Ultralow background one-pot detection of Lead(II) using a non-enzymatic double-cycle system mediated by a hairpin-involved DNAzyme

Abstract

A double-cycle system has been developed for specifically detecting trace amounts of Pb²⁺ by significantly decreasing the background signal. The detection involves two types of RNA cleavage reactions: one using a Pb²⁺-specific GR5 DNAzyme (PbDz) and the other utilizing a newly constructed 10-23 DNAzyme with two hairpins embedded in its catalytic center (hpDz). The ring-structured hpDz (c-hpDz) exhibits significantly lower activity compared to the circular 10-23 DNAzyme without hairpin structures, which plays a crucial role in reducing the background signal. When Pb²⁺ is present, PbDz cleaves c-hpDz to its active form, which then disconnects the molecular beacon to emit the fluorescent signal. The method allows for rapid and sensitive Pb²⁺ detection within 40 min for 10 fM of Pb²⁺ and even as short as 10 min for 100 nM of Pb²⁺. Additionally, visual detection is possible through the non-crosslinking assembly of Au nanoparticles. The entire process can be performed in one pot and even one step, making it highly versatile and suitable for a wide range of applications, including food safety testing and environmental monitoring.

Keywords: DNAzyme; Hairpin structure; Lead; Low background; Rapid detection.