pH and ionic strength effect on single fibrinogen molecule adsorption on mica studied with AFM

Although several investigations have been reported on the effect of pH or ionic strength on protein adsorption, most of them have been carried out with protein monolayers and not with single molecules. We have used atomic force microscopy to image, in phosphate buffer, single fibrinogen molecules adsorbed on mica and compare the surface coverage at variable pH (7.4, 5.8, 3.5) or ionic strength (15, 150, 500 mM) conditions. The images obtained and the statistical analysis of the surface coverage indicate adsorption enhancement at the IEP of fibrinogen (pH 5.8) and minimum adsorption at pH 3.5. On the other hand, more protein was adsorbed when the salt concentration of the buffer at pH 7.4 was increased from 15 to 150 mM. However, further increase of salt concentration up to 500 mM resulted in decreased adsorption. To confirm the aforementioned results an approaching bare Si3N4 tip was used as an electrostatic analogue to a protein molecule and interaction force curves between it and the substrate were recorded. The results were in consistence with the double layer theory which justifies the screening of electrostatic repulsion as the salt concentration increases.