Total internal reflection fluorescence: a technique for examining interactions of macromolecules with solid surfaces

Abstract

Total internal reflection fluorescence (TIRF) is a technique that may be used to examine the interaction of macromolecules with solid surfaces. In this report, a TIRF apparatus and its application to the adsorption of proteins from flowing solutions on polymer films are described. Bovine serum albumin (BSA)/crosslinked polydimethylsiloxane (silicone rubber) is the protein/surface pair considered herein. To relate TIRF fluorescence signals to actual surface concentrations, a calibration technique using BSA labeled with both tritium and fluorescein isothiocyanate (FITC) was developed. Unambiguous calibration results require a linear relationship between observed fluorescence intensity and protein surface concentration, reproducible evanescent field intensities, and constant fluorescence quantum efficiencies. The contribution of bulk solution fluorescence to the total TIRF signal must be ascertained to ensure accurate quantitative interpretation of TIRF data. There are two sources of solution fluorescence: one arising from excitation of fluorescent solution protein by scattered light, the second from solution protein within the evanescent field. Solution protein within the evanescent field is distinguished from reversibly adsorbed proteins by comparing its exchange dynamics with that of nonadsorbing FITC-dextran together with the predictions of a hydrodynamic model. By removing the solution protein contributions to the total TIRF signal, surface-adsorbed protein fluorescence, and thus the amount of protein adsorbed, can be obtained. Bulk solution contribution to the total TIRF signal increases with solution concentration. Experiments were conducted at solution concentrations of 2 and 200 mg%, both corresponding to an equilibrium surface concentration of 0.14 μg/cm 2, the saturated surface concentrations for BSA adsorbing on silicone rubber. Proteins in the 2 mg% solution contributed negligibly to the total fluorescence, while fluorescence from the 200 mg% solution constituted a significant fraction of the total TIRF signal.