Hyperbranched Rolling Circle Amplification Enabled Nanopore Sensing Platforms for Ultrasensitive Multiplex MicroRNA...

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.