A fusion-mediated FRET approach for rapid analysis of extracellular vesicle miRNA as a diagnostic biomarker in ischemic stroke

Ischemic stroke is an acute neurological emergency caused by cerebral blood flow obstruction, with current diagnosis heavily dependent on time-consuming neuroimaging techniques that often delay critical intervention. Extracellular vesicles (EVs) have emerged as promising biomarker carriers for brain disorders due to their ability to cross the blood-brain barrier, yet their clinical translation has been hindered by complex isolation and detection requirements. Here, we develop a novel wash-free analytical platform leveraging liposome-EV fusion mediated by membrane lipid fluidity and integrated with Förster resonance energy transfer (FRET) technology. This innovative approach enables direct, rapid quantification of both EV concentration and EV-encapsulated miRNA in native biofluids without requiring prior EV separation, overcoming key limitations of conventional methods. When applied to clinical plasma samples, our method demonstrated high sensitivity with detection limits of 4.272 × 1011 particle/mL for EVs and 0.779 nM for miRNA. While no significant difference in total EV concentration was observed between ischemic stroke patients and hypertensive controls, EV-derived miRNA-21 levels were markedly elevated in patient samples, showing exceptional diagnostic performance (AUC = 0.915). This fusion-mediated FRET platform represents a significant advancement in EV-based diagnostics, offering a rapid, sensitive tool with substantial potential for point-of-care stroke diagnosis and timely intervention.