Escherichia coli fatality dependence on oxidation level of graphite: Insights into membrane permeabilization and protein leakage

Background: Graphene oxide (GO) with different oxidation levels were evaluated for their bactericidal activities, with the aim to investigate the antibacterial mechanism by correlating the bactericidal efficacy with GO oxidation level. Methods: The GO samples were synthesized and labelled as GO1, GO2, GO3 and GO4 using graphite/KMnO₄ ratios of 1:3, 1:4, 1:5 and 1:6, respectively. Those samples were characterized and tested for antibacterial activity against Escherichia coli (E. coli). Significant findings: This study revealed graphite transformation from multilayer stacks to monolayer wrinkled GO nanosheets with increasing KMnO₄ dosage. The higher oxidized GO exhibited a lower negative zeta potential value, and higher O/C ratio. GO4 exhibited the lowest half maximal inhibitory concentration value at 161 µg mL⁻¹, followed by GO3 (307 µg mL⁻¹), GO2 (652 µg mL⁻¹), and GO1 (1077 µg mL⁻¹), indicating a strong correlation (R²=0.94) between oxidation level and bactericidal effectiveness. GO primarily interacts with the outer membrane through its hydrophobic surface, leading to permeabilized membrane (˃ 50 %), protein leakage (˃ 40 µg mg⁻¹), significant morphological alterations, triggering reactive oxygen species production, and resulting in extensive damage to the entire bacterial cell. In summary, highly oxidized GO4 (O/C ratio of 0.54) demonstrated antibacterial potential for clinical use.