MECHANISTIC STUDIES OF POSTCAPILLARY AFFINITY DETECTION FOR CAPILLARY-ZONE-ELECTROPHORESIS BASED ON THE BIOTIN-STREPTAVIDIN SYSTEM

Postcapillary affinity detection is developed to analyze structurally related compounds following capillary zone electrophoresis separation. In this work, biotin and biotin derivatives are resolved by capillary zone electrophoresis and selectively identified on the basis of their cross-reactivity with streptavidin-fluorescein isothiocyanate, added via a postcapillary reactor. Direct sensing of the affinity reaction i s achieved bq monitoring the resulting fluorescence enhancement of str eptavidin-fluorescein isothiocyanate upon biotin binding. The effects of postcapillary reactor parameters, including capillary gap distance, dilution ratio, and reaction distance, on peak area and peak efficien cy of the biotin-streptavidin complex are investigated. A reaction kin etic model is presented for evaluating the dynamic range and detection limits of postcapillary affinity detection as a function of streptavi din concentration in the postcapillary reactor. The detection limit fo r postcapillary affinity detection of biotin is on the order of 3 nM, or 4 fg.