Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
7682762 | Talanta | 2013 | 6 Pages |
Abstract
Herein, a novel label-free fluorescent assay has been developed to detect the activity of thrombin and its inhibitor, based on a recombinant enhanced green fluorescence protein (EGFP) and Ni2+ ions immobilized nitrilotriacetic acid-coated magnetic nanoparticles (Ni2+-NTA MNPs). The EGFP, containing a thrombin cleavage site and a hexahistidine sequence (His-tag) at its N-terminal, was adsorbed onto Ni2+-NTA MNPs through Ni2+-hexahistidine interaction, and dragged out of the solution by magnetic separation. Thrombin can selectively digest EGFP accompanied by His-tag peptide sequence leaving, and the resulting EGFP cannot be captured by Ni2+-NTA MNPs and kept in supernatant. Hence the fluorescence change of supernatant can clearly represent the activity of thrombin. Under optimized conditions, such assay showed a relatively low detection limit (3.0Ã10â4Â UÂ mLâ1), and was also used to detect the thrombin inhibitor, Hirudin, and further applied to detect thrombin activity in serum. Combined with the satisfactory reusability of Ni2+-NTA MNPs, our method presents a promising candidate for simple, sensitive, and cost-saving protease activity detecting and inhibitor screening.
Related Topics
Physical Sciences and Engineering
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Authors
Ming Wang, Chunyang Lei, Zhou Nie, Manli Guo, Yan Huang, Shouzhuo Yao,