Reduced Graphene Oxide-Zirconium Dioxide-Thionine Nanocomposite Integrating Recognition, Amplification, and Signaling for an Electrochemical Assay of Protein Kinase Activity and Inhibitor Screening

Protein kinase activity analysis is essential and important for elucidation of many fundamental biological processes, disease diagnosis, and drug discovery. Herein, a novel electrochemical biosensing method for protein kinase (PKA) activity was demonstrated by the use of a reduced graphene oxide-zirconium dioxide-thionine (rGO-ZrO2-Thi) nanocomposite, which interestingly served as an integral phosphopeptide-recognizing, signal amplifying and reporting platform. The ZrO2 nanoparticle-decorated reduced graphene oxide (rGO-ZrO2) was first prepared by a hydrothermal reaction route, and then the thionine was conjugated onto the rGO surface via pi-pi stacking as an excellent electrochemical probe. The prepared rGO-ZrO2-Thi nanocomposites were well-characterized by various techniques. With the full advantage of specific recognition of ZrO2 nanoparticles for the phosphate group, signal amplification, and transduction of abundant thionines onto the rGO surface, and excellent conductivity of rGO, the rGO-ZrO2-Thi nanocomposite endowed a label-free and one-step electrochemical analysis of kemptide phosphorylation catalyzed by PKA. The detection limit for PKA activity was experimentally achieved as 0.005 U/mL, which was evidently lower than most of the reported methods. The proposed sensing strategy could be also applied for an efficient inhibitor evaluation. Therefore, it offered an excellent pathway for a generic and sensitive electrochemical assay of PKA activity and inhibitor.