Terminal-Optimized Aptamer–Guide-DNAzyme Triplex Boosts Clostridium butyricum Argonaute-Driven Multiplex Foodborne Pathogen Detection without Nucleic Acid Amplification

Multiplexed and sensitive detection of foodborne pathogens is important and necessary to control foodborne diseases. ‌Mesophilic Clostridium butyricum Argonaute ( Cb Ago) exhibits endonuclease activity, enabling precise guide DNA (gDNA)-directed target cleavage without sequence constraints, thus holding potential for multiplexed foodborne pathogen detection. The intrinsic activity constraints of Cb Ago could be addressed by engineered modification of its gDNA in combination with synergistically enzymatic mechanisms, potentially generating highly efficient cooperative cleavage activity. ‌In this study, the gDNA was engineered to incorporate a DNAzyme-functionalized fragment (designated as cDNA) for substrate strand cleavage, followed by systematic investigation of terminal modification group preferences. The synergistic integration of these dual enzymatic activities ultimately enhanced signal amplification efficacy. Furthermore, we developed a DNAzyme– Cb Ago synergistic fluorescence aptamer biosensor, enabling multiplexed detection of three pathogenic bacteria with a detection limit down to 69 CFU mL −1 . This work provides a potential platform for the simultaneous detection of multiplex foodborne pathogenic bacteria without DNA extraction and amplification.