Myco-fabricated CuO NPs and chitosan-conjugated CuO nanoparticles using endophytic Aspergillus sp. JAWF3: Targeting E. coli outer membrane for enhanced antibacterial and biofilm inhibition

• Sustainable Biosynthesis of CuO Nanoparticles Using Endophytic Aspergillus sp. • Chitosan-Conjugated CuO Nanoparticles Enhance Antibacterial Efficiency Against E. coli . • Targeted Disruption of E. coli Outer Membrane Through OmpF Protein Interaction. • Strong Biofilm Inhibition Potential of Chitosan-Conjugated CuO Nanoparticles. Myco-assisted nanomaterial biosynthesis has been established as a sustainable and eco-friendly method to fabricate bioactive nanoparticles with various applications. In this study, the plant-associated fungus strain Aspergillus sp. JAWF3 was obtained from Juncus acutus’ leaves. The obtained fungus’s identity was confirmed by amplifying its ITS region, sequencing, and analysis. The biosynthesis of CuO NPs was carried out using fungal filtrate cell filtrate, which contains bioactive metabolites that facilitated Cu 2+ reduction as confirmed by color change and a characteristic SPR band at 320 nm. The biogenic CuO NPs were successfully conjugated with chitosan to enhance biocompatibility, stability, and drug delivery properties. TEM and FESEM Showed cuboid CuO NPs and aggregated chitosan/CuO nanoconjugates (150.6–250.9 nm). At the same time, the crystallinity of the biosynthesized nanoparticles and functional groups that participated in the formation was also confirmed by XRD and FTIR, respectively. The biogenic CuO NPs and Ch-CuO nanoconjugate were biologically tested toward four pathogenic bacteria, and results showed that E. coli was the most susceptible bacteria to chitosan-CuO nanoconjugates with inhibition percentage reached (90.2 %) compared to CuO NPs (86.8 %) and the crude fungal extract (76.5 %). TEM micrograph clearly showed the accumulation of chitosan/CuO Nanoconjugates and adhesion to the bacterial membrane, while molecular docking supported selective interactions with OmpF , suggesting a potential antibacterial mechanism. Additionally, the formed chitosan/CuO Nanoconjugates remarkably inhibit the biofilm formation of E. coli with an inhibition percentage (90.47 %). These findings highlight the promise of biogenic nanomaterials in therapeutic progress.