Exonuclease I assisted fluorometric aptasensor for adenosine detection using 2-AP modified DNA

A novel sensitive fluorescence method was developed for adenosine detection by using adenosine aptamer, Exo I and a 2-aminopurine-modified DNA (2-AP probe). 2-AP probe was completely complementary to adenosine aptamer. This strategy took advantage of the high binding affinity of adenosine aptamer and the susceptibility of 2-aminopurine (2-AP) to the local base stacking environment. In the absence of the adenosine, the formation of double-stranded DNA (dsDNA) through the hybridization of the aptamer with the 2-AP probe prevented the digestion of 2-AP probe by Exo I. The fluorescence of 2-AP was significantly quenched due to the base-stacking interaction. In the presence of adenosine, the binding of adenosine with the aptamer prevented the hybridization between aptamer and 2-AP probe, leading to the digestion of 2-AP probe by Exo I and the release of 2-AP. The fluorescence was recovered. A limit of detection of 0.30 μM and a linear range from 10 μM to 600 μM (R2 = 0.9933) were achieved by this assay. Moreover, adenosine analogs did not produce any significant change in the fluorescence intensity response, indicating good selectivity of this sensor for adenosine detection. Finally, it was used for the diluted serum sample.