A triple-amplification differential pulse voltammetry for sensitive detection of DNA based on exonuclease III, strand displacement reaction and terminal deoxynucleotidyl transferase

In this research, a sensitive and specific electrochemical biosensor for DNA detection was constructed. The highly sensitivity of this biosensor is due to the exploitation of exonuclease III-assisted double recycling and toehold-mediated strand displacement recycling to achieve the target triple recycling amplification, thus generating a large amount of Y-shaped DNA structures. Combination with a terminal deoxynucleotidyl transferase (TDT)-mediated cascaded signal amplification strategy can catalyze the repetitive incorporation of biotin-dUTP to the 3'-OH of the Y-shaped DNA. Via biotin-streptavidin interaction, multiple streptavidin-alkaline phosphatases were conjugated to the surface of an Au electrode and generated a sharply increasing electrochemical signal in a 1-naphthyl phosphate (1-NP) solution. In this method, an impressive detection limit of 0.05 fM was obtained, presenting outstanding selectivity with a dynamic response scope between 0.1 fM and 1 nΜ. Thus, the designed biosensor opens an avenue for DNA detection in clinical molecular diagnostics, pathogen detection, gene therapy, food safety and environmental monitoring.