Label-Free Fluorescence Biosensor Constructed Based on <i>Pyrococcus furiosus</i> Argonaute System and Programmable DNAzyme for Sensitive Detection of Foodborne Pathogen

<i>Salmonella typhimurium</i> (<i>S. typhimurium</i>) is one of the major foodborne pathogenic bacteria, posing a serious threat to human health and food safety. The sensitive and reliable <i>S. typhimurium</i> detection methods are crucial for preventing disease outbreaks and ensuring food safety. Recently, Argonaute proteins have emerged as a highly effective tool for pathogenic bacteria detection owing to their programmability and exceptional specificity. In this study, we developed a novel label-free fluorescent biosensing platform by innovatively integrating <i>Pyrococcus furiosus</i> Argonaute (<i>Pf</i>Ago) with programmable Aurora DNAzyme for sensitive detection of <i>S. typhimurium</i>. Specifically, a label-free signaling probe (engineered-Aurora, E-Aurora) was designed by introducing the target sequence into the catalytic core sequence of Aurora DNAzyme. The E-Aurora could bind to the 4-methylumbelliferyl phosphate (4-MUP) and transfer its phosphoryl group to its own 5'-OH terminus, thereby generating the strong fluorescent compound 4-MU. In this biosensing platform, the <i>Pf</i>Ago could be activated by <i>S. typhimurium</i> and was able to cleave the E-Aurora, thereby destroying the structural integrity of E-Aurora. The fragmentary E-Aurora was incapable of catalyzing 4-MUP to generate fluorescent signals. The concentration of <i>S. typhimurium</i> was inversely proportional to the fluorescent signal. Benefiting from the specific recognition and cleavage ability of <i>Pf</i>Ago and the outstanding catalytic ability of E-Aurora, the established biosensor achieved high sensitivity for <i>S. typhimurium</i> with a limit of detection as low as 1 CFU/mL. This work provided a new insight for designing a novel label-free <i>Pf</i>Ago-based fluorescence biosensor for the detection of pathogenic bacteria.