## 3 Results and Discussion
### Determination of the S-Layer Ion Permeability by Cyclic Voltammetry  
Cyclic Voltammetry (CV), the measurement of the running electrical current intensity as the potential varies cyclically between two extreme values, is an electrochemical tool of high usefulness whenever the packing of a layer has to be determined. In our particular case, this technique was  
Figure 4: Fe-SbpA crystalline film electrical responsiveness. EQCDD measurements allow monitoring the real time \(\Delta\)f variation upon potential shifts between oxidation (+1 V) and reduction (\(-1\) V) inducing conditions. A calcium-containing S-layer was used as negative control. 0.1 M HEPES was employed as running buffer. envisaged to complement the observations from the previous section. Indeed, more light might be shed on the different features of S-layers assembled from two types of iron salts, such as chloride or sulphate. Since S-layers act, by definition, as protective outermost membranes (in bacteria) and so does their technological application meant to be for, characterization of their ion permeability turns certainly relevant (among others). Figure 5 shows the respective Cyclic Voltammograms (three cycles each) recorded from both Fe\({}^{2+}\)-containing SbpA films, when sweeping the +1/-1 V range. The octanethiol-coated gold system represents the current intensity measured in the absence of a protective layer. At first glimpse, the current intensity damping caused by the presence of any of the S-layers under study is detected straightforward. Such behaviour confirms the great isolating capability of these films in spite of their nanometric thickness. Obviously, the presence of multiple pores all over the structure (at the central position in between the four subunits forming the square) enables a higher transfer of sufficiently small ions than, for instance, a homogeneous polymer film [31; 32]. A more detailed observation of the plots, as offered by the insets on top, allows a clear distinction between films built in the presence of different counterions. Hence, SbpA crystalline film assembled from FeCl\({}_{2}\) attenuates much more effectively the passage of ions from the bulk (around a 50% lower current at the extreme potentials), compared to its sulphate-based homologous structure. Such a performance totally matches the QCMD/AFM results from Section 3.2, where the S-layer built in the presence FeSO\({}_{4}\) already appeared as a more loosely packed film. Since in their natural environment S-layers usually appear attached to an underlying secondary cell-wall polymer (SCWP) film, which contributes to their high stability (because of specific protein-carbohydrate interactions) and homogeneity, it was decided to employ such a biopolymer as hosting substrate for the recrystallization of SbpA with iron salts. The enhancement induced in  
Figure 5: Cyclic Voltammetry measurements (\(-1\) to +1 V) reflecting the ion permeability variation of the Fe\({}^{2+}\) containing S-layers, attending to the corresponding counterion (Cl\({}^{-}\), black to grey solid lines versus SO\({}_{4}\)\({}^{2-}\), red to orange open circles). Experiments were performed in 0.1 M HEPES and the octanethiol-gold system (yellow to green solid lines) was employed as signal reference. the final structure by the presence of SCWP has already been reported by our group [22]. Following the abovementioned measuring protocol, the ion permeability of the prepared samples was studied by means of Cyclic voltammetry and the obtained voltammograms (three cycles each) are depicted in Figure 6.  
As main outcome, the presence of an underlying SCWP seems to pair the performance of both SbpA films in terms of ion permeability. The resulting attenuation of the current intensity shown by the iron (II) sulphate is obviously a contribution of not only a better packing of the S-layer (as shown by the topography micrograph and the derived Fourier spectra analysis) but also the presence of the polymer underneath.