Abstract
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 (Kf) for the immobilized antibody, determine the effects of storage time on Kf and the excited-state 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.
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