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.
A CbAgo-CRISPR/Cas12a-powered cascade amplification sensing strategy was proposed. • It combined CbAgo's precise cleavage with the Cas12a's robust trans-cleavage activity. • An amplified fluorescent biosensor was developed for detecting Salmonella Typhimurium . • The proposed biosensor demonstrated comparable analytical performance to qPCR assay. Sensitive and accurate detection of foodborne pathogens constitutes a critical imperative for safeguarding food safety. Herein, we proposed a Clostridium butyricum argonaute (Cb A go)-CRISPR/ C as12a-powered synergistic amplification strategy and developed a DNA amplification-free f luorescent ( ACF ) biosensor for ultrasensitive detection of Salmonella Typhimurium . In this biosensor, magnetic beads-phage conjugates enabled the selective capture of viable Salmonella Typhimurium , followed by rapid DNA extraction within 5 min using commercial lysis buffers. A synergistic signal amplification strategy was designed for efficient biosensing by utilizing the primary cleavage product (tDNA) of the CbAgo system as a molecular bridge to integrate CbAgo and Cas12a systems. Specifically, CbAgo performed primary cleavage of target DNA to generate tDNA fragments. These tDNA subsequently activated the CRISPR/Cas12a system, initiating its trans-cleavage activity to degrade fluorescent reporter probes for quantitative analysis. This tDNA-mediated integration synergizes CbAgo's precision cleavage capability with Cas12a's efficient trans-cleavage activity, enabling exponential amplification. Under optimized conditions, the ACF biosensor exhibited a linear detection range of 10 2 –10 7 CFU/mL and a detection limit of 24.8 CFU/mL for Salmonella Typhimurium without DNA amplification steps. Moreover, the ACF biosensor demonstrated comparable analytical performance to the qPCR assay and achieved satisfactory results in the detection of Salmonella Typhimurium in 20 food samples, highlighting its potential as an ultrasensitive tool for foodborne pathogen monitoring. Download: Download high-res image (107KB) Download: Download full-size image
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