Evanescent Detection of Adsorbed Protein Concentration-Distance Profiles: Fit of Simple Models to Variable-Angle Total Internal Reflection Fluorescence Data

Total internal reflection fluorescence spectroscopy (TIRF) is an established technique for following the course of interfacial reactions. Theoretically, by gathering TIRF data as a function of observation angle, one can obtain the density of fluorophores with respect to distance away from a solid/liquid interface. In order that the practical application of the theory might be explored, variable observation angle data from solutions of fluorescein and from adsorbed layers of fluorescein isothio-cyanate labeled immunoglobulin have been analyzed in terms of simple concentration-distance profiles. In all cases the general shape of the data curves was found to conform to the theoretical expectation. Layer thickness determinations varied over a range of 20 to 100 nm, with concentrations in the layer ranging from 12 to 61 mg/mL. The theoretical background, sources of error, and system improvements are also discussed.