Antibody-biotin-streptavidin-horseradish peroxidase (HRP) sensor for rapid and ultra-sensitive detection of fumonisins

Highlights

• A NMB probe was synthesized.

• An antibody-biotin-streptavidin-HRP sensor was designed for detection of FBs.

• The sensor showed satisfactory recoveries, detection limit and linear dynamic ranges.

Abstract

Fumonisins (FBs) exist widely in crops, foods and feeds. Consumption of FBs contaminated corn can cause oesophageal cancer. So it is necessary to develop sensitivity methods for its detection. Here, we report an enhanced assay for rapid and ultra-sensitive detection of FBs based on nanomagnetic beads (NMBs) and antibody-biotin-streptavidin-HRP. Because antibody-BNHS can bind with several number of streptavidin-HRP, the signal amplification of the catalytical oxidation of TMB was enhanced. The detection limit of sensor was down to 0.21 ng mL⁻¹ with a linear range from 0.31 to 162.42 ng mL⁻¹. Since NMBs provide a nearly “in solution” reaction, they lead to a shortened reaction time (22 min) than that of flat solid-phase based traditional assay. Furthermore, the recoveries obtained by standard FBs spiked to maize samples were from 100.6 to 107.3%. This enhanced assay supplied a rapid, sensitive and reliable method for detection of FBs in maize.

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.