Click Detect: A Rapid and Sensitive Assay for Shiga Toxin 2 Detection

Shiga toxin-producing <i>Escherichia coli</i> (STEC) is a major foodborne pathogen, responsible for severe gastrointestinal disease and hemolytic uremic syndrome (HUS). Here, we report Click Detect, a novel diagnostic platform that leverages click display to efficiently produce sensing probes for sandwich-style antigen detection. Click display is an in vitro protein display technology that generates uniform and covalently linked protein-cDNA conjugates in a simple one-pot reaction format within 2 h. The captured sensing probe can be quantified by standard nucleic acid amplification assays. Using click displayed DARPin (D_#20) as the sensing probe and a high-affinity nanobody (N_G1) as the capture reagent, Click Detect reliably detected Shiga toxin 2 (Stx2) at 600 fM by quantitative PCR (qPCR) and 6 pM by loop-mediated isothermal amplification (LAMP). The assay maintained comparable sensitivity in matrices containing up to 40% public swimming pool water or lettuce extract, highlighting robustness for real-world surveillance applications. Key advantages of Click Detect include simple, rapid, and cost-effective (~USD 0.04 per assay) sensing probe preparation, as well as a versatile plug-and-play probe format for detecting other targets. We believe that Click Detect has great potential as a novel sensing platform for food/environmental monitoring and point-of-care diagnostics, with potentially broad applicability to other toxins and protein targets.