Citation: FU Xin, ZOU Ting, ZHANG He, ZHANG Pei-Rou, JIANG Xu-Chun. Ultrasensitive Colorimetric Sensor for Detection of Thrombin Based on Loop Mediated Isothermal Signal Amplification Triggered by Aptamer Sandwich-Mediated Enzymatic Digestion[J]. Chinese Journal of Analytical Chemistry, ;2022, 50(7): 1022-1031. doi: 10.19756/j.issn.0253-3820.210793 shu

Ultrasensitive Colorimetric Sensor for Detection of Thrombin Based on Loop Mediated Isothermal Signal Amplification Triggered by Aptamer Sandwich-Mediated Enzymatic Digestion

Hunan Provincial Key Laboratory of Environmental Catalysis and Waste Recycling, College of Materials and Chemical Engineering, Hunan Institute of Engineering, Xiangtan 411104, China

Corresponding author: ZHANG He, mzhang_he@126.com
Received Date: 14 October 2021
Revised Date: 20 December 2021

Fund Project: Supported by the National Natural Science Foundation of China (No.21005067) and the Natural Science Foundation of Hunan Province, China (No.2019JJ40055).

An ultrasensitive colorimetric sensor for detection of thrombin was designed based on loop mediated isothermal signal amplification (LAMP) triggered by aptamer sandwich-mediated enzymatic digestion. In the presence of thrombin, aptamer capable of recognizing thrombin heparin site and fibrinogen site was used to recognize thrombin and construct a sandwich structure. The Nb.BsrDI nicking endonuclease could recognize and cleave this complementary sequence to start-up loop mediated isothermal amplification. G-quadruplex-hemin DNAzyme could catalyze the oxidation of ABTS to ABTS^·+ in the presence of H₂O₂, and the color changed to green. Under the optimal experimental conditions, the linear detection range of this method for thrombin was 0.01-1.0 ag/mL, the detection limit (3σ) was 0.008 ag/mL, and the regression equation was ΔA_{420 nm}=0.187C_thrombin+0.171 (R²=0.991). When there were a large number of other interfering proteins in the serum sample, the sensor still had a high selectivity to thrombin. When this method was applied to the detection of thrombin content in serum samples, the recoveries were 96.1%-103.2%. This method innovatively integrated aptamer-based sandwich assay, loop-mediated isothermal amplification technology, and G-quadruplex-hemin DNAzyme enzymatic signal amplification technology that caused the ultrasensitive detection of thrombin. The method showed many advantages such as lower cost, simple operation and good stability, and could be used for highly sensitive clinical detection of thrombin in human serum.
- Thrombin,
- Loop mediated isothermal amplification,
- G-quadruplex-hemin DNAzyme,
- Colorimetric analysis
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