A DNA Nanoplatform with Rolling Circle-DNAzyme Feedback Programming for Autonomous Exponential Amplification Enables Multiplexed Orthogonal miRNA Profiling toward Precise Diagnosis of Bladder Cancer

Abstract Simultaneous and sensitive detection of multiple microRNAs in complex biological and clinical serum samples is critical for early and accurate bladder cancer (BC) diagnosis. Here, by integrating I-R3-mediated self-cleavage-coupled feedback amplification into three independently programmed circular templates, we developed an orthogonal multiplex exponential rolling circle amplification (RCA) fluorescent sensing platform (termed the 3E-RCA system) for the simultaneous detection of three BC-associated miRNAs (miR-21, miR-155, and miR-183). Target miRNA recognition initiated the RCA reaction to generate tandem products rich in the I-R3 DNAzyme, which achieved Zn2+-mediated site-specific cleavage and released numerous secondary primers to drive cyclic amplification for exponential signal enhancement. The released primers specifically hybridized with molecular beacons to unfold hairpin structures and restore orthogonal multichannel fluorescent signals, enabling interference-free multi-target detection. Importantly, the three amplification circuits were independently programmed and operated in parallel, with each circuit coupled to a corresponding molecular beacon and a spectrally distinct fluorescence channel, enabling target-specific amplification and orthogonal FAM, Cy3, and Cy5 signal readout within a single reaction system. Benefiting from this cascade cyclic amplification strategy, the system exhibited excellent single-base discrimination ability and multi-target detection orthogonality, with low limits of detection of 0.38 pM, 0.31 pM, and 0.28 pM for the three miRNAs, which greatly improved the detection sensitivity compared with conventional linear RCA assays. Clinical serum sample validation verified that this multi-miRNA combinatorial signature effectively distinguished BC patients from healthy individuals and improved diagnostic AUC values (0.966), which provided a high-performance and non-invasive tool for early BC molecular diagnosis and exhibited promising clinical translation potential.