In Situ , Fusion-Free, Multiplexed Detection of Small Extracellular Vesicle miRNAs for Cancer Diagnostics.

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