Department of Chemistry, Liaocheng University, Liaocheng 252059, PR China.
Department of Chemistry, Liaocheng University, Liaocheng 252059, PR China.
Biosens Bioelectron. 2014 Jun 15;56:231-6. doi: 10.1016/j.bios.2014.01.021. Epub 2014 Jan 22.
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 100 nM for thrombin with a detection limit of 0.20 nM (3.29 SB/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 (0.26 nM), 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.
本工作提出了一种新的适体生物传感器，用于检测凝血酶，该传感器基于荧光偏振（FP），使用硅纳米粒子作为增强探针。被链霉亲和素覆盖的硅纳米粒子被标记有凝血酶适体（5'-生物素-GGTTGGTGTGGTTGG-3'），通过链霉亲和素和生物素之间的特异性相互作用结合到硅纳米粒子表面。在凝血酶存在下，它诱导适体形成四链体结构。当另一个带有荧光素（5'-FAM-AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3'）标记的凝血酶适体被添加到上述体系中时，可以在硅纳米粒子表面形成三明治结构。因此，荧光偏振增强，建立了荧光偏振信号与凝血酶浓度之间的定量关系。该传感器在均相介质中提供了 0.6 至 100 nM 的线性范围，检测限为 0.20 nM（3.29 SB/m，根据 IUPAC 的最新建议）。在添加了人血清样本中也获得了相同的线性范围，只是检测限略高（0.26 nM），表明该传感器在复杂的生物样本基质中有很高的抗干扰能力。并且该传感器可以用于监测真实人血浆中添加的凝血酶水平，结果令人满意。
