Site-Specific DNA methylation detection using nonlinear hybridization chain reaction

DNA methylation at specific CpG sites plays a critical role in the diagnosis and treatment of human diseases. In this study, we introduce a site-specific DNA methylation detection biosensor that leverages immune recognition and nonlinear hybridization chain reactions (NHCR). Since anti-methylcytosine antibodies are unable to recognize methylcytosine within a DNA duplex, we specifically target the CpG site by removing the guanine complementary to the target methylcytosine in the capture probe, thereby creating a protruding region accessible for antibody recognition. We utilized NHCR as a signal amplification strategy to enable site-specific DNA methylation detection under isothermal and enzyme-free conditions. The proposed method achieves a detection limit of 0.15 pM and can differentiate methylation levels as low as 0.1 %, even in the presence of a large excess of unmethylated DNA. This approach is capable of detecting DNA methylation at specific CpG sites in genomic DNA extracted from human hepatocellular carcinoma (HCC) tissues, showcasing its considerable potential for biological research and disease diagnosis.