Novel strategy to improve the sensing performances of split ATP aptamer based fluorescent indicator displacement assay through enhanced molecular recognition

Split aptamer strategy was often used to improve the sensitivity of aptasensor. However, traditional split aptamer strategy can not be directly used to improve the label-free aptamer based Thioflavin T (ThT) displacement assay for ATP because the split ATP aptamer display much lower enhancement effects on the fluorescence of ThT than intact aptamer. In order to address this issue, this is the first report using G-rich DNA sequence to enhance the affinity of the two split ATP aptamer halves to ThT and offer lower limit of detection (LOD), wider linear range and higher selectivity through the enhanced molecular recognition. Compared to the intact aptamer/ThT complex, the ensemble of two G-rich split ATP aptamer fragments/ThT are higher fluorescent. Consequently, G-rich sequences would improve the fluorescent signal and thus the sensing performance of the proposed assay. In the optimized conditions, the LOD of the proposed fluorescent ATP aptasensor is 2 nM, which is lower than the reported ThT/ATP aptamer based methods. Additionally, our aptasensor has a wider dynamic linear range (0.1 μM - 120 μM) and higher selectivity. The proposed aptasensor has been successfully applied to detect ATP in 15% human serum. More importantly, the current study not only provides a novel method for ATP assay but also presents a way to construct a label-free split aptamer based fluorescent sensor for other species where aptamer can be generated.