Fluorescence polarization assay

Abstract

The present invention relates to methods for detecting the presence of one or more analytes of interest in a sample by measuring changes in fluorescence anisotropy as a result of binding of the analytes to specific aptamers. The aptamers are immobilized on a solid support and may be in the form of an array.

1 . A method for detecting an analyte in a sample comprising: 
 (a) contacting a sample with a fluorophore-labeled aptamer bound to a solid support;    (b) directly illuminating the aptamer with polarized light whereby the direct illumination of the fluorophore directly excites the fluorophore;    (c) measuring the fluorescence anisotropy of the fluorophore; and    (d) identifying a presence or amount of the analyte when said fluorescence anisotropy measurement is greater than an anisotropy measurement obtained in the absence of the analyte.    
     
     
         2 . The method of  claim 1  wherein the aptamer comprises between about 10 and about 100 nucleotides.  
     
     
         3 . The method of  claim 1  wherein the aptamer is labeled with a fluorophore selected from the group consisting of fluorescein derivatives, eosin derivatives, coumarin derivatives, and rhodamine derivatives.  
     
     
         4 . The method of  claim 3  wherein the aptamer is labeled with carboxyfluorescein (FAM).  
     
     
         5 . The method of  claim 1  wherein the aptamer is part of an array of aptamers.  
     
     
         6 . The method of  claim 5  wherein the array comprises two or more addressable locations.  
     
     
         7 . The method of  claim 6  wherein each addressable location comprises a single type of aptamer.  
     
     
         8 . The method of  claim 6  wherein each addressable location comprises multiple types of aptamers.  
     
     
         9 . The method of  claim 8  wherein each type of aptamer is labeled with a fluorophore with unique spectral characteristics.  
     
     
         10 . The method of  claim 1  wherein the polarized light is laser light.  
     
     
         11 . The method of  claim 1  wherein the analyte is associated with a disease or disorder.  
     
     
         12 . The method of  claim 1  wherein the sample is obtained from a patient suspected of suffering from a disease or disorder.  
     
     
         13 . The method of  claim 1  wherein the analyte is a protein.  
     
     
         14 . The method of  claim 1  wherein the analyte is a metabolite.  
     
     
         15 . The method of  claim 1  wherein the sample is from a human patient and the analyte is associated with a disease or disorder.