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Citation: ZHAI Kun, Li Feng-Quan, SHI Bo-An, XIANG Dong-Shan. Detection of Thrombin Using Double Quenching Molecular Beacon[J]. Chinese Journal of Analytical Chemistry, ;2017, 45(10): 1462-1466. doi: 10.11895/j.issn.0253-3820.170162 shu

Detection of Thrombin Using Double Quenching Molecular Beacon

1.
Key Laboratory of Biologic Resources Protection and Utilization of Hubei Province, Hubei University for Nationalities, Enshi 445000, China;2 School of Chemical and Environmental Engineering, Hubei University for Nationalities, Enshi 445000, China;3 Medical School, Hubei University for Nationalities, Enshi 445000, China

Received Date: 17 March 2017
Revised Date: 28 August 2017

Fund Project: This work was supported by the National Natural Science Foundation of China (Nos.21465010, 31460172).

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.
- Double quenching molecular beacon,
- Thrombin,
- Fluorescence,
- Quantitative detection,
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