SHIELD: An Argonaute-powered immobilization-free homogeneous electrochemical biosensor for ultrasensitive viable pathogen detection

Accurate identification of viable foodborne pathogens is critical for preventing foodborne disease outbreaks. Herein, we developed a SHIELD platform (Sensitive Homogeneous Immobilization-free Electrochemical Live-bacteria Detection based on Argonaute). This strategy uses Argonaute-guided cleavage as the main electrochemical transduction mechanism, bypassing the reproducibility problems related to surface-fixed sensors. Crucially, the system effectively eliminates false positives from dead bacteria by incorporating Propidium Monoazide (PMA) pretreatment, which selectively blocks residual DNA from dead bacteria. Combined with the highly specific DNA cleavage capability of Argonaute, this ensures the exclusive detection of viable Salmonella typhimurium. In the detection workflow, the target DNA undergoes isothermal amplification (LAMP) to generate abundant amplicons. Subsequently, target-activated Argonaute programmatically cleaves dual methylene blue (MB)-biotin labeled electrochemical probes. Unreacted probes are removed via streptavidin magnetic separation, generating a significantly enhanced "signal-on" response. This platform achieves a remarkable limit of detection down to 1 CFU/mL within 70 min. Synergizing the specificity of PMA and Argonaute with the efficiency of an immobilization-free design, SHIELD demonstrates a reliable and innovative electrochemical biosensing paradigm, offering a promising solution for the rapid, sensitive screening of viable foodborne pathogens.