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Magnetite (Fe3O4) nanoparticles, are promising inorganic nanomaterials for future biomedical applications due to their low toxicity and unique magnetic properties. However, the synthesis of these particles can often be expensive, energy intensive, and non-scalable, requiring the addition of surfactants to stabilize the material to control the particle size and avoid agglomeration. We wish to report a simple, green, surfactant-free electrochemical synthesis of these materials using a closed aqueous system at ambient temperature. Particle diameter, between 19 and 33 nm, was controlled by simply modifying the distance between the electrodes. These magnetite nanoparticles were then fully characterized using both spectroscopy and microscopy. Vibrational magnetometry indicates that as the size of the particle decreases, the magnetic hysteretic gap decreases, although for samples below 25 nm no inter-sample difference was observed. To support this experimental data, we carried out a Density Functional Theory (DFT) analysis of magnetite containing more than three iron atoms in the cluster, an essential proposition as magnetite contains three distinct iron species. These calculations were used to support the experimental observations, and closely reproduced both the experimental IR spectrum, and the XRD pattern. In vitro cytotoxicity assays showed dose responsive behavior for the nanoparticles, and demonstrated that they are non-toxic at clinically relevant concentrations; below 200 µg/mL we observed no toxicity in a 48-hour standard assay. This work represents the first DFT based simulation of this detailed magnetite cluster, and …