Lysis of Extracellular Vesicles and Multiplexed Protein Detection via a Reverse Phase Immunoassay Using a...

Extracellular vesicles (EVs) are cell-derived membrane-bound particles with molecular cargo reflective of their cell of origin. Analysis of disease-related EVs and associated cargo from biofluids is a promising tool for disease management. To facilitate the analysis of intravesicular molecules, EV lysis is needed. Moreover, highly sensitive and multiplexed detection methods are required to achieve early diagnostics. While cell lysis approaches have been well studied, the analysis of EV lysis methods and their effects on downstream molecular detection is lacking. In this work, we analyzed chemical, thermal, and mechanical EV lysis methods and determined their efficiency based on EV particle concentration and immunoassay activity. We, for the first time, discovered that vortex was an efficient EV lysis method and used it for detection of surface and intravesicular markers in a highly sensitive multiplexed reverse phase immunoassay on a gold-nanoparticle-embedded membrane. In phosphate-buffered saline, detection limits up to 3 orders of magnitude lower than enzyme-linked immunosorbent assay were achieved. In spiked human plasma, detection limits as low as 7.27 × 10⁴ EVs/mL were achieved, making it suitable for early diagnostics. These results demonstrated an effective pipeline for lysing and molecular analysis of EVs from complex biofluids, paving the way for their broad applications in biomedicine.