title: Label-free fluorescent detection of thrombin activity based on a recombinant enhanced green fluorescence protein and nickel ions immobilized nitrilotriacetic acid-coated magnetic nanoparticles.
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 Ni(2+) ions immobilized nitrilotriacetic acid-coated magnetic nanoparticles (Ni(2+)-NTA MNPs). The EGFP, containing a thrombin cleavage site and a hexahistidine sequence (His-tag) at its N-terminal, was adsorbed onto Ni(2+)-NTA MNPs through Ni(2+)-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 Ni(2+)-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 Ni(2+)-NTA MNPs, our method presents a promising candidate for simple, sensitive, and cost-saving protease activity detecting and inhibitor screening.
authors.affiliation: State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, PR China.
authors.forename: Ming
authors.initials: M
authors.lastname: Wang
authors.forename: Chunyang
authors.initials: C
authors.lastname: Lei
authors.forename: Zhou
authors.initials: Z
authors.lastname: Nie
authors.forename: Manli
authors.initials: M
authors.lastname: Guo
authors.forename: Yan
authors.initials: Y
authors.lastname: Huang
authors.forename: Shouzhuo
authors.initials: S
authors.lastname: Yao
mesh_terms: D000991:Antithrombins / Q000097:blood* / Q000737:chemistry; D017743:Equipment Reuse; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D006629:Hirudins / Q000097:blood* / Q000737:chemistry; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000235:genetics; D057230:Limit of Detection; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics; D009942:Organometallic Compounds / Q000737:chemistry*; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods; D013917:Thrombin / Q000032:analysis* / Q000737:chemistry
chemical_list: D000991:Antithrombins; D006629:Hirudins; C471213:His-His-His-His-His-His; D055767:Immobilized Proteins; D058185:Magnetite Nanoparticles; D009842:Oligopeptides; D009942:Organometallic Compounds; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C088321:nickel nitrilotriacetic acid; D006639:Histidine; D013917:Thrombin; D009571:Nitrilotriacetic Acid
keywords: Fluorescence assay; Magnetic nanoparticles; Recombinant enhanced green fluorescent protein; Thrombin