Multiplexed and Ultrasensitive Pathogens Detection with an Orthogonal Argonaute Circuit Based on Encoded Microsphere Microscopic Imaging

Multiplexed and sensitive detection of pathogens is crucial for controlling risks of public health. Here, we present a mesophilic Clostridium butyricum Argonaute (CbAgo)-based guide DNA (gDNA)-target DNA-circuit (GTC) platform based on encoded microsphere microscopic imaging for multiplexed and ultrasensitive detection of pathogens. A user-friendly interactive interface enables rapid customized screening of orthogonal gDNAs and rational design of cleavage circuits from pathogen genome sequences, reducing cross-hybridization to below 0.032% and enhancing the cleavage efficiency of CbAgo by up to 157%. Encoded polystyrene microspheres by particle size and color serve as programmable multiprobes in microimaging bioassay, allowing simultaneous and ultrasensitive detection of multiple pathogens through spatial confinement and facilitating straightforward signal decoding via computer vision. The platform demonstrated excellent performance in multiplexed detection of three pathogenic bacteria (10 1 to 10 7 CFU/mL) across 60 real-world samples within 70 min without DNA amplification. Compared with quantitative polymerase chain reaction, the GTC platform shows a 10-fold improvement in sensitivity with a limit of detection down to 1 CFU/mL and exhibits higher accuracy in the detection of real samples. This work establishes an intelligent biosensing strategy for multiplexed pathogen detection and provides a promising next-generation digital platform for public health monitoring.