Primer exchange reaction catalyzed DNA walker cooperating target recycling for sensitive and reliable microRNA analysis

MicroRNAs (miRNAs) have gained significant attention as potential biomarkers and therapeutic targets for cardiovascular diseases (CVDs). However, the sensitive detection of miRNAs in complex biological samples remains challenging due to their low abundance and interference from non-specific targets, despite its critical importance for biopsy analysis and disease diagnosis. In this study, we developed a self-priming DNA polymerization-initiated PER-driven DNA walking system for the highly sensitive detection of miRNA targets in complex biological matrices. The sensing system was immobilized on a plate surface, which not only provides a three-dimensional interface for DNA walkers but also enhances the enrichment and isolation of RNA targets from complex samples such as serum. The DNA walking system is activated by target recognition-mediated self-priming DNA polymerization, enabling the walker to traverse the plate surface through primer exchange reaction (PER) catalysis, thereby converting RNA targets into amplified fluorescence signals. This platform offers ultra-high sensitivity with a detection limit of 0.76 fM for miRNA-21, enabling precise measurement even in complex biological samples. It also provides excellent specificity through a label-free ThT-based fluorescence mechanism, effectively distinguishing target miRNAs from homologous sequences without the need for complex labelling. This platform holds significant promise for applications in RNA bio-sensing and biomedical diagnostics.