title: A sensitive electrochemical biosensor for detection of protein kinase A activity and inhibitors based on Phos-tag and enzymatic signal amplification.
abstract: A simple, highly sensitive and selective electrochemical assay is developed for the detection of protein kinase A (PKA) activity based on the specific recognition utility of Phos-tag for kinase-induced phosphopeptides and enzymatic signal amplification. When the substrate peptide was phosphorylated by PKA reaction, they could specifically bind with Phos-tag-biotin in the presence of Zn(2+) through the formation of a specific noncovalent complex with the phosphomonoester dianion in phosphorylated peptides. Through the further specific interaction between biotin and avidin, avidin functionalized horseradish peroxidase (HRP) can be captured on the electrode surface. Under the catalytic effect of HRP, a sensitive electrochemical signal for benzoquinone was obtained, which was related to PKA activity. Under the optimal experiment conditions, the proposed electrochemical method presented dynamic range from 0.5 to 25 unit/mL with low detection limit of 0.15 unit/mL. This new detection strategy was also successfully applied to analyze the inhibition effect of inhibitors (ellagic acid and H-89) on PKA activity and monitored the PKA activity in cell lysates. Therefore, this Phos-tag-based electrochemical assay offers an alternative platform for PKA activity assay and inhibitor screening, and thus it might be a valuable tool for development of targeted therapy and clinical diagnosis.
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China.
authors.forename: Huanshun
authors.initials: H
authors.lastname: Yin
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China.
authors.forename: Mo
authors.initials: M
authors.lastname: Wang
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China.
authors.forename: Bingchen
authors.initials: B
authors.lastname: Li
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China.
authors.forename: Zhiqing
authors.initials: Z
authors.lastname: Yang
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China. Electronic address: zhyl@sdau.edu.cn.
authors.forename: Yunlei
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018 Taian, Shandong, PR China. Electronic address: ashy@sdau.edu.cn.
authors.forename: Shiyun
authors.initials: S
authors.lastname: Ai
mesh_terms: D016227:Benzoquinones / Q000737:chemistry; D015374:Biosensing Techniques; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D004610:Ellagic Acid / Q000494:pharmacology; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D057230:Limit of Detection; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D013379:Substrate Specificity; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: D016227:Benzoquinones; D007546:Isoquinolines; D010455:Peptides; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D004610:Ellagic Acid; C004532:quinone; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide
keywords: Elelctrochemical biosensor; Enzymatic signal amplification; Inhibitors; Phos-tag-biotin; Protein kinase A