Antibacterial and Antibiofilm Efficacy of Graphene Oxide Nanomaterials against Gram-Positive Bacteria: Mechanistic Approaches to Understand the Membrane Damage and Oxidative Stress in Cells

The global rise of antimicrobial resistance (AMR) underscores the urgent need for novel antibacterial agents, particularly those based on two-dimensional (2D) nanomaterials, such as graphene oxide (GO). In this study, GO was synthesized from graphite using an improved Hummer's method optimized for scalability, safety, and environmental sustainability. The protocol eliminates hazardous sodium nitrate and substitutes highly corrosive sulfuric acid with milder phosphoric acid, which serves dual roles as an oxidizing and intercalating agent. This facilitates edge and surface etching, resulting in an oxo-functionalized GO with high aqueous stability and uniform dispersion. The antibacterial efficacy of the synthesized GO was evaluated against two Gram-positive bacteria, <i>Enterococcus faecalis</i> (ATCC 29212) and <i>Staphylococcus aureus</i> (ATCC 29213). The concentration and time-dependent killing efficacy of GO demonstrated the rapid killing of 5 log₁₀ CFU/mL bacterial cells at 200 μg/mL within 1 h. The biofilm eradication potential of GO was explored, where ∼92% reduction in biofilm cell viability was noted at 100 μg/mL against both bacteria. The oxidative stress induction was assessed by Ellman's assay, and a concentration-dependent loss of glutathione was noted up to 36.01% at 200 μg/mL. The membrane permeabilization ability was analyzed by the propidium iodide (PI) uptake assay, where significant permeabilization was observed at 50 μg/mL or higher. The antibacterial mechanisms were further corroborated by atomic force microscopy and scanning electron microscopy, which revealed the morphological distortions attributed by the nanoknife-like edges of GO. The hemolytic assay against mice red blood cells indicated minimum to negligible toxicity of GO when tested up to 400 μg/mL, confirming its safety toward mammalian cells. Overall, these findings reinforce the potential of this safely synthesized GO as a multifunctional nanomaterial with significant potential for biomedical applications like antimicrobial coatings and wound dressings and for environmental applications including water purification and disinfection.