Rapid and ultrasensitive detection of botulinum neurotoxin serotype A1 in human serum and urine using single-molecule array method


Botulinum neurotoxin (BoNT) is one of the most poisonous substances ever known. It is produced by Clostridium botulinum, which causes botulism, a persistent paralysis of peripheral nerve termini. Clostridium botulinum bacteria, the source of BoNTs, are widely distributed in the environment. BoNT poses a significant risk as a bioweapon and is classified as a Category A agent by the Centers for Disease Control and Prevention because of its ready availability and extreme potency. In this paper, a rapid and ultrasensitive method using a single-molecule array assay was developed for the quantitative analysis of BoNT serotype A1 (BoNT/A1), the most common toxin serotype. This method can detect as little as 400 fg/mL of solid powder toxin resulting in a broad quantitation range from 0.4 to 100 pg/mL and can be accomplished in approximately an hour. Additionally, to show that it would be useful for clinical samples during a potential exposure and to test for any possible matrix interference, the method was applied to the analysis of spiked human serum and urine samples. The limits of detection were 200 fg/mL for serum (in 25 % dilution) and 1.00 pg/mL for urine (in 10 % dilution), with sample quantitation ranges from 0.8 to 400 and from 10 to 1000 pg/mL, respectively.

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This study was funded in part by the Defense Threat Reduction Agency (Contract Number W911SR-10-D-0011).

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Correspondence to David R. Walt.

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Conflict of interest

The authors declare the following competing financial interest(s): David R. Walt is the scientific founder and a board member of Quanterix Inc. All remaining contributing authors declare no competing financial interests.

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Research does not involve human participants and/or animals. Unidentified human serum and urine samples were purchased commercially. No informed consent was needed by researchers, according to the guidelines of National Institutes of Health (NIH).

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Dinh, T.L., Ngan, K.C., Shoemaker, C.B. et al. Rapid and ultrasensitive detection of botulinum neurotoxin serotype A1 in human serum and urine using single-molecule array method. Forensic Toxicol 35, 179–184 (2017). https://doi.org/10.1007/s11419-016-0336-7

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  • Botulinum neurotoxin
  • Immunoassay
  • Single-molecule detection
  • Single-molecule array method
  • Human serum
  • Human urine