High-Sensitivity Magnetic Levitation Reveals Hidden Heterogeneity in Nanoparticle Protein Coronas

The protein corona (PC)—a layer of biomolecules that adsorbs onto nanoparticles (NPs) surfaces upon contact with biological fluids—remains a major challenge in nanomedicine due to its complexity and variability. Even identical NPs exposed to the same biological source can form heterogeneous coronas with distinct compositions, which are difficult to resolve using conventional characterization methods. Here, we use a high-sensitivity magnetic levitation (MagLev) system with 1–3 orders of magnitude greater density resolution than standard configurations (i.e., ~ 10⁻³ g/cm³). This enhanced platform detects extremely small variations in PC coverage and composition—down to 10⁻⁵ g/cm³. Our results show that PC-coated NPs, indistinguishable in conventional standard MagLev systems, exhibit clearly distinct levitation profiles in the high-sensitivity configuration, confirming nanoscale heterogeneity among nominally identical particles. Complementary gel electrophoresis and proteomic analyses further reveal differences in both the number and type of proteins adsorbed on NP surfaces. Together, these findings establish MagLev as a powerful, label-free analytical tool to uncover PC diversity and advance the clinical translation of nanomaterials.