Ultrasensitive aptamer-based thrombin assay based on metal enhanced fluorescence resonance energy transfer

A sensitive "on-off" fluorescent protocol for thrombin detection is demonstrated. Firstly, thrombin aptamers which hybridize with labeled help DNA were immobilized on the surface of Ag-SiO₂ nanoparticles (NPs). The silver core causes the label Cy5 to display strong metal-enhanced fluorescence. On addition of thrombin and graphene oxide (GO), thrombin (with its high affinity for the aptamers) displaces the Cy5-labeled help DNA which then binds to the surface of GO via π-stacking, causing fluorescence quenching of Cy5. The findings were used to design a thrombin assay that has a 0.05 nM detection limit and excellent selectivity. It was applied to the quantification of thrombin in spiked serum samples where is showed recoveries ranging from 97 % to 107 %, with relative standard deviations between 2.2 and 4.5 %. (author)