Simple and rapid profiling of tumor EVs for differential diagnosis of NSCLC via orthogonal barcode-driven CRISPR/Cas12a

Tumor-derived extracellular vesicles (tEVs), a class of nanoscale vesicles actively released by malignant cells, have emerged as attractive biomarkers for non-invasive cancer diagnosis. However, their clinical translation remains challenging due to low abundance, molecular heterogeneity, and the requirement for multiplexed surface marker discrimination. Here, we report a dual aptamer-mediated CRISPR/Cas12a-assisted sensing platform (DA-CAS) for rapid and orthogonally programmable dual-marker profiling of tEVs, enabling differential diagnosis of non-small cell lung cancer (NSCLC) using only 10 µL of plasma within 100 min. The DA-CAS system integrates proximity ligation-based dual-marker recognition with hyperbranched rolling circle amplification (HRCA) to generate programmable DNA barcodes, which selectively trigger Cas12a trans-cleavage in an orthogonal manner. Using EpCAM and PD-L1 as representative surface markers, the platform achieves subtype-specific detection of NSCLC-derived tEVs with minimal background activation and a detection limit as low as 75 particles/mL. Moreover, a portable lateral flow readout enables accurate, instrument-free visual detection at concentrations down to 406 particles/mL. Under the condition of free-ultracentrifugation, clinical validation using a cohort of 45 plasma samples demonstrated a sensitivity of 97%, specificity of 88%, and overall diagnostic accuracy of 96%, outperforming conventional ELISA assays and multi-marker serum panels in both analytical sensitivity and subtype resolution. In addition, this platform demonstrated preliminary potential for discriminating between benign and malignant lung diseases and for dynamically monitoring radiotherapeutic responses. The orthogonal barcode design effectively eliminates inter-channel crosstalk and enzymatic interference, enabling orthogonal dual-target recognition with high subpopulation specificity. Overall, DA-CAS provides a robust, rapid, and point-of-care-compatible strategy for tEV-based cancer diagnostics, offering strong translational potential for non-invasive tumor profiling and dynamic immune status monitoring.