title: Aptameric peptide for one-step detection of protein kinase.
abstract: Protein kinases are significant regulators in the cell signal pathway, and it is difficult to achieve quick kinase detection because traditional kinase assays normally rely on a time-consuming kinase phosphorylation process. Herein, we present a novel one-step strategy to detect protein kinase by using a kinase-specific aptameric peptide-functionalized quartz crystal microbalance (QCM) electrode, in which the detection can be finished in less than 10 min. A peptide kinase inhibitor (IP(20)) was used as the aptameric peptide because of its selective and strong interaction with the target protein kinase (cyclic adenosine monophosphate-dependent protein kinase A, PKA), high stability, and ease of inexpensive synthesis, presenting a new direct recognition element for kinase. The aptameric peptide was immobilized on the Au-coated quartz electrode through dual-thiol anchoring and the binding of His-tagged peptide with a nitrilotriacetic acid/Ni(II) complex, fabricating a highly specific and stable detection platform. The interaction of aptameric peptide with kinase was monitored with the QCM in real time, and the concentration of protein kinase was sensitively measured by the frequency response of the QCM with the low detection limit for PKA at 0.061 mU μL(-1) and a linear range from 0.64 to 22.33 mU μL(-1). This method is rapid and reagentless and does not require a phosphorylation process. The versatility of our aptameric peptide-based strategy has also been demonstrated by the application in kinase assay using electrochemical impedance spectroscopy. Moreover, this method was successfully applied to detect the forskolin/3-isobutyl-1-methylxanthine-stimulated activation of PKA in cell lysate.
authors.affiliation: State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, People's Republic of China.
authors.forename: Xiahong
authors.initials: X
authors.lastname: Xu
authors.forename: Jiang
authors.initials: J
authors.lastname: Zhou
authors.forename: Xin
authors.initials: X
authors.lastname: Liu
authors.forename: Zhou
authors.initials: Z
authors.lastname: Nie
authors.forename: Meng
authors.initials: M
authors.lastname: Qing
authors.forename: Manli
authors.initials: M
authors.lastname: Guo
authors.forename: Shouzhuo
authors.initials: S
authors.lastname: Yao
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D052158:Aptamers, Peptide / Q000737:chemistry*; D015374:Biosensing Techniques; D045744:Cell Line, Tumor; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D058266:Dielectric Spectroscopy; D004566:Electrodes; D006046:Gold; D006639:Histidine; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry*; D007700:Kinetics; D057230:Limit of Detection; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D009842:Oligopeptides; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D058525:Quartz Crystal Microbalance Techniques
chemical_list: D052158:Aptamers, Peptide; C471213:His-His-His-His-His-His; D055767:Immobilized Proteins; D009842:Oligopeptides; D047428:Protein Kinase Inhibitors; D005576:Colforsin; D006639:Histidine; D006046:Gold; D017868:Cyclic AMP-Dependent Protein Kinases; D009571:Nitrilotriacetic Acid; D015056:1-Methyl-3-isobutylxanthine