Unraveling diffusion-controlled bimolecular reactions using fluorescence correlation spectroscopy

Diffusion-controlled bimolecular reactions play a central role in solution-phase chemistry, but it remains challenging to characterize the interplay of diffusive molecular encounters and the criteria that govern successful reactions. In molecular biophysics, fluorescence correlation spectroscopy (FCS) has been widely used to study the dynamics of diffusion-controlled reactions, but little work has been done to extend this technique to the study of small-molecule chemistry. Here, we describe design principles for studying diffusion-controlled bimolecular reactions with FCS and characterize a model system that meets a series of experimental criteria. The reversible hydrogen-bond mediated binding of coumarin 153 with Schreiner's thiourea catalyst is investigated in a variety of organic solvents, and a detailed kinetic analysis is presented in tetrachloroethylene. By extending conventional FCS formalism to account for the variation of diffusion coefficients and molecular brightness among reactants and products, we are able to isolate translational diffusion coefficients, association and dissociation rate constants, and equilibrium constants from a single FCS measurement. These advances illustrate how FCS and related single-molecule fluorescence techniques can be used to probe coupled diffusive and reactive dynamics in small-molecule solution-phase chemistry.