(150d) Evaluation of Azure a Redox Dye As DNA Hybridization Indicator Using Electrochemical Methods

Over the last two decades, DNA-based electrochemical biosensors have captured an increasing attraction as base-pairing interactions between two complementary strands of nucleic acids offer high sensitivity and selectivity for biosensing applications. An electrochemical DNA biosensor can be designed either as label-based or label-free. The target DNA sequence or probe DNA receptor is labeled with redox-active molecules in label-based sensors while redox molecules such as intercalator dyes are used as DNA hybridization indicators in the label-free detection method. To date, various intercalator molecules have been used in biosensing applications such as methylene blue, ethidium bromide, doxorubicin, and meldola’s blue. Here, we investigated the use of Azure A redox molecule, a phenothiazinium dye, in the intercalation-based DNA biosensor system developed to detect the ctxA gene of pathogenic V. cholerae bacteria.

For electrochemical sensing, the 5’ of capture DNA probe was functionalized with biotin. High-affinity binding chemistry of streptavidin-biotin was used for attaching the biotinylated capture DNA probe onto the screen-printed carbon electrode surface. Selective hybridization between capture probe and complementary target DNA strands results in double stranded DNA sequences. The intercalation of redox dyes between the base pairs of DNA double helix allows long-range electron transfer between the Azure A and the carbon electrode surface, which results in a unique concentration dependent current change, distinct from the redox potential of Azure A, as measured by square wave voltammetry (SWV). Capture probe density, intercalator concentration, and intercalation duration were optimized. All SWV measurements were employed at 25 mV amplitude and 50 Hz frequency.

The results of this work suggest that Azure A may provide superior limits of detection for electrochemical intercalation-based biosensor systems.