## 3 Results and Discussion  
### Optimization of Chemical Conditions for EPD and Co-Crosslinking  
As pointed out in Section 1, only charged particles can migrate under an electrical field in EPD and then be deposited onto the target electrode. Glucose oxidase (GOD) and bovine serum albumin (BSA) have isoelectric points at pH 4.3 and 4.7, respectively [10], so they are positively charged in an acidic solution while being negatively charged in neutral and alkaline solutions. Since low pH values significantly reduce the GOD activity and induce the denaturation of both proteins, EPD was performed in neutral solutions (i.e., a phosphate buffer at pH 7, 1 0.1 M), so both proteins were expected to be in their negatively charged forms and be attracted by the anode of EPD cell.  
Crosslinking [24; 25] is a conventional method for enzyme immobilization able to produce insoluble polymeric structures of enzyme molecules covalently linked by a crosslinking agent. By using, e.g., glutaraldehyde (GLU) as a bifunctional crosslinker, both aldehyde groups react with primary amino group residues of proteins, initially creating Schiff bases, followed by the formation of stable secondary amine linkages. The kinetics and the degree of crosslinking are usually controlled by the concentration of both glutaraldehyde and enzyme, reaction time, pH, and temperature: usually, higher concentrations of protein and crosslinker result in stronger crosslinking, which adversely affects the catalytic activity of the immobilized structure due to enzyme crowding. For this reason, co-crosslinking [24; 25], i.e., inert protein-assisted enzyme crosslinking, was preferred; indeed, the use of an inert protein like BSA significantly reduces the crowding of GOD, resulting in a immobilized structure with sufficient catalytic activity. Typical co-crosslinking chemical conditions were already studied and optimized elsewhere, e.g., for GOD [26], by varying enzyme, BSA, and GLU concentrations as well as buffer type and pH. Anyway, the aim of the present research was to drastically slow down the kinetics of the co-crosslinking reaction in the bath solution to avoid any gelation process in the cell while inducing co-crosslinking exclusively on the electrode surface during the EPD process. With this aim in mind, a carefully study showed that it is more effective to reduce the concentrations of both proteins than the concentration of the crosslinker agent; in fact, reducing the GLU concentration to 0.5 \(\div\) 1% \(v/v\), to avoid any co-crosslinking in the solution, did not lead to any deposition, even if, for example, the protein concentration was higher than 10% \(w/v\). Typically, a GOD concentration of 0.5% \(w/v\), a BSA concentration of 1% \(w/v\), and a GLU concentration of 2.5% \(v/v\) in phosphate buffer at pH 7, 1 0.1 M, guaranteed the absence of any gelation processes in the cell for as long as several hours but permitted in situ co-crosslinking of GOD and BSA under suitable EPD conditions (vide infra). It is worth noting that a buffer with high ionic strength was preferred due to its influence on the size of the diffusion layer (i.e., the electrical double layer) surrounding the protein molecules; in fact, as Scheme S1 shows, higher ionic strengths, reducing the size of the diffusion layer, permit fewer retardation effects on the electrophoretic mobilities of proteins and constitute a proper approach for molecules in the co-crosslinking reaction.