Coenzyme-Mediated Electro-RAFT Polymerization for Amplified Electrochemical Interrogation of Trypsin Activity

Trypsin is a key proteolytic enzyme in the digestive system and its abnormal levels are indicative of some pancreatic diseases. Taking advantage of the coenzyme-mediated electro-grafting of ferrocenyl polymers as a novel strategy for signal amplification, herein, a signal-on cleavage-based electrochemical biosensor is reported for the highly selective interrogation of trypsin activity at ultralow levels. The construction of the trypsin biosensor involves (i) the immobilization of peptide substrates (without free carboxyl groups) via the N-terminus, (ii) the tryptic cleavage of peptide substrates, (iii) the site-specific labeling of the reversible addition-fragmentation chain transfer (RAFT) agents, and (iv) the grafting of ferrocenyl polymers through the electro-RAFT (eRAFT) polymerization, which is mediated by potentiostatic reduction of nicotinamide adenine dinucleotide (NAD(+)) coenzymes. Through the NAD+-mediated eRAFT (NAD(+)-eRAFT) polymerization of ferrocenylmethyl methacrylate (FcMMA), the presence of a few tryptic cleavage events can eventually result in the recruitment of a considerable amount of ferrocene redox tags. Obviously, the NAD(+)-eRAFT polymerization is low-cost and easy to operate as a highly efficient strategy for signal amplification. As expected, the as-constructed biosensor is highly selective and sensitive toward the signal-on interrogation of trypsin activity. Under optimal conditions, the detection limit can be as low as 18.2 mu U/mL (similar to 72.8 pg/mL). The results also demonstrate that the as-constructed electrochemical trypsin biosensor is applicable to inhibitor screening and the interrogation of enzyme activity in the presence of complex sample matrices. Moreover, it is low-cost, less susceptible to false-positive results, and relatively easy to fabricate, thus holding great potential in diagnostic and therapeutic applications.

Automated summary

A novel signal-on cleavage-based electrochemical biosensor for highly selective interrogation of trypsin activity at ultralow levels is reported, utilizing coenzyme-mediated electro-grafting of ferrocenyl polymers. The constructed trypsin biosensor involves immobilization of peptide substrates, tryptic cleavage, labeling of RAFT agents, and grafting of ferrocenyl polymers mediated by potentiostatic reduction of NAD(+) coenzymes. This strategy is cost-effective and highly efficient for signal amplification, leading to a highly selective and sensitive biosensor with potential applications in diagnostics and therapeutics.