Pattern-Verifiable Heterowalkers Scaffolded on a DNA Origami Nanosheet for Multiplexed Profiling of miRNAs

Multiplexed profiling of microRNAs (miRNAs) is central to refining diagnosis and prognosis, yet conventional fluorescence-based strategies remain vulnerable to environmental interference and signal crosstalk and rarely provide ground-truth validation of nanoprobe conformation. Here, we report pattern-verifiable heterowalkers (HW-TDONs) that integrate three DNAzyme walking modules confined on a triangular DNA origami nanosheet, each tethered to a size-distinct gold nanoparticle (10/15/20 nm) for the concurrent sensing of miRNA-10b, miRNA-21, and miRNA-155. Target recognition triggers autonomous DNAzyme walking that cleaves substrate strands, activating orthogonal fluorophores, and reconfiguring the origami pattern via AuNP detachment from predesignated sites. This dual-mode output quantitatively couples tricolored fluorescence with atomic force microscopy (AFM)─resolvable nanopattern statistics, thereby self-validating signal generation and mitigating false interpretations. This reliable and versatile platform enables combinatorial miRNA quantification in cell lysates and permits simultaneous imaging of multiple miRNAs in living cells, thereby supporting precise cell subtype discrimination. By coupling nanoscale conformational inspection to orthogonal optical readouts, HW-TDONs provide a programmable and scalable toolkit for facilitating multiplexed nucleic acid analysis and marker-associated pathological exploration with expansible potential for integrating patient-specific biomarkers in biomedical applications.