The multifrequency phase and modulation (MPM) technique is used in concert with total internal reflection fluorescence (TIRF) to probe <i>in situ</i> the distribution of free and analyte-bound sites of a fluorescently labeled, silica-immobilized <i>F</i><sub>(ab')</sub> antibody fragment. Using this new technique, one is able to recover the affinity constant (<i>K</i><sub><i>f</i></sub>) for the immobilized antibody, determine the effects of storage time on <i>K</i><sub><i>f</i></sub> 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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