A terminal protection system for the detection of adenosine triphosphate via enzyme-assisted signal amplification

In this study, we have developed a biosensor to detect adenosine triphosphate (ATP), based on fluorescence resonance energy transfer (FRET) and making use of the activities of exonuclease I (EXO I) and exonuclease III (EXO III). In the absence of ATP in the biosensor reaction system, the aptasensor is hydrolyzed by EXO I. When ATP is present, it conjugates with the aptasensor and protects it from hydrolysis by EXO I; the aptasensor can then hybridize with a fluorescent sequence linked to graphene oxide (GO). The dsDNA formed by the interaction between the aptasensor and the fluorescent sequence is then recognized and cleaved by EXO III. The increased distance between the fluorescent particle (FAM, 6-carboxyfluorescein) and the GO significantly hinders the FRET and increases the fluorescence of FAM. By incorporating EXO III into the process, the fluorescence signals of the biosensor are therefore greatly amplified and they were found to displayed a good linear relationship with ATP concentration, in the range from 0 to 3 mu M. This system can be widely employed for the detection of other biological molecules.