Work

Analysis of bio-nano interactions by electrical asymmetric flow field-flow fractionation with multiple online detectors

Year: 2026

Type: article

Abstract: • EAF4 enables in situ size and surface charge assessment in protein rich media. • EAF4 gently separates nanoparticle–protein complexes, avoiding matrix interference. • Protein exposure alters size, shape, and zeta potential of silk nanoparticles. • EAF4 delivers deeper physicochemical profiling than DLS or PTA in biological media. • EAF4 pipeline supports studying bio-nano interactions for nanomedicines. Understanding bio-nano interactions and protein corona formation is critical for advancing nanomedicines towards clinical translation. However, conventional methods for nanoparticle analysis have limited utility for in situ analysis due to interference from unbound proteins present in bulk biological media. Electrical asymmetric flow field-flow fractionation (EAF4), which integrates AF4 with an applied electrical field, enables size and surface charge-based separation, and when coupled with online detectors, provides simultaneous measurement of particle size, electrophoretic mobility, and zeta potential, key parameters governing bio-nano interactions. Here, we report the first application of multiplexed EAF4 with online detection for evaluating biophysical changes occurring in polystyrene latex and silk nanoparticles, used as model nanomedicine systems, following exposure to serum under conditions that mimic the protein composition of cell culture media. Our findings reveal significant alterations in particle physical attributes, including particle size, shape factor, zeta potential, and electrophoretic mobility following exposure to protein-containing media. Furthermore, we demonstrate that EAF4 enables gentle fractionation of complex biological samples, providing comprehensive physicochemical profiling of diverse particulate and macromolecular species within nanoparticle–protein complexes. This work establishes EAF4 as a powerful analytical platform for resolving nano–bio interactions and guiding the rational design of next-generation nanomedicines.

Source: Journal of Chromatography A

DOI: 10.1016/j.chroma.2026.466879