Fig. 8
The ROS assays. After a, c E. coli and b, d B. subtilis were treated with GO, GO-POAA, and GO-chitosan (up to 20 μm wide) in either a, b nutrient or c, d PBS alone at 37 °C for 3 h, superoxide radical anion (O ·−2 ) were generated. XTT was used to monitor the generated superoxide radical anion and the absorbance at 470 nm was recorded. Singlet Oxygen Sensor Green Reagent was used to directly detect singlet oxygen. Singlet oxygen (1O2) measurements were conducted by monitoring similarly treated e, g—E. coli and f, h—B. subtilis in either e, f nutrient or g, h PBS alone with the same treatment. For E. coli, a p = 0.266, p < 0.001 and p < 0.001, and c p < 0.001, p < 0.001 and p < 0.001 are for treatment with GO, GO-POAA and GO-chitosan, respectively; for B. subtilis, b p = 0.179, p < 0.001 and p < 0.001, and d p < 0.001, p < 0.001 and p < 0.001 are for treatment of GO, GO-POAA and GO-chitosan, respectively; for E. coli, e p = 0.845, p = 0.337 and p = 0.341, and g p = 0.297, p = 0.281 and p = 0.266 are for treatment of GO, GO-POAA and GO-chitosan, respectively; for B. subtilis, f p = 0.415, p = 0.360 and p = 0.329, and h p = 0.305, p = 0.311 and p = 0.282 are for treatment of GO, GO-POAA and GO-chitosan, respectively. Data are mean ± SD (n = 6). *p value obtained by Student’s t-test. CM-H2DCFDA was used to detect the generated hydroxyl peroxide (H2O2) via flow cytometry. Measurements were conducted for similarly treated (i–k, o–q—E. coli and l–n, r–t—B. subtilis in either i–n nutrient or o–t PBS with the same treatment. The higher the percentage (histogram moves to the right), the greater is the oxidative stress induced. Negative control (0 μg mL−1): bacteria alone without any treatment