SERS detection of Clostridium botulinum neurotoxin serotypes A and B in buffer and serum: Towards the development of a biodefense test platform

Botulinum neurotoxins (BoNTs) are classified at a highest degree of threat in biodefense, due largely to their high lethality. With the growing risk of biowarfare, the shortcomings of the gold standard test for these neurotoxins, the mouse bioassay, have underscored the need to develop alternative diagnostic testing strategies. This paper reports on the detection of inactivated Clostridium botulinum neurotoxin serotype A (BoNT-A) and serotype B (BoNT-B), the two most important markers of botulism infection, by using a sandwich immunoassay, gold nanoparticle labels, and surface-enhanced Raman scattering (SERS) within the context of two threat scenarios. The first scenario mimics part of the analysis needed in response to a “white powder” threat by measuring both neurotoxins in phosphate-buffered saline (PBS), a biocompatible solvent often used to recover markers dispersed in a powdered matrix. The second scenario detects the two neurotoxins in spiked human serum to assess the clinical potential of the platform. The overall goal is to develop a test applicable to both scenarios in terms of projections of required levels of detection. We demonstrate the ability to measure BoNT-A and BoNT-B in PBS at a limit of detection (LoD) of 700 pg/mL (5 pM) and 84 pg/mL (0.6 pM), respectively, and in human serum at 1200 pg/mL (8 pM) and 91 pg/mL (0.6 pM), respectively, with a time to result under 24 h. The steps required to transform this platform into an onsite biodefense screening tool that can simultaneously and rapidly detect (<1 h) these and other agents are briefly discussed.