Multimodal Detection of α-Amanitin Using a Cell-Free Biosensing Platform

This study developed a transcription-based, multimodal cell-free biosensing platform for the sensitive detection of α-amanitin, a deadly mushroom toxin. The core design of the platform converts α-amanitin-dependent RNA polymerase inhibition into tunable, genetically encoded outputs. The system uses programmable DNA templates to produce specific RNA sequences, which are then processed in parallel by three independent signal transduction modules: fluorescence using the Malachite Green (MG) light-up RNA aptamer; colorimetry through RNA-directed gold nanoparticle aggregation; and enzymatic catalyzed by an in vitro transcribed G-quadruplex/hemin (G4/hemin) RNAzyme. This multimodal output strategy provides built-in self-validation for a single detection event, significantly enhancing result reliability. Evaluations demonstrated that the biosensor exhibits high sensitivity, with limits of detection (LODs) of 1.30 μg/mL for fluorescence detection, 0.69 μg/mL for AuNP-based colorimetric detection, and 6.29 μg/mL for RNAzyme-based colorimetric detection, and showed good selectivity and robust performance in complex matrices. Lyophilization tests confirmed operational stability for potential on-site applications. This work establishes a versatile, modular framework for transcription-based biosensing, with potential extension to other RNA polymerase inhibitors.