Chelating Surfaces for Native State Proteins Patterning: The Human Serum Albumin Case

The paper reports a new soft surface functionalization strategy, based on a highly selective ion metal chelation process. The proposed stepwise methodology implies at first the construction of a monolayer of terpyridine-based thiol (Tpy), whose highly packed structuring has been followed in situ by using quartz crystal microbalance (QCM-D) measurements, showing that the monolayers consist of about 2.7 x 10(14) Tpy/cm(2). Then, the tridentate sites of the each Tpy moiety are employed to partially chelate divalent metal ions, providing an effective platform to anchoring proteins by completing the metal ion coordination with an available site on the protein of interest. We report the case study of the application of the process to the HSA immobilization onto various surfaces, including Tpy Fe(II) and Tpy Cu(II) complexes, as well as hydrophilic bare gold substrates and hydrophobic self-assembled Tpy-based monolayers. It is shown that the chelation interaction between Tpy Cu(II) complexes and HSA produces the highest and most robust HSA immobilization, with an adsorbed mass at the steady state of similar to 800 ng/cm(2), with respect to an average adsorption of similar to 350 ng/cm(2) for the other surfaces. Furthermore, Cu(II)-chelated surfaces seem to promote a sort of protein soft landing, preventing the ubiquitous surface-induced major unfolding and transmitting an orientation information to the protein, owing to the highly specific symmetry coordination of the Tpy Cu(II) protein complex. Indeed, the interaction with a specific monoclonal antiboby (anti-HSA) indicated the lack of a significant protein denaturation, while a massive reorientation/denaturation process was found for all the remaining surfaces, including the Tpy Fe(II) complex. Finally, the metal-ion-dependent HSA immobilization selectivity has been exploited to obtain micropatterned surfaces, based on the strikingly different strength of interaction and stability observed for Fe(II) and Cu(II) complexes.