Development of real-time assays for impedance-based detection of microbial double-stranded DNA targets

A real-time, label free assay was developed for microbial detection, utilizing double-stranded DNA targetsand employing the next generation of an impedimetric sensor array platform designed by Sharp Labo-ratories of America (SLA). Real-time curves of the impedimetric signal response were obtained at fixedfrequency and voltage for target binding to oligonucleotide probes attached to the sensor array surface.Kinetic parameters of these curves were analyzed by the integrated data analysis package for signal quan-tification. Non-specific binding presented a major challenge for assay development, and required assayoptimization. For this, differences were maximized between binding curve kinetic parameters for probesbinding to complementary targets versus non-target controls. Variables manipulated for assay opti-mization included target concentration, hybridization temperature, buffer concentration, and the use ofsurfactants. Our results showed that (i) different target–probe combinations required optimization of spe-cific sets of variables; (ii) for each assay condition, the optimum range was relatively narrow, and had to bedetermined empirically; and (iii) outside of the optimum range, the assay could not distinguish betweenspecific and non-specific binding. For each target–probe combination evaluated, conditions resulting ingood separation between specific and non-specific binding signals were established, generating highconfidence in the SLA impedimetric dsDNA assay results.