Electrochemically Controlled RAFT Polymerization for Highly Sensitive Electrochemical Biosensing of Protein Kinase Activity

Phosphorylation of proteins catalyzed by protein kinases (PKs) is essential to many biological processes; the sensitive detection of PK activity and the screening of PK inhibitors are thus integral to disease diagnosis and drug discovery. Herein, a highly sensitive biosensor has been fabricated for the electrochemical detection of PK activity by exploiting the electrochemically controlled reversible addition–fragmentation chain transfer (eRAFT) polymerization as a novel amplification strategy. The fabrication of the eRAFT-polymerization-based electrochemical biosensor involves (1) the immobilization of substrate peptides onto a gold electrode by way of gold–sulfur self-assembly, (2) the site-specific phosphorylation of substrate peptides by PKs, (3) the anchoring of carboxyl-group-containing chain transfer agents (CTAs) to the phosphorylated sites, and (4) the eRAFT polymerization under a potentiostatic condition, using ferrocenylmethyl methacrylate (FcMMA) as the monomer. Through the eRAFT polymerization, long polymer chains containing numerous electroactive Fc tags can be de novo grafted from each phosphorylated site, resulting in significant amplification of the electrochemical detection signal. The as-fabricated biosensor is highly selective and features a very low detection limit of 1.02 mU mL^–1, in the presence of adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent PK (PKA) as the model target. Results also demonstrate that it can be applied to the screening of PK inhibitors and the detection of PK activity in complex serum samples and cell lysates. Moreover, it holds the merits of easy fabrication, high efficiency, and low cost, which make it a promising tool for the detection of PK activity and the screening of potential PK inhibitors.