A sensitive strategy for label-free and time-resolved fluorescence assay of thrombin using Tb-complex and unmodified gold nanoparticles

Abstract

Gold nanoparticles (GNPs) can effectively differentiate the unfolded and folded aptamer, and quench the fluorescence of terbium ternary complexes (Tb-complexes), thus the authors herein report a sensitive strategy for protein detection, using label-free aptamer, Tb-complexes and GNPs. In the presence of thrombin, the aptamer is inclined to form G-quartet, and the folded aptamer cannot adsorb on the surface of GNPs, inducing the GNPs aggregation in the presence of 0.5 mol L⁻¹ salt. After centrifugation at low speed to remove the aggregated GNPs, the quenching capability of the supernatant for Tb-complexes is decreased. The fluorescence intensity of Tb-complexes increases as the concentration of thrombin increases. Due to the highly specific recognition ability of the aptamer for thrombin and the strong quenching property of GNPs for Tb-complexes, the proposed protocol has good selectivity and high sensitivity for thrombin. Under the optimum conditions, a linear range from 1.0 × 10⁻⁹ M to 1.0 × 10⁻⁸ M is obtained with a detection limit of 0.14 nM, which is much lower than those commonly obtained for colorimetric sensors and some fluorescent sensors. The signal of Tb-complexes can be measured by time-resolved manner which made most of the unspecific fluorescent background signals be eliminated. The proposed sensor has been successfully applied in complicated biological samples for thrombin detection, and it can provide a promising potential for label-free aptamer-based protein detection.