Bioconjugation and Magnetic Separation Performance of Cellulose Nanofiber/Fe₃O₄ Nanoparticle Composites with Tunable Fe₃O₄ Sizes for Biomedical Applications

Magnetic separation-based materials have emerged as powerful tools in biomedical applications such as clinical diagnostics, targeted drug delivery, and biosensing. In this study, we developed magnetic cellulose nanofiber composite particles by integrating TEMPO-oxidized cellulose nanofiber (TOCN), a renewable biopolymer enriched with carboxyl groups, with magnetite (Fe3O4) particles of three different sizes─10 nm, 50 nm, and 230 nm─using spray drying for rapid and reusable magnetic bioseparation. During solvent evaporation of the spray drying process, colloidal science principles were applied to control mass transfer, whereby smaller Fe3O4 nanoparticles migrated more readily to the droplet surface while larger ones remained embedded within the composite structure, enabling size-dependent migration and localization of particles within the composites. Morphological and structural analyses showed that smaller Fe3O4 (10 nm) tended to localize at the composite surface, reducing the availability of TOCN’s functional sites, while larger Fe3O4 (230 nm) allowed greater TOCN exposure, leading to resulting in a higher negative surface charge (zeta potential: −60 mV). This enhanced surface charge led to strong electrostatic repulsion, providing excellent redispersibility and consistent performance over thirteen separation cycles in phosphate-buffered saline. Magnetization measurements also showed a size-dependent increase in saturation magnetization, with 230 nm Fe3O4-based composites reaching 48 emu/g, enabling magnetic separation 2.5 times faster than those containing 10 nm Fe3O4 (in deionized water at pH ≈ 7). To evaluate surface bioconjugation capability, chemiluminescent immunoassays based on streptavidin–biotin interactions were performed, demonstrating over a 2-fold increase in signal intensity for 230 nm Fe3O4 composites compared to 10 nm, attributed to improved exposure of TOCN functional groups. This study reveals that controlling Fe3O4 sizes via colloidal mass transfer during drying is critical for enhancing magnetic responsiveness and surface biofunctionality, offering significant potential for biomedical composites in biomolecule immobilization and diagnostic applications.