Abstract
Rapid and sensitive detection of botulinum neurotoxins (BoNTs) is important for immediate treatment with proper antitoxins. However, it is difficult to detect BoNTs at the acute phase of infection, owing to its rarity and ambiguous symptoms. To resolve this problem, we developed a surface-enhanced Raman scattering (SERS)-based immunoassay technique for the rapid and sensitive detection of BoNTs. Magnetic beads and SERS nanotags as capture substrates and detection probes, respectively, and Nile Blue A (NBA) and malachite green isothiocyanate (MGITC) as Raman reporter molecules were used for the detection of two different types of BoNTs (types A and B), respectively. The corresponding limits of detection (LODs) were determined as 5.7 ng/mL (type A) and 1.3 ng/mL (type B). Total assay time, including that for immunoreaction, washing, and detection, was less than 2 h.
Highlights
Botulinum neurotoxins (BoNTs) are regarded as one of the most serious high-risk biological agents used in bioterrorism
Gold (III) chloride trihydrate (HAuCl4 ·3H2 O), tri-sodium citrate (Na3 -citrate), 1-ethyl-3-(3-[dimethylamino]propyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), thiol-PEG-COOH (HS-PEG-COOH, MW∼3500), bovine serum albumin (BSA), Nile blue A (NBA), 3,3’,5,5’-tetramethylbenzidine (TMB), liquid substrate system for enzyme-linked immunosorbent assay (ELISA), horseradish peroxidase (HRP)-conjugated goat anti-rabbit polyclonal antibody, and HRP-conjugated goat anti-mouse polyclonal antibody were purchased from Sigma-Aldrich
We developed a surface-enhanced Raman scattering (SERS) immunoassay technique for the rapid and sensitive
Summary
Botulinum neurotoxins (BoNTs) are regarded as one of the most serious high-risk biological agents used in bioterrorism. The gold standard method for the detection and identification of BoNTs is the mouse toxicity and neutralization bioassay (e.g., mouse bioassay), which is the only FDA-approved method to confirm the presence of active BoNTs [9,10] This method has several drawbacks, including labor intensiveness, cost ineffectiveness, animal use, and time consumption (longer than four days). To resolve these problems, we developed a surface-enhanced Raman scattering (SERS)-based immunoassay technique using magnetic beads [11,12,13,14,15]. Magnetic beads and gold nanoparticles (AuNPs) were used to capture antibody-supporting materials and detect antibody-conjugated sensing probes, respectively This magnetic bead-based assay offers several advantages over conventional SERS assays using two-dimensional substrates [21,22,23].
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