MBD-Functionalized Magnetic Nanoparticles for Direct Enrichment of Plasma Methylated DNA in High-Sensitivity Liquid Biopsy

DNA methylation serves as a crucial biomarker for early cancer detection, holding great promise in liquid biopsy. However, its low abundance in plasma and interference from complex matrices pose significant challenges to clinical detection sensitivity. To this end, this study developed a novel direct enrichment platform based on methyl-binding domain (MBD) protein-functionalized magnetic nanoparticles (MBD@MNPs). Through systematic optimization, the methylated DNA (mDNA) capture efficiency of MBD@MNPs reached 86.6% with an elution efficiency of 44.9% in simulated systems, while bypassing the cumbersome step of total cell-free DNA (cfDNA) extraction and overcoming plasma matrix interference. In comparison, this strategy enhanced the sensitivity of methylation-specific PCR by 25-fold, with a detection limit as low as 0.04 ng/mL. Applied to plasma samples from patients with various tumors (including colorectal, lung, breast, cervical, liver, and gastric cancers), the technology significantly improved the detection rates up to 100% of tumor-specific methylation biomarkers, such as SDC2, SHOX2, RASSF1A, ZNF671, Septin9, and BMP3. Therefore, this study provides an efficient, universal and user-friendly enrichment and accurate detection of mDNA, laying a methodological foundation for early cancer screening and prognosis assessment.