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This work aims to optimize the antibacterial activity of iron oxide nanoparticles (IONPs) against both Gram-positive and Gram-negative bacteria. IONPs were greenly biosynthesized using Moringa oleifera leaves extract, and surface methodology (RSM) based on central composite design (CCD) was employed to investigate the combined effect of various experimental factors on the antibacterial activity of IONPs. The reaction and annealing temperatures besides precursor concentration were set as independent variables, while the antibacterial activity was set as a response to obtain the optimal conditions that maximizes IONPs antibacterial activity. Different characterization techniques such as UV–Vis, FTIR, XRD, SEM, and EDX were employed to study the properties of the biosynthesized nanoparticles. Meanwhile, the antibacterial activity was tested using the disk diffusion method. The characterizations results have confirmed the biosynthesis of Hematite (α-Fe2O3) nanoparticles of rhombohedral structure. The generated model has exhibited predicted values very close to the actual proving its validity to analyze and optimize the studied process. The model indicated that all the investigated parameters and their interactions have significantly affected IONPs antibacterial activity. An optimal antibacterial activity was achieved when biosynthesis factors at their lower levels (? 1). Furthermore, the effect of IONPs size on the antibacterial activity was studied and the results shown that the latter is significantly related to the nanoparticles size.

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