Detection of Thrombin Using Double Quenching Molecular Beacon
10.11895/j.issn.0253-3820.170162
- VernacularTitle:基于双重猝灭原理的分子信标定量检测凝血酶
- Author:
Kun ZHAI
1
;
Quan Feng LI
;
An Bo SHI
;
Shan Dong XIANG
Author Information
1. 生物资源保护与利用湖北省重点实验室
- Keywords:
Double quenching molecular beacon;
Thrombin;
Fluorescence;
Quantitative detection
- From:
Chinese Journal of Analytical Chemistry
2017;45(10):1462-1466
- CountryChina
- Language:Chinese
-
Abstract:
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 △I=24. 63C (nmol/L)+ 13. 06 (R2 =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.
