Label-free fluorescent detection of thrombin activity based on a recombinant enhanced green fluorescence protein and nickel ions immobilized nitrilotriacetic acid-coated magnetic nanoparticles

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 (3.0×10−4 U mL−1), 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.