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Zhao, Z.; Zhang, Z.; Zhang, H.; Liang, Z. Applications of Small Peptides in Mycotoxin Detection. Encyclopedia. Available online: (accessed on 22 April 2024).
Zhao Z, Zhang Z, Zhang H, Liang Z. Applications of Small Peptides in Mycotoxin Detection. Encyclopedia. Available at: Accessed April 22, 2024.
Zhao, Zitong, Zhenzhen Zhang, Haoxiang Zhang, Zhihong Liang. "Applications of Small Peptides in Mycotoxin Detection" Encyclopedia, (accessed April 22, 2024).
Zhao, Z., Zhang, Z., Zhang, H., & Liang, Z. (2023, February 23). Applications of Small Peptides in Mycotoxin Detection. In Encyclopedia.
Zhao, Zitong, et al. "Applications of Small Peptides in Mycotoxin Detection." Encyclopedia. Web. 23 February, 2023.
Applications of Small Peptides in Mycotoxin Detection

Mycotoxins pose significant risks to humans and livestock. In addition, contaminated food- and feedstuffs can only be discarded, leading to increased economic losses and potential ecological pollution. Mycotoxin removal and real-time toxin level monitoring are effective approaches to solve this problem.Small peptides derived from phage display peptide libraries, combinatorial peptide libraries, and rational design approaches can act as coating antigens, competitive antigens, and anti-immune complexes in immunoassays for the detection of mycotoxins. Furthermore, as a potential approach to mycotoxin degradation, small peptides can mimic the natural enzyme catalytic site to construct artificial enzymes containing oxidoreductases, hydrolase, and lyase activities. In summary, with the advantages of mature synthesis protocols, diverse structures, and excellent biocompatibility, also sharing their chemical structure with natural proteins, small peptides are widely used for mycotoxin detection and artificial enzyme construction, which have promising applications in mycotoxin degradation. 

Small peptides mycotoxin detection antigens

1. Introduction

Peptides have emerged as a promising approach to synthetic biomimetics [1]. The excellent properties of synthetic peptides in small-molecule contaminant (SMCs) recognition make them a potential alternative to antibodies and natural receptors in mycotoxin biosensor applications [2]. Small peptides show high-affinity binding to these small analytes and have stability, playing an increasingly important role in the rapid detection of mycotoxins [3]. Among immunologically based mycotoxin detection methods, peptides can serve as competing antigens, coating antigens, and anti-immune complexes (Table 1).

2. Peptides as Competing Antigens

As stated earlier, competitive antigens are essential in competitive immunoassays, but the obtaining of specificity antigens always involves the use of mycotoxins. Peptides can serve as competing antigens and competitively bind with specific monoclonal antibody (mAb), which can avoid the use of poisonous mycotoxins [11][14]. In 1999, Yuan et al. [32] identified two phage-displayed mimotopes (SWGPFPF and SWGPLPF) and used them to detect DON firstly. Subsequently, mimotopes as competitive antigens for the detection of mycotoxins have been increasingly reported, involving OTA, AFB1, ZEN, FB1, and other mycotoxins [33]. Peltomaa et al. [26] reported the selection of a novel dodecapeptide (VTPNDDTFDPFR) from a 12-mer peptide library; then, a biotinylated synthetic derivative of this mimotope (VTPNDDTFDPFRGGGSK-Biotin) was used for the detection of FB1 by a competitive binding inhibition assay. Its 50% inhibitory concentration (IC50) was 37.1 ng/mL, with a detection limit (LOD) of 11.1 ng/mL, and a dynamic range from 17.3 to 79.6 ng/mL. Recently, the authors used the same method to obtain a ZEN mimetic epitope peptide (GWWGPYGEIELL); the peptide was used to create fusion proteins with the bioluminescent Gaussia luciferase (GLuc) that were directly used as tracers for mycotoxin detection in a competitive immunoassay [21]. Meanwhile, using the same small peptide sequence developed a competitive upconversion-linked immunosorbent assay (ULISA) for ZEN monitoring with a LOD of 20 pg mL−1 [22].
Chen et al. [18] constructed a peptide@Tyr-RMC probe by selecting a mimotope peptide from a phage display library and labeling it a Tyr-RMC composite, which was used to develop a competitive enzyme-linked immunosorbent assay (ELISA) for the ultrasensitive detection of ZEN. It has a linear range of 10−6–1.0 ng/mL, and a low detection limit of 10−6 ng/mL. Zhao et al. [11], using AFB1 as a model system, selected a mimotope (YSWHEWYIPQLS) from Ph.D-12 phage display peptide library, and the rapid magnetic-beads-based directed competitive ELISA (MB-dcELISA) was developed by mimotope ME17. The IC50 and LOD of the MB-dcELISA were 0.75 and 0.13 ng/mL, respectively, with a linear range of 0.24–2.21 ng/mL. Zou et al. [9] obtained a mimotope from the commercial Ph.D.-7 phage display peptide library and connected it with biotin (GMVQTIF-GGGSK-biotin). The biotinylated 12-mer peptide was used as a competing antigen to develop a competitive peptide ELISA for OTA detection and showed a wide linear range of 0.005–0.2 ng/mL with the detection limit of 0.001 ng/mL. The IC50 was 0.024 ng/mL, which is approximately five times more sensitive as a competing antigen than the OTA-HRP conjugates used in the conventional ELISA .

