Protein-induced fluorescence enhancement as aptamer sensing mechanism for thrombin detection

Aptamer-based protein sensing can be implemented using a variety of optical and non-optical detection methods. In this report, we present protein-induced fluorescence enhancement (PIFE) based detection of DNA aptamer binding to thrombin. We demonstrate that PIFE reports on direct binding of thrombin to the aptamer strand carrying the fluorophore and hence is unaffected by salt based stabilization of the aptamer conformations as observed in fluorescence resonance energy transfer (FRET) assays. PIFE based thrombin detection displayed excellent linearity in the range of 0.25 pM-25 nM with a detection limit of 8.9 pM. In an alternate scheme, PIFE was demonstrated by placing the fluorophore on a connector strand thus bypassing the requirement to label the aptamer directly. This strategy allowed us to examine thrombin binding by variously modified thrombin aptamers and can serve as a platform for aptamer screening. We finally show that PIFE based thrombin aptamer assay displays high specificity even in the presence of 'natural' background such as blood plasma. We hence propose PIFE based aptamer sensing of protein ligands as a facile yet sensitive and general strategy for biosensing.