Modulating nanoconfined space charge for PCR-free and bisulfite-free detection of cfDNA methylation in ovarian cancer

Aberrant DNA methylation in circulating cell-free DNA (cfDNA) represents a critical epigenetic biomarker for noninvasive diagnosis of ovarian cancer. However, conventional methodologies are inherently limited by the destructive nature of bisulfite conversion and the operational complexities of PCR thermal cycling. Herein, we report a bisulfite-free and PCR-free electrochemical nanochannel biosensor that modulates nanoconfined space charge to directly translate discrete epigenetic marks into measurable ion flux. Specifically, rigid DNA tetrahedrons (TDs) are covalently assembled onto anodic aluminum oxide (AAO) nanochannels, providing a sterically unhindered interface for highly efficient cfDNA capture. Target discrimination is achieved via HhaI endonuclease, which selectively cleaves unmethylated cfDNA (UME) while leaving methylated cfDNA (ME) structurally intact due to methylation-induced steric hindrance. The retained ME targets subsequently initiate an in situ hybridization chain reaction (HCR) cascade. Through this isothermal assembly, densely packed polyanionic DNA networks form at the outer surface of the nanochannels, reshaping the local electrostatic field into a high-density negative space-charge layer. This designed space charge enriches cations and reduces the access resistance, yielding a significantly amplified transmembrane ion current. Benefiting from this dual-amplified transduction mechanism, the biosensor achieves a limit of detection of 208 aM with high specificity. Furthermore, this platform successfully discriminated ovarian cancer patients from those with benign gynecological diseases using clinical plasma samples, demonstrating great translational potential for precise epigenetic liquid biopsy.