Development of a Cell-Free, Toehold Switch-Based Biosensor for Rapid and Sensitive Zika Virus Detection

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.