Monitoring Non-Specific Adsorption at Solid-Liquid Interfaces by Supercritical Angle Fluorescence Microscopy

Abstract

Supercritical angle fluorescence (SAF) microscopy is a novel imaging tool based on the use of distance-dependent fluorophore emission patterns to provide accurate locations of fluorophores relative to a surface. This technique has been used extensively to construct accurate cellular images and to detect surface phenomena in a static environment. However, the capability of SAF microscopy in monitoring dynamic surface phenomena and changes in millisecond intervals is underexplored. Here we report on a hardware add-on for a conventional inverted microscope coupled with a post-processing Python module that extends the capability of SAF microscopy to monitor dynamic surface phenomena thereby greatly expanding the range of potential applications of this tool. We first assessed the performance of the system by probing the specific binding of biotin-fluorescein conjugates to a neutravidin-coated cover glass in the presence of non-binding fluorescein. The SAF emission was observed to increase with the quantity of bound fluorophore on the cover glass. However, high concentration of unbound fluorophore also contributed to overall SAF emission, leading to over-estimation in surface-bound fluorescence. To expand the applications of SAF in monitoring surface phenomena, we monitored the non-specific surface adsorption of BSA and non-ionic surfactants on a Teflon-AF surface. Solution mixtures of BSA and nine Pluronic/Tetronic surfactants were exposed to a Teflon-AF surface. No significant BSA adsorption was observed in all BSA-surfactant solution mixture with negligible SAF intensity. Finally, we monitored the adsorption dynamics of BSA onto the Teflon-AF surface and observed rapid BSA adsorption on Teflon-AF surface within 10 seconds of addition. The adsorption rate constant (ka) and half-life of BSA adsorption on Teflon-AF were determined to be 0.2093{+/-}0.002 s-1 and 3.312{+/-}0.032 s respectively using a pseudo-first-order adsorption equation.