Integrated Low-Speed Isolation and Ultrasensitive In-Situ Detection of Glioblastoma EVs via UiO-66-NH 2 @Au Nanocomposite with Temperature Synergistic Membrane Fusion and Y-EXPAR Assay

Extracellular vesicles (EVs) secreted by glioblastoma (GBM) cells carry tumor-specific biomarkers such as microRNA-1246 (miR-1246), offering significant potential for noninvasive GBM detection. However, the efficient isolation of EVs from complex biofluids and ultrasensitive detection of their low-abundance miRNAs encapsulated in EVs remain formidable challenges. To address this, we designed a multifunctional nanoplatform of UiO-66-NH 2 @Au@MSDC (UAM), which integrates EV capture and in situ miRNA analysis. The initial construction of UiO-66-NH 2 @Au (UA) provided a high density of thiol-binding sites for the attachment of mercapto (SH)-spacer-DNA-cholesterol (MSDC) probes to form UiO-66-NH 2 @Au@MSDC (UAM). The abundant cholesterol motifs on these probes subsequently enabled highly efficient EV capture, achieving a recovery rate of 84.8 ± 2.4% under a low centrifugal force of 8000 g while preserving EV structural integrity and bioactivity. For the downstream analysis of EVs, we developed an innovative EXPAR-based strategy, termed Y-EXPAR, to address the specificity challenge in detecting miR-1246. The entire detection system employs a temperature-synergistic strategy at 55 °C, which simultaneously triggers the membrane fusion of EVs with synthetic cationic liposomes (preloaded with Y-EXPAR reagents) and activates the Y-EXPAR. The resulting fusion creates confined nanoreactors that concentrate trace amounts of miR-1246, thereby enabling ultrasensitive detection with detection limits of 1.4 fM for miR-1246 and 22 particles/μL for EVs. This integrated platform overcomes key bottlenecks in EV-based liquid biopsy, providing a practical, low-equipment-demand approach for the early diagnosis of GBM and laying a foundation for advancing precision oncology diagnostics.