Botulinum neurotoxins (BoNTs)—the molecular agents behind botulism—remain among the deadliest substances known, capable of paralyzing muscles and causing death even in microscopic doses. In addition to their role in foodborne and clinical cases, BoNTs are classified as Tier 1 agents on the U.S. Federal Select Agent list because of their potential misuse in bioterrorism. While outbreaks are relatively rare, their high fatality rate and frequent misdiagnosis continue to make botulism a persistent public health, food safety, and biosecurity challenge. Recent research published in Toxins provides a comprehensive review of detection technologies that may replace the decades-old mouse bioassay (MBA) as the gold standard.
The work, led by researchers at the National Institutes for Food and Drug Control in Beijing, synthesizes progress across microbiology, immunology, bioengineering, and analytical chemistry. Their review highlights how new methods are not only advancing scientific understanding but also edging closer to practical deployment in clinics, food monitoring, and environmental surveillance.
Why Faster and Smarter Detection Matters
The clinical course of botulism underscores the urgency: patients often present with blurred vision, muscle weakness, and difficulty breathing, and misdiagnoses are common. In China, more than a quarter of foodborne botulism cases from 2004–2020 were initially misdiagnosed, and Canadian data show that 70% of patients require mechanical ventilation. Early administration of antitoxins is critical, but only possible if diagnosis is rapid and reliable.
Traditional mouse bioassay (MBA) remains sensitive and broad, but it is slow (up to four days), and costly, potentially delaying treatment and understanding of the threat situation. The search for alternatives aims to balance accuracy, speed, and practicality, while also enabling serotype discrimination.
Cell-Based Assays: Toward an Ethical Standard
Cell-based assays (CBA) replicate the toxin’s interaction with neuronal receptors and intracellular machinery, allowing researchers to measure actual toxin activity without animal testing. Models based on cell lines such as SiMa, human induced pluripotent stem cells, and engineered neuromuscular junction organoids have reached sensitivities rivaling or exceeding MBA. The U.S. FDA has already approved CBA for potency testing of commercial BoNT/A products, demonstrating its regulatory feasibility.
However, CBAs face hurdles in standardization, throughput, and reproducibility across labs. The authors argue that future development must focus on engineered stable cell lines and automated platforms to realize their potential as a global diagnostic standard.
Immunological Methods: Breadth and Speed, With Caveats
Antibody-based approaches such as ELISA, AlphaLISA, and lateral flow assays remain popular due to their affordability and rapid turnaround. Novel variants—such as SpinDx, a centrifugal microfluidic immunoassay, and immuno-PCR—achieve detection limits far surpassing traditional ELISA. Flow cytometry and advanced lateral flow strips using nanoparticle labels are pushing toward multiplexed, field-deployable formats.
Yet, immunological assays cannot distinguish between active and inactive toxin, leaving uncertainty in clinical or food safety contexts. Antibody cross-reactivity and matrix interference also contribute to false positives.
Mass Spectrometry: Precision at the Molecular Level
Techniques combining chromatography with liquid chromatography–tandem mass spectrometry (LC-MS/MS) have delivered high-resolution identification of toxin subtypes and associated proteins. Endopeptidase–mass spectrometry (Endopep-MS), in particular, directly measures BoNT enzymatic cleavage of SNARE proteins, yielding results in under eight hours while differentiating serotypes with high specificity.
Optimizations in peptide substrates and antibody purification now allow detection of trace toxins at levels surpassing animal bioassays. The review positions Endopep-MS as a strong candidate for future reference standards in public health laboratories.
Biosensors: Portability and On-Site Promise
Perhaps the most forward-looking category, biosensors leverage nanomaterials, plasmonics, electrochemical platforms, and even wearable devices to convert toxin–receptor interactions into measurable signals. Recent advances include quantum-dot FRET systems, paper-based electrochemical strips, and localized surface plasmon resonance sensors with picogram-per-milliliter sensitivity. Some wearable prototypes even detect early botulism signatures in exhaled breath.
The challenge remains scaling these technologies from “proof-of-concept” devices into robust, validated platforms for clinical and field use. Sample preparation, reproducibility, and multi-center validation are key bottlenecks.
Looking Forward: Public Health and Security Implications
The authors emphasize that no single method is likely to replace MBA across all use cases. Instead, a layered toolkit—rapid biosensors for field screening, immunoassays for high-throughput monitoring, and cell-based or Endopep-MS platforms for confirmatory diagnostics—may form the backbone of future detection infrastructure.
The increasing recognition of underreported cases in low-resource regions and the opportunity for novel chimeric BoNTs highlight the urgency of this transition. But beyond clinical medicine and food safety, the biodefense dimension cannot be overlooked. Rapid, reliable detection is central to countering potential deliberate misuse. For public health agencies, food safety regulators, and security stakeholders alike, investment in next-generation detection technologies will be critical to reducing diagnostic delays, improving outbreak response, and strengthening global resilience against both natural and intentional threats.
Wang S, Zhang H, Xue Y, et al. Research Progress on the Detection Methods of Botulinum Neurotoxin. Toxins, 8 September 2025