Abstract
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 (Fe(3)O(4)) 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 using varying nanoparticle amounts (10-30 mg) and initial enzyme concentrations (25-250 µg mL(-1)). Catalytic activity was evaluated using pyrogallol oxidation assays. The Fe(3)O(4)/5ASA-HRP system exhibited a maximum activity of 2.5 U per 20 mg of support (approximately 125 U g(-1)), whereas Fe(3)O(4)/CA showed minimal activity under the same conditions. Enzyme loading studies confirmed that 5ASA-enabled covalent attachment resulted in significantly higher immobilization efficiency (up to 1068 mg g(-1)) compared to the CA system. Reusability tests demonstrated that the Fe(3)O(4)/5ASA system retained high absolute catalytic activity during the initial reaction cycles and consistently outperformed the non-covalently immobilized Fe(3)O(4)/CA system upon repeated reuse. The magnetic properties of Fe(3)O(4) allowed rapid recovery of the biocatalysts using an external magnetic field. These results highlight the effectiveness of ICT-based functionalization for enzyme immobilization, positioning Fe(3)O(4)/5ASA as a promising platform for robust and reusable biocatalysts in environmental and industrial applications.