DUAL-COLOR FLUORESCENCE CROSS-CORRELATION SPECTROSCOPY FOR MULTICOMPONENT DIFFUSIONAL ANALYSIS IN SOLUTION

The present paper describes a new experimental scheme for following di ffusion and chemical reaction systems of fluorescently labeled molecul es in the nanomolar concentration range by fluorescence correlation an alysis. In the dual-color fluorescence cross-correlation spectroscopy provided here, the concentration and diffusion characteristics of two fluorescent species in solution as well as their reaction product can be followed in parallel. By using two differently labeled reaction par tners, the selectivity to investigate the temporal evolution of reacti on product is significantly increased compared to ordinary one-color f luorescence autocorrelation systems. Here we develop the theoretical a nd experimental basis for carrying out measurements in a confocal dual -beam fluorescence correlation spectroscopy setup and discuss conditio ns that are favorable for cross-correlation analysis. The measurement principle is explained for carrying out DNA-DNA renaturation kinetics with two differently labeled complementary strands. The concentration of the reaction product can be directly determined from the cross-corr elation amplitude.