College of Environmental Science and Engineering, Key Laboratory of Environmental Biology and Pollution Control, Hunan University, Ministry of Education, Hunan University, Changsha 410082, China.
Analyst. 2012 Dec 7;137(23):5607-13. doi: 10.1039/c2an36117e. Epub 2012 Oct 16.
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.
金纳米颗粒 (GNPs) 可以有效地区分未折叠和折叠的适体，并猝灭铽三元配合物 (Tb-complexes) 的荧光，因此本文报道了一种使用无标记适体、Tb-complexes 和 GNPs 的灵敏蛋白质检测策略。在凝血酶存在下，适体倾向于形成 G-四链体，而折叠的适体不能吸附在 GNPs 表面，在 0.5 mol L(-1) 盐存在下诱导 GNPs 聚集。在低速离心去除聚集的 GNPs 后，上清液对 Tb-complexes 的猝灭能力降低。随着凝血酶浓度的增加，Tb-complexes 的荧光强度增加。由于适体对凝血酶具有高度特异性识别能力和 GNPs 对 Tb-complexes 的强猝灭特性，该方案对凝血酶具有良好的选择性和高灵敏度。在最佳条件下，获得了从 1.0 × 10(-9) M 到 1.0 × 10(-8) M 的线性范围，检测限为 0.14 nM，远低于比色传感器和一些荧光传感器通常获得的检测限。可以通过时间分辨方式测量 Tb-complexes 的信号，从而消除大部分非特异性荧光背景信号。该传感器已成功应用于复杂生物样品中的凝血酶检测，为无标记适体基蛋白质检测提供了有前景的潜力。
