基于双重猝灭原理的分子信标定量检测凝血酶

Detection of Thrombin Using Double Quenching Molecular Beacon

利用G碱基和有机猝灭基团对荧光基团的双重猝灭作用构建了分子信标,建立了一种基于双重猝灭原理的检测凝血酶的简单方法。此分子信标中荧光基团设计为羧基荧光素(FAM),有机猝灭基团设计为Black Hole Quencher 1(BHQ-1),BHQ-1连接3个含有G碱基的核苷酸,分子信标的环设计为凝血酶的核酸适配体。体系中没有凝血酶时,分子信标呈茎环结构,荧光基团FAM与有机猝灭基团BHQ-1及G碱基相互靠近,FAM的荧光在BHQ-1及G碱基的双重猝灭下,其荧光信号很弱;当体系中有凝血酶存在时,分子信标与凝血酶特异性结合,形成G-四联体结构,茎-环结构被破坏,FAM远离猝灭基团BHQ-1及G碱基,其荧光得到恢复。在最适条件下,体系的荧光强度(ΔI)与凝血酶的浓度(C)在0.4~40 nmol/L范围内具有良好的线性关系,线性回归方程为ΔI=24.63C(nmol/L)+13.06(R^2=0.9972),检出限为0.18 nmol/L(3σ,n=9)。实际血样加标回收率为96.3%~98.7%。

A double quenching molecular beacon（MB） with simple structure was designed based on organic quencher and G bases,and a simple detection method for thrombin was developed using this MB.In this MB,FAM and BHQ-1 were selected as fluorophore and organic quencher,three continuous nucleotides with G base were connected with BHQ-1,and the loop of MB was designed as a nucleic acid aptamer of thrombin.In the absence of thrombin,the MB was in the stem-loop structure,the fluorophore FAM was close to BHQ-1 and G bases,the fluorescence of FAM was dual quenched by BHQ-1 and G bases,and the fluorescence signal of FAM was very weak.In the presence of thrombin,MB specifically bound thrombin and formed a G-quadruplex structure.The stem-loop structure of the MB was destroyed,and FAM was separated with BHQ-1 and G bases,leading to recovery of fluorescence of FAM.Under the optimal conditions,the fluorescence intensity of FAM exhibited a good linear relationship with concentration of thrombin in the range of 0.4-40.0 nmol/L,and regression equation was ΔI = 24.63C（nmol/L）＋13.06（R^2=0.9972） with the detection limit of 0.18 nmol/L（3σ,n=9）.The average recoveries of this method in serum samples were 96.3%-98.7%,which indicated that the method had high accuracy.