A self-reinforced DNAzyme nanomachine for multiplexed miRNA imaging and breast cancer staging

MicroRNAs (miRNAs) precisely coordinate complex gene regulatory networks and serve as critical biomarkers for disease diagnosis. However, simultaneous monitoring of intracellular multiple miRNAs remains constrained by inefficient delivery and insufficient signal gain. Herein, we construct a self-reinforced DNAzyme nanomachine (Dz@hMNS) by integrating a honeycomb MnO₂ nanosponge (hMNS) with a miRNA-responsive DNAzyme system for simultaneous visualization of miR-21, miR-155, and miR-10b in living cells. Upon encountering the glutathione (GSH)-rich intracellular environment, the hMNS scaffold efficiently delivers miRNA-responsive DNAzyme system into cells and releases Mn²⁺ as an essential cofactor, facilitating target miRNAs-activated cyclic cleavage of reporter probes to generate amplified fluorescence signals. This self-reinforced mechanism operates without either protein enzymes or exogenous amplification, achieving a detection limit of 50.13 fM for miR-21, 34.78 fM for miR-155, and 49.10 fM for miR-10b. By simultaneously profiling three miRNAs, this Dz@hMNS nanomachine can discriminate breast cancer patients from healthy donors and distinguish among stages I-III breast cancers with 100% accuracy (AUC = 1.000), superior to single-miRNA analysis. Moreover, it enables real-time imaging of multiple miRNAs in living cells with minimal crosstalk and can be reprogrammed to detect other RNA species (e.g., piRNAs, lncRNAs, and circRNAs), with promising applications in biological research and cancer diagnosis.