Precise Mutation Detection in Low-Abundance Tumor-Derived Extracellular Vesicles via Microfluidic Immunomagnetic Reversible Affinity Capture

Abstract

Molecular diagnostic technologies based on liquid biopsy are increasingly vital for clinical tumor molecular profiling. Compared to highly fragmented and easily degradable cell-free DNA (cfDNA), extracellular vesicles (EVs) offer distinct advantages by protecting nucleic acids from degradation. Enrichment of tumor-derived EVs using specific surface markers significantly enhances the relative abundance of mutant nucleic acids, establishing a foundation for highly sensitive mutation detection. To fully leverage this enrichment strategy, we developed the MIRACLE platform, an integrated system combining microfluidic magneto-controllable reversible affinity capture with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for efficient, multiplexed detection of tumor-derived EV DNA mutations. The platform utilizes anti-EpCAM-functionalized magnetic beads to construct a reversible capture interface within a herringbone-structured microfluidic chip, achieving over 98% capture efficiency of tumor-derived EVs under turbulence-enhanced conditions. Following EV lysis, a 7-plex multiplex single base extension assay coupled with MALDI-TOF-MS analysis enables simultaneous and accurate identification of seven mutation sites with single-nucleotide resolution. Furthermore, the platform achieved a detection limit of 0.05% for tumor-derived EVs within a background of wild-type EVs. Validation using plasma samples from 26 colorectal cancer patients demonstrated 96.2% accuracy and 93.3% sensitivity, successfully detecting key mutations including G12V, G13D, and G12D. The tumor EV enrichment-guided mutation detection strategy established in this study provides a precise, multiplexed, and clinically applicable approach for molecular profiling in liquid biopsy.