Engineering of Copper-Doped Zinc Oxide Nanoparticles: Unlocking Synergistic Antibacterial Mechanisms and Biofilm Eradication for Sustainable Aquaculture

The overuse of antibiotics in aquaculture has accelerated the emergence of drug-resistant bacteria and biofilm-associated infections, creating an urgent need for alternative antimicrobial agents. Zinc oxide nanoparticles (ZnO NPs) are promising candidates owing to their low toxicity and cost, but their intrinsic antibacterial activity is often inadequate. Herein, we report a series of copper-doped ZnO nanoparticles (Cu@ZnO NPs, 1–5 mol% Cu) synthesized via a facile solvothermal method. The 2% Cu@ZnO NPs, comprising well-dispersed spherical particles of approximately 200 nm with a highly positive surface charge (+20 mV), exhibited the strongest antibacterial activity, outperforming pristine and commercial ZnO, with a minimum inhibitory concentration (MIC) of 0.2 mg/mL and a minimum bactericidal concentration (MBC) of 1.0 mg/mL against both Escherichia coli and Staphylococcus aureus. Mechanistic studies suggested that bactericidal activity may arise from the synergy of electrostatic adsorption, membrane disruption accompanied by protein leakage, intracellular reactive oxygen species (ROS) generation, and sustained metal-ion release. The nanoparticles were shown to reduce biofilm formation by 80.96% and maintained 88.4% viability of L929 fibroblasts at 20 μg/mL, indicating good biocompatibility. In a zebrafish larvae infection model, 5.0 μg/mL of 2% Cu@ZnO increased the 72 h survival rate from 6.25% (Vibrio-challenged) to 65.0%. Cu@ZnO-loaded filter materials further demonstrated practical applicability for aquaculture water pretreatment. This work elucidates the structure–activity relationship of Cu-doped ZnO nanomaterials and offers a low-toxicity, filter-integrable alternative to antibiotics for sustainable aquaculture.