Phosphate Group-Derivated Bipyridine-Ruthenium Complex and Titanium Dioxide Nanoparticles for Electrochemical Sensing of Protein Kinase Activity.

Monitoring of protein kinase activity is of significance for fundamentals of biochemistry, biomedical diagnose, and drug screening. To reduce the usage of a relatively complicated bio-labeled signal probe, the phosphate group-derivated bipyridine-ruthenium (Pbpy-Ru) complex and titanium dioxide nanoparticles (TiO NPs) were employed as signal probes to develop an electrochemical sensor for evaluating the protein kinase A (PKA) activity. Through the specific interaction between the phosphate groups and TiO NPs, the preparation of a Pbpy-Ru-TiO NP signal probe and its linkage with the phosphorylated PKA substrate peptides could be performed in a simple and effective way. The tethering of Pbpy-Ru onto the TiO NP surface does not degrade the electrochemical property of the complex. The Pbpy-Ru-TiO NP probe exhibits well-defined redox signals at about 1.0 V versus Ag/AgCl reference and notably has about fivefold current response than that of the TiO NPs with physically adsorbed tris-(bipyridine)-Ru. The PKA activity evaluation was realized by measuring the electrochemical response of the Pbpy-Ru-TiO NPs at the phosphorylated peptide-assembled electrode. Operating at optimal conditions, the cathodic signals at the potential of 1.03 V exhibit a good linearity with the PKA concentrations of 0.5-40 U mL. The electrochemical sensor shows good selectivity, low detection limit (0.2 U mL, signal/noise = 3), qualified reproducibility, and satisfactory applicability for PKA determination in the cell lysate. The Pbpy-Ru-TiO NPs/electrode system would be an excellent electrochemical platform for protein phosphorylation monitoring and sensing.