Coenzyme-mediated electro-grafting for ultrasensitive electrochemical DNA biosensing

Abstract

Signal amplification through the grafting of polymers has attracted increasing attention in biodetection at ultralow concentrations. Herein, an ultrasensitive peptide nucleic acid (PNA)-based electrochemical DNA biosensor is reported by using the coenzyme-mediated electro-grafting of polymers as a novel amplification method. For the capture of DNA target, PNA is used as the probe. After hybridization, reversible addition-fragmentation chain-transfer (RAFT) agents are site-specifically tethered to the deoxyribose phosphate backbone of the captured DNA targets via the carboxylate-Zr(IV)-phosphate cross-linking chemistry. Subsequently, electro-grafting of polymers through the nicotinamide adenine dinucleotide (NAD+) coenzyme-mediated electro-RAFT (NAD+-eRAFT) polymerization of ferrocenylmethyl methacrylate (FcMMA) recruits numerous ferrocene redox tags for voltammetric measurement. As the electro-grafting of polymers involves simply i) the tethering of RAFT agents and ii) the NAD+-eRAFT polymerization, it is highly efficient, easy to use, and low-cost. Under optimal conditions, the voltammetric signal correlates linearly with the logarithm of DNA concentration over the range from 0.1 fM to 0.1 nM (R2 = 0.996), with a detection limit of 0.067 fM. The PNA-based electrochemical DNA biosensor can also differentiate even single base mismatch and is applicable to DNA detection in the presence of complex serum matrices, thus showing great promise in the sequence-specific detection of DNA targets at ultralow concentrations.