Small molecules, often defined as low molecular weight organic molecules typically less than 1000 Da in size, include of a wide variety of different chemical compounds, of either natural or pharmaceutical origin, many of which are biologically, pharmacologically, or environmentally relevant molecules. Myriad of different biosensors and bioanalytical assays have been reported for the diverse group of small molecule analytes, including mycotoxins, although owing to their small size they are often challenging analytes. To begin with, small molecules might be problematic targets for many recognition elements, in particular for antibodies.93 While large analytes, such as proteins, can be detected with two-site or sandwich immunoassays, small molecules that inherently are not capable of binding two antibodies simultaneously must be conventionally analyzed using a competitive assay format. The important role of the recognition element is particularly eminent in such assay format because the recognition depends exclusively on the specificity of a single binder, and the sensitivity of the system is mainly defined by the equilibrium constant of the recognition element.93,221 Moreover, the competitive assay format requires conjugation of the target to a carrier molecule or a label to enable immobilization or detection of this competitor. Synthesis of these conjugates can be challenging and time- consuming or result in randomly cross-linked or unstable molecules. Labeling of the analyte may also alter the epitope reducing or even abolishing the biorecognition event, which can compromise the assay sensitivity. Moreover, lot-to-lot variations of the conjugates, or even false positives caused by the release of the analyte moiety from the conjugate, are known to affect the assay reproducibility and accuracy.93,222,223
Instead of using the analyte-conjugate as the competitor, the so-called epitope mimics have been introduced as an intriguing alternative. The exceptional ability of these molecules to mimic the epitope of the analyte, and thus bind to the same antibody paratope, has been witnessed in several fields, including immunotherapy, epitope mapping, and allergy treatment.224,225 For biosensor development, epitope mimics can provide significant advantages as they can replace the analyte-conjugate and thus overcome the limitations related to target conjugation.193 Antibodies themselves, known as anti-idiotypic antibodies,226 or short peptides, also called mimotopes, have been widely used for the detection of small molecules, mycotoxins in particular.193,222
Reported mimotopes, for example for the detection of DON,227 FB1,228–230 OTA,121,231–234 and ZEA,235 vary in length and structure; use of linear 7-mer121,231 and 12-mer230 peptides, as well as cyclic mimotopes,228 has been described. After the discovery of mimotopes from phage- displayed libraries, the phage-borne peptides have been used directly in ELISAs with colorimetric231 and chemiluminescent234 detection, as well as in dipstick234 and dot- immunoassays.236 Alternatively, the phage-borne peptides can be replaced with their synthetic or recombinant counterparts, thus avoiding the use of the phage in the assay. Short peptides, which are relatively simple and cheap to synthesize chemically, have been used in various immunoassays, for example for the detection OTA121 and FB1.228,230 Interestingly, recombinant proteins, on the other hand, which can be produced in bacteria cost-effectively even in large
quantities, can be constructed of the mimotope in fusion with a protein ideal for coating, such maltose-binding protein (MBP),229,233 or for detection, for example, fluorescent237,238 or bioluminescent proteins.239
Nanobodies are the most widely used form of anti-idiotypic antibodies for mycotoxin detection. This special class of antibodies is derived from naturally occurring heavy chain antibodies in camelids and sharks and is renowned for the complete lack of the light chain. Hence, the antibody paratope is formed by a single VHH domain with three hypervariable loops, as opposed to six in the IgG.169,240 The small size of nanobodies and their high stability to harsh conditions have gained significant attention in the field of biotechnology and biosensing.169,241 Mostly because of their unsuitability for small molecule detection as a consequence of the special paratope, the majority of applications using nanobodies for mycotoxin detection employ them as epitope mimics. In fact, the exceptional convex-like structure of the paratope is known to bind well to clefts and cavities, making them highly suitable for molecular mimicry of haptens.93,242 Detection of mycotoxins, including aflatoxin (Figure 3D),243 citrinin,244,245 DON,246 FB1,124,242 and OTA,247 using anti-idiotypic nanobodies has been reported to increase the sensitivity, at best 20-fold in comparison with the conventional assays using the toxin- conjugates.
