Probing biosensor interfaces by multifrequency phase and modulation total internal reflection fluorescence (MPM-TIRF)

The multifrequency phase and modulation (MPM) technique is used in concert with total internal reflection fluorescence (TIRF) to probe in situ the distribution of free and analyte-bound sites of a fluorescently labeled, silica-immobilized F_(ab) antibody fragment. Using this new technique, one is able to recover the affinity constant (K_f) for the immobilized antibody, determine the effects of storage time on K i and the excitedstate fluorescence decay parameters, and follow time-dependent changes in antibody conformation at the silica substrata. This new technique adds a powerful tool to the study of biosensor interfaces.