On-Chip Profiling of Bacterially Infected Host Cell-Derived Small Extracellular Vesicles by Surface-Enhanced Raman Scattering

Small extracellular vesicles (sEVs) are membrane-bound nanoparticles (<200 nm) released into bodily fluids, carrying proteins, lipids, and nucleic acids that reflect the state of their parent cells. In lung cancer, bacterial infections can worsen disease progression by altering sEV composition, influencing immune responses, metastasis, and therapy resistance. However, current tools are limited in profiling host-derived sEVs during host-pathogen interactions. Here, we present an integrated microfluidic platform for on-chip analysis of sEVs from A549 lung epithelial cells infected with <i>Pseudomonas aeruginosa</i> strain PAO1. This platform integrates host-pathogen coculture with on-chip sEV capture and multiplexed molecular profiling of host cell-derived sEVs. A two-layer modular design enables stepwise cell loading, controlled coculture, and efficient sEV capture through hydrophobic interactions between the sEV membrane and silane-modified polymer that modified on the chip. Multiplexed surface-enhanced Raman scattering (SERS) nanotags allow simultaneous detection of key sEV biomarkers from A549 cells (CD81, EGFR, EpCAM), and PAO1 (oprF). Through SERS mapping, molecular signatures across sEVs are spatially resolved, providing insights into compositional changes in sEVs following PAO1 exposure. By minimizing direct bacterial contact and relying on diffusion-driven capture, the system more closely mimics physiological infection processes.