We herein report a novel electrochemical method in this paper to monitor protein phosphorylation and to assay protein kinase activity based on Zr4+ mediated signal transition and rolling circle amplification (RCA). First, substrate peptide immobilized on a gold electrode can be phosphorylated by protein kinase A. Then, Zr4+ links phosphorylated peptide and DNA primer probe by interacting with the phosphate groups. After the introduction of the padlock probe and phi29 DNA polymerase, RCA is achieved on the surface of the electrode. As the RCA product, a very long DNA strand, may absorb a large number of electrochemical speices, [Ru(NH3)(6)](3+), via the electrostatic interaction, localizing them onto the electrode surface, initiated by protein kinase A, a sensitive electrochemical method to assay the enzyme activity is proposed. The detection limit of the method is as low as 0.5 unit/mL, which might promise this method as a good candidate for monitoring phosphorylation in the future.
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