Magnetically Recoverable ICT-Functionalized Fe₃O₄ Nanoparticles for Efficient Horseradish Peroxidase Immobilization

The formation of interfacial charge transfer (ICT) complexes between phenolic ligands and metal oxide surfaces enables surface functionalization strategies with potential applications in catalysis and bioconjugation. In this study, magnetite (Fe₃O₄) nanoparticles were modified with two phenolic ligands, 5-aminosalicylic acid (5ASA) and caffeic acid (CA), to generate ICT complexes capable of covalent or non-covalent enzyme immobilization, respectively. The modified nanomaterials were structurally characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM), and Fourier-transform infrared spectroscopy (FTIR). Horseradish peroxidase (HRP) was immobilized on these functionalized supports. Catalytic activity was evaluated using pyrogallol oxidation assays, with systematic variations in nanoparticle mass and enzyme concentration. The Fe₃O₄/5ASA–HRP system exhibited a maximum activity of 2.5 U per 20 mg of support (approximately 125 U/g), whereas Fe₃O₄/CA showed minimal activity under the same conditions. Data from enzyme loading studies confirmed that 5ASA-enabled covalent attachment resulted in significantly higher immobilization efficiency (up to 1068 mg/g) compared to the CA system. The magnetic properties of Fe₃O₄ allowed for rapid recovery of the biocatalysts using an external magnetic field. These results highlight the effectiveness of ICT-based functionalization for enzyme immobilization, positioning Fe₃O₄/5ASA as a promising platform for robust and reusable biocatalysts in environmental and industrial applications.