期刊
SENSORS AND ACTUATORS B-CHEMICAL
卷 206, 期 -, 页码 728-734出版社
ELSEVIER SCIENCE SA
DOI: 10.1016/j.snb.2014.09.017
关键词
Photoelectrochemical assay; Biotinylated Phos-tag; Protein kinase A activity; Bismuth sulfide; Phosphorylation
资金
- National Natural Science Foundation of China [21105056, 21375079]
- China Postdoctoral Science Foundation [2014M550369]
- Development of Science and Technology of Shandong Province, China [2013GZX20109]
A novel, sensitive and label-free photoelectrochemical assay for monitoring the activity and inhibition of protein kinase A (PKA) was presented based on the specific recognition ability of biotinylated Phos-tag for kinase-induced phosphopeptides. When the assembled substrate peptides were phosphorylated by PKA, they could bind specifically to the biotinylated Phos-tag in the presence of Zn2+. Then streptavidins could be further captured on the electrode surface through their specific interaction towards biotin, resulting in a significant decrease on the photocurrent of substrate Bi2S3 modified ITO electrode. The decreased photocurrent was proportional to the PKA concentration ranging from 0.05 to 100 unit/mL with a detection limit of 0.017 unit/mL (S/N= 3). Moreover, this photoelectrochemical method could be also used to quantitative analysis of kinase inhibition. In the light of the inhibitor concentration dependant photocurrent signal, the IC50 value for ellagic acid and HA-1077 was estimated to be 3.23 and 1.77 mu M, respectively. With changing the sequence of substrate peptide, this method could also be applied to detect other kinase activity and inhibitors. Therefore, the developed protocol provides a new and promising platform for assay of kinase activity and its inhibitors with low cost and high sensitivity. (C) 2014 Elsevier B.V. All rights reserved.
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