Low-Cost Platform for Multiplexed Electrochemical Melting Curve Analysis

Detection and identification of single nucleotide polymorphisms (SNPs) have garnered increasing interest in the past decade, finding potential application in detection of antibiotic resistance, advanced forensic science, as well as clinical diagnostics and prognostics, moving toward the realization of personalized medicine. Many different techniques have been developed for genotyping SNPs, and ideally these techniques should be rapid, easy-to-use, cost-effective, flexible, scalable, easily automated, and requiring minimal end-user intervention. While high-resolution melting curve analysis has been widely used for the detection of SNPs, fluorescence detection does not meet many of the desired requirements, and electrochemical detection is an attractive alternative due to its high sensitivity, simplicity, cost-effectiveness, and compatibility with microfabrication. Herein, we describe the multiplexed electrochemical melting curve analysis of duplex surfaces tethered to electrodes of an array. In this approach, thiolated probes designed to hybridize to a DNA sequence containing the SNP to be interrogated are immobilized on gold electrodes. Asymmetric PCR using a ferrocene-labeled forward primer is used to generate this single-stranded redox-labeled PCR amplicon. Following hybridization with the probe immobilized on the electrode surface, the electrode array is exposed to a controlled ramping of temperature, with concomitant constant washing of the electrode array surface while simultaneously carrying out voltammetric measurements. The optimum position of the site complementary to the SNP site in the immobilized probe to achieve maximum differentiation in melting temperature between wild-type and single base mismatch, thus facilitating allelic discrimination, was determined and applied to the detection of a cardiomyopathy associated SNP.