Total Internal Reflection with Fluorescence Correlation Spectroscopy: Nonfluorescent Competitors

Abstract

Total internal reflection with fluorescence correlation spectroscopy is a method for measuring the surface association/dissociation rate constants and absolute densities of fluorescent molecules at the interface of a planar substrate and solution. This method can also report the apparent diffusion coefficient and absolute concentration of fluorescent molecules very close to the surface. Theoretical expressions for the fluorescence fluctuation autocorrelation function when both surface association/dissociation kinetics and diffusion through the evanescent wave, in solution, contribute to the fluorescence fluctuations have been published previously. In the work described here, the nature of the autocorrelation function when both surface association/dissociation kinetics and diffusion through the evanescent wave contribute to the fluorescence fluctuations, and when fluorescent and nonfluorescent molecules compete for surface binding sites, is described. The autocorrelation function depends in general on the kinetic association and dissociation rate constants of the fluorescent and nonfluorescent molecules, the surface site density, the concentrations of fluorescent and nonfluorescent molecules in solution, the solution diffusion coefficients of the two chemical species, the depth of the evanescent field, and the size of the observed area on the surface. Both general and approximate expressions are presented.

Size Dependence of Protein Diffusion Very Close to Membrane Surfaces: Measurement by Total Internal Reflection with Fluorescence Correlation Spectroscopy

“…[18][19][20] A number of experimental TIR-FCS studies have also been carried out. Initially, TIR-FCS was used to examine the reversible kinetics of tetramethylrhodamine-labeled immunoglobulin and insulin interacting with fused silica coated with serum albumin 21 and of rhodamine 6G interacting with C-18-modified silica surfaces.…”

Fluorescence

“…In some applications, an objective lens with a large numerical aperture acts as the medium with the higher refractive index, and also serves to collect fluorescence emitted by the sample. Excitation schemes utilizing total internal reflection have been used to measure binding of fluorescent ligands to protein adsorbed on a glass surface [205][206][207][208], and to probe the orientation and motions of molecules in planar lipid bilayers deposited on such a surface [164,165,209].…”

Protein–surfactant interactions at hydrophobic interfaces studied with total internal reflection fluorescence correlation spectroscopy (TIR-FCS)

“…The measured autocorrelation functions (ACFs) were fitted to a model based on the theoretical description of surface interaction kinetics and axial diffusion in the evanescent field by Thompson and co-workers [33,34]. Triplet state kinetics and transversal diffusion through the laterally restricted detection volume were included in the model [35,36].…”

“…(iii) When the solution also contains a concentration of nonfluorescent molecules A n , a competition for binding sites will take place, forming densities of fluorescent C f and nonfluorescent C n surface bound molecules. The reaction mechanism for the later case can be written as A n + B C n and the fraction of unoccupied binding sites can be written as β = B/(B + C f + C n ), which gives [24,29,34,37] (4)…”