基于双重猝灭原理的分子信标定量检测凝血酶

发布时间：2018-02-20 00:50

本文关键词： 双重猝灭分子信标凝血酶荧光定量检测　出处：《分析化学》2017年10期 　论文类型：期刊论文

【摘要】：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%。
[Abstract]:Molecular beacons were constructed by double quenching of fluorescent groups by G base and organic quenching groups. A simple method for the detection of thrombin was established based on the principle of double quenching. The fluorescence group in the molecular beacon was designed as carboxyl fluorescein, and the organic quenching group was designed as Black Hole Quencher 1 BHQ-1BHQ-1 to bind three nucleotides containing G base. The ring of molecular beacon was designed as the aptamer of nucleic acid of thrombin. When there was no thrombin in the system, the molecular beacon showed stem ring structure. The fluorescence of fluorescence group FAM, organic quench group BHQ-1 and G base group were close to each other under the double quenching of BHQ-1 and G base. When there is thrombin in the system, the molecular beacons bind to thrombin specifically to form a G-quadruplex structure, and the stem-loop structure is destroyed away from the quenched groups BHQ-1 and G bases. Under the optimum conditions, the fluorescence intensity (螖 I) of the system has a good linear relationship with the concentration of thrombin in the range of 0.4 ~ 40 nmol/L. The linear regression equation is 螖 I ~ (24. 63) C ~ (mol / L) ~ (13. 06) ~ (2) ~ 0.9972 ~ (2), and the detection limit is 0. 18 nmol/L(3.
【作者单位】：湖北民族学院生物资源保护与利用湖北省重点实验室;湖北民族学院化学与环境工程学院;湖北民族学院医学院;
【基金】：国家自然科学基金项目(Nos.21465010,31460172) 生物资源保护与利用湖北省重点实验室开放基金(No.PKLHB1532) 湖北民族学院博士启动基金(No.MY2015B015) 湖北省林学一级学科资助~~
【分类号】：O657.3;R446.1

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