Xiaoqin Tang,Xinyue Li,Yuhan Tian,Qiang Zhang,Shuli Man,Weipan Peng,Long Ma
Foodborne illnesses and contamination are becoming increasingly severe global public health issues. In recent years, the resistance of pathogenic bacteria has increased significantly, which makes it particularly urgent to develop advanced detection technologies beyond traditional methods. Pf Ago ( Pyrocus furiosus Argonaute), a novel programmable nuclease with specific sequence targeting and cleavage capabilities, offers significant advantages over traditional CRISPR diagnostics (CRISPR-Dx) in molecular detection, particularly in flexible design and multiplexed detection. In this work, Pf Ago was innovatively integrated with the nucleic acid reverse-phase enhanced fluorescent lateral flow test strip (rLFTS) and smartphone for the first time, enabling ultrasensitive, portable and multiplexed molecular detection of pathogenic bacteria in a cross-validating manner. Pf Ago precisely recognized LAMP amplicons, triggering linker DNA cleavage that generated dual colorimetric/fluorescent signals on rLFTS. A 3D-printed visualizer equipped with a homemade App-enabled smartphone achieved single-cell sensitivity (1 CFU/mL) within 45 min, two orders of magnitude lower than traditional qPCR (∼100 CFU/mL). In summary, a conceptually novel cross-validating manner integrating Pf Ago with dual-mode rLFTS was constructed. It eliminated the reliance on large instruments, representing a significant step forward in developing an ultrasensitive, portable, accurate and multiplexed molecular detection technology for foodborne pathogens.
Foodborne pathogens have posed a great threat on food safety and public health, and thus the development of accurate and sensitive methods for the detection of foodborne pathogens is of great significance. In this study, a novel Pyrococcus furiosus Argonaute ( Pf Ago) biosensing platform based on metal-organic framework (MOF) nanozyme (namely AgoZyme) was developed for the detection of Salmonella typhimurium . Inspired by the catalytic characteristics of horseradish peroxidase, we synthesized a Hemin@ZIF-90 nanozyme with peroxidase-like activity by encapsulating hemin into a zeolitic imidazolate framework-90 through a self-assembly strategy. By integrating the specific recognition and cleavage capabilities of Pf Ago with the highly catalytic performance of Hemin@ZIF-90, the AgoZyme biosensor exhibited excellent detection sensitivity and specificity. This biosensor had a limit of detection of 1 CFU/mL in a linear range from 10 1 to 10 8 CFU/mL. This work provided a new insight for designing and developing novel biosensors for food safety detection.
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.
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