title: Fluorescent detection of protein kinase based on zirconium ions-immobilized magnetic nanoparticles.
abstract: We report here an affinity separation-based fluorometric method for monitoring the activity and inhibition of protein kinase. In this assay, when the fluorescein isothiocyanate (FITC) labeled substrate peptides (S-peptide) are phosphorylated by kinase, the product peptides (P-peptide) will be adsorbed and concentrated onto the surface of Zr(4+)-immobilized nitrilotriacetic acid-coated magnetic nanoparticles (Zr-NTA MNPs) through the chelation of Zr(4+) and phosphate groups. After magnetic separation, the fluorescence intensity of the homogeneous solution changes dramatically. Hence the fluorescence response allows this MNPs-based method to easily probe kinase activity by a spectrometer. The feasibility of the method has been demonstrated by sensitive measurement of the activity of cAMP-dependent protein kinase (PKA) with a low detection limit (0.5 mU μL(-1)). Moreover, the system is successfully applied to estimate the IC50 value of PKA inhibitor H-89 and detect the Forskolin/3-isobutyl-1-methylxanthine (IBMX) stimulated activation of PKA in cell lysate. Additionally, Zr-NTA MNPs are reusable by stripping Zr(4+) ions from NTA-coated MNPs and rechelating again. This method, which relies on the surface-functionalized MNPs, presents a promising candidate for simple and cost-effective assay of kinase activity 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: Penglong
authors.initials: P
authors.lastname: Tan
authors.forename: Chunyang
authors.initials: C
authors.lastname: Lei
authors.forename: Xin
authors.initials: X
authors.lastname: Liu
authors.forename: Meng
authors.initials: M
authors.lastname: Qing
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: D000327:Adsorption; D045744:Cell Line, Tumor; D006801:Humans; D008280:Magnetics; D053758:Nanoparticles / Q000737:chemistry*; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D015040:Zirconium / Q000737:chemistry*
chemical_list: D015040:Zirconium; D011494:Protein Kinases