Electrochemically controlled grafting of polymers for ultrasensitive electrochemical assay of trypsin activity.

As one of the most important proteolytic enzymes, trypsin is useful as a reliable and specific biomarker for the diagnosis of pancreatitis and other pathological conditions. In this paper, a novel signal-on electrochemical biosensor based on the use of electrochemically controlled grafting of polymers as an amplification strategy is described for the ultrasensitive assay of trypsin activity. The carboxyl-group-free peptide, serving as the substrate for the recognition of trypsin, is first immobilized via its N-terminus. The tryptic cleavage of peptide substrate can generate a free carboxyl group at the C-terminus of the truncated peptide, to which through the carboxylate-Zr(IV)-carboxylate linkage the carboxyl-group-containing initiator for atom transfer radical polymerization (ATRP) can be conjugated. The subsequent surface-initiated grafting of polymers (SI-GOP) based on electrochemically controlled ATRP (eATRP), with ferrocenylmethyl methacrylate (FcMMA) as the monomer, can bring a large amount of Fc tags to electrode surface, resulting in the generation of a very high detection signal. The eATRP-based SI-GOP is easy to operate and low-cost as an amplification strategy. Under optimal conditions, the detection limit for trypsin activity can be down to 0.016 mU mL (~2.68 pM or ~0.064 ng mL). As the current signal increases with trypsin activity, this trypsin biosensor is less susceptible to false positives due to the signal-on mode. Moreover, it is highly selective and applicable to inhibitor screening and the assay of trypsin activity in the presence of complex biological matrices. Taking together, this electrochemical trypsin biosensor may hold great potential in diagnostic applications.