On the other hand, since non-competitive immunoassays are usually considered superior to the competitive assays because of their higher sensitivity and specificity, novel methodologies have been implemented in order to benefit of these properties also in applications for small molecule detection. For such, non-conventional antibodies developed by recombinant antibody technology have been reported for small molecule detection. For example, the open sandwich immunoassay (OS-IA) is based on the association of separated VH and VL chains in the presence of the analyte and has been reported to outperform competitive assay in terms of sensitivity, working range, and assay time.248 OS-IAs have been reported for the detection of ZEA using the VH and VL chains of a monoclonal anti-ZEA antibody. The non-competitive assays showed superior performance compared to the competitive assay,249 although OS-IA in which the recognition still relies on only one antibody can suffer from cross-reactivity.248,250 Alternatively, non-competitive immunoassays for small molecules have been reported using two antibodies, one of which, known as the anti-immune complex or anti-metatype antibody, binds to the primary antibody only when it is in complex with the target analyte. Such non-competitive immunoassays based on anti-immune complex antibodies have the added advantage of increased specificity due to the use of two antibodies instead of one. For example, anti-immune complex antibodies for the detection of HT-2 were selected from phage display libraries, and the developed immunoassays showed superior performance in comparison with the competitive ELISA (Figure 3A).125,136
Figure 3. Examples of mycotoxin analysis based on different recognition elements and detection schemes. (A) Non-competitive HT-2 toxin assay based on the anti-immune complex Fab and HT-2 specific primary antibody. In the presence of the toxin, FRET (Förster resonance energy transfer) can occur due to the short distance between the two fluorophores. A, acceptor (Alexa Fluor); D, donor (europium dye). (B) Localized surface plasmon resonance (LSPR) sensor using aptamer- modified gold nanorods chemically attached to an optical fiber core. OTA binding to the aptamer induces an LSPR peak shift. (C) Detection of AFB1 by DNA aptamer–based fluorescent assay using
graphene oxide (GO) nanosheets to bind the labeled aptamer in the absence of the toxin and quench the fluorescence. Signal amplification was achieved using DNase I for regeneration. (D) Anti-idiotypic nanobody was used in a phage-based real-time immuno-PCR for the detection of aflatoxin. Figures adapted from A, Arola et al. (2016);136 B, Lee et al. (2018);111 C, Zhang et al.
Table 3. Examples of biosensors and bioaffinity assays for mycotoxin detection.
Target toxin Recognition element Assay Solid phase Label Measurement LOD LR Samples Ref.
OTA MAb Competitive (mimotope) Microtiter plate HRP CL 0.005 ng mL–1 0.006–0.245 ng mL–1 Coffee Corn
Rice
234
AF MAb Competitive (AI-Nb) Microtiter plate TaqMan PD-IPCR 0.02 ng mL–1 n.d. Corn Rice
Peanut
243
HT-2 toxin Fab Non-competitive (IC) – Eu + AF647 FRET 0.38 ng mL–1 n.d. Wheat 136
AFB1 Nb Competitive (AFB
1–BSA) Microtiter plate HRP A n.d. 0.117–5.676 ng mL–1
Corn Rice Peanut
251
OTA Aptamer (RCA) Direct Gold electrode MB CV 0.065 ppt 0.1 ppt–5 ppb Wine 213
FB1 Aptamer Direct Microcantilever – Cantilever deflection 33 ng mL–1 100–40000 ng mL–1 – 252
OTA Aptamer Direct GNR on optical fiber – LSPR 0.006 ng mL–1 5.1–50718 ng mL–1 Grape juice 111
AFB1 AChE Direct SPE – A 468 ng mL–1 625–62500 ng mL–1 Olive oil 162
AFB1 MIP Direct Gold electrode – LSV 0.3 fg mL–1 1 fg mL–1–1 μg mL–1 – 217
ZEA MIP Direct – QD F 0.64 ng mL–1 9.55–99330 ng mL–1 Corn Rice
Wheat flour
253
Abbreviations: A, amperometry; AChE, acetylcholinesterase; AF, AlexaFluor; AI-Nb, anti-idiotypic nanobody; BSA, bovine serum albumin; CL, chemiluminescence; CV, cyclic voltammetry; F, fluorescence; FRET, Förster resonance energy transfer; GNR, gold nanorod; HRP, horse-radish peroxidase; IC, immune complex; LOD, limit of detection determined the blank + 3 × standard deviation of the blank; LR, linear range; LSPR, localized surface plasmon resonance; LSV, linear sweep voltammetry; MAb, monoclonal antibody; MB, methylene blue; MIP, molecularly imprinted polymer; Nb, nanobody; n.d., not determined; QD, quantum dot; PD-IPCR, phage display mediated immuno polymerase chain reaction; RCA, rolling circle amplification; SPE, screen-printed electrode.