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

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.