## 3 Results and Discussion
### S_p_u_ Recrystallization in Fe (II): Electrical Control and Real Time Monitoring  
The electrochemical version of a QCMD chamber (EQCM) allows establishing a three-electrode system. Obviously, such a feature turned highly relevant when attempting to exert full control over the oxidation state of the Fe\({}^{2+}\) containing solutions. The use of this module, in addition to its capability of setting an external voltage throughout the SbpA adsorption and recrystallization steps (-1 V in our case, as established by the Fe\({}^{2+}\)/Fe\({}^{3+}\) redox potential), permits the real-time monitoring of the process in terms of kinetics and changes in both adsorbed mass and film viscoelasticity, as defined by variations in frequency (\(\Delta\)f) and dissipation (\(\Delta\)D) factors, respectively. As aforementioned, this second approach of QCMD technique has extensively been employed by our group to follow-up the formation of S-layers with CaCl\({}_{2}\) on top of very diverse substrates [6; 22] and, therefore, it constitutes a perfect tool for comparative purposes between Ca\({}^{2+}\) and Fe\({}^{2+}\) derived films. The resulting \(\Delta\)f versus time and \(\Delta\)D versus time plots of the SbpA recrystallization on hydrophobic-gold, under a controlled potential, are depicted in Figure 2.  
Figure 2: EQCMD measurements showing \(\Delta\)f (**a**) and \(\Delta\)D (**b**) time evolution for the recrystallization of SbpA protein in the presence of Fe\({}^{2+}\) (chloride and sulphate) and of Ca\({}^{2+}\) divalent ions. Injection was performed on octanethiol-modified gold chips and under controlled bulk potential (–1 V). The black arrow (above) indicates the exact protein injection time. The inset highlights the kinetics of the diffusion/adsorption process. The arrow below points out the appearance of the characteristic structure transition peak observed in \(\Delta\)D.  
The formation of the SbpA layer is observed to exhibit certain differences between the Ca\({}^{2+}\) film acting as reference and those generated from Fe (II) salts, even though the general trend seems to be rather similar. Overall, the most relevant outcomes would be the following: (1) the total mass adsorbed differs depending on the divalent ion employed and also the counterion (Cl\({}^{-}\) vs. SO\({}_{4}\)\({}^{2-}\)) turns crucial for the final performance recorded, (2) dissipation values show in all the three cases the transition state-indicating peak towards a more rigid film, which connects to first crystal formation and (3) the kinetics of the process are slowed down in the presence of Fe\({}^{2+}\) salts. Attending to the combined analysis of time-dependent frequency and dissipation factors (see also Appendix A, Figure A3), films produced from FeCl\({}_{2}\) yielded lower adsorption in wet mass at the end of the binding process, when compared to their SO\({}_{4}\)\({}^{2-}\)-containing partners (ca. 70%). On the contrary, the former system underwent a surprisingly faster structural arrangement, as derived from the continuous post-peak drop in dissipation down to 1 \(\times\) 10\({}^{6}\) at the end of the incubation time, which resembles the properties of Ca\({}^{2+}\)/SbpA layers. In turn, the transition peak for S-layers derived from FeSO\({}_{4}\) led to a much smoother drop in viscosity as incubation went by. At this point and since the employed divalent ion is the same, the role played by either chloride or sulphate is undoubtedly of main relevance. Thus, ferrous sulphate (FeSO\({}_{4}\)) is known to easily dissociate in the individual molecules and to form water-complexes when dissolved in water. This means that strong water coordination takes place, which might explain the larger mass deposition sensed by QCMD. Furthermore, SO\({}_{4}\)\({}^{2-}\)appears as one of the most _kosmotropic_ ions, attending to the Hofmeister series [29; 30], which reinforces the high hydration theory.