DNA Tetrahedral Nanoprobes-Based Logic-Gated FRET Sensor for in Situ Analysis of Dual MicroRNAs in Extracellular Vesicles

Abstract Conventional analysis of extracellular vesicle (EV)-derived microRNAs relies on vesicle lysis and RNA extraction, which disrupt native vesicular structure and eliminate intravesicular molecular cargoes. Here, we report a rigid DNA tetrahedral nanoprobes-based logic-gated Förster resonance energy transfer (FRET) platform for in situ, amplification-free detection of dual miRNAs directly within intact EVs. Tetrahedral DNA nanostructures (TDNs) targeting miR-21 and miR-141 were engineered with concealed sticky ends that become exposed upon target binding, triggering conditional interparticle assembly and FRET activation exclusively under dual-input conditions. This design physically implements an AND logic operation, converting miRNA coexpression into unified ratiometric output. The rigid tetrahedral scaffold constrains fluorophore spacing and reduces conformational entropy, enabling precise distance-modulated signal transduction with a detection limit of 12.86 pM for dual miRNAs without enzymatic amplification. In clinical plasma samples from 20 prostate cancer (PCa) patients and 20 healthy controls, the ratiometric FRET signal achieved an area under the curve (AUC) of 0.903 and 85.0% diagnostic accuracy, outperforming single-channel measurements. By integrating structural programmability with molecular logic sensor, this strategy transforms EV biomarker analysis from independent signal acquisition into intrinsic nanoscale information processing, establishing a robust platform for noninvasive liquid biopsy.