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The need for rapid and sensitive diagnostic tools is emphasized by the significant impact of infectious diseases on global health. This study presents a cell-free biosensor utilizing toehold switch technology, combined with nucleic acid sequence-based amplification (NASBA), for high specificity and sensitivity in Zika virus detection. The toehold switch, a denovo-designed regulator of gene expression, forms the crux of our detection system, offering a versatile and programmable approach to nucleic acid–based diagnostics. The cell-free system based on Escherichia coli extract served as the platform for sensor expression, enabling real-time monitoring and optimization of the reaction conditions for minimal background leakage and maximal activation efficiency. The performance of the toehold switch sensor was rigorously evaluated through a series of tests, revealing that switch S23 demonstrated the most promising activation effects and sequence specificity. Notably, the integration of NASBA technology significantly enhanced the detection sensitivity, achieving a remarkable limit of 2.9 aM, thus addressing the intrinsic limitation of toehold switches in detecting low-abundance targets. The detection system’s low cost, simplicity, and adaptability to various pathogens render it a valuable asset in the global health toolkit. This study presents a significant advancement in the field of synthetic biology, offering a robust, sensitive, and rapid diagnostic solution for Zika virus detection.
Chen et al. (Mon,) studied this question.