LAUSR

Argonaute (Ago) as a powerful enzyme has provided new insights into biosensing due to its programmability, high sensitivity, and user‐friendly operation. However, current strategies mainly rely on phosphorylated guide DNA to modulate the cleavage activity of Ago, which is limited in versatility and simplicity. Herein, the authors report the Mn2+‐enhanced cleavage activity of Ago and employ Mn‐ions with variable valence to regulate the activity of Pyrococcus furiosus Ago (PfAgo) for biosensing applications. The conversion of Mn ions with different valence states through MnO2 nanoflowers enables the sensitive detection of ascorbic acid, alkaline phosphatase, and arsenic with limits of detection of 2.5 nmol L−1, 0.009 U L−1, and 0.4 ng mL−1, respectively. A PfAgo‐based immunoassay is further developed that allows for the detection of diverse targets, thus providing a promising toolbox to broaden PfAgo‐based sensors into versatile bioanalytical and biomedical applications.