A graphene oxide-based multiplexed fluorescence assay for the detection of protein kinase activity in cell lysates and the evaluation of protein kinase inhibition

作者：Huang, Yong^*; Huang, Huakui; Qin, Jian; Liu, Xiaoqian; Zhao, Shulin^*; Chen, Zhen-Feng; Liang, Hong^*

来源：Sensors and Actuators B: Chemical , 2017, 238: 908-916.

DOI：10.1016/j.snb.2016.07.140

• Graphene oxide
• Multiplexed detection
• Protein kinase
• Cell lysates

We have developed a novel graphene oxide (GO) sensing platform based on enzymatic phosphorylation for the multiplexed detection of protein kinases. The dye-labeled peptide substrate exhibits strong fluorescence in the absence of kinases. When the biotinylated adenosine 5'-triphosphate co-substrate and kinase are present, the peptide substrate is phosphorylated by kinase, and the phosphorylated peptide product carrying both the biotin site and the dye can be assembled onto streptavidin-coated GO via streptavidin-biotin chemistry, leading to fluorescence quenching. Importantly, since GO is a highly efficient quencher for multiple different fluorophores, the platform allows simultaneous detection of multiple protein kinases using different peptide substrates labeled with corresponding dyes. As a proof of-concept, we demonstrate that this GO sensing platform can simultaneously and selectively detect protein kinase A, protein kinase Abl and protein kinase Src with low detection limits of 0.005 U/mL, 0.02 U/mL and 0.05 U/mL, respectively. Furthermore, the application of this method for detection of multiple protein kinases in biological samples and protein kinase inhibitor screening has also been demonstrated. This approach holds great promise as a routine tool for the high-throughput screening of protein kinases and their inhibitors.

• 出版日期2017-2
• 单位广西师范大学