3. Peptides as Coating Antigens

Peptides also can serve as competing antigens in immunological-based mycotoxin detection. He et al. [16] selected a phage display dodecapeptide (ESYWATVPWTRH) as a substitute for coating antigens and applied it for the rapid detection of ZEN by dot-immunoassay . The cut-off level for detecting ZEN in cereal samples was 50 mg/kg and the results can be accomplished within 10 min. With the phage display peptide library technology, Zhou et al. [23] reported a phage mimotope-based direct competitive fluorescence immunosorbent assay (P-dcFLISA); the IC50 of P-dcFLISA was 0.301 ng/mL, which was lower than the phage-based indirect competitive enzyme-linked immunosorbent assay (P-icELISA) under the same conditions. The LOD and detection range of P-dcFLISA was 0.023 ng/mL and 0.060–1.531 ng/mL, respectively. However, there is no corresponding amino acid sequence information in the article. Liu et al. [24], utilizing mimotope peptide-bovine serum albumin conjugate as a coating antigen, developed a peptide ELISA for detecting FB1, in which the IC50 and LOD were 6.06 ng/mL and 1.18 ng/mL, respectively. Except for phage display peptides, small synthetic peptides by rational design are also used as coating antigens for mycotoxin detection. Bazin et al. [34] designed an OTA-binding peptide (VYMNRKYYKCCK) derived from an oxidoreductase and developed a peptide-based competitive enzyme-linked immunosorbent assay (peptide-based competitive ELISA) in which the peptide was the coating antigen.
Mimotope-based fusion proteins can also be used as synthetic coating antigens for the detection of mycotoxins [35]. Xu et al. [25], using FB1 as a model hapten, screened two mimotopes (F1: NNAAMYSEMATD, F15: TTLQMRSEMADD) that have affinity to the anti-FB1 antibody from a 12-mer peptide library and developed a new method for the development of a sensitive and environmentally friendly immunoassay for FB1 based on the peptide–MBP (maltose-binding protein) fusion protein. Quantitative immunoassay for FB1 using F1-MBP and F15-MBP showed the LOD was 0.32 and 0.21 ng/mL, respectively, and the IC50 of the assay was 2.15 and 1.26 ng/mL, which was 10 times more sensitive than the conventional FB1-BSA conjugate-based ELISA. Meanwhile, using the same strategy, they also constructed a small peptide–MBP fusion protein-coated antigen that can be used to detect OTA and DON with good sensitivity and stability [7][15]. Recently, to monitor the co-contamination of mycotoxins in agricultural products and foods, Yan et al. [36] developed a mimotope–MBP fusion protein-based multiplex immunochromatographic assay (mICA) that can quickly and simultaneously detect FB1, ZEN, and OTA without the building-up process of mycotoxin conjugates. The LOD of peptide–MBP-based mICA for FB1, ZEN, and OTA were 0.25, 3.0, and 0.5 ng/mL, respectively.

4. Anti-Immune Complex Peptides

As small molecule contaminants (SMCs), mycotoxins usually only have one immunological binding site and are not suitable for detection by conventional sandwich non-competitive immunoassays [37]. Alternatively, the development of non-competitive immunoassays using specific recognizers for the immune complex of anti-SMC antibodies is a feasible strategy. Anti-immune complex peptide (AIcP) is a peptide that specifically binds to immune complexes, neither antibodies nor antigen monomers [38]. Biopanning the phage display random peptides library using antigen–antibody conjugates allows the screening of peptides that can bind specifically to immune complexes and can be used to establish non-competitive immunoassays [39]. Unlike mimotopes, anti-immune complex peptides recognize only complexes of antigens and antibodies and do not bind to antibodies or antigens alone, and because of this dual site recognition pattern, anti-immune complex peptides are often thought to improve the specificity of assays.
González-Sapienza’s team achieved promising results in the detection of pesticide contaminants using anti-immune complex peptides, demonstrating the advantages of the method in the detection of SMCs [31][39][40][41]. Zou et al. [10], using AFB1 and anti-AFB1 nanobody conjugates as the immune complex, screened anti-immune complex peptides from a phage display random linear 8-mer peptide library; the best binding peptide was biotinylated and coupled with horseradish peroxidase-labeled streptavidin (SA-HRP) for developing the magnetic-phage anti-immune complex immunoassay (MPHAIA) detection of AFB1. Phage-peptide p13 (DLLWVPST)-based MPHAIA, showing the lowest SC50 (50% signal saturation concentration) value (0.12 ng/mL) and the highest ODmax/SC50 ratio (12.75), was selected for further study. Under the ultimate condition, the LOD was 0.006 ng/mL, with a linear range of 0.019–0.407 ng/mL. Lassabe et al. [42] used a verotoxin (VTX) of Escherichia coli as a scaffold for multivalent display anti-immunocomplex peptides, and, among these peptides, ICX09m (CLEAPNVEAC) showed 10-fold increased sensitivity and excellent recovery than the competitive ELISA for clomazone detection.
Other studies use this method for residual pesticide and veterinary drug detection, but with few applications in mycotoxin detection. Although the non-competitive assay has advantages, the technique is still in the early stages of development, and the biggest challenge in developing this assay is the screening of anti-immune complex peptides. Therefore, improving the success rate of peptide screening for anti-immune complexes will significantly enhance the development of non-competitive immunoassays for small-molecule compounds, such as mycotoxins and pesticides. The anti-immune complex peptides of mycotoxins, pesticides, and other small molecules are summarized in Table 1.


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