An electrochemical biosensor based on ARGET ATRP with DSN-assisted target recycling for sensitive detection of tobacco mosaic virus RNA.

Herein, an electrochemical biosensor for detecting tobacco mosaic virus (TMV) RNA is constructed by activator regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) combined with duplex-specific nuclease (DSN)-assisted target recycling. First, the captured DNA (cDNA) is self-assembled on the electrode surface and hybridizes with the TMV RNA (tRNA) to form cDNA/tRNA hybrids. And then the initiator of ARGET ATRP (α-bromoisobutyric acid, BMP) is attached to the cDNA via an amide bond and later triggers ARGET ATRP. Many electroactive monomers (ferrocenylmethyl methacrylate, FMMA) are polymerized and a remarkable electrical signal response of ferrocene (Fc) is obtained. However, with the present of DSN, DSN cleaves the cDNA/tRNA hybrid and releases tRNA to hybridize with another cDNA, thereby causing significant shortening of the length of the cDNA. The number of polymer chains on the electrode surface is drastically reduced, which is followed by a noticeable reduction in the signal of Fc. The method shows high sensitivity, superior selectivity, excellent stability and good reproducibility under optimal conditions with the limit of detection (LOD) of 2.9 fM. Furthermore, the biosensor showed satisfactory applicability in detecting tRNA in real samples, thereby demonstrating the potential of the method for practical TMV RNA detection.