Electrochemical/Electrochemiluminescent Detection Of Protein Kinase Activity Utilizing Nanomaterial-based Signal Enhancement

Protein kinase-catalyzed phosphorylation plays a significant role in many vitalbiological processes, including metabolism, cellular signal transduction, cellproliferation and differentiation, and so on. Up to30%of human proteins aremodified by protein kinases and then could regulate the physiological functions ofcells. Abnormal protein phosphorylation can be closely related to many diseases suchas cancer, diabetes, Alzheimer’s and chronic inflammatory disease. Therefore, theidentification of kinase activities and their potential inhibitors is not only necessaryfor understanding many fundamental biological metabolism processes, but also veryimportant for the research of cancer development, diagnosis and treatment. There aremany traditional assay methods for protein kinase activities, which are effective buttime-consuming, laborious, complex and costly. The development of novel methodsfor the detection of protein kinase activities is greatly significant. Electrochemicaland electrochemiluminescence biosensors have received increasing attention due totheir advantages of high sensitivity, simple operation and good selectivity. With thedevelopment of nanotechnology, various nanomaterials have been gradually adoptedto construct electrochemical and electrochemiluminescence biosensing systems.Because of small sizes, large surface areas, excellent biocompatibility, and convenientsurface modification, nanomaterials show excellent signal enhancement effect in thedetection of DNA, proteins, small molecules, which holds great potential for thefurther improvement and optimization of electrochemical and electrochemi-luminescence biosensors. Aiming at the development of novel methods for proteinkinase activity assay，this thesis mainly focuses on constructing novel electrochemicaland electrochemiluminescence biosensors for the protein kinase activity detection andthe related inhibitors research by utilizing nanomaterials-based signal enhancement.The main works are listed as follows:1. TiO₂/MWNTs nanocomposites-based electrochemical strategy for label-freeassay of casein kinaseⅡactivity and inhibitionA novel label-free electrochemical strategy has been developed for assay ofcasein kinaseⅡ（CK2） activity and inhibition using TiO₂/MWNTs nanocomposites.This detection system takes advantage of specific binding of the phosphate groups with TiO₂nanoparticles and fast electron transfer rate of MWNTs. In this strategy, thesynthesized TiO₂/MWNTs nanocomposite was firstly deposited on the surface of aglassy carbon electrode （GCE）. The presence of MWNTs not only increased thesurface area of the electrode but also promoted electron-transfer reaction. In thepresence of CK2, the kinase reaction resulted in the phosphorylation of peptidesubstrates. The phosphorylated peptides were subsequently captured to the surface ofGCE modified with TiO₂/MWNTs nanocomposite through specific binding of thephosphate groups with TiO₂nanoparticles. Then the access of redox probe[Fe（CN）₆]^3-/4- to electrode surface was blocked. As a result, the decrease peakcurrents were related to the concentrations of the CK2, providing a se nsingmechanism for monitoring peptides phosphorylation. The electrochemical strategycan be employed to assay CK2activity with a low detection limit of0.07U/mL. Thelinear range of the assay for CK2was0U/mL to0.5U/mL. Furthermore, theinterferences experiments of PKA and inhibition of CK2have been also studied byusing this strategy.2. Highly sensitive electrochemiluminescent assay of protein kinase A activitybased on signal amplification of Rubpy-PSiNPsA highly sensitive and label-free ECL sensing strategy has been developed forassay of protein kinase A （PKA） activity and inhibition using Rubpy-PSiNPs as asignal indicator. In this method, Rubpy-PSiNPs are firstly synthesized by the reversemicroemulsion method. The substrate peptides self-assembled on the electrode arephosphorylated by PKA in the presence of ATP. Zirconium cations, which possess astrong coordination interaction with phosphate groups, can mediate the linkagebetween the phosphorylated peptides and Rubpy-PSiNPs. Rubpy-PSiNPs containing agreat deal of Ru（bpy）32+, can be covalently attached to the surface of Au electrode,thus generating the high ECL signal. The ECL intensity is proportional to the activityof PKA. This strategy can be employed to assay PKA activity with a low detectionlimit of0.005U/mL. The linear range of the assay for PKA was0.01U/mL to1U/mL.Furthermore, the interferences experiments of CK2and inhibition have been alsostudied by using this strategy.