Fluorescence polarization studies of affinity interactions in capillary electrophoresis

Abstract

Capillary electrophoresis (CE) combined with molecular recognition for ultrasensitive bioanalytical applications often requires the formation of stable complexes between an analyte and its binding partner. Previous studies of binding interactions using CE involve multiple-step titration experiments and are time-consuming. We describe a simple method based on laser-induced fluorescence polarization (LIFP) detection for CE separation, which allows for on-line monitoring of affinity complex formation. Because fluorescence polarization is sensitive to changes in the rotational diffusion arising from molecular association or dissociation, it is capable of providing information on the formation of affinity complexes prior to or during CE separation. Applications of the CE/LIFP method to three binding systems including vancomycin and its antibody, staphylococcal enterotoxin A and its antibody, and trp operator and trp repressor were demonstrated, representing peptide-protein, protein-protein, and DNA-protein interactions. The affinity complexes were readily distinguished from the unbound molecules on the basis of their fluorescence polarization. The relative increase in fluorescence polarization upon complex formation varied with the molecular size of the binding pairs.