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

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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.

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