Simple and Rapid Tumor EV Enrichment Enabled by Long DNA Probe‐Guided Entanglement

Specific subpopulations of extracellular vesicles (EVs) hold significant clinical potential for biomarker discovery, disease diagnosis, and therapeutic agents. However, this field remains underutilized due to the lack of straightforward and versatile techniques for isolating EV subpopulations from biofluids. Here, we present LODGE, a long DNA probe‐guided EV entanglement strategy for the simple, rapid, and selective enrichment of tumor‐derived EVs (tEVs) from clinical specimens. LODGE uses two long DNA affinity probes to recognize specific subpopulations, causing them to aggregate with the assistance of splint strands, thereby achieving non‐destructive, high‐yield, and high‐purity separation of tEVs within a short period. Proteomic analysis revealed that the isolated tEVs contributed to the identification of tumor‐associated biomarkers compared to total EVs. Additionally, by incorporating a split G‐quadruplex‐containing molecular trap domain, a novel structure that significantly improves the fluorescence emission of thioflavin T (ThT), into DNA affinity probes, we developed an innovative LODGE‐ThT sensing strategy for the highly sensitive profiling of multiple tEV subpopulations. Using data from the tEVs alongside clinical indicators processed with machine learning algorithms, we effectively classified five tumor types. Our results show that LODGE is a promising tool for identifying specific EV subpopulations, fostering their biomedical applications.