Abstract
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 (k(d)) between perfect match (PM) and mismatch (MM) duplexes at constant temperature, we achieved statistically significant differentiation of homozygous and heterozygous alleles (>2σ, n = 7). As proof of concept, the biosensor was applied to detect the CYP2C19*17 allele in human saliva samples (n = 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.