Adsorption of bovine serum albumin on fused silica: Elucidation of protein-protein interactions by single-molecule fluorescence microscopy

The adsorption of bovine serum albumin (BSA) on fused silica at pH 4.7 was studied at the single molecules level by total-internal-reflection fluorescence microscopy. This pH value was the isoelectric point of BSA. At low [BSA] of 20 pM, protein molecules adsorbed as monomers. At intermediate [BSA] of 500 pM, protein molecules adsorbed as clusters of about five monomers on average. Both monomers and clusters had adsorption rate coefficients of the order 10^-7 m s^-1 and desorption rate coefficients of about 2 × 10^-2 s^-1. The respective steady-state coverage was about 10× higher than that at neutral pH, presumably because of the more favorable BSA-silica electrostatics. At pH 4.7 and with [BSA] higher than 100 nM, adsorption begot further adsorption to produce nonlinear isotherms. The coverage at 1 μM BSA was 2.5× that of the linearly extrapolated coverage. This suggests that at pH 4.7, solute-adsorbate affinity was the dominant factor that explains the enhanced adsorption observed in ensemble measurements.