Mechanisms of fibrinogen adsorption at the silica substrate determined by QCM-D measurements

Abstract

Adsorption kinetics of fibrinogen at a silica substrate was thoroughly studied in situ using the QCM-D method. Because of low dissipation, the Sauerbrey’s equation was used for calculating the wet mass per unit area (wet coverage of the protein). Measurements were done for various bulk suspension concentrations, flow rates and pHs. These experimental data were compared with the theoretical dry coverage data derived from the solution of the mass transfer equation. In this way, the hydration functions and water factors of fibrinogen monolayers were quantitatively evaluated for various pHs. In the case of pH 7.4 and ionic strength of 0.15M, the hydration function changed from 0.75 to 0.6 for the dry coverage Γd equal to 0 and 4mgm−2, respectively. Interestingly, for pH 7.4 and 4.5 (ionic strength of 10−2M) a minimum of the hydration function appeared at Γd ca. 2mgm−2. Analytical polynomial expressions were formulated for the interpolation of the experimental results. By using the hydration functions, the fibrinogen adsorption/desorption runs derived from QCM-D measurements were converted to the Γd vs. the time relationships. This allowed to precisely determine the maximum coverage that varied between 1.2mgm−2 at pH 3.5 and 4.2mgm−2 at pH 7.4 for ionic strength of 0.15M. These results agree with theoretical modeling and previous experimental data derived by using ellipsometry, OWLS and TIRF. Various fibrinogen adsorption mechanisms were revealed by exploiting the maximum coverage data whose validity was also confirmed by the dissipation vs. the dry mass relationships. Beside significance to basic science, these results enable to develop a robust technique, based on the QCM-D measurements, suitable for precisely determining the dry mass of protein monolayers adsorbed under various physicochemical conditions.