A new method for the detection of adenosine based on time-resolved fluorescence sensor

In this work, we report a thrombin binding aptamer complex based time-resolved fluorescence sensor for small molecule detection. The sensor employs two strands (DNA1 and DNA2) of oligonucleotides. This two strands of oligonucleotides contain two aptamer (α-aptamer and β-aptamer) respectively. DNA1 and DNA2 were labeled with biotin and DIG at the 3′-end, respectively. Binding of the α-aptamer and β-aptamer to the thrombin promotes the hybridization between the complementary stem sequences attached to the two oligonucleotide sequences. The hybridization then brings biotin to be hidden in the shield part on DNA1, shielding biotin from being approached by the streptavidin modified on the microplate due to the steric hindrance effect of the shield part of DNA1. Result in the thrombin-aptamer complex cannot be modified on the surface of microplate which further leads to no signal reported. The strategy integrates the distinguishing features of aptamer and fluorescent techniques. As a proof-of-principle, adenosine in serum was detected with a detection limit of 0.5 nM. A nice detection limit and linear relationship were obtained.