Botulinum neurotoxins (BoNT) are the most potent toxins, which are produced by Clostridium 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−1 while presenting a detection limit of 4.8 pg mL−1. 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.

Botulinum neurotoxins (BoNTs) are the most potent toxins known in nature produced by Clostridium botulinum strains, which can cause life-threatening diseases in both humans and animals. The latter is of serious environmental and economic concern, resulting in high mortality, production losses, and rejection of contaminated animal feed. The available in vivo mouse assay is inadequate for real-time and on-site assessment of outbreaks. Herein, we present a reflective-based approach for the detection of BoNT/C while estimating its activity. Two adjacent porous Si Fabry–Pérot interferometers are simultaneously utilized to quantify minute BoNT/C concentrations by a competitive immunoassay and to assess their endopeptidase activity. The reflectivity signals of each interferometer are amplified by biochemical reaction products infiltration into the scaffold or by peptide fragments detachment from the nanostructure. The optical assay is highly sensitive in compliance with the in vivo approach by presenting a detection limit of 4.24 pg mL–1. The specificity and selectivity of the designed platform are cross-validated against BoNT/B and BoNT/D, also relevant to animal health. Finally, the analytical performances of both interferometers for real-life scenarios are confirmed using actual toxins while depicting excellent compliance to complex media analysis. Overall, the presented sensing scheme offers an efficient, rapid, and label-free approach for potential biodiagnostic elucidation of botulism outbreaks.