Programmable AIESTA: All-in-One Isothermal Enzymatic Signal Transduction Amplifier for Portable Profiling

Abstract

The Argonaute (Ago) protein exhibits high specificity in nucleic acid recognition and cleavage, making it highly promising for biosensing applications. Its potential is further enhanced by its independence from protospacer adjacent motif (PAM) requirements and the cost-effectiveness of using short DNA guides. Both Ago and CRISPR/Cas systems face challenges in signal amplification, which limit their ability to detect targets at ultralow concentrations. To overcome this limitation, a thermostable quadratic amplification system (T-QAS) was constructed by integrating a thermostable nicking-enzyme-mediated amplification (NEMA) strategy with TtAgo. The system leverages the high stability of T-QAS at elevated temperatures to enhance guide–target interactions and decrease false positives caused by nonspecific amplification. Additionally, nanozyme is integrated with T-QAS to construct the AIESTA platform (all-in-one isothermal enzymatic signal transduction amplifier), which is a single-tube visual sensing platform. Within the AIESTA system, T-QAS improves specificity through high operational temperatures and offers programmable functions, enabling the sensitive detection of miRNA and foodborne toxins. The combination of T-QAS and nanozyme makes AIESTA a candidate of point-of-care testing (POCT) field, showcasing the potential for biosensing in resource-limited and complex environments.