Exonuclease III-based target recycling for ultrasensitive homogeneous monitoring of HIV DNA using Ag+-coordinated hairpin probe

Abstract

A new homogeneous electrochemical sensing strategy based on exonuclease III-assisted target recycling amplification was utilized for simple, rapid and highly sensitive detection of human immunodeficiency virus (HIV) DNA on an immobilization-free Ag(I)-assisted hairpin DNA through the cytosine-Ag(+)-cytosine coordination chemistry. The assay involved target-induced strand-displacement reaction accompanying dissociation of the chelated Ag(+) in the hairpins and exonuclease III-triggered target recycling. Initially, the added target DNA hybridized with hairpin DNA to disrupt the Ag(I)-coordinated hairpin probe and releases the coordinated Ag(+) ion. Then, the newly formed DNA double-stranded DNA could be cleaved by exonuclease III, and released target HIV DNA, which retriggered the strand-displacement reaction with the hairpin for target recycling, thereby resulting in formation of numerous free Ag(+) ions in the detection cell. The released Ag(+) ions can be readily captured by the negatively charged electrode, and subsequent anodic-stripping voltammetric detection of the captured Ag(+) ions are conducted to form the anodic current for the production of the electronic signal within the applied potential. Under optimal conditions, the exonuclease III-based sensing system exhibited good electrochemical responses for the detection of HIV DNA at a concentration as low as 23 fM.