Unlocking all activation modes of Thermus thermophilus Argonaute for comprehensive gold nanoparticle sensor

Rapid and accurate molecular diagnosis is crucial for evaluating detection methods. Thermus thermophilus Argonaute (TtAgo) has gained prominence in molecular diagnosis as a state-of-the-art tool, owing to its low-level sensitivity, single-base specificity, and rapid turnover. However, TtAgo-based detection strategies are currently mainly limited to the detection of nucleic acid mutations and lack of research on coupled nanomaterials. Thus, the development of a comprehensive TtAgo sensing platform is needed. In this study, we fabricated functionalized gold nanoparticles capable of utilizing various sources of guide DNA for TtAgo activation, including phosphate transfer, enzymatic cleavage, and direct introduction. The versatile platform enables the detection of three types of biomarkers: small molecules, proteins, and nucleic acids. Furthermore, we thoroughly analyzed all three assays, showcasing their exceptional specificity and sensitivity with the advantages of functionalized gold nanoparticle reporter. Introducing TtAgo as an effective tool offers valuable insights that can advance future molecular diagnosis.