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

Shiga toxin–producing Escherichia coli (STEC) are major foodborne pathogens responsible for severe gastrointestinal disease and hemolytic uremic syndrome (HUS). Here we report Click Detect, a novel diagnostic platform that leverages click display – an in vitro protein display technology – to generate uniform and covalently linked protein–cDNA conjugates for toxin detection. Using a DARPin (D#20) as the signal probe and a high-affinity nanobody (NG1) as the capture reagent, Click Detect achieves sandwich-style recognition of Shiga toxin 2 (Stx2). The captured protein–cDNA conjugates can be quantified by standard nucleic acid amplification assays. Click Detect reliably detected Stx2 at 600 fM by qPCR and 6 pM by LAMP. The assay maintained comparable sensitivity in matrices containing up to 40% public swimming pool water or lettuce extract, highlighting robustness in real-world contamination scenarios. Key advantages of Click Detect include simple, rapid, and cost-effective (~$0.04 per assay) probe preparation as well as versatile plug-and-play probe format for sensing other targets. We believe Click Detect has great potential as a novel sensing platform for food safety monitoring, environmental surveillance, and point-of-care diagnostics, with potentially broad applicability to other toxins and protein targets.