Magnetic Surface Molecularly Imprinted Fe ₃ O ₄ Nanoparticles for Highly Selective Recognition and Separation of Osteopontin

Osteopontin (OPN) is a multifunctional protein with diverse physiological roles, but its low content in bovine milk and poor selection of existing extraction methods pose a serious challenge to its efficient separation. Therefore, this study developed magnetic molecular imprinted polymers (MIPs) using methacrylic acid (MAA)-functionalized Fe₃O₄ nanoparticles as matrix and <i>N</i>-isopropylacrylamide/N-(3-(dimethylamino)propyl) methacrylamide (NIPAM/DMAPMA) as functional monomers via surface free radical polymerization. The synthesized magnetic surface molecularly imprinted materials exhibited a core-shell structure with Fe₃O₄ nanoparticles as the core and a thin imprinted polymer shell. FTIR and XPS confirmed the presence of amide groups involved in specific interactions. XRD indicated the crystalline nature of Fe₃O₄, while TGA demonstrated good thermal stability for repeated use. The MIPs exhibited a maximum adsorption capacity of 96.2 mg/g for OPN with an imprinting factor of 2.57, after pseudo-second-order kinetics and Langmuir isotherm model fitting with an <i>R</i>² > 0.98. Optimal performance occurred at 33 °C. Selective adsorption experiments with OPN, BSA, LF, α-LA, and β-LG demonstrated that MIPs had 2-3 times higher adsorption capacity for OPN compared to the interfering proteins. Competitive adsorption studies indicated preferential binding toward OPN in mixed protein systems, demonstrating superior molecular recognition. The MIPs also maintained 75% of their initial adsorption capacity after four regeneration cycles. These magnetic MIPs represent the first application of surface molecular imprinting for OPN and offer a promising strategy for selective separation with advantages in selectivity, magnetic separability, and reusability for practical applications.