Assay methods and system
AssigneeCaliper Technologies Corp.
InventorTheo T. Nikiforov
Life of this patent
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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.
Claims56
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.
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 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.
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 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.
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 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.
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 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.
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 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.
A method for quantifying a reaction parameter 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 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.
The system of claim 58, wherein the multiwell plate comprises 1536 wells.
Dependencies are inferred from the claim text.