KINETIC INVESTIGATIONS BY FLUORESCENCE CORRELATION SPECTROSCOPY - THEANALYTICAL AND DIAGNOSTIC POTENTIAL OF DIFFUSION STUDIES

This review demonstrates the large analytical and diagnostic potential of fluorescence correlation spectroscopy applied to freely diffusing biomolecules in solution. All applications discussed here in detail ar e based on changes in the diffusion characteristics of fluorescenctly labeled complementary strands of nucleic acids when they associate. Ho wever, the principle of the measurement can be extended to many differ ent reactions with characteristic association times between several mi nutes up to several hours. If the reaction significantly affects the d iffusion constants of at least one partner, single-color autocorrelati on analysis is sufficient to extract kinetic parameters. If the observ ed binding process has only a moderate effect on diffusion coefficient s, the detection selectivity and sensitivity can be improved by dual-c olor cross-correlation analysis. Finally, we show that diffusional ana lysis on the single-molecule level even opens up diagnostic applicatio ns, such as the detection of minute amounts of infectious agents like HIV-1 viruses in blood. (C) 1997 Published by Elsevier Science B.V.