Ultra-sensitive nucleic acid detection based on target cycling of triple helix molecular switch and ATRP double signal amplification

We propose a novel electrochemical biosensor platform with triple helix structure as switch, Exonuclease III (Exo III)-mediated target cycle at 2-D level and electrochemical-mediated atom transfer radical polymerization (ATRP) at 3-D level as double signal amplification strategy for nucleic acid detection with ultra-sensitivity and high-selectivity. In this strategy, triple-helical DNA modified with N3 was first constructed on the gold electrode (AuE) surface. Exo III mediated target cycle was then triggered in the presence of Target DNA. N3 was released and exposed on the AuE surface, owing to the triple helix structure was destroyed in this process. propargyl-2bromoisobutyrate (PBIB), the initiator of ATRP, was then introduced to the biosensor surface via click chemistry. Finally, electrochemically mediated ATRP polymerizes a large amount of electroactive monomer, ferrocene methacrylate (FMMA), onto the AuE surface. The target circulates to make target DNA reused at the 2-D level, and ATRP polymerizes a large number of electrochemical signal molecules at the 3-D level, making the biosensor detection limit as low as 1.954 aM. Moreover, the linear range of target DNA detection reaches 7 orders of magnitude (10 aM-10 fM, R2 = 0.993). In addition, the biosensor shows excellent anti-interference ability when analyzing DNA in serum samples. In short, the preparation method is rapid, simple, easy to operate, and has potential applications in biological analysis.