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

Salmonella typhimurium (S. typhimurium) is one of the major foodborne pathogenic bacteria, posing a serious threat to human health and food safety. The sensitive and reliable S. typhimurium 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 Pyrococcus furiosus Argonaute (PfAgo) with programmable Aurora DNAzyme for sensitive detection of S. typhimurium. 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 PfAgo could be activated by S. typhimurium 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 S. typhimurium was inversely proportional to the fluorescent signal. Benefiting from the specific recognition and cleavage ability of PfAgo and the outstanding catalytic ability of E-Aurora, the established biosensor achieved high sensitivity for S. typhimurium with a limit of detection as low as 1 CFU/mL. This work provided a new insight for designing a novel label-free PfAgo-based fluorescence biosensor for the detection of pathogenic bacteria.