of the second and third channels are fluidly connected to first and second reservoirs, the first and second reservoirs having the first and second reagents disposed therein, respectively.
- 23. The method of claim 1, further comprising:
directing an excitation light at the first reagent and measuring a level of fluorescence polarization in the first reagent; directing an excitation light at the second reagent mixture and measuring a level of fluorescence polarization from the second reagent mixture; and wherein the comparing step comprises determining a first difference in the level of fluorescence polarization from the first reagent and the level of fluorescent polarization of the second reagent mixture.
- 24. The method of claim 23, further comprising introducing a test compound into at least one of the first reagent and second reagent mixture, and performing the comparing step in the presence and absence of the test compound, a decrease or increase in the first difference in the level of fluorescence polarization in the presence of the test compound relative to the difference in the absence of the test compound being indicative that the test compound is an inhibitor or enhancer of a reaction between the first and second reagents.
- 25. The method of claim 24, wherein the second mixture is transported along a first channel, and the test compound is introduced into the first channel.
- 26. The method of claim 25, wherein the first channel is fluidly connected to or is an integral portion of a capillary element, and the test compound is drawn into the capillary element from a source of the test compound.
- 27. The method of claim 24, wherein a plurality of different test compounds are separately introduced into separate volumes of the at least one of the first reagent and second reagent mixture.
- 28. The method of claim 24, wherein the second reagent mixture is transported along a first channel, and the plurality of test compounds are introduced into the first channel as discrete fluid regions.
- 29. A method of identifying the presence of a subsequence of nucleotides in a target nucleic acid, the method comprising:
contacting the target nucleic acid sequence with a substantially uncharged or positively charged, fluorescently labeled nucleic acid analog in a first reaction mixture, the nucleic acid analog being complementary to the subsequence whereby the nucleic acid analog is capable of specifically hybridizing to the subsequence to form a first hybrid; contacting the first reaction mixture with a polyion; and comparing a level of fluorescence polarization of the first reaction mixture in the presence of the polyion to a level of fluorescence polarization of the nucleic acid analog in the absence of the target nucleic acid sequence, an increase in the level of fluorescence polarization indicating a presence of the first hybrid.
- 30. The method of claim 29, wherein the nucleic acid analog is selected from a peptide nucleic acid (PNA), a methyl phosphonate polymer, and a cationic nucleic acid.
- 31. The method of claim 29, wherein the polyion is a polycation.
- 32. The method of claim 31, wherein the polycation is a polyamino acid.
- 33. The method of claim 32, wherein the polyamino acid comprises a protein that associates with one of the fluorescent first reagent or product in a charge dependent manner.
- 34. The method of claim 32, wherein the polyamino acid is selected from polylysine, polyhistidine, polyarginine and copolymers thereof.
- 35. The method of claim 29, wherein the target nucleic acid sequence comprises at least one locus for a single nucleotide polymorphism.
- 36. The method of claim 29, wherein the nucleic acid analog is complementary to one allele of the single nucleotide polymorphism in the target nucleic acid sequence.
- 37. A method of detecting the phosphorylation of a phosphorylatable compound, comprising:
providing the phosphorylatable compound with a fluorescent label; contacting the phosphorylatable compound with a kinase enzyme in the presence of a phosphate donor in a first mixture; contacting the first mixture with a polyion; comparing a level of fluorescence polarization from the first mixture in the presence of the polyion to a level of fluorescence polarization from the phosphorylatable compound with the fluorescent label in the absence of the kinase enzyme.
- 38. The method of claim 37, wherein the polyion associates with the product.
- 39. The method of claim 37, wherein the polyion associates with the first reagent.
- 40. The method of claim 37, wherein the polyion bears a charge that is opposite to a charge of the charged product.
- 41. The method of claim 37, wherein the polyion is a polycation.
- 42. The method of claim 41, wherein the polycation is a polyamino acid.
- 43. The method of claim 42, wherein the polyamino acid comprises a protein that associates with one of the fluorescent first reagent or product in a charge dependent manner.
- 44. The method of claim 42, wherein the polyamino acid is selected from polylysine, polyhistidine, polyarginine, and copolymers thereof.
- 45. A method of detecting the phosphorylation of a phosphorylatable compound, comprising:
providing the phosphorylatable compound with a fluorescent label; contacting the phosphorylatable compound with a kinase enzyme in the presence of a phosphate group in a first mixture; contacting the first mixture with a second reagent mixture comprising a protein having a chelating group associated therewith, and a metal ion selected from Fe3+, Ca2+, Ni2+ and Zn2+; comparing a level of fluorescence polarization from the first mixture in the presence of the second mixture to a level of fluorescence polarization from the phosphorylatable compound with the fluorescent label in the absence of the kinase enzyme.
