Real-time observation of plasma protein film formation on well-defined surfaces with scanning force microscopy

We report here the results of a scanning force microscopic (SFM) investigation into the structure of adsorbed bovine fibrinogen films on well-defined surfaces. SFM images show that a monolayer of fibrinogen assembles on both highly oriented pyrolytic graphite (HOPG) and mica. We find, however, that the film morphology varies as a function of the substrate. To unravel the nature of the observed differences, we tracked the growth of bovine fibrinogen layers at both surfaces in real-time. Consistent with the differences observed in the final film structure, we find that film formation on HOPG and mica proceeds through distinct mechanisms. A network is observed during fibrinogen film formation on HOPG, implying that protein-protein interactions are involved in the growth mechanism and likely govern the final film structure. The growth mechanism on mica proceeds by the homogeneous increase in the number of nuclei across the surface and gives no indication of significant intermolecular interactions. Surfactant elution studies show that films on HOPG are more tightly bound than those on mica, as predicted on the basis of the observed growth mechanisms. An adsorption mechanism consistent with our observations and based on the known structure of fibrinogen is proposed.