Electrochemical detection of protein kinase activity based on carboxypeptidase Y digestion triggered signal amplification

An effective assay method for monitoring protein kinase activity and screening inhibitors is greatly beneficial to kinase-related drug discovery, early diagnosis of diseases, and therapeutic effect evaluation. Herein, we develop a simple electrochemical method for detecting the activity of casein ki...

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Bibliographic Details
Published in:Biosensors & bioelectronics 2015-04, Vol.66, p.77-83
Main Authors: Yin, Huanshun, Wang, Xinxu, Guo, Yunlong, Zhou, Yunlei, Ai, Shiyun
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amplification
Assaying
Biosensing Techniques - methods
Biosensors
Carboxypeptidase Y
Casein Kinase II - analysis
Casein Kinase II - antagonists & inhibitors
Casein Kinase II - metabolism
Cathepsin A - metabolism
Corrosion inhibitors
Digestion
Drug Evaluation, Preclinical - methods
Electrochemical biosensor
Electrochemical Techniques - methods
Enzyme Assays - methods
Hep G2 Cells
Humans
Inhibition
Kinases
Limit of Detection
Peptides
Peptides - chemistry
Peptides - metabolism
Phosphorylation
Protein kinase
Protein Kinase Inhibitors - pharmacology
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Summary:An effective assay method for monitoring protein kinase activity and screening inhibitors is greatly beneficial to kinase-related drug discovery, early diagnosis of diseases, and therapeutic effect evaluation. Herein, we develop a simple electrochemical method for detecting the activity of casein kinase II (CK2) based on phosphorylation against carboxypeptidase Y (CPY) digestion triggered signal amplification, where CK2 catalyzed phosphorylation event protects the substrate peptide from the digestion of CPY, maintains the repulsive force of the substrate peptide towards the redox probe, and results in a weak electrochemical signal. Whereas, without phosphorylation, the substrate peptide is digested by CPY and a strong electrochemical signal is obtained. The detection feasibility is demonstrated for the assay of CK2 activity with low detection limit of 0.047unit/mL. Moreover, the biosensor was used for the analysis of kinase inhibition. Based on the electrochemical signal dependent inhibitor concentration, the IC50 value of ellagic acid was estimated to be 39.77nM. The proposed method is also successfully applied to analyze CK2 activity in cell lysates, proving the applicability in complex biological samples. [Display omitted] •A novel electrochemical biosensor is fabricated for kinase activity assay.•Carboxypeptidase Y digestion of peptide is inhibited due to phosphorylation.•This method shows high sensitivity with detection limit of 0.047unit/mL.•The developed method can also be applied to screen inhibitors.
ISSN:0956-5663
1873-4235
DOI:10.1016/j.bios.2014.11.014