- 46. An assay system, comprising:
a fluid receptacle comprising:
a first reaction zone having disposed therein: a first reagent mixture which comprises a first reagent having a fluorescent label; a second reagent that reacts with the first reagent to produce a fluorescently labeled product having a substantially different charge than the first reagent; and a polyion; and a detection zone; and a detector disposed in sensory communication with the detection zone, the detector being configured to detect a level of fluorescence polarization of reagents in the detection zone.
- 47. The assay system of claim 46, wherein the fluid receptacle comprises a body structure having at least a first channel disposed therein, a portion of the first channel comprising the reaction zone and a portion of the first channel comprising the detection zone.
- 48. The assay system of claim 47, further comprising a material transport system for transporting the first reagent, second reagent and polyion from the reaction zone portion of the first channel to the detection zone portion of the first channel.
- 49. The assay system of claim 48, wherein the material transport system comprises a pressure or vacuum source applied to one terminus of the first channel to push or draw fluid through from the reaction zone to the detection zone.
- 50. The assay system of claim 48, wherein the material transport system comprises an electrical power source operably coupled to different points in the first channel for creating an electric field across at least a portion of a length of the first channel, the electric field causing movement of material from the reaction zone to the detection zone.
- 51. The assay system of claim 46, wherein the reaction receptacle comprises a well in a multiwell plate.
- 52. The assay system of claim 46, wherein the reaction receptacle comprises a test tube.
- 53. An assay system, comprising:
a first channel disposed in a body structure, the first channel being fluidly connected to:
a source of a first reagent mixture which comprises a first reagent having a fluorescent label; a source of a second reagent that reacts with the first reagent to produce a fluorescently labeled product having a substantially different charge than the first reagent; and a source of a polyion; a material transport system for introducing the first reagent, the second reagent and the polyion into the first channel; and a detector disposed in sensory communication with the first channel, the detector being configured to detect a level of fluorescence polarization of reagents in the detection zone.
- 54. The assay system of claim 53, wherein the source of first reagent, the source of second reagent and the source of polyion comprise first, second and third reservoirs disposed in the body structure, respectively, the first, second and third reservoirs being fluidly connected to the first channel.
- 55. The assay system of claim 54, further comprising an external sampling capillary having a capillary channel disposed therethrough, the capillary channel having first and second ends, the capillary channel being fluidly connected to the first channel at the first end, and open at the second end.
- 56. The assay system of claim 53, further comprising a computer operably coupled to the detector, the computer being programmed to receive fluorescence polarization data from the detector, and compare a level of fluorescence polarization of the first reagent mixture to a level of fluorescence of a second reagent mixture which comprises the first reagent, the second reagent and the polyion.
- 57. A kit, comprising:
a volume of a first reagent which comprises a fluorescent label; a volume of a second reagent which reacts with the first reagent to produce a fluorescent product having a substantially different charge from the first reagent; a volume of a polyion; and instructions for:
determining a level of fluorescence polarization of the first reagent; mixing the first reagent, the second reagent and the polyion in a first mixture; determining a level of fluorescence polarization of the first mixture; and comparing the level fluorescence polarization of the first reagent to the level of fluorescence polarization of the first mixture.
- 58. The kit of claim 57, further comprising a fluid receptacle.
- 59. The kit of claim 58, wherein the fluid receptacle comprises at least a first well in a multiwell plate.
- 60. The kit of claim 57, wherein the fluid receptacle comprises a body structure having at least a first channel disposed therein.
- 61. The kit of claim 60, wherein the fluid receptacle comprises a body structure having a plurality of intersecting channels disposed therein.
- 62. In an assay system for quantifying a reaction parameter which comprises providing a first reagent mixture, which comprises a first reagent having a fluorescent label, introducing a second reagent into the first reagent mixture to produce a second reagent mixture, the second reagent reacting with the first reagent to produce a fluorescently labeled product having a substantially different charge than the first reagent, and introducing a polyion into at least one of the first and second reagent mixtures, a computer implemented process, comprising the steps of:
determining a first level of fluorescence polarization of the first reagent mixture; determining a second level of fluorescence polarization of the second reagent mixture; and comparing the first and second levels of fluorescent polarization; and calculating the reaction parameter.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent application Ser. No. 09/316,447, filed May 21, 1999, and also claims priority to Provisional Patent Application Nos. 60/139,562, filed Jun. 16, 1999 and 60/156,366, filed Sep. 28, 1999. The disclosure of each of these references is incorporated by reference in its entirety for all purposes.
Provisional Applications (2)
|
Number |
Date |
Country |
|
60139562 |
Jun 1999 |
US |
|
60156366 |
Sep 1999 |
US |
Continuation in Parts (1)
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Number |
Date |
Country |
Parent |
09316447 |
May 1999 |
US |
Child |
09569193 |
May 2000 |
US |