## 3 Results and Discussion
### Potentiodynamic Deposition
The double potential step experiments shown in Figure 6 clearly evidenced that both the chronoamperometric curves, the positive ones due to Pt oxide stripping and the negative ones due to Pt oxide formation, increased in terms of current values and time extension upon increasing the final potential \(E_{\rm f}\). Nevertheless, this concurrent increase was biased for the high values. Considering that the areas under these curves correspond to the charges due to reduction (positive curves) and oxidation processes (negative curves), plots of these charge values, as shown in Figure 7, better highlight this occurrence: in fact, the net charge due to the oxidation processes increased to a greater extent than that due to the reduction processes upon increasing the final potential starting from values higher than \(0.7\) V vs. Ag/AgCl. Since this charge hysteresis means that the produced Pt oxide persists onto the electrode surface (thus catalyzing water discharge) even after the reduction-stripping step, care should be taken in choosing the right timing of the potential pulse sequence: in fact, \(t_{1}\) should be sufficiently higher than \(t_{2}\), and the latter should be significantly lower to assure the almost complete removal of Pt oxide formed during the application of \(E_{\rm f}\) to induce the electrophoretic movement of proteins; on the other hand, the value of the latter potential should be sufficiently high to promote efficient electrophoresis in the time scale of the deposition experiment while minimizing Pt oxide formation and hence gas evolution. Further experiments performed using pulsed voltammetry permitted us to optimize the potential and timing parameters of the potential pulse sequence shown in Scheme 2. Using an \(E_{\text{i}}\) value of \(-0.1\) V vs. Ag/AgCl, as previously optimized, it was found that an \(E_{\text{f}}\) value of \(1.8\) V was a good compromise between efficient and quick protein deposition and low dioxygen evolution. Regarding the timing of the potential pulse sequence, \(t_{2}\) was reduced at 12 ms (a value much lower than that used in galvanodynamic deposition), while \(t_{1}\) was set to 5 s; indeed, \(t_{1}\) could be set even higher, up to 20 s, assuring the complete absence of any side effects, but higher \(t_{1}\) values were not adequate for promoting sufficient electrophoresis/co-crosslinking in the time scale of the experiment in order to produce a deposit that had good mechanical properties and was firmly attached to the deposition electrode. Figure 6: Typical chronoamperometric curves resulting from double potential step experiments at the Pt deposition electrode in phosphate buffer a pyH \(7\), \(I\) 0.1 M. \(E_{\text{i}}\) was \(-0.1\) V vs. Ag/AgCl; \(t_{2}\) is 1 s and \(t_{1}\) is 5 s in both panels; \(E_{\text{f}}\) was in the range \(0.1\div 1\) V vs. Ag/AgCl (left panel) and in the range of \(1.3\div 2\) V vs. Ag/AgCl (right panel). Outer curves in both panels correspond to higher voltage values. Figure 7: Charge due to the oxidation processes (upper curve) and reduction processes (lower curve) versus the final potential \(E_{\text{f}}\) (V vs. Ag/AgCl) as obtained by integrating the areas of the chronoamperometric curves in Figure 6. Each measurement refers to 5 replicates. In these optimized conditions, the growth of the proteic layer somewhat resembled that already described for the galvanodynamic deposition technique. Even using this potentiodynamic approach, after just a few seconds, an opalescent foamy-like layer was evident onto the electrode surface; on the contrary, after less than 1 min and more, a visual inspection showed a nearly \"double\" proteic layer, with the inner layer much more compact and adherent to the electrode, while the outer layer was foamy and easily detachable via a simple water washing procedure. Further insights were gained via an EQCM study. Figure 8 shows a typical EQCM profile of the growth of the proteic layer using this approach.