## 4 Conclusions  
EPD is a powerful and promising technique for enzyme immobilization but suffers from concomitating electrochemical processes, which, by inducing gas evolution at the deposition electrode, significantly depress the deposition process and physically damage the deposition layer. Since gas evolution due to water discharge also produces a severe pH drop at the deposit electrode/solution interface, this undesirable coupled process further damages the quality of the enzymatic layer under EPD production. The present paper demonstrates that current or potential pulse techniques can be used to minimize these undesirable effects. After an appropriate study and optimization of the current or potential pulse sequences proposed herein, water discharge and dioxygen evolution were sufficiently minimized to allow for the production of intact proteic deposits. Notably, in the present study, EPD was also used, mainly as a novel tool for performing an electrophoretically assisted, classical enzyme immobilization technique like co-crosslinking, thus permitting the immobilization of the desired protein in situ, i.e., exclusively onto the deposition electrode. This novel approach permitted the coupling of the advantages arising from classical enzyme immobilization techniques, like high activity and stability of their enzymatic products, with those expected from electrochemically assisted deposition, i.e., the precise spatial control of the deposition process and the thickness of the deposit and the ability to coat any complexly shaped substrates as well. Unfortunately, the potentiodynamic approach, as investigated herein, apparently did not permit the control of the mass of the deposit and its thickness, even if the quick formation of a proteic deposit was observed, a feature highly desirable in biosensor fabrication. Since preliminary experiments revealed that mass and thickness could also be controlled by varying some electrical parameters like the final potential, further studies are required to optimize this latter electrical perturbation method. Of course, this original EPD approach, studied here and proposed for the first time, allowed us to fabricate novel amperometric biosensors. Further insights about their production and their analytical performances will be described elsewhere. The following supporting information can be downloaded at [[https://www.mdpi.com/article/10.3390/app14010212/s1](https://www.mdpi.com/article/10.3390/app14010212/s1)]([https://www.mdpi.com/article/10.3390/app14010212/s1](https://www.mdpi.com/article/10.3390/app14010212/s1)). Scheme S1: The influence of ionic strength on (a) the double layer size of the proteins, (b) their electrophoretic retardation, and (c) their interaction for successful crosslinking; Figure S1: Chronopotentiometric curves observed under EPD galvanostatic deposition of GOD (0.5% \(w/v\)) and BSA (1% \(w/v\)) at GLU level of 2.5% \(v/v\) in phosphate buffer at pH 7, \(I\) 0.1 M. Curves (bottom to top) refer to 1, 3, 5, 7, and 9 mA/cm\({}^{2}\) current densities, respectively; Figure S2: Cyclic voltammograms at the Pt deposition electrode in phosphate buffer at pH 7, \(I\) 0.1 M, at low (left panel) and high (right panel) scan rates (in both panels, inner curves refer to the lower scan rate values). **Author Contributions:** Conceptualization, A.G.; methodology, A.G. and R.C. ; investigation, A.G., R.C., G.B., A.D.C. and M.A.A. ; resources, A.G.; data curation, A.G. and R.C. ; writing--original draft preparation, A.G.; writing--review and editing, A.G.; visualization, A.G.; supervision, A.G.; project administration, A.G.; funding acquisition, A.G. All authors have read and agreed to the published version of the manuscript. **Funding:** The Authors gratefully acknowledge financial support from Ministero dell'Istruzione, dell'Universita e della Ricerca Italiana (MIUR). **Data Availability Statement:** The data presented in this study are available in the article and in supplementary material here.