Zhang Ge, Liu Jingwen, Liu Chengying, Ding Fan, Li Yingqian, Tang Hao, Ma Ming
Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, People's Republic of China.
ACS Sens. 2021 Dec 24;6(12):4451-4460. doi: 10.1021/acssensors.1c01908. Epub 2021 Dec 6.
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.
监测蛋白激酶活性对于生物化学基础、生物医学诊断和药物筛选具有重要意义。为了减少相对复杂的生物标记信号探针的使用，采用磷酸基团衍生的联吡啶钌（Pbpy-Ru）配合物和二氧化钛纳米颗粒（TiO NPs）作为信号探针，开发了一种用于评估蛋白激酶A（PKA）活性的电化学传感器。通过磷酸基团与TiO NPs之间的特异性相互作用，可以以简单有效的方式制备Pbpy-Ru-TiO NP信号探针并将其与磷酸化的PKA底物肽连接。将Pbpy-Ru连接到TiO NP表面不会降低配合物的电化学性质。Pbpy-Ru-TiO NP探针在相对于Ag/AgCl参比电极约1.0 V处表现出明确的氧化还原信号，并且其电流响应比物理吸附三（联吡啶）钌的TiO NPs的电流响应明显高约五倍。通过测量Pbpy-Ru-TiO NPs在磷酸化肽组装电极上的电化学响应来实现PKA活性评估。在最佳条件下操作，1.03 V电位下的阴极信号与0.5-40 U mL的PKA浓度呈现良好的线性关系。该电化学传感器具有良好的选择性、低检测限（0.2 U mL，信号/噪声 = 3）、合格的重现性以及在细胞裂解物中测定PKA的令人满意的适用性。Pbpy-Ru-TiO NPs/电极系统将是用于蛋白质磷酸化监测和传感的出色电化学平台。
