<i>In\nSitu</i>, Fusion-Free, Multiplexed\nDetection of Small Extracellular Vesicle miRNAs for Cancer Diagnostics

Tumor-derived small extracellular vesicle (sEV) microRNAs\n(miRNAs)\nare emerging biomarkers for cancer diagnostics. Conventional sEV miRNA\ndetection methods necessitate the lysis of sEVs, rendering them laborious\nand time-consuming and potentially leading to damage or loss of miRNAs.\nMembrane fusion-based <i>in situ</i> detection of sEV miRNAs\ninvolves the preparation of probe-loaded vesicles (<i>e.g.</i>, liposomes or cellular vesicles), which are typically sophisticated\nand require specialist equipment. Membrane perforation methods employ\nchemical treatments that can induce severe miRNA degradation or leaks.\nInspired by previous studies that loaded nucleic acids into EVs or\ncells using hydrophobic tethers for therapeutic applications, herein,\nwe repurposed this strategy by conjugating a hydrophobic tether onto\nmolecular beacons to aid their transportation into sEVs, allowing\nfor <i>in situ</i> detection of miRNAs in a fusion-free\nand multiplexing manner. This method enables simultaneous detection\nof multiple miRNA species within serum-derived sEVs for the diagnosis\nof prostate cancer, breast cancer, and gastric cancer with an accuracy\nof 83.3%, 81.8%, and 100%, respectively, in a cohort of 66 individuals,\nindicating that it holds a high application potential in clinical\ndiagnostics.