Constant Temperature Electrochemical Biosensor for SNP Detection in Human Genomic DNA Based on DNA Melting Analysis

Detection of single-nucleotide polymorphisms (SNPs) is critical in both bioanalytical science and clinical diagnostics. We present an electrochemical biosensor capable of SNP detection in human genomic DNA immediately following a polymerase chain reaction, eliminating the need for temperature gradients. The biosensor employs a "sandwich" hybridization format in which a target DNA strand binds to an electrode-anchored probe and is interrogated by two allele-specific reporters. By analyzing the kinetic differences in melting rates (<i>k</i>_d) between perfect match (PM) and mismatch (MM) duplexes at constant temperature, we achieved statistically significant differentiation of homozygous and heterozygous alleles (>2σ, <i>n</i> = 7). As proof of concept, the biosensor was applied to detect the <i>CYP2C19*17</i> allele in human saliva samples (<i>n</i> = 6), with results confirmed using sequencing. Additionally, melting-rate-derived Gibbs free energy differences allowed for the identification of previously undetected mismatches, suggesting a novel pathway for electrochemical sequencing.