Multiplex Immunoassay Techniques for On-Site Detection of Security Sensitive Toxins

Abstract

Biological toxins are a heterogeneous group of high molecular as well as low molecular weight toxins produced by living organisms. Due to their physical and logistical properties, biological toxins are very attractive to terrorists for use in acts of bioterrorism. Therefore, among the group of biological toxins, several are categorized as security relevant, e.g., botulinum neurotoxins, staphylococcal enterotoxins, abrin, ricin or saxitoxin. Additionally, several security sensitive toxins also play a major role in natural food poisoning outbreaks. For a prompt response to a potential bioterrorist attack using biological toxins, first responders need reliable, easy-to-use and highly sensitive methodologies for on-site detection of the causative agent. Therefore, the aim of this review is to present on-site immunoassay platforms for multiplex detection of biological toxins. Furthermore, we introduce several commercially available detection technologies specialized for mobile or on-site identification of security sensitive toxins.

Keywords: electrochemical biosensor; low molecular weight toxins; multiplex immunoassay platforms; on-site detection; optical biosensor; proteotoxins; security sensitive toxins.

Figure 1

General overview of immunosensor components for detection of security sensitive toxins as analyte (3D structure model of ricin (pdb: 2AAI [30]) as well as a ball-and-stick model of saxitoxin are depicted). Because this review is focused on immunosensors, antibodies (or fragments of antibodies) (VL: variable light chain; VH: variable heavy chain; CL: constant light chain; CH: constant heavy chain; Fab: antigen binding fragment; scFv: single-chain variable fragment; VHH: variable domain of heavy chain antibody; Nb: nanobody; sdAb: single domain antibody) are depicted as biorecognition elements only (Adapted from [31]. MDPI (2014)). For the sake of completeness, it should be noted that alternative biorecognition elements, such as aptamers, natural receptor proteins, carbohydrates or cell-based receptors, could also be employed for toxin detection. Several examples of possible transduction mechanisms are noted as well as signal processing.

Figure 2

(A) Picture of the portable, four channel fluorimetric RAPTOR^TM assay system (Reprinted from Biosensors and Bioelectronics, 14, Anderson, G.P.; King, K.D.; Gaffney, K.L.; Johnson, L.H., Multi-analyte interrogation using the fiber optic biosensor, 771–777, Copyright (2000), with permission from Elsevier). (B) Scheme of a RAPTOR^TM assay coupon depicting orientation of optical fibers (Reprinted from Biosensors and Bioelectronics, 14, Anderson, G.P.; King, K.D.; Gaffney, K.L.; Johnson, L.H., Multi-analyte interrogation using the fiber optic biosensor, 771–777, Copyright (2000), with permission from Elsevier).

Figure 3

Portable MBio MQ reader and disposable cartridge (Top). Schematic representation of LightDeck^® technology (Bottom) (Reprinted with permission from [85]. Copyright (2018) American Chemical Society).

Figure 4

(A) Schematic layout of the microcytometer for multiplex detection of security sensitive toxins. The syringe pump provides sheath flow, while the CAVRO^® syringe pump was used to inject samples into the microfabricated channel of the polydimethylsiloxane (PDMS) chip. Depicted cables into the PDMS chip guided 635 and 532 nm laser light into the interrogation region and guided excess light out of the beam stops. Additional fiber optics directed emission light to four separate photo-multiplier tubes (PMTs) to collect microsphere ID fluorescences (670 ± 10 nm and ≥700 nm), light scatter (635 ± 5 nm) and phycoerythrin fluorescence (565 ± 10 nm). Sizes are not to scale (Reprinted with permission from [127]. Copyright (2009) American Chemical Society). (B) Schematic representation of conventional sandwich immunoassay performed on microspheres (Reprinted with permission from [127]. Copyright (2009) American Chemical Society). (C) Signal amplification approach applying additional biotin-labeled anti-streptavidin antibodies for enhanced streptavidin–phycoerythrin binding (Reprinted with permission from [127]. Copyright (2009) American Chemical Society).

Figure 5

(A) Chemiluminescence instrument MCR3 for multiplex microarray analysis (Reprinted by permission from Springer Customer Service Centre GmbH: Springer, Analytical and Bioanalytical Chemistry [132], Copyright (2014)). (B) Schematic representation of sandwich (for SEB and ricin) and indirect competitive (for saxitoxin) immunoassays combined in the same microarray chip by using anti-idiotypic antibodies (Reprinted from [131]. Published by The Royal Society of Chemistry, 2014).

Figure 6

(A) Portable BioDetector integrated (pBDi) with sample holder and reagents holder as well a Tablet PC with control software running. (B) Electrochemical biochip layout with interdigitated electrode structure (Reprinted with permission from [141]. Copyright (2006) American Chemical Society). (C) Assay scheme of multiplex competitive immunoassay for detection of security relevant low molecular weight toxins (Reprinted from [94]. MDPI (2019)).

Figure 7

Schematic assay workflow for an “active” sandwich immunoassay relying on magnetic bead labels. Step 1: electrophoretic capturing of bacterial proteotoxins on a microarray solid surface from flow-through. Step 2: active electrophoretic labeling of the captured analyte by addition of corresponding biotinylated detection antibodies. Step 3: Microscopic detection of the microarray-bound biotin labels by addition of streptavidin-coated magnetic beads in a shear-flow and magnetic field (Reprinted with permission from [149]. Copyright (2012), American Chemical Society).