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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.<\/jats:p>","DOI":"10.1021\/acs.analchem.6c05088","type":"journal-article","created":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T15:45:45Z","timestamp":1788450345000},"page":"27226-27234","source":"Crossref","is-referenced-by-count":0,"title":["Precise Mutation\nDetection in Low-Abundance Tumor-Derived\nExtracellular Vesicles via Microfluidic Immunomagnetic Reversible\nAffinity Capture"],"prefix":"10.1021","volume":"98","author":[{"given":"Xing","family":"Xu","sequence":"first","affiliation":[{"name":"Fujian Medical University , , ,","place":["Fuzhou, China, 350005"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shanshan","family":"Liang","sequence":"additional","affiliation":[{"name":"The First Affiliated Hospital of Xiamen University, Xiamen University , , ,","place":["Xiamen, China, 361005"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hengkai","family":"Chen","sequence":"additional","affiliation":[{"name":"First Affiliated Hospital of Fujian Medical University , , ,","place":["Fuzhou, China, 350001"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tianchen","family":"Lan","sequence":"additional","affiliation":[{"name":"The First Affiliated Hospital of Xiamen University, Xiamen University , , ,","place":["Xiamen, China, 361005"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinyan","family":"Peng","sequence":"additional","affiliation":[{"name":"Union Hospital of the Fujian Medical University , , ,","place":["Fuzhou, China, 350001"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yupeng","family":"Chen","sequence":"additional","affiliation":[{"name":"First Affiliated Hospital of Fujian Medical University , , ,","place":["Fuzhou, China, 350001"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guanlong","family":"Wei","sequence":"additional","affiliation":[{"name":"Fujian Medical University , , ,","place":["Fuzhou, China, 350005"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Canjie","family":"Liao","sequence":"additional","affiliation":[{"name":"Fujian Medical University , , ,","place":["Fuzhou, China, 350005"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ao","family":"Li","sequence":"additional","affiliation":[{"name":"First Affiliated Hospital of Fujian Medical University , , ,","place":["Fuzhou, China, 350001"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhiwei","family":"Liu","sequence":"additional","affiliation":[{"name":"Union Hospital of the Fujian Medical University , , ,","place":["Fuzhou, China, 350001"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rui","family":"Su","sequence":"additional","affiliation":[{"name":"The First Affiliated Hospital of Xiamen University, Xiamen University , , ,","place":["Xiamen, China, 361005"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinfu","family":"Zhuang","sequence":"additional","affiliation":[{"name":"First Affiliated Hospital of Fujian Medical University , , ,","place":["Fuzhou, China, 350001"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2374-5342","authenticated-orcid":true,"given":"Chaoyong","family":"Yang","sequence":"additional","affiliation":[{"name":"The First Affiliated Hospital of Xiamen University, Xiamen University , , ,","place":["Xiamen, China, 361005"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,2]]},"reference":[{"issue":"7800","key":"2026091511075739200_cit1","doi-asserted-by":"publisher","first-page":"S9","DOI":"10.1038\/d41586-020-00844-5","article-title":"Perspective:\nThe future of liquid biopsy","volume":"579","author":"Alix-Panabieres","year":"2020","journal-title":"Nature"},{"issue":"12","key":"2026091511075739200_cit2","doi-asserted-by":"publisher","first-page":"E550","DOI":"10.1016\/S1470-2045(20)30687-2","article-title":"The future of liquid biopsy","volume":"21","author":"Biswas","year":"2020","journal-title":"Lancet Oncol."},{"issue":"9","key":"2026091511075739200_cit3","doi-asserted-by":"publisher","first-page":"531","DOI":"10.1038\/nrclinonc.2017.14","article-title":"Integrating liquid\nbiopsies into the management of cancer","volume":"14","author":"Siravegna","year":"2017","journal-title":"Nat.\nRev. 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