Potentialities of confocal fluorescence for investigating protein adsorption on mica and in ultrafiltration membranes

In conditions of tangential flow, the confocal fluorescence technique is applied to the determination of the concentration profile of a fluorescent protein in the direction normal to a sheet of mica and to the surface of sulfonated membrane (AN69((R)); acrylonitrile-methallylsulfonate copolymer) as a function of time. The penetration of the protein inside the nanoporous membrane with its large area available for adsorption is put into evidence. In addition, by means of the time resolved fluorescence technique in the confocal configuration, we compared the photophysical properties (fluorescence lifetime and correlation time) of the adsorbed labelled protein with those of the same protein in solution: (i) the fluorescence lifetime of the labelled protein in solution (4.1 ns) was found to be unchanged (4.0 ns) when adsorbed on the membrane and significantly smaller (3.2 ns) when adsorbed on mica, suggesting on the latter surface some quenching due to protein-surface or protein-protein interactions; (ii) the correlation time determined on both surfaces, mica and membrane, was found very large (> 300 ns) with respect to the value in solution (33 ns). As the labelling was performed in such a way that the rotational motion of the label corresponded to that of the protein, these results exhibited the low rotational freedom of the protein in its adsorbed state. As a summary this technique should be helpful to characterize the membrane functioning under conditions similar to those encountered in applications, by the determination of both concentration profile as a function of time and rotational mobility. (c) 2006 Elsevier B.V. All rights reserved.