Molecular Crowding‐Driven RNA Condensates Enable Förster Resonance Energy Transfer‐Enhanced Small‐Molecule Sensing

ABSTRACT Fluorescent RNA aptamers offer promising opportunities for next‐generation biosensing but are often limited by low signal‐to‐background ratios and unstable folding kinetics. In this work, a label‐free Förster resonance energy transfer (FRET)‐enhanced fluorescent artificial RNA condensate (F‐FARCON) is developed for small‐molecule sensing, leveraging neutral molecular crowders (e.g., polyethylene glycol 8K), and RNA structural motifs to induce multivalent interactions and drive dynamic self‐assembly. As a demonstration, a label‐free FRET system is constructed by integrating a histamine‐responsive RNA aptamer with thioflavin T (ThT) as the fluorescence donor, which increases the signal‐to‐noise ratio while reducing sequence complexity and production costs. Molecular crowders optimize the thermodynamic environment of RNA–ligand and RNA–RNA multivalent interactions, thereby improving folding stability, signal amplitude (dynamic range of up to ∼970‐fold), and target affinity. The platform exhibits fast kinetics (<15 min), an adjustable detection range (0.1–200 and 5–1000 µM), and high sensitivity (limit of detection, 15.36 nM), with robust performance in complex biological matrices. The platform is further integrated into a freeze‐dried paper‐based portable device that enables dual‐channel fluorescence readout for on‐site rapid detection without sophisticated instrumentation. To further validate the modularity of F‐FARCON beyond histamine, we reprogrammed the recognition module to target S‐adenosyl‐L‐methionine (SAM), achieving nanomolar limits of detection. By linking crowding‐guided assembly to hierarchical photophysical enhancement and analytical performance, the work delineates a generalizable aggregate‐science route to versatile, low‐cost, and field‐deployable fluorescence sensing across food safety, environmental monitoring, and biomedical diagnostics.