Electrochemical strategy for detection of phosphorylation based on enzyme-linked electrocatalysis

Abstract

In this paper, we report an electrochemical strategy for the detection of phosphorylation based on enzyme-linked electrocatalysis. In this strategy, the substrate peptides modified on a gold electrode surface are firstly phosphorylated by protein kinase, and then biotinylated via the recognition and combination with the specific biotin-labeled antibody. After that, gold nanoparticles (Au-NPs) which are modified with horseradish peroxidase (HRP) loaded streptavidin are immobilized on the surface of the electrode through biotin–avidin interaction. As a result, HRP on the electrode surface may take catalytic reaction towards the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB), and the generated electrochemical signals can be used to monitor phosphorylation. This proposed electrochemical strategy can be employed to detect kinase activity with a satisfactory detection limit of 1 × 10 −3 unit/mL and linear range from 1 × 10 −3 to 1 × 10 −2 unit/mL. Furthermore, the inhibition of protein kinase has been also studied by using this strategy.