## 3 Results
### EPR Spectroscopy  
EPR spectroscopy was used to evaluate the improvement on ROS generation due to the influence of NPs in water, according to different US powers. The samples ([NPs] = 200 \(\upmu\)g/mL) were irradiated with US for 1 min, at a frequency of 1 MHz, a Duty Cycle of 100%. Figure 5 reports the EPR spectra after US stimulation of the NPs and clearly shows the presence of characteristics DMPO-OH spin adducts due to the presence of ROS (Figure 5c,d). For output power higher than 0.9 W/cm\({}^{2}\) a qualitative increase in ROS generation was observed when pristine a-TiO\({}_{2}\) and a-TiO\({}_{2}\)-NH\({}_{2}\) NPs were present. Figure 5: EPR spectra of amine-functionalized (graphs on top) and pristine (graphs on the bottom) a-TiO\({}_{2}\) based NPs (200 \(\upmu\)g/mL) in water using DMPO (10 mM) as spin trap, irradiated with different US powers: (**a**) irradiation of 0.3 W/cm\({}^{2}\) of US, (**b**) irradiation of 0.6 W/cm\({}^{2}\) of US; (**c**) irradiation of 0.9 W/cm\({}^{2}\) of US; (**d**) irradiation of 1.2 W/cm\({}^{2}\) of US. Black spectra: control sample—water without NPs; red spectra: water with 200 \(\upmu\)g/mL of a-TiO\({}_{2}\) based NPs. For quantitative evaluation, Table 2 shows the concentration of DMPO-OH spin adduct measured in all the analyzed conditions. Those data are directly correlated to ROS production, especially to the hydroxyl and superoxide anions. Concerning both pristine a-TiO\({}_{2}\) and a-TiO\({}_{2}\)-NH\({}_{2}\) NPs measured under daylight conditions, an increase in ROS generation was detected for US output powers of 0.9 and 1.2 W/cm\({}^{2}\). The EPR spectra of biomimetic a-TiO\({}_{2}\)-NH\({}_{2}\)/DOPC-DOTAP NPs suspension (Figure 6) show that no generation of ROS occurs, even when the sample is stimulated with the highest values of US irradiation powers (at daylight). In this case, even for US powers for which ROS were detected on the control samples (US powers of 0.9 and 1.2 W/cm\({}^{2}\)), no ROS production was observed for a-TiO\({}_{2}\)-NH\({}_{2}\)/DOPC-DOTAP NPs suspension (Figure 6 and Table 2). Although all the synthesized a-TiO\({}_{2}\) based NPs exhibited band gaps in the UV region, the influence of visible light on ROS generation was studied. This evaluation was carried out only for the highest responses (corresponding to the highest values of US irradiation, i.e., 0.9 and 1.2 W/cm\({}^{2}\), see Table 2). \begin{table}
\begin{tabular}{c c c c c} \hline \hline  & **a-TiO\({}_{2}\) NPs** & **a-TiO\({}_{2}\)-NH\({}_{2}\) NPs** & **a-TiO\({}_{2}\)-NH\({}_{2}\) NPs** & **a-TiO\({}_{2}\)-NH\({}_{2}\)/DOPC-DOTAP** \\
**US Output Power** & **(Daylight** & **(Daylight** & **a-TiO\({}_{2}\)-NH\({}_{2}\) NPs** & **a-TiO\({}_{2}\)-NH\({}_{2}\)/DOPC-DOTAP** \\
**(W/cm\({}^{2}\))** & **Conditions)** & **Conditions)** & **(Dark Conditions)** & **(Daylight Conditions)** \\  & **[-OH] M** & **[-OH] M** & **[-OH] M** & **[-OH] M** \\ \hline
0.3 & 0 & 0 & - & 0 \\
0.6 & 0 & 0 & - & 0 \\
0.9 & \(1.81\times 10^{-5}\) & \(3.297\times 10^{-5}\) & \(1.73\times 10^{-6}\) & 0 \\
1.2 & \(1.54\times 10^{-5}\) & \(3.295\times 10^{-5}\) & \(1.98\times 10^{-5}\) & 0 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Concentrations of DMPO-OH spin adduct obtained by using Bruker SpinFit software. All the experiments were performed in daylight. In the case of the highest ROS production, i.e., concerning the sample a-TiO\({}_{2}\)-NH\({}_{2}\) NPs, tests in dark conditions were also performed. Figure 6: EPR spectra of a-TiO\({}_{2}\)-NH\({}_{2}\) based NPs (200 \(\mu\)g/mL) in water using DMPO (10 mM) as spin trap, irradiated with different US powers: (**a**) 0.3 W/cm\({}^{2}\) of US output power, (**b**) 0.6 W/cm\({}^{2}\) of US output power; (**c**) 0.9 W/cm\({}^{2}\) of US output power; (**d**) 1.2 W/cm\({}^{2}\) of US output power. Black spectra: control sample—water without NPs; red spectra: water with 200 \(\mu\)g/mL of a-TiO\({}_{2}\)-NH\({}_{2}\) NPs; blue spectra: water with 200 \(\mu\)g/mL of a-TiO\({}_{2}\)-NH\({}_{2}\)/DOPC-DOTAP NPs.