## 3 Results and Discussion
subtilis_ after the cells were treated with C/TiO\({}_{2}\) hybrid dots in the presence and absence of ROS scavenger L-histidine, and **(C)** in the absence and presence of ROS scavenger \(t\)-butanol, along with those of untreated control cells. Treatment: 0.1 mg/mL PEI&PEG-C/TiO\({}_{2}\)-Dots with visible light illumination for 2 h. Scavenger concentration: 60 mM. the disruption of cell membranes [41; 42]. Often accompanying cell membrane damage is the lipid peroxidation [43], a multiple-step process in which lipid carbon-carbon double bonds are attacked by free radicals and/or strong oxidants [44; 45; 46]. Some studies have demonstrated that free radicals can induce cell membrane damage [47], and the oxidative stress can lead to lipid peroxidation, inhibiting bacterial growth [48]. Other studies have found that the membrane damage by photoexcited TiO\({}_{2}\) is often associated with lipid peroxidation [49]. The lipid peroxidation produces lipid hydroperoxides (LOOH) and various aldehydes including especially malondialdehydedehyde (MDA) [50], which is commonly used as an indicator of lipid peroxidation. In this study, the extent of lipid peroxidation in _B. subtilis_ cells upon the treatments of C/TiO\({}_{2}\)-Dots with visible light was measured by quantifying MDA with a commercial kit (TBARS Assay), which is based on the reaction of MDA with thiobarbituric acid (TBA) to form a red fluorescent adduct [51; 52]. Shown in Figure 5A is the increased amount of MDA measured in _Bacillus_ cells after the treatment with 0.1 mg/mL PEI&PEG-C/TiO\({}_{2}\)-Dots and visible light for 2 h. The amount is significantly higher than that in the untreated control samples, indicating the cell membrane lipid peroxidation caused by the highly reactive species produced in the treatment. The damage to the cell membrane can be assessed more directly with the live/dead bacterial viability kit, which contains two nucleic acid dyes, SYTO 9 and propidium iodide (PI), to stain the live and dead cells for their significant differences in the membrane permeability. The green SYTO 9 dye can penetrate membranes of both live and dead cells, but the red PI dye can only penetrate the damaged cell membranes of dead cells. Thus, the kit stains the live cells with intact membranes in green, and the dead cells with damaged membranes in red. Figure 5B-D illustrates the changes in cell permeability due to the treatment with PEG&PEI-C/TiO\({}_{2}\)-Dots and visible light. In control samples, almost all cells were viable and stained green (Figure 5B), but in the treated cells there was obvious damage to the cell membrane, more evident when higher dot concentrations were used  
Figure 5: (**A**) The increased amount of MDA in _B. subtilis_ cells after they were treated with PEI&PEG-C/TiO\({}_{2}\)-Dots at 0.1 mg/mL with visible light for 2 h. (**B–D**) The fluorescent images of _B. subtilis_ cells without dots treatment, with 0.02 mg/mL, and 0.1 mg/mL PEG&PEI-C/TiO\({}_{2}\)-Dots treatment with visible light for 2 h, respectively, and stained with live/dead bacterial viability kit. in the treatments, as demonstrated by the increase in the number of red-stained cells (Figure 5C,D). The live/dead staining results clearly confirmed that the treatments with C/TiO\({}_{2}\) hybrid dots damaged bacterial membrane to result in higher permeability. The cells post-treatment with the hybrid dots were also examined by TEM imaging. Shown in Figure 6 are TEM images of untreated control cells and the treated cells. For the untreated control cells, their structures show regularly situated genomic materials surrounded by the cytoplasmic area, with clearly organized cell membrane and wall and the periplasmic spaces. For the treated cells, there is shrinkage of the cytoplasm, with a separation of the cytoplasm from the cell wall, and there are also some distortions and collapse in the membrane-wall continuity, most likely due to the cell membrane damage.