Kinetic investigations by fluorescence correlation spectroscopy: the analytical and diagnostic potential of diffusion studies

Abstract

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 are based on changes in the diffusion characteristics of fluorescenctly labeled complementary strands of nucleic acids when they associate. However, the principle of the measurement can be extended to many different reactions with characteristic association times between several minutes up to several hours. If the reaction significantly affects the diffusion constants of at least one partner, single-color auto-correlation analysis is sufficient to extract kinetic parameters. If the observed binding process has only a moderate effect on diffusion coefficients, the detection selectivity and sensitivity can be improved by dual-color cross-correlation analysis. Finally, we show that diffusional analysis on the single-molecule level even opens up diagnostic applications, such as the detection of minute amounts of infectious agents like HIV-1 viruses in blood.