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

Abstract

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.