The interaction between the antigen (peptide, protein) and the surface is a crucial step in the development of immunoassays. Site-oriented chemical adsorption, obtained by modifying the surface, the antigen or both, provides a strong and stable interaction with the appropiate orientation to favor the immunoreaction. In some techniques to produce recombinant antigens, a hexahistidine peptide is added to the primary sequence of the molecule to facilitate the purification. Since the histidine residues bind with high affinity to metal ions (Ni2+, Cu2+) immobilized on a sorbent surface, they are used to achieve site-oriented adsorption of peptides and proteins. The aim of this work is to elucidate in detail the mechanisms of physical and chemical adsorption of (hexa)histidine on Ni2+ modified silica surfaces. The obtained results provide a useful insight to better understand the site-oriented adsorption process and will be used as the starting point to adsorb recombinant peptides on solid surfaces.
The whole adsorption process was followed by reflectometry that is very suitable to measure on the time scale from seconds to hours. Therefore, with this technique both the first steps of the process (transport and attachment) and the adsorbed amount in steady state conditions were determined. The transport from the solution to the interface and the first molecules-surface attachment were studied at different pH and (hexa)histidine concentrations. After reaching steady state conditions, adsorption isotherms were evaluated at different pH values. Physical and chemical adsorptions were compared by performing experiments on bare and modified silica surfaces and by inducing desorption with surfactants that selectively remove physically adsorbed molecules.
