Fluorescent detection of protein kinase based on zirconium ions-immobilized magnetic nanoparticles

•Present a novel method for PKA activity analysis based on magnetic separation.•Zr-NTA MNPs are implemented in detection of PKA activation in cell lysates.•Specific adsorption between zirconium ions and phosphate group.•Highly selective, sensitive and fast recognition of phosphorylated peptides.•The MNPs are recyclable. 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 Zr4+-immobilized nitrilotriacetic acid-coated magnetic nanoparticles (Zr-NTA MNPs) through the chelation of Zr4+ 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 (0.5mUμL−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 Zr4+ 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.