Detecting low-frequency mutations within a high-abundance wild-type (WT) background is essential for precision cancer diagnostics. Standard methods like qPCR and NGS, while effective, are hindered by high costs, complexity, and lengthy workflows. Electrochemical biosensors using mutation-specific capture probes offer a simpler, cost-effective alternative but lack the selectivity needed for detecting low-abundance mutations. To address this, we developed a high-throughput platform for systematically evaluating the hybridization affinity between biorecognition elements (capture probes) and target DNA under varied conditions.