A convenient sandwich assay of thrombin in biological media using nanoparticle-enhanced fluorescence polarization

Abstract

A new aptamer biosensor was presented for the detection of thrombin in this work, which was based on fluorescence polarization (FP) using silica nanoparticles as enhancement probe. The silica nanoparticles covered by streptavidin were tagged with a thrombin aptamer (5'-biotin-GGTTGGTGTGGTTGG-3'), which was bound to the surface of silica nanoparticle through the specific interaction between streptavidin and biotin. In the presence of thrombin, it induced the aptamer to form quadruplex structure. When the other thrombin aptamer labeled with fluorescein (5'-FAM-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3') was added to the above system, a sandwich structure can form at the surface of silica nanoparticles. The fluorescence polarization was therefore enhanced and quantification between fluorescence polarization signal and concentration of thrombin was built. The sensor provided a linear range from 0.6 to 100nM for thrombin with a detection limit of 0.20nM (3.29SB/m, according to the recent recommendation of IUPAC) in a homogeneous media. The same linear range was obtained in spiked human serum samples with a slightly higher detection limit (0.26nM), demonstrating high anti-interference of the sensor in a complex biological sample matrix. And the sensor can be used to monitor spiked concentration of thrombin level in real human plasma with satisfactory results obtained.