Mesophilic Argonaute‐Based Single Polystyrene Sphere Aptamer Fluorescence Platform for the Multiplexed and Ultrasensitive Detection of Non‐Nucleic Acid Targets

Abstract The rapid, simultaneous, and accurate identification of multiple non‐nucleic acid targets in clinical or food samples at room temperature is essential for public health. Argonautes (Agos) are guided, programmable, target‐activated, next‐generation nucleic acid endonucleases that could realize one‐pot and multiplexed detection using a single enzyme, which cannot be achieved with CRISPR/Cas. However, currently reported thermophilic Ago‐based multi‐detection sensors are mainly employed in the detection of nucleic acids. Herein, this work proposes a Mesophilic Argonaute Report‐based single millimeter Polystyrene Sphere (MARPS) multiplex detection platform for the simultaneous analysis of non‐nucleic acid targets. The aptamer is utilized as the recognition element, and a single millimeter‐sized polystyrene sphere (PSmm) with a large concentration of guide DNA on the surface served as the microreactor. These are combined with precise Clostridium butyricum Ago (CbAgo) cleavage and exonuclease I (Exo I) signal amplification to achieve the efficient and sensitive recognition of non‐nucleic acid targets, such as mycotoxins (<60 pg mL−1) and pathogenic bacteria (<102 cfu mL−1). The novel MARPS platform is the first to use mesophilic Agos for the multiplex detection of non‐nucleic acid targets, overcoming the limitations of CRISPR/Cas in this regard and representing a major advancement in non‐nucleic acid target detection using a gene‐editing‐based system.