Electrodes based on Cytochromes and nanostructured oxides with variable crystalline structure for the detection of Cyclophosphamide.

Within the huge family of sensors and biosensors technology, electrochemical sensors allow the transduction of a chemical information to an electrical signal to precisely characterize complex chemical phenomena, such as redox reactions, or to quantify the concentration of a molecule in a solution. Nanostructured metal oxides, with spinel crystalline structure, are attractive for electroanalytical methodologies because they could improve the catalytic activity of the redox process of some molecules: they could have the ability to enhance the detection' sensibility. Therefore, the aim of this work is to study the electrochemical performance of nanostructured metal oxides (i.e. zinc chromite, zinc ferrite and copper ferrite) in the detection of Cyclophosphamide, a very well-known anticancer drug, whose metabolism relies on Cytochrome P450. The metal oxide nanoparticles were produced by adopting the auto-combustion synthesis technique and they were morphologically and chemically characterized by scanning electron microscopy and Raman spectroscopy. Nanostructured metal oxides were then used to tailor the surface of screen-printed carbon electrodes by drop casting technique with or without the addition of Cytochrome P450 in his two isoforms of 3A4 or 2B6. Drug detection was acquired in phosphate-buffered solutions. This thesis project aimed thus to unveil the effect of electrode surface functionalization on the electrochemical behaviour of the drug, considering different crystal lattice of the nanoparticles. The analyses were carried out with drug concentrations between 250 µM and 1 mM, using cyclic voltammetry and differential pulsed voltammetry techniques. The results of bare electrode and spinel nanomaterials functionalized electrodes are comparable, providing a maximum Faradic current peak of 0.18 ± 0.08 μA with a sensitivity of 0.09 ± 0.04 nA/μM, obtained by the linear calibration of the sensor. Surface functionalization only with enzymes leads to an increase in the maximum current peak and in the sensitivity by an order of magnitude. However, the best results are obtained with the synergetic coupling of nanostructured metal oxide and Cytochromes: the functionalization with zinc chromite nanoparticles and Cytochrome P450 2B6 leads to a maximum of current peak of 9.24 ± 0.65 μA and a sensitivity of 6.90 ± 0.41 nA/μM. Instead, concerning the limit of detection, the best performance is with zinc ferrite nanoparticles and Cytochrome P450 3A4, with a value of 20.04 ± 0.98 μM. From the analyses carried out so far, it is possible to conclude that the presence of nanostructured oxides has a catalytic action on the reductive process of the Cytochromes, and consequently of the drug, intensifying the signal that is detected. Metal oxides with a normal spinel lattice with Cytochrome P450 2B6 seem to guarantee the best performance for the detection of Cyclophosphamide. Promising results obtained suggest the necessity to conduct further research and investigations to optimize the application of these hybrid surface functionalizations in the field of biosensing.