DNA Nanoarray for Multiplexed RNA Detection With Single‐Molecule Readout

ABSTRACT Sensitive and multiplexed RNA analysis at the single‐molecule level remains a key challenge in molecular diagnostics. Conventional fluorescence microarrays provide high throughput but lack molecular resolution, whereas single‐molecule sensors such as nanopores offer exquisite precision but limited scalability. Here, we present a DNA carrier‐based nanoarray that integrates programmable nucleic acid hybridization with solid‐state nanopore readout for direct, multiplexed RNA detection. Each modular DNA carrier is pre‐assembled with spatially defined probe sites that sequence‐specifically capture RNA targets of varying lengths and conformations, in which poly(dT) sequences both structurally facilitate target binding and enhance signal strength without the need for fluorescent or protein labeling. Upon nanopore translocation, hybridized carrier‐target complexes generate binary ionic current signatures, allowing single‐molecule identification of target occupancy at each sensing site. Using a ternary coordinate encoding system, we constructed a nanoarray comprising 27 carriers with 81 addressable sensing sites, enabling simultaneous detection of multiple bacterial and viral RNA targets within a single assay directly from total RNA extracts without target‐specific isolation, amplification, or enrichment. By combining the multiplexing capability of traditional microarrays with the single‐molecule precision of nanopore sensing, this carrier‐based nanoarray establishes a scalable, programmable, and universally adaptable framework for high‐throughput molecular diagnostics in complex biological backgrounds.