DNA encoded multi-round profiling of extracellular vesicle membrane proteins for cancer diagnostics

The rising global burden of cancer underscores the urgent demand for minimally invasive precision diagnostic methods. Extracellular vesicles (EVs) are emerging cancer liquid biopsy biomarkers carrying promising molecular markers such as membrane proteins. However, conventional approaches to EV membrane protein profiling remain limited by low multiplexing capability, high sample consumption, and complex operational workflows. Herein, we report a DNA encoded multi-round profiling of extracellular vesicle membrane proteins for cancer diagnostics (DETECT) strategy that enables detection of EV membrane proteins with high sensitivity and scalability. This method leverages engineered aptamer probes to facilitate the capture and multi-round in situ detection of 9 EV surface proteins. DETECT integrates aptamer recognition with hybridization chain reaction (HCR) for signal amplification, followed by enzymatic cleavage for complete signal erasure, thereby enabling cyclic detection of multiple protein targets on the same EVs population. Clinical validation with EVs isolated from 48 serum samples of three cancer (gastric, breast, and prostate) demonstrated DETECT's capability to uncover cancer-specific membrane protein fingerprints, which achieved 100 % accuracy in differentiating cancers from noncancers and 83.3 % classification accuracy in differentiating three cancer types. DETECT represents a feasible, robust, and scalable technical platform for profiling EV surface proteins that shall hold great application potential in cancer diagnostics and beyond.