A novel exonuclease III-aided amplification assay based on a graphene platform for sensitive detection of adenosine triphosphate

We have developed a novel exonuclease III (Exo III)-aided amplification assay based on a graphene platform for sensitive detection of adenosine triphosphate (ATP). This system consists of fluorescein isothiocyanate (FITC)-labeled ATP aptamers, graphene and Exo III. In the presence of target ATP, the target molecule binding the aptamer leads to the formation of aptamer-target complexes. Subsequently, Exo III selectively catalyzes the stepwise removal of mononucleotides from aptamer-target complexes, resulting in the removal of FITC and the release of target ATP. The released target ATP then binds with another FITC-labeled ATP aptamer probe, and the cycle starts anew, resulting in the continuous cleavage of aptamer-target complexes. Consequently, each target ATP can initiate the cleavage of many FITC-labeled ATP aptamer probes, generating a concomitant increase in the fluorescence. Finally, graphene is added to quench the fluorescence of rudimental FITC-labeled aptamer probes, and the fluorescence from the free FITC is detected. With this approach, the fluorescence intensity of the system increases with the increase in the concentration of ATP over a range of 10-1000 nM with a detection limit of 5 nM. The present method was successfully applied for the detection of ATP in human serum.