Phd in PHARMACEUTICAL SCIENCES

Thesis title: Nanostructure-based enzymatic biosensors and biofuel cells: characterization and applications

The enhancement of protein direct and mediated electron transfer (DET and MET, respectively) represents one of the main challenges in enzyme-based biosensors and biofuel cells development. This thesis aims to realize new and simple nanostructured platforms able to effectively improve the enzyme-electrode communication and increase the electron transfer (ET) rate. In the first part of the thesis, star-shaped gold nanoparticles (AuNSs) have been employed as protein DET-promoters. To the best of our knowledge, this was the first time that non-spherical metal nanoparticles were used for this purpose. Specifically, DET of cytochrome c (cyt c) was improved at AuNSs interfaces compared with AuNSphs based electrodes. The adsorption of cyt c-AuNSs bioconjugates onto pyrolytic graphite (PG) electrodes have shown the ritention of the protein native properties. On the contrary, the cyt c direct adsorption onto PG electrodes induced the formation of a non-native species with peroxidase activity, wich was enhanced in presence of AuNSs. This latter platform proved to be suitable for further improvements in order to realize a DET-based biosensor for H2O2. In the second part of the thesis, three multi-walled carbon nanotubes (MWCNTs)-modified screen-printed platforms were realized to promote the ET of the following enzymes: recombinant anionic tobacco peroxidase (r-TOP), bilirubin oxidase (BOx) and FAD-dependent glucose dehydrogenase (FAD-GDH). In the case of r-TOP and BOx, MWCNTs have been combined with diazonium salts, making a site-oriented immobilization of both the enzymes and promoting DET at the electrode. Differently, for FAD-GDH, methylene blue (MB) was electropolymerized onto the MWCNTs-modified electrode, in order to promote a MET process. Finally, the r-TOP bioelectrode was used to realize a biosensor for the H2O2 detection, resulting a promising candidate as a screening analytical method for the detection of H2O2 in real samples. BOx and r-TOP platforms were employed as biocathode and bioanode respectively in the development of a hybrid glucose/oxygen BFC. Optimal results were achieved in terms of OCV and Pout by connecting in series two EFCs, each one composed by a double-sided screen-printed electrode (SPE). The biodevice was tested in human serum and saliva, achiving a good performance and proving to be a promising candidate for the powering of miniaturized implantable devices.