Short communicationAntibody-biotin-streptavidin-horseradish peroxidase (HRP) sensor for rapid and ultra-sensitive detection of fumonisins
Introduction
Fumonisins (FBs), a family of food-borne carcinogenic mycotoxins, are produced by fusarium fungi. They exist widely in crops, foods and feeds. In naturally contaminated samples, FB1, FB2, and FB3 are the most abundant occurring FBs and account for 75, 20 and 5% of the total FBs, respectively (Rheeder, Marasas, & Vismer, 2002). Consumption of FB1 contaminated corn can cause oesophageal cancer in humans (Deepa and Sreenivasa, 2017, Khan et al., 2018, Khayoon et al., 2010). The allowed maximum limits of total FBs in maize products for human foods should be less than 2 mg/kg recommended by the United States Food and Drug Administration (FDA) (Food and Drug Administration, 2001) and less than 5 mg/kg in unprocessed maize grain proposed by Codex Coordinating Committee for Latin America and the Caribbean (CCLAC) (FAO/WHO CCLAC, 2010).
Considering the risks associated with the consumption of maize and maize-based foods contaminated by FBs, it is necessary to develop sensitivity methodologies for its detection in maize. Several techniques have been reported for the detection of FBs in food samples, such as electrochemical immunosensor (Lu, Seenivasan, Wang, Yu, & Gunasekaran, 2016), high performance liquid chromatography (HPLC) (Ndube, van der Westhuizen, Green, & Shephard, 2011), liquid chromatography coupled to various detectors like mass spectrometry (MS) (Gazzotti et al., 2009), fluorescence detection (FLD) (Smith, Francis, Johnson, & Gaskill, 2017) etc. Although these techniques provide good dynamic range and low detection limit. They require expensive instruments, skilled analysts and time-consuming sample pre-treatment processes. Immunoassays offer significant advantages over these techniques, such as enzyme-linked immunosorbent assay (ELISA) (Wang, Wang, et al., 2014) and nanoparticle probe based strips (Li et al., 2012). The integration of nanotechnology with immunoassay provides a possibility for advanced development of rapid and sensitive detection technique (Tansil & Gao, 2006).
Nanomagnetic beads (NMBs) have unique biocompatibility and large surface areas that can be separated fast from reaction mixture by external magnet and re-dispersed immediately following removal of the magnet (Ma, Wang, Yang, Shi, & Cao, 2011). NMBs based solid-phase enable a nearly “in solution” reaction which lead to a shorter reaction time than that of flat solid-phase based traditional assay (Song et al., 2014). Therefore, NMBs have been integrated into immunoassays for various target detection, such as antibody against ApxIVA (Wei, Li, Yang, Yu, Zhao, Zhou et al., 2012), aptamer (Pan et al., 2010), heme (Jones & Allen, 2011), aflatoxin B1 (Wang, Niessner, & Knopp, 2014), cyclosporine A antibody (Ahn et al., 2016) and enterobacter sakazakii (Zhu & Wang, 2016).
The affinity of biotin to streptavidin is more than one million times higher than that of antigen–antibody binding (Li, Kong, Gao, & Yan, 2008). The interaction is rapid and one biotinylated antibody (antibody-BNHS) can bind tightly with several HRP-streptavidins (Lin et al., 2008). Therefore, biotin-streptavidin system has been used to enhance the sensitivity of the assay for various targets, such as human thyroid stimulating hormone (Lin et al., 2008), progesterone (Dong et al., 2017), expression of platelet membrane glycoproteins (Zhang et al., 2010), inflammatory biomarkers (Wu et al., 2017), N6-methyladenosine (Yin, Wang, Jiang, Zhou, & Ai, 2017) and Escherichia coli O157:H7 (Guo et al., 2016).
In this study, NMBs were employed for loading FB1-ovalbumin (FB1-OVA) as solid-phase probe (Fig. 1). An antibody specific for FBs was labelled with biotin-NHS (BNHS), and streptavidin was employed as a bridge for loading horseradish peroxidase (HRP) to form antibody-BNHS-streptavidin-HRP system to enhance the signal amplification of catalytic oxidization. The NMB probe dispersed in solution, competed with FBs in sample for binding with antibody-BNHS. Then streptavidin-HRP was captured by antibody-BNHS. The HRP catalytically oxidized the substrate and generated optical signals that were correlated to the concentration of FBs and could be measured by microplate reader. Each reaction step was in a homogeneous phase.
Section snippets
Chemicals and apparatus
FB1, FB2 and FB3 were purchased from Sigma Aldrich (Milwaukee, USA). Bovine serum albumin (BSA) and ovalbumin (OVA) were supplied by Fermentek Ltd (Jerusalem, Israel). NMBs were obtained from BioCanal (China). N-Hydroxysuccinimide (NHS), dimethyl formamide (DMF), biotin-NHS (BNHS), ethylenediamine (EDA), 3,3′,5,5′-tetramethylbenzidine (TMB) and 2-(N-Morpholino)ethanesulfonic acid (MES) were obtained from Aladdin (Shanghai, China). Dicyclohexylcarbodiimide (DCC), carbodiimide hydrochloride
Analysis of antibody-BNHS and NMB probe
Different structures of substance have different ultraviolet and visible spectrum (UV–vis) absorption and the shift of absorptions before and after target conjunction is the way to show whether the product is successful prepared or not (Meng et al., 2015, Zhou et al., 2012). The antibody-BNHS and NMB probe were confirmed by UV–visible spectroscopy. The maximum absorption peaks of antibody, antibody-BNHS and BNHS were 230, 232, and 258 nm, respectively (Fig. 2A). The maximum absorption peaks of
Conclusion
A signal amplified assay based on NMB solid-phase probe and antibody-biotin-streptavidin-HRP sensor was developed for detection of FBs. Because one antibody-BNHS can bind tightly with several HRP-streptavidins, the signal amplification of the catalytical oxidation of TMB was enhanced. Compared with reported methods, this technique showed satisfactory detection limit and linear dynamic ranges. The assay can be accomplished in 22 min without complicated sample pretreatment and handling
CRediT authorship contribution statement
Hualin Yang: Conceptualization, Methodology, Writing - original draft. Qi Zhang: Methodology, Data analysis. Xiaolei Liu: Data analysis, Writing - original draft. Yuying Yang: Data analysis. Yong Yang: Writing - review & editing. Mingyuan Liu: Writing - review & editing. Peiwu Li: Supervision, Validation, Investigation. Yu Zhou: Validation, Resources, Writing - review & editing, Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The authors are thankful to the financial support of the National Key R&D Program of China (grant numbers 2017YFC1601205) and the National Natural Science Foundation of China (NSFC, No. 31601536 & No. 31871888).
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These authors contributed equally to this work.