## 3 Results and Discussion
In a subsequent experiment, we examined how the traditional ROS scavengers, L-histidine and \(t\)-butanol, could affect the antimicrobial outcome of C/TiO\({}_{2}\) hybrid dot treatments. Generally, L-histidine and \(t\)-butanol are known as being able to scavenglet molecular oxygen and hydroxyl radicals, respectively. Compared in Figure 4B are the viable cell number changes in _B. subtilis_ cells due to the treatment of PEG&PEI-C/TiO\({}_{2}\)-Dots with visible light in the absence and presence of L-histidine. The treatment with the dot concentration of 0.1 mg/mL under visible light resulted in \(\sim\)4.6 log reduction in viable cells in the absence of L-histidine, but only \(\sim\)1.9 log reduction in the presence of L-histidine, consistent with the known protective effect of L-histidine as an ROS scavenger. However, the protection by L-histidine was limited, reducing the magnitude of log reduction in viable cells by \(\sim\)60% only even at the high L-histidine concentration of 60 mM (near the solubility limit), far from being complete. The same experiments with \(t\)-butanol as ROS scavenger were performed, with the results showing no protective effect even at the high \(t\)-butanol concentration of 60 mM. The outcomes for both L-histidine and \(t\)-butanol are essentially the same as those found in similar experimental evaluations on the protective effects of these scavengers in the treatment of bacterial cells with neat CDots and visible light exposure, namely some limited protection by L-histidine and none by \(t\)-butanol [37]. In the mechanistic framework that the antibacterial activities of light-activated CDots are due to the combined actions of two sets of highly reactive species, the initially formed electron-hole pairs and the classical ROS generated in the emissive excited states [37], the different \"lifetimes of killing\" between the two sets of photo-generated reactive species in CDots could be used to rationalize the observed inadequate protection by the scavengers, such that L-histidine is only capable of quenching the longer lived set of reactive species (the classical ROS generated in the emissive excited states, whose lifetimes are in nanoseconds [40]). Interestingly, the scavenging outcomes were not affected in any fundamental fashion by the presence of colloidal TiO\({}_{2}\) in the hybrid dots, suggesting the preservation of the basic photoexcited state processes and the actions of the different reactive species on different time scales in the structurally and composition-wise more complex hybrid dots. However, the likely \"sharing\" of the excitation energy between the initially excited nanoscale carbon domains (heat CDot-like, Figure 1) and the photo-sensitized TiO\({}_{2}\) nanoparticles in the hybrid dots, both contributing to the observed antimicrobial activities, represents added complications mechanistically. Important questions such as the time scale of the photo-sensitization and potentially differential quenchings of the reactive species at the carbon and TiO\({}_{2}\) sides by the scavengers remain to be addressed in further dedicated investigations. The obvious major difference between the two scavengers L-histidine and \(t\)-butanol is puzzling, beyond the mechanistic rationale discussed above. However, it might be speculated that the difference could be due to different lifetimes of their targeted classical ROS, namely that the hydroxyl radicals in aqueous medium are too short lived for \(t\)-butanol to scavenger. The consequences of the treatment with the C/TiO\({}_{2}\) hybrid dots and visible light include the suppression of the intrinsic antioxidant defense capacity of the cells, causing various adverse effects and damage to the cells, such as the oxidation of proteins and  
Figure 4: **(A)** The fold increase in intracellular ROS generation; and **(B)** the viable cell number change in _B.