A sensitive ligase-based ATP electrochemical assay using molecular beacon-like DNA

A sensitive and selective ligase-based signal-on electrochemical sensing method for adenosine-5′-triphosphate (ATP) detection had been developed using molecular beacon (MB)-like DNA. In this method, the biotin-tagged MB-like DNA was self-assembled onto a gold electrode to form a stem-loop structure by means of facile gold-thiol chemistry, which resulted in blockage of electronic transmission. It was eT OFF state. In the presence of ATP, two nucleotide fragments which were complementary to the loop of the MB-like DNA could be ligated by the ATP-dependent T4 DNA ligase. Hybridization of the ligated DNA with the MB-like DNA induced a significant conformational change in this surface-confined DNA structure, which in turn released the biotin from the surface allowing free exchange of electrons with the electrode generating a measurable electrochemical signal (eT ON). The resulting change in electron transfer efficiency was readily measured by differential pulse voltammetry at target ATP concentrations as low as 0.05 nM and with linear response range from 0.1 to 1000 nM. Moreover, it was also able to discriminate ATP from its analogues. The proposed method had been successfully applied to the determination of ATP in the Escherichia coli O157:H7 extracts of water samples, and the linear response was found between the concentrations of 103 and 107 cfu/mL.