Bisulfite-free detection of SEPT9 DNA methylation using a novel dipyridamole-based fluorometric assay

Circulating tumor DNA (ctDNA) methylation profiling is rapidly emerging as a promising approach for the early detection of cancer. In colorectal cancer (CRC), aberrant methylation of the SEPT9 gene has been established as a reliable biomarker for early-stage diagnosis in clinical settings. However, current detection methods for DNA methylation rendered by bisulfite conversion along with probe based quantitative real-time polymerase chain reaction (qPCR) are labor-intensive and time-consuming. In this study, we present a novel Dipyridamole-based fluorometric nanobiosensor using gold-coated silica (Au@SiO 2 ) nanoparticles (NPs) for sensitive and specific detection of DNA methylation of SEPT9 gene for CRC. The sensor leverages the fluorescence quenching and enhancement properties of Dipyridamole upon hybridization with methylated and unmethylated DNA sequences. By optimizing hybridization conditions and utilizing Au@SiO 2 NPs functionalized with single-stranded DNA probes, we achieved superior sensitivity and selectivity in distinguishing methylated from unmethylated sequences. The nanobiosensor demonstrated a detection limit as low as 1.5 × 10 −16 M for unmethylated and 2.25 × 10 −16 M for methylated, and was successfully applied to human plasma samples, showing an average recovery rate of ~80 %. This study presents a novel approach to ctDNA methylation testing, offering several potential advantages over traditional techniques, including real-time detection, reduced sample processing time for scalable diagnostic applications in early cancer detection.