Fibrinogen adsorption on mica studied by AFM and in situ streaming potential measurements

Adsorption of fibrinogen from aqueous solutions on mica was studied using AFM and in situ streaming potential measurements. In the first stage, bulk physicochemical properties of fibrinogen and the mica substrate were characterized for various ionic strength and pH. The zeta potential and number of uncompensated (electrokinetic) charges on the protein surfaces were determined from microelectrophoretic measurements. Analogously, using streaming potential measurements, the electrokinetic charge density of mica was determined for pH range 3-10 and the NaCl background electrolyte concentration of 10(-3) and 10(-2) M. Next, the kinetics of fibrinogen adsorption at pH 3.5 and 7.4 in the diffusion cell was studied using a direct AFM determination of the number of molecules per unit area of the mica substrate. Then, streaming potential measurements were performed to determine the apparent zeta potential of fibrinogen-covered mica for different pH and ionic strength in terms of its surface concentration. A quantitative interpretation of these streaming potential measurements was achieved in terms of the theoretical model postulating a side-on adsorption of fibrinogen molecules as discrete particles. On the basis of these results, the maximum coverage of fibrinogen Θ close to 0.29 was predicted, in accordance with previous theoretical predictions. It was also suggested that anomalous adsorption for pH 7.4, where fibrinogen and the mica substrate were both negatively charged, can be explained in terms of a heterogeneous charge distribution on fibrinogen molecules. It was estimated that the positive charge was 12 e (for NaCl concentration of 10(-2) M and pH 7.4) compared with the net charge of fibrinogen at this pH, equal to -21 e. Results obtained in this work proved that the coverage of fibrinogen can be quantitatively determined using the streaming potential method, especially for Θ < 0.2, where other experimental methods become less accurate.