▎ 摘　　要

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. (C) 2016 Elsevier B.V. All rights reserved.