The principle of FP centers on the fact that, plane polarized excitation light would be re-emitted by a fluorophore in the same plane as the excitation plane, if the fluorophore remained stationary during its fluorescence lifetime. However, molecular rotation and Brownian motion cause the emission to occur out of the apparent initial plane of polarization.

Larger molecules or aggregates of ligands and their specific receptors (e.g., antigen-antibody complexes with an attached fluorophore) rotate slower in solution than the individual components, and therefore exhibit a greater degree of emission “polarization” or less “randomization”. Unbound small molecules (free ligands or analytes of interest) labeled with a fluorophore rotate faster and show a more randomized, less polarized, fluorescence emission. The FP analysis principle works for many types of binding assays including aptamer assays, PCR amplification and of course immunoassays if one fluorescently labels ligands, probes or primers and looks at the bound versus unbound intensity ratio.

Mathematically, FP is defined as the ratio of fluorescence intensity (I) parallel to the incident excitation plane minus the intensity of the perpendicular plane divided by the sum of these two intensities as expressed in Equation 1.

Equation 1: FP = (I par allel - I perpendicular) / (I parallel + I perpendicular)

With some simple mathematical derivations, FP can also be shown to be proportional to the ratio of fluorescence intensity (I) of bound to unbound analytes in immunoassays or hybridized versus unhybridized fluorophore-labeled nucleic acid (DNA or RNA) probes in Equation 2, where k1 and k2 are constants associated with a given type of assay.

Equation 2: FP = k1(Ibound / Iunbound), or P = k2(Ihybridized / Inonhybridized probes)

Another key factor in utilizing FP fully is consideration of the fluorophores “polarizability.” In order to be “polarizable,” a fluorophore must possess a lifetime sufficient for some molecular rotation to occur before the fluorophore re-emits the light energy it has absorbed. It is preferable to have a lifetime on the order of 4-5 nsec or even microseconds in the case of some coordination complexes. The longer lifetime fluorophores allow greater time for rotating dyes to emit and reveal their change in position. If the dye molecules emit too quickly, they may not be perceived to have rotated at all.

Principle of Fluorescence Polarization (FP) as it applies to a competitive immunoassay for small molecules. This displacement or competitive format has been popularized by Abbott Laboratories (Chicago) for many clinical diagnostic assays used on their TDx clinical analyzers. FP has also been adapted to detect and quantitative Aptamer assays

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