Assay methods and system
DCFirst Claim
1. A method of detecting a reaction, comprising:
- providing a first mixture which comprises a first reagent having a fluorescent label;
introducing a second reagent into the first mixture to produce a second mixture, the second reagent reacting with the first reagent to produce a fluorescently labeled product having a substantially different charge than the first reagent;
introducing a polyionic polymer into at least one of the first and second mixtures; and
comparing a fluorescent polarization in the second mixture relative to the first mixture.
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Abstract
Methods, systems, kits for carrying out a wide variety of different assays that comprise providing a first reagent mixture which comprises a first reagent having a fluorescent label. A second reagent is introduced into the first reagent mixture to produce a second reagent mixture, where the second reagent reacts with the first reagent to produce a fluorescently labeled product having a substantially different charge than the first reagent. A polygon is introduced into at least one of the first and second reagent mixtures, and the fluorescent polarization in the second reagent mixture relative to the first reagent mixture is determined, this fluorescent polarization being indicative of the rate or extent of the reaction.
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Citations
56 Claims
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1. A method of detecting a reaction, comprising:
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providing a first mixture which comprises a first reagent having a fluorescent label;
introducing a second reagent into the first mixture to produce a second mixture, the second reagent reacting with the first reagent to produce a fluorescently labeled product having a substantially different charge than the first reagent;
introducing a polyionic polymer into at least one of the first and second mixtures; and
comparing a fluorescent polarization in the second mixture relative to the first mixture. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 51)
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 mixture and measuring a level of fluorescence polarization from the second 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 mixture.
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21. The method of claim 20, further comprising introducing a test compound into at least one of the first reagent and second 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.
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22. The method of claim 21, wherein the second mixture is transported along a first channel, and the test compound is introduced into the first channel.
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23. The method of claim 22, 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.
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24. The method of claim 21, wherein a plurality of different test compounds are separately introduced into separate volumes of the at least one of the first reagent and second mixture.
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25. The method of claim 21, wherein the second mixture is transported along a first channel, and the plurality of test compounds are introduced into the first channel as discrete fluid regions.
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51. The method of claim 1, wherein the first reagent mixture is provided and the second reagent is introduced into a well of a multiwell plate.
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26. A method of identifying the presence of a subsequence of nucleotides in a target nucleic acid, the method comprising:
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contacting the target nucleic acid sequence with an uncharged, 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 polyionic polymer; and
comparing a level of fluorescence polarization of the first reaction mixture in the presence of the polyionic polymer to a level of fluorescence polarization of the uncharged 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. - View Dependent Claims (27, 28, 29, 30, 31, 52)
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32. A method of detecting the phosphorylation of a phosphorylatable compound, comprising:
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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 to produce a phosphorylated product;
contacting the first mixture with a polyionic polymer;
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. - View Dependent Claims (33, 34, 35, 36, 37, 38, 39)
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40. A method of detecting the phosphorylation of a phosphorylatable compound, comprising:
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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 he group consisting of 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.
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41. An assay system, comprising:
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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 polyionic polymer; 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. - View Dependent Claims (42, 43, 44, 45, 53, 54, 55)
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46. An assay system, comprising:
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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 polyionic polymer;
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. - View Dependent Claims (47, 48, 49)
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50. A method for quantifying a reaction parameter comprising:
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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;
introducing a polyionic polymer into at least one of the first and second reagent mixtures; and
initiating a computer implemented process that comprises 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;
comparing the first and second levels of fluorescent polarization; and
calculating the reaction parameter.
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56. The system of claim 58, wherein the multiwell plate comprises 1536 wells.
Specification