Rapid, high-throughput isolation of tumor specific small extracellular vesicles using radial flow microfluidic chip with IEDDA chemistry (ExoOnco ChipEpCAM−TCO)

Extracellular vesicles (sEVs) are promise as biomarkers for early cancer diagnostics and prognostics. Immunoaffinity-based isolation techniques using antibodies for specific sEV surface proteins offer high specificity and purity. However, researchers struggle with isolating rare sEV subtypes, achieving sufficient throughput and managing harmless release. To address these challenges, we developed the ExoOnco chip, a microfluidic device featuring a radial flow design with bean-shaped micro-posts that create a varying shear rate profile for efficient sEV capture. This device integrates the catalyst-free, biocompatible, and biorthogonal Inverse electron demand Diels-Alder (IEDDA), conjugated with antibodies for the rapid and precise isolation of rare sEV subtypes. Additionally, our modified chemistry incorporates a reducible disulfide bridge for simple release of captured sEVs. We successfully captured and released sEVs expressing high levels of epithelial cellular adhesion molecule (EpCAM) from cell line media and non-small cell lung cancer (NSCLC) patient plasma. Following captured, we show the potential for characterizing isolated sEV using WB and micro-bicinchoninic acid assay. We have illustrated our device's specificity towards tumor derived sEVs (TDEs) utilizing patient and healthy plasma to show a significant difference in TAA expression level using dPCR analysis. By implementing the rapid IEDDA chemistry and functional disulfide bridge, the improved ExoOnco Chip facilitates the isolation and release of rare TDEs, enabling further investigation of their bioactive constituents. This technology opens avenues for advancements in early-stage cancer diagnosis and adaptive immunotherapies based on these bioactive constituents.