A versatile immunoassay based on functionalized nanoparticles for botulinum neurotoxin detection and sensor development

Abstract

Botulinum neurotoxins (BoNTs) serotypes A and B are the two most common of the four BoNTs that cause the high mortality botulism disease in individuals consuming contaminated foods. The gold standard assay for BoNT detection is the live mouse bioassay, which has several major disadvantages, including tedious procedures and animal sacrifice requirements. In this study, we developed an immuno-based assay using magnetic streptavidin nanoparticles (mSNP) functionalized with specific synthetic biotinylated, 6xHis-tagged peptide substrates (Peptides A, PA and B, PB) designed for BoNT/A and BoNT/B proteolytic reactions, respectively. In the presence of active toxins that possess endopeptidase activity, upon cleavage, the released fragments with His-tag were dotted on a blotting membrane, ultimately producing color signals after incubation with anti-His antibody, alkaline phosphatase (AP)-conjugated antibody, and then AP substrates. The results showed that the efficiency of peptide-mSNP complex formation reached up to 81%, and the dot blot immunoassay allowed peptide detection from 10 ng of His-tagged peptides. Preliminary testing with the extracellular extracts from the isolated Clostridium botulinum strains indicated that the botulinum toxin in the 2020 botulism outbreak in Vietnam belonged to serotype A, the most potent BoNT. The established assay could be applied to construct a portable biosensor for BoNT detection and a high throughput device to screen potential BoNT inhibitors for drug development.