Controlling non-specific adsorption in electrochemical DNA methylation assays by probe architecture and surface blocking protein type

DNA methylation is a clinically relevant biomarker, but its reliable electrochemical detection is limited by low target abundance and non-specific adsorption of enzyme-labelled immunoreagents. Here, we developed an affinity-based electrochemical DNA hybridization assay for methylated DNA (5-mC DNA), focusing on the suppression of non-specific adsorption of rabbit anti-mouse HRP-IgG used for signal generation. Four thiolated DNA probes of different lengths were evaluated in combination with protein blocking agents, including BSA, casein, and gelatin. Methylated DNA was recognized by anti-5-mC and HRP-IgG, followed by amperometric detection at -0.25 V using the H₂Q/H₂O₂ system. The optimized Probe 1/BSA interface enabled selective discrimination between methylated and non-methylated DNA, with good repeatability for 1.00 × 10^-9 M 5-mC DNA, expressed as an RSD of 4.63%. Distinguishable responses were obtained down to 1.00 × 10^-11 M, the lowest concentration tested. The sensor also retained its response in a synthetic biological matrix containing 5-mC DNA, a 100-fold excess of non-methylated DNA, fragmented salmon sperm DNA, human serum albumin, and human IgG. SPR measurements under microfluidic flow confirmed the role of probe architecture and blocking chemistry in controlling HRP-IgG adsorption. Overall, these results demonstrate that rational interfacial design is essential for reliable affinity-based electrochemical methylated DNA detection.