Immobilized-free miniaturized electrochemical sensing system for Pb 2+ detection based on dual Pb 2+ -DNAzyme assistant feedback amplification strategy.

We presented a novel dual-DNAzyme feedback amplification (DDFA) strategy for Pb2+ detection based on a micropipette tip-based miniaturized homogeneous electrochemical device. The DDFA system involves two rolling circle amplification (RCA) processes in which two circular DNA templates (C1 and C2) have been designed with a Pb2+ -DNAzyme sequence (8-17 DNAzyme, anti-GR-5 DNAzyme) and an antisense sequence of G-quadruplex. And a linear DNA (L-DNA), which consists of a primer sequence and a Pb2+ -DNAzyme substrate sequence, could hybridize with C1 and C2 to form two DNA complexes. In presence of Pb2+ , the Pb2+ -DNAzyme exhibited excellent cleavage specificity toward the substrate sequence in L-DNA, leaving primer sequence to trigger two paths of RCA process and finally resulting in massive long nanosolo DNA strands with reduplicated G-quadruplex sequences. And then, methylene blue (MB) could selectively intercalate into G-quadruplex to reduce the free MB concentration in the solution. Thereafter, a carbon fiber microelectrode-based miniaturized electrochemical device was constructed to record the decrease of electrochemical signal due to the much lower diffusion rate of MB/G-quadruplex complex than that of free MB. Therefore, the concentration of Pb2+ could be correctively and sensitively determined in a homogeneous solution by combining DDFA with miniaturized electrochemical device. This protocol not only exhibited high selectivity and sensitivity toward Pb2+ with a detection limit of 0.048 pM, but also reduced sample volume to 10 µL. In addition, this sensing system has been successfully applied to Pb2+ detection in Yangtze River with desirable quantitative manners, which matched well with the atomic absorption spectrometry (AAS).