Ultrasensitive peptide-based electrochemical detection of protein kinase activity amplified by RAFT polymerization

Since the oversecretion of protein kinases is indicative of multiple human diseases, the screening of their activities is quite important to clinical diagnosis and targeted therapy. In this work, an ultrasensitive peptide-based electrochemical biosensor was presented for the detection of protein kinase activity by using the reversible addition-fragmentation chain transfer (RAFT) polymerization technique as a signal amplification strategy. First, the substrate peptides were tethered to a gold electrode surface via the thiol terminals. After the phosphorylation of substrate peptides by protein kinases, the carboxyl group-containing dithiobenzoates were labeled to the phosphorylated sites via the robust phosphate-Zr⁴⁺-carboxylate linkages. Finally, the RAFT polymerization was initiated using ferrocenylmethyl methacrylates (FcMMAs) and dithiobenzoates as the monomers and the RAFT agents, respectively. The grafting of ferrocenyl polymer chains efficiently recruits a great number of electro-active Fc probes to each phosphorylated site, leading to a drastic amplification of the electrochemical signal. With PKA (protein kinase A) as the target, the detection limit of the peptide-based biosensor can be as low as 1.05 mU mL^-1. Moreover, it can selectively differentiate the target from other interferents and is applicable for the screening of potential inhibitors as well as the detection of protein kinase activity in complex cell lysates. Therefore, the peptide-based biosensor shows great promise as a universal tool for protein kinase activity detection and inhibitor screening.