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

Abstract

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 electrografting 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 μU/mL (∼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.

Bioinspired Electro-RAFT Polymerization for Electrochemical Sensing of Nucleic Acids

“…41 The surface-tethered R • radicals can react continuously with numerous ferrocenyl monomers, 41 leading to the dynamic growth of polymers, 32 thereby recruiting plenty of Fc electroactive tags for amplified sensing. 32,33 3.2. Redox Properties of the MNA Cation and the CPPA Agent.…”

“…The CV of MNA features two reduction peaks, corresponding to the formation of an organic radical (−0.36 V) and a dihydropyridine species (−0.6 V). 33,38 As the peak potential corresponding to the oneelectron reduction of CoI is located at −0.75 V, 33 the use of the MNA organic cation as the mediator holds the advantage of milder reduction potential. The CV of CPPA features a reduction peak at −1.02 V. 32 As the reduction potential corresponding to the electrogeneration of the organic radical is significantly higher (by about 0.66 V) than that of the CPPA agent, the electroreduction of the MNA cations would not be accompanied by the irreversible consumption of the CPPA agents.…”

“…BERP-Based Electrochemical Sensing of NAs. After the sequential pretreatment according to the previous report, 33 a cleaned Au electrode was incubated at 37 °C with 7.5 μL of the PNA recognition probe (0.5 μM) for 0.5 h. After soaking the Au/PNA…”

In Situ Assembly of Nanomaterials and Molecules for the Signal Enhancement of Electrochemical Biosensors

“…However, the utilization of Cu 2+ ions as catalysts may interfere with the next electrochemical experiments because of the non-specific interaction with nucleic acids and the electrochemical deposition of metal on the electrode. For this consideration, they further explored novel surface-initiated electrochemically controlled reversible-addition-fragmentation-chain-transfer (SI-eRAFT) polymerization without the use of transition metal ions for assays [90][91][92]. For instance, they reported a signal-amplified electrochemical sensing of thrombin activity by SI-eRAFT polymerization [93].…”

“…aterials 2021, 11, x 12 of 19 use of transition metal ions for assays [90][91][92]. For instance, they reported a signal-amplified electrochemical sensing of thrombin activity by SI-eRAFT polymerization [93].…”