Amplified Electrochemical Biosensing of Thrombin Activity by RAFT Polymerization

As a serine protease, thrombin is a pivotal component in coagulation cascade and has been frequently screened as an informative biomarker for the diagnosis of coagulation disorder-related diseases. Herein, a "signal-on" electrochemical biosensor is described for the highly sensitive and selective detection of thrombin activity, by exploiting a thrombin-specific substrate peptide (Tb peptide) as the recognition element and reversible addition-fragmentation chain transfer (RAFT) polymerization for signal amplification. Specifically, the carboxyl-group-free Tb peptides are self-assembled onto gold electrode surface via the N-terminal cysteine residue and are used for the specific recognition of thrombin molecules. After the proteolytic cleavage of the Tb peptides, the carboxyl-group-containing RAFT agents (4-cyano-4-(phenylcarbonothioylthio)pentanoic acid, CPAD) are tethered to the free carboxyl termini of the truncated peptide fragments via the carboxylate-zirconium-carboxylate chemistry. The subsequent RAFT polymerization leads to the grafting of a polymer chain from each proteolytically cleaved site, enabling the recruitment of a large number of electroactive ferrocene (Fc) tags to the electrode surface when ferrocenylmethyl methacrylate (FcMMA) is used as the monomer. Under optimal conditions, the detection limit of the described thrombin biosensor is as low as 2.7 μU mL^-1 (∼0.062 pM), with a linear response over the range of 10-250 μU mL^-1 (R² = 0.997). Results also indicate that the biosensor is highly selective and applicable to the detection of thrombin activity in complex serum samples and the screening of thrombin inhibitors. The described biosensor is low-cost and relatively easy in preparation and thus shows great promise for the highly sensitive and selective detection of thrombin activity.