Label-free fluorescent aptasensor for thrombin detection based on exonuclease I assisted target recycling and SYBR Green I aided signal amplification

We developed a label-free fluorescence aptasensor strategy for the ultrasensitive determination of thrombin by exonuclease I (Exo I)-assisted target recycling and SYBR Green I (SGI) dye-aided fluorescence-signal amplification. The thrombin aptamer was hybridized with its complementary DNA (cDNA) to form double-stranded DNA (dsDNA) probe, resulting in a weak fluorescence signal of SGI. In the present of thrombin, the thrombin aptamer was removed from the dsDNA probe, forming an aptamer-thrombin complex with a G-quadruplex structure. As a result, an enhancement fluorescence intensity of SGI can be found. After adding a small amount of Exo I, partial G-quadruplex structure was destroyed and the released thrombin participated in a new binding with the rest of dsDNA probe to form another G-quadruplex complex. With the Exo I-catalyzed target recycling, increasing SGI intercalates into G-quadruplex, deeply amplifying the fluorescence emission. At optimum conditions, the fluorescence signal increased linearly with thrombin concentration from 0.28 nM to 86 nM. The detection limit of thrombin was 30 pM, which is lower than those of commonly used fluorescent aptamer sensors. Moreover, the proposed method was successfully employed to detect thrombin in real sample analysis. Our finding suggested its great potential to achieve disease diagnosis.