Shiga toxin-producing Escherichia coli (STEC) are major foodborne pathogens responsible for severe infections, including the deadly hemolytic uremic syndrome (HUS). However, the current diagnostic methods lack the sensitivity and speed required for effective clinical and food safety applications. Early detection of Shiga toxin 2 (Stx2), a primary virulence factor of STEC, could potentially offer critical benefits for timely intervention. In this work, gold nanoparticles (AuNPs) are functionalized with a pair of high-affinity, designed ankyrin repeat proteins (DARPins) targeting the A and B subunits of Stx2, and used as multifunctional signal transductors for rapid and electronic detection (RED). This DARPin-RED platform leverages active centrifugal forces and vortex agitation for signal enhancement within a short turnaround time (<30 minutes), achieving highly sensitive (attomolar to femtomolar) detection of Stx2 spiked in food matrices, such as milk, lettuce extract, and ground beef extract, as well as biological fluids, including whole blood, and serum. Additionally, DARPin-RED is capable of detecting multiple Stx2 subtypes without serious background interference, and successful in both differentiating high-toxin-producing E. coli strain (RM5856) from low toxin producer (RM9872) (p < 0.001) and analyzing different bacterial inoculation stages (p = 0.011) from STEC culture within 8 hours post-inoculation. The ability of DARPin-RED to detect Stx2 from food and human specimens at a high sensitivity and specificity using a point-of-care (POC) readout circuit presents a significant advancement for mitigating foodborne outbreaks and effective management of HUS progression.

The authors have declared no competing interest.

This project was supported in part by the National Science Foundation (NSF) under grant no. 1847324, U.S. Department of Agriculture AFRI 2022-67021-37013, and by the National Institute of Health under grant no. R21AI169098, R21AI186134, and DP2GM149552.

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