## 1 Introduction
Electrodes are represented by the large rectangles; circles and ellipses represent the protein and the enzyme molecules, respectively, whose concentration profiles from the electrode surface are shown on the right of each panel; and upper and lower right panels illustrate the concentration profiles of the molecules when an electrical field is applied between electrodes (for the case of negatively charged molecules). techniques have been investigated for reducing these adverse effects while optimizing enzyme deposition: accordingly, the use of EPD for biosensor fabrication requires the careful study of the electrical perturbation to be used for promoting suitable electrophoresis and hence deposition of the enzyme molecules, minimizing the undesired but inevitable side effects of water electrolysis. Furthermore, it is worth noting that the deposition process and the nature of the deposit are still not well explained [11], complicating the effectiveness of the EPD approach. Even if electrosorption, multilayer adsorption, and electrocoagulation are sometimes employed, a few studies [22; 23] apparently show that the significant pH decrease at the electrode/solution interphase occurring due to water electrolysis is responsible for enzyme precipitation and deposition. Anyway, since low pH values and precipitation essentially signify the denaturation of the enzyme, it is unclear how a denatured enzyme, as declared, can be used as an effective sensing element in biosensors. Not less importantly, a fully compact deposit of a pure enzyme is expected to limit biosensor performance (e.g., induce a poor linear range) due to the low permeability/high activity of the immobilized enzyme. Therefore, a different deposition approach should be explored to allow for the efficient use of EPD in biosensor fabrication. Conventional enzyme immobilization usually relies on the use of, e.g., suitable chemical reactions that transform soluble enzyme molecules in insoluble enzyme aggregates firmly attached to the desired support [24; 25]. For fabricating amperometric enzyme electrodes, enzyme crosslinking and co-crosslinking [24; 25] have turned out to be quite popular thanks to the simplicity of the approach, the high stability of the immobilized enzyme, and the ability to obtain a thin enzyme membrane strongly adherent (tight) to the electrode surface and mechanically very stable even in stirred and flowing solutions. As an example, in our laboratory, enzyme co-crosslinking with bovine serum albumin proved successful in fabricating glucose-oxidase- [26], choline-oxidase- and acetylcholine- esterase- [27; 28], and lysine-oxidase [29; 30; 31]-based amperometric biosensors. Unfortunately, as already pointed out above, this enzyme immobilization procedure, however powerful, requires careful casting of the crosslinking solution onto the electrode surface, and for this reason, it is limited to macroscopic electrodes with simple geometries. Surely, this limitation could be overcome if enzyme co-crosslinking could be electrochemically assisted so as to induce crosslinking only and exclusively on the electrode surface, without requiring any manual casting of the enzyme-crosslinking solution. In the present paper, an original method, namely, electrophoretic-protein-deposition-assisted in situ co-crosslinking enzyme immobilization, was investigated in depth. In this approach, EPD was performed in a solution containing the enzyme of interest, an inert protein like bovine serum albumin with glutaraldehyde as a crosslinker, at concentration levels low enough to kinetically decelerate the co-crosslinking process. When polarization occurs at the electrodes, the concentrations of both proteins greatly increase at the electrode/solution interface due to their electrophoretic movements, thus starting co-crosslinking in situ, exclusively on the electrode surface (see Figure 1). This novel method assured the accurate spatial control of the deposition process while using a classical enzyme immobilization methodology, regardless of the shape of the deposition electrode.