Multi-attribution characterization of metallic nanoparticles in biological media by electrical asymmetrical flow field-flow fractionation coupled to a hybrid detection platform.

For the first time, the potential of electrical asymmetrical flow field-flow fractionation (EAF4) coupled online to a multidetector platform (UV-vis, MALS, and ICP-MS) for the comprehensive characterization of metallic nanoparticles (NPs) in biomedically relevant media is demonstrated. Beyond size determination, this technique provides insights into surface charge, a critical parameter governing NP's stability and behaviour. The inclusion of ICP-MS significantly enhances its selectivity, enabling discrimination among different NP populations within complex, polydisperse matrices at low, biomedically relevant concentrations. Furthermore, the data are obtained in a size-resolved way, surpassing the limitations of currently available batch techniques. As a case study, gold (AuNPs) and platinum (PtNPs) NPs have been analysed after incubation in biological media representing different levels of complexity, i.e., bovine serum albumin (BSA), fetal bovine serum (FBS), and Dulbecco's Modified Eagle Medium (DMEM) to evaluate their transformations (e.g., protein corona formation) and their impact on physicochemical properties. The proposed approach enables simultaneous determination of hydrodynamic radius (Rh), gyration radius (Rg), shape-related parameters, electrophoretic mobility (μ), and zeta potential (Z-potential) under mild, biocompatible fractionation conditions, achieving recoveries >70%. Size-resolved data revealed broader NP populations and significant modifications in surface charge due to protein corona formation, showing differences between simple protein scenarios and complex biological matrices. Overall, the study highlights the unique capability of EAF4 hyphenated with complementary detectors as a powerful analytical tool for multidimensional NP characterization in biological environments, providing valuable insights into NP-bioentity interactions and supporting the development and safety assessment of nanomaterials for biomedical applications.