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

Area of Science:

• Biotechnology
• Biosensing
• Molecular Diagnostics

Background:

• α-amanitin is a potent toxin found in certain mushrooms, posing a significant health risk.
• Accurate and sensitive detection methods for α-amanitin are crucial for public safety and clinical diagnostics.

Purpose of the Study:

• To develop a transcription-based, multimodal cell-free biosensing platform for sensitive α-amanitin detection.
• To convert α-amanitin-dependent RNA polymerase inhibition into detectable outputs.
• To enhance the reliability of toxin detection through a multimodal output strategy.

Main Methods:

• Utilized programmable DNA templates to generate RNA sequences.
• Employed three parallel signal transduction modules: Malachite Green aptamer fluorescence, RNA-directed gold nanoparticle colorimetry, and G-quadruplex/hemin RNAzyme catalysis.
• Integrated a multimodal output strategy for built-in self-validation.

Main Results:

• Achieved high sensitivity with limits of detection (LODs) of 1.30 μg/mL (fluorescence), 0.69 μg/mL (AuNP colorimetry), and 6.29 μg/mL (RNAzyme colorimetry).
• Demonstrated good selectivity and robust performance in complex matrices.
• Confirmed operational stability through lyophilization, indicating potential for on-site applications.

Conclusions:

• Established a versatile and modular transcription-based biosensing framework.
• The developed platform offers sensitive, reliable, and self-validated detection of α-amanitin.
• The framework has potential for extension to detect other RNA polymerase inhibitors.