An Integrated Cascade‐Amplified Microfluidic Biosensor for Reconfigurable Profiling of CRC‐Associated Faecal Extracellular Vesicle Surface Proteins

Faecal extracellular vesicles (EVs) provide disease‐relevant molecular information for non‐invasive colorectal cancer (CRC) analysis. However, sensitive EV surface protein profiling remains challenging because low‐abundance targets are obscured by heterogeneous vesicles and complex matrices. Here, an integrated cascade‐amplified microfluidic biosensor was developed for reconfigurable profiling of EV surface proteins, combining EV capture, signal amplification, and fluorescence detection in a single workflow. CD63 aptamer‐functionalized Y‐shaped micropillar arrays capture EVs. Target‐specific aptamer‐initiator conjugates convert protein recognition into nucleic‐acid outputs to trigger entropy‐driven catalysis (EDC) and DNAzyme amplification. After magnetic removal of residual probes, output strands activate secondary DNAzyme‐mediated amplification in the detection chamber to generate amplified fluorescence signals. Based on this modular target‐trigger design, the platform enables reconfigurable multi‐marker profiling of EpCAM, EGFR, and HSP70, with limits of detection at 98, 115, and 103 particles mL −1 , respectively. In faecal samples from 13 CRC patients and 12 healthy donors, the three‐marker combined analysis yielded exploratory signal separation (AUC = 0.987) within this limited cohort, demonstrating the platform's analytical capability for EV surface protein profiling in complex faecal specimens. This modular microfluidic system provides a versatile analytical tool for multiplex EV protein measurement, which can be readily adapted to other biomarker panels and biological specimens.