Separation-Free Extracellular Vesicle Microribonucleic Acid Profiling Using Structurally Oriented Membrane Fusion and Spatially Confined Amplification.

Current methods for profiling of extracellular vesicle (EV) microRNAs (miRNAs) involve either PCR or sequencing analysis of EV lysate or delivery of RNA probes into EVs for in situ hybridization, which suffer from complex protocols and low delivery efficiency/limited sensitivity, respectively. To overcome this limitation, we present a programmable, aptamer-mediated membrane fusion strategy that integrates aptamer-functionalized liposomes with nucleic acid nanostructures-Dual-color, Bone-shaped Biosensor-loaded Aptamer-Liposome (DBS-Alip) for rapid, one-step, in situ detection of multiple tumor-specific EVs-derived miRNAs. DBS-Alip selectively fuses with intact EVs, enabling direct intravesicular delivery of DBS that triggers nonenzymatic signal amplification within confined nanospaces, producing an enhanced fluorescence signal within 30 min. This platform achieved an ultralow detection of 0.5 fM using only 5 μL of plasma, eliminating the need for RNA extraction or amplification while maintaining high sensitivity and specificity. In clinical validation, DBS-Alip accurately distinguished early-stage nonsmall cell lung cancer patients from healthy donors (100% accuracy) and pneumonia cases (90% accuracy). This isolation-free, multiplexed detection platform offers a robust and programmable EV-based liquid biopsy strategy with a strong potential for early cancer diagnosis.