title: A label-free and sensitive fluorescent assay for one step detection of protein kinase activity and inhibition.
abstract: In this paper, a label-free, highly sensitive and simple assay for one step detection of protein kinase (PKA) activity and inhibition that avoids the fluorescent dye process has been established. The detection was based on the fluorescence (FL) quenching of peptide-Ag nanoclusters (Ag NCs) caused by antibody modified Au nanoparticles (anti-Au NPs) via fluorescence resonance energy transfer (FRET). With PKA and adenosine 5'-triphosphate (ATP) introduced, the substrate peptide of Ag NCs could react with PKA via targeted phosphorylation, and followed by the linking interactions between peptide-Ag NCs and anti-Au NPs. According to the fluorescence quenching of Ag NCs, the activity of protein kinase can be facilely monitored in the range of 0.1-2000 mU/μL with high sensitivity. The detection limit for PKA is 0.039 mU/μL. We further explored the inhibitory effect of H-89 for protein kinase activity. The developed method was also applied to the investigation of drug-induced PKA activation in HeLa cells, which provides a promising means for screening of kinase-related drugs and the clinical diagnosis of disease.
authors.affiliation: Department of Analytical Chemistry, College of Chemistry, Jilin University, Changchun, 130012, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.affiliation: Department of Analytical Chemistry, College of Chemistry, Jilin University, Changchun, 130012, China.
authors.forename: Xu
authors.initials: X
authors.lastname: Yan
authors.affiliation: Department of Analytical Chemistry, College of Chemistry, Jilin University, Changchun, 130012, China. Electronic address: suxg@jlu.edu.cn.
authors.forename: Xingguang
authors.initials: X
authors.lastname: Su
mesh_terms: D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D006046:Gold / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D053768:Metal Nanoparticles / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000032:analysis* / Q000378:metabolism; D012834:Silver / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D010455:Peptides; D047428:Protein Kinase Inhibitors; D012834:Silver; D006046:Gold; D011494:Protein Kinases
keywords: Fluorescence; Inhibitor; PKA activity; Peptide-Ag NCs