SAFE: a Mix‐and‐Read Assay for miRNA Detection in Extracellular Vesicles From Unprocessed Plasma Toward Clinical Disease Diagnosis

ABSTRACT Rapid and accurate diagnosis of cardiovascular disease (CVD) is essential for timely intervention, yet current workflows often rely on hospital‐based instrumentation that can delay decision‐making. We present SAFE (Sonication‐Assisted liposome Fusion with Extracellular vesicles (EVs)), a rapid, direct‐from‐plasma assay that quantifies microRNAs (miRNAs) encapsulated within EVs in approximately 10 min, providing a molecular signature of myocardial injury. SAFE delivers molecular beacons (MBs) into endogenous EVs through liposome fusion, eliminating the need for EV isolation and RNA extraction while minimizing interference from plasma proteins by confining hybridization within the EV lumen. Brief sonication accelerates fusion kinetics, shortening the assay time to 10 min. In a pilot clinical evaluation involving patients with CVD ( n = 20) and healthy controls ( n = 15), SAFE detected elevated levels of two cardiac‐associated miRNAs (miR‐133a and miR‐208a), achieving a diagnostic accuracy of 91.4% for each. These findings position SAFE as a rapid and minimally processed platform for the direct quantification of EV‐associated miRNAs, well suited for time‐critical CVD triage and ready for implementation studies across diverse clinical environments.