Aptamer Biosensor Based on Fluorescence Resonance Energy Transfer from Upconverting Phosphors to Carbon Nanoparticles for Thrombin Detection in Human Plasma

We presented a new aptamer biosensor for thrombin in this work, which was based on fluorescence resonance energy transfer (FRET) from upconverting phosphors (UCPs) to carbon nanoparticles (CNPs). The poly(acrylic acid) (PAA) functionalized UCPs were covalently tagged with a thrombin aptamer (5'-NH2- GGTTGGTGTGGTTGG-3'), which bound to the surface of CNPs through pi-pi stacking interaction. As a result, the energy donor and acceptor were taken into close proximity, leading to the quenching of fluorescence of UCPs. A maximum fluorescence quenching rate of 89% was acquired under optimized conditions. In the presence of thrombin, which induced the aptamer to form quadruplex structure, the pi-pi interaction was weakened, and thus, the acceptor was separated from the donor blocking the FRET process. The fluorescence of UCPs was therefore restored in a thrombin concentration-dependent manner, which built the foundation of thrombin quantification. The sensor provided a linear range from 0.5 to 20 nM for thrombin with a detection limit of 0.18 nM in an aqueous buffer. The same linear range was obtained in spiked human serum samples with a slightly higher detection limit (0.25 nM), demonstrating high robustness of the sensor in a complex biological sample matrix. As a practical application, the sensor was used to monitor thrombin level in human plasma with satisfactory results obtained. This is the first time that UCPs and CNPs were employed as a donor acceptor pair to construct FRET-based biosensors, which utilized both the photophysical merits of UCPs and the superquenching ability of CNPs and thus afforded favorable analytical performances. This work also opened the opportunity to develop biosensors for other targets using this UCPs-CNPs system.