Jie Zhou, Yuwei Wu, Yaqin Chai, Pu Zhang, Ruo Yuan
A constitutional dynamic DNA network (CDN) integrated with catalytic hairpin assembly (CHA) was developed for the accurate ratiometric electrochemical detection of multiple microRNAs (miRNAs). To overcome the limitations of conventional single-signal multiplex assays, this strategy relies on a [2×2] equilibrium system that undergoes target-triggered dynamic reconfiguration in the presence of miRNA-21 or miRNA-221. This reconfiguration regulates the cleavage of specific signal probes at the electrode interface, while distinct dynamic states initiate orthogonal CHA pathways. Subsequently, generated target mimics adaptively amplify the DNA assembly, producing opposing methylene blue (MB) and ferrocene (Fc) electrochemical responses for dual-channel ratiometric readout. Under optimal conditions, a broad linear range from 1 fM to 1 nM was achieved, with detection limits of 0.57 fM and 0.68 fM for miRNA-21 and miRNA-221, respectively. The analytical reliability of the method was further demonstrated through the successful discrimination of endogenous miRNA expression levels in HeLa, MCF-7, and MHCC97L cell lysates. Ultimately, this adaptive dynamic DNA system provides a highly precise and robust platform for multiplexed biomarker analysis in complex biological samples.