| Summary: | Botulinum neurotoxins (BoNT) are the most potent toxins, which are produced by <i>Clostridium</i> bacteria and cause the life-threatening disease of botulism in all vertebrates. Specifically, animal botulism represents a serious environmental and economic concern in animal production due to the high mortality rates observed during outbreaks. Despite the availability of vaccines against BoNT, there are still many outbreaks of botulism worldwide. Alternative assays capable of replacing the conventional in vivo assay in terms of rapid and sensitive quantification, and the applicability for on-site analysis, have long been perused. Herein, we present a simple, highly sensitive and label-free optical biosensor for real-time detection of BoNT serotype C using a porous silicon Fabry–Pérot interferometer. A competitive immunoassay coupled to a biochemical cascade reaction was adapted for optical signal amplification. The resulting insoluble precipitates accumulated within the nanostructure changed the reflectivity spectra by alternating the averaged refractive index. The augmented optical performance allowed for a linear response within the range of 10 to 10,000 pg mL<sup>−1</sup> while presenting a detection limit of 4.8 pg mL<sup>−1</sup>. The practical aspect of the developed assay was verified using field BoNT holotoxins to exemplify the potential use of the developed optical approach for rapid bio-diagnosis of BoNT. The specificity and selectivity of the assay were successfully validated using an adjacent holotoxin relevant for farm animals (BoNT serotype D). Overall, this work sets the foundation for implementing a miniaturized interferometer for routine on-site botulism diagnosis, thus significantly reducing the need for animal experimentation and shortening analysis turnaround for early evidence-based therapy.
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