title: Fluorescent detection of protein kinase based on positively charged gold nanoparticles.
abstract: Herein, we report a fluorometric method for monitoring the activity and inhibition of protein kinase based on positively charged gold nanoparticles, (+)AuNPs. In this assay, when the cationic substrate peptide (S-peptide) is phosphorylated by protein kinase, the resulting negatively charged product peptide (P-peptide) will be adsorbed onto (+)AuNPs through electrostatic interaction, and the fluorescence of fluorescein isothiocyanate (FITC) on the peptide will be quenched by (+)AuNPs. Thus, the fluorescence of solution can respond to the activity of protein kinase. The feasibility of this (+)AuNPs-based 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)). Furthermore, the system is successfully applied to estimate the IC50 value of PKA inhibitor H-89. The fast mix-and-readout detection process as well as the simple synthesis of the unmodified (+)AuNPs makes this proposed method a promising candidate for simple and cost-effective kinase activity detection and a good potential in high-throughput screening of kinase-related drugs.
authors: State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China.
authors: Guoyan
authors: G
authors: Lu
authors: State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China.
authors: Penglong
authors: P
authors: Tan
authors: State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China.
authors: Chunyang
authors: C
authors: Lei
authors: State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China.
authors: Zhou
authors: Z
authors: Nie
authors: State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China. Electronic address: huangyan.hnu@gmail.com.
authors: Yan
authors: Y
authors: Huang
authors: State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China.
authors: Shouzhou
authors: S
authors: Yao
mesh_terms: D000838:Anions / Q000737:chemistry; D002412:Cations / Q000737:chemistry; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D005240:Feasibility Studies; D005453:Fluorescence; D006046:Gold / Q000737:chemistry*; D007546:Isoquinolines / Q000494:pharmacology; D053768:Metal Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: D000838:Anions; D002412:Cations; D007546:Isoquinolines; D010455:Peptides; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D006046:Gold; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide
keywords: Fluorometric method; Phosphorylation; Positively charged gold nanoparticles; Protein kinase