Hyperbranched Rolling Circle Amplification Enabled Nanopore Sensing Platforms for Ultrasensitive Multiplex MicroRNA Detection and Logic-Gated Molecular Diagnostics.

MicroRNAs (miRNAs) have emerged as crucial biomarker candidates and therapeutic targets for various diseases and cancers, where precise trace-level detection holds significant value for early clinical diagnosis. However, the development of ultrasensitive, isothermal, cost-effective, and reliable miRNA detection platforms remains challenging in clinical applications. Herein, we present a nanopore sensing strategy based on hyperbranched rolling circle amplification (HRCA) for label-free detection and logic-gated diagnosis of miR-122 and miR-141. This method synergistically integrates the high amplification efficiency of HRCA with the single-molecule sensitivity of nanopore sensors, enabling ultrasensitive miRNA detection at ultralow concentrations. The results demonstrate that the HRCA-nanopore system achieves a remarkable detection limit of 0.1 fM, representing a three-order-of-magnitude improvement compared to conventional HRCA-based fluorescence methods, while maintaining excellent specificity to distinguish target miRNAs from interfering sequences. Leveraging the programmable hybridization characteristics and conformational switching of nucleic acids, we further constructed a four-input parallel cascade logic circuit (INHIBIT//INHIBIT-OR) for simultaneous dual-miRNA analysis. This logic architecture establishes a novel conceptual framework for implementing molecular computing in nanopore-based biosensing, particularly for multiplex biomarker diagnosis. The proposed platform not only addresses the critical need for clinical miRNA detection but also provides a versatile paradigm for developing intelligent diagnostic systems through the integration of nucleic acid amplification techniques, nanoscale sensing, and molecular logic operations.