Analytical Derivatization Reactions Based on Changes in Fluorescence Polarization: A Worthwhile Idea?

Abstract

When a fluorophor is coupled to an analyte it will necessarily increase in size and, therefore, will depolarize fluorescence less rapidly than fluorophor by itself. Thus, it is possible to distinguish analyte fluorescence from excess reagent fluorescence on the basis of polarization even if the analytical reaction does not otherwise cause a change in fluorescence behavior. This would permit the use of sensitive fluorogenic reagents. However, sensitivity is lost when analyte is distinguished from excess reagent on the basis of polarization because the measured analytical signal is a relatively small difference between large signals. We have calculated detection limits for analyte resolved from excess reagent on the basis of fluorescence polarization relative to the detection limit for fluorophor by itself. Detection limits are calculated as a function of rotational relaxation times for analyte and excess reagent, intrinsic polarization, excess reagent concentrations, and nature of the noise. It is assumed that coupling the fluorophor to analyte does not affect fluorescence efficiency or wavelength distribution. The calculations serve to define the conditions under which it may be practical to resolve analyte from excess reagent on the basis of polarization.