## 3 Results and Discussion
### Galvanodynamic Deposition
Accordingly, the application of a current pulse sequence at low frequency values and low duty cycles constitutes mandatory conditions for the optimal electrophoresis of both proteins and in situ co-crosslinking: a current pulse sequence with \(t_{1}\) 5 s and \(t_{2}\) 0.5 s yielded the best protein deposit at a current density of 2 mA/cm\({}^{2}\). In these optimized conditions, after just a few seconds, an opalescent layer was observed on the electrode surface, without any evidence of gas evolution. After 1 min and more, a yellowish proteic layer was clearly visible; it did not have any gas bubbles  
Figure 3: Chronopotentiometric curves at the deposition electrode due to the application of current pulse sequence (see Scheme 1) for a fixed duty cycle (\(t_{1}\) 5 s, \(t_{2}\) 0.5 s) while varying \(I\): (**a**) \(-\)0.5 mA/cm\({}^{2}\), (**b**) \(-\)2 mA/cm\({}^{2}\), and (**c**) \(-\)5 mA/cm\({}^{2}\). entrapped on it and was quite resistant to being washed with water when the electrode was removed from the bath solution. More insights into the growth of protein deposition and in situ co-crosslinking were gained via an electrochemical quartz crystal microbalance (EQCM) study. Here, a platinum-plated quartz disk electrode was used as the deposition electrode in the electrochemical cell: by measuring the frequency change of the quartz crystal resonator, the growth of the proteic deposit could be followed and eventually quantified [34]. Since EQCM measurements allowed for the measurement of mass changes as low as about 1 ng/cm\({}^{2}\) and are sensitive to viscosity changes, preliminary experiments were performed to exclude significant frequency changes due to protein adsorption at the surface of the deposition electrode and/or changes in the viscosity of the solution due to crosslinking in a bath solution. Using a 1 h time window, the frequency changes in a pure buffer reached a maximal value of about 0.1 kHz, while those observed in the presence of both proteins and a crosslinker but without any current pulse application were on the order of 1 kHz; finally, frequency changes due to protein adsorption, induced in the absence of GLU in the solution and without any current pulse application at the deposition electrode, were about 0.1 kHz as well. These frequency changes were surely lower than those observed for EPD experiments wherein protein deposition and in situ co-crosslinking were expected. In fact, as reported in Figure 5, the frequency changes due to EPD/co-crosslinking were significantly higher, increasing with the time of protein deposition and starting and stopping with the current pulse sequence application as expected for a process under electrostatic control; further, the EQCM profiles were almost reproducible, as curves (b) and (c) in Figure 5 show, since they represented a couple of replicate experiments differing only in their deposition time. Notably, at the end of the deposition time, as shown in Figure 5 for both curves (b) and (c), which depict the cessation of current pulse application, the frequency changes reversed (i.e., the resonance frequency increased). This is likely due to the disruption of the concentration gradient of these proteins, still not crosslinked, near the electrode surface/solution interface, originally produced by their electrophoretic movements towards the deposition electrode; anyway, these frequency changes soon reached a plateau, demonstrating the presence of a deposit on the electrode surface and further detachment from it. Figure 5 also shows that frequency changes varied almost linearly with the low deposition time (case (b)) and in a hyperbolic fashion at longer durations, with an overall frequency change of about 0.3 kHz/min; using the well-known Sauerbrey equation [34], this corresponded to about 3 \(\times\) 10\({}^{-7}\) g/min, so, e.g., in the case (c) of Figure 5, a proteic deposition of about 10 \(\upmu\)g (corresponding to nearly 500 ng/mm\({}^{2}\) and roughly to a one-micron thickness) was observed in less than an hour.