Dimer peptidomimetics-based non-toxic immunoassay for bongkrekic acid in food and biosamples compatible with smartphone demonstration

Abstract

Non-toxic immunoassays are effective analytic methods that offer enhanced safety and cost efficiency by substituting toxins with peptidomimetics. Bongkrekic acid (BA), a highly lethal bacterial toxin, has recently garnered significant attention due to its toxicity. This work isolated a phage-borne peptide (CFSFQVGDC) with a strong binding affinity to an anti-BA monoclonal antibody (mAb) from a peptide phage library. Monomer and dimer peptidomimetics were synthesized to achieve a phage-free peptide. ELISA revealed a detection sensitivity of 0.63 ng/mL for the dimer, which is 120-fold higher than that of the monomer. Meanwhile, computer-aided simulations revealed that the enhancement of the hydrogen bonding network of the dimer was the key factor driving its stronger binding affinity. Then, a sensitive immunochromatographic assay (ICA) based on the dimer peptidomimetic was developed to detect BA, compatible with smartphone demonstration. The visible limit of detection (vLOD) was 2.50 μg/kg, while the quantitative limit of detection (qLOD) was 0.12 μg/kg. The recovery rates for real samples, including blood and urine, ranged from 85.00 % to 115.20 %, consistent with standard LC-MS/MS. Importantly, the dimer restored binding capacity to levels comparable to the native toxin, laying a robust foundation for further optimization of toxin-free peptide designs via directed evolution. The developed ICA platform also provided an alternative method for the non-toxic, sensitive, and on-site detection of BA.