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| - Extending the Reach of Immunoassays to OpticallyDense Specimens by Using Two-Photon ExcitedFluorescence Polarization
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| - Fluorescence anisotropy/polarization measurements represent a powerful tool for quantifying biomolecule/ligandcomplexation. These types of measurements are also atthe heart of a wide variety of commercial homogeneousfluoroimmunoassays. In this note, we demonstrate thepower of two-photon excited fluorescence anisotropy (2-PEFA) measurements as a tool for quantifying hapten/antibody association in the presence of a strongly absorbing, nonfluorescent dye. The results of these experimentsshow that 2-PEFA measurements are intrinsically moresensitive when compared to traditional one-photon excitedfluorescence anisotropy (1-PEFA) strategies and 2-PEFA-based measurements allow one to perform accuratehapten/antibody binding measurements in strongly absorbing samples directly under conditions where 1-PEFAmeasurements fail completely. Overall, the 2-PEFA approach offers significant advantages when compared totraditional 1-PEFA methods especially in strongly absorbing samples. 2-PEFA also opens the door to perform morerapid and reliable polarization/anisotropy-based measurements with minimal sample preparation.
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