Novel magnetic nanobeads-based fluoroimmunoassays for zearalenone detection in cereals using protein G as the recognition linker

Highlights

• Two novel MNBs-based fluoroimmunoassays were developed for detecting zearalenone.

• Protein G was used as recognition binder to capture anti-zearalenone IgG.

• CdTe/CdS/ZnS QDs were modified as novel signal probes.

• The assays were applied in the sensitive detection of zearalenone in cereal samples.

Abstract

Zearalenone (ZEN) is a type of estrogenic mycotoxin commonly found in cereals. In order to satisfy the need for ultrasensitive detection of ZEN, we developed two novel magnetic nanobeads (MNBs)-based fluoroimmunoassays using protein G (PG) as recognition binder on the sensing interface. One proposed facile strategy is based on a first capture last react (FCLR) procedure while the other is a first react last capture (FRLC) format. Specifically, CdTe/CdS/ZnS quantum dots were synthesized and modified to antigen (OVA-ZEN) as the signal probes. The PG modified MNBs specifically captured the fragment crystallizable region of immunoglobulin G (IgG) with a level of orientation while avoiding the destruction of antibody’s binding sites caused by chemical coupling. Under the optimized conditions, the detection limits of 0.019 ng mL⁻¹ and 0.049 ng mL⁻¹ in the extract solution were obtained for the FCLR and FRLC, respectively. Furthermore, the established methods proved to be successful in detecting ZEN in real cereal samples with the detection limits being 0.6 μg kg⁻¹ and 1.5 μg kg⁻¹ in the FCLR and FRLC, respectively. The performance of the proposed assays was evaluated utilizing commercial ELISA kits with satisfactory results.

Introduction

Zearalenone (ZEN) is a type of low molecular weight estrogenic mycotoxin produced mainly by fungi belonging to the genus Fusarium [1]. It is most commonly distributed in cereal grains worldwide such as maize, sorghum, wheat and rice, but also treated as a contaminant in cereal products like flour, plant oil, bread and beer [2]. The estrogenic activity of ZEN can enter the food chain causing several reproductive disorders in both humans and animals, for instance central precocious puberty, reproduction problems [3], as well as increasing age-adjusted incidence rates of breast cancer [4]. To date, many countries and regions have established maximum residue levels (MRLs) for this toxin in commodities and foods. For example, the European Union set the MRLs of 350 μg kg⁻¹ for ZEN in unprocessed corn (Commission Regulation, EC No.1126/2007). More recently, China set the MRLs of 60 μg kg⁻¹ for ZEN in cereal and cereal-based products (National Food Safety Standard GB2761-2011). Consequently, developing effective methods able to measure ZEN contaminates in cereals is urgently required and is essential for governments and farmers to monitor the cereal foods or feeds for their safety.

Based on antigen-antibody interaction, various traditional or novel immunoassays, for example enzyme-linked immunosorbent assays (ELISA) [5], fluoroimmunoassays (FLISA) [6], fluorescence polarization immunoassays (FPIA) [7], electrochemical immunosensors [8] and lateral flow immunoassays [9] have been developed for sensitive analysis of ZEN due to their high sensitivity, low cost, rapid action and being suitable for analyzing numerous samples in a short period of time. Of these immunoassays, the recently developed magnetic nanobeads (MNBs)-based fluoroimmunoassays have been successfully utilized in monitoring target biomolecules [10,11] or small molecular substances [12]. Compared with conventional liquid-solid phase 2D assays, such as the 96-wells system, the nanobeads in a liquid-liquid phase system provide a high surface area, contributing to the increase of reaction rate and exhibited superiorities in terms of specificity, sensitivity and wide detection range [10]. However, most established MNBs-based immunoassays require immune probes which are either: firstly, antibodies that have been immobilized onto the surface of MNBs; or secondly, antibodies labeled with tracers like enzymes, fluorophores or other nanoparticles (in cases where a toxin-protein conjugate is immobilized). Furthermore, the conjugation between tracer and antibody mostly involves a chemical coupling reaction, which may potentially destroy the antibody’s binding sites, leading to a reduction in the stability and binding affinity of the antibody to analytes, and consequently the analytical performance is compromised [13,14]. Much attention has consistently been paid by researchers to develop molecular binders that can recognize and capture the non-binding sites of the antibody to avoid the reduction of the stability and binding affinity in the immobilization process [14,15]. The IgG-binding protein G (PG), a kind of protein expressed at the cell surface of certain group C and group G streptococcal strains, shows high affinity and selectivity to the fragment crystallizable (Fc) region of immunoglobulin G (IgG) antibodies sourced from mouse, goat and rabbit, particularly the IgG₁ type of mouse monoclonal antibodies [[16], [17], [18]]. As the specific molecular binder against the Fc domain of IgG, PG has already been widely used in IgG purification [19], antibody identification [20], cell imaging [21] and immunoassays [[22], [23], [24]].

Compared with core or core/shell quantum dots (QDs) such as CdTe and CdTe/CdS QDs, CdTe/CdS/ZnS QDs are a kind of relatively new ternary QDs with higher fluorescence intensity. Coating the shells of CdTe with CdS and ZnS not only increased the fluorescence intensity and stability but also prevented the release of toxic Cd²⁺ [25]. The CdTe/CdS/ZnS QDs have demonstrated huge potential in cell imaging [26] and biomacromolecule detection [27,28]. Benefiting from their reduced toxicity, enhanced stability, and increased fluorescence intensity, this kind of core/shell/shell QDs were applied as signal probes in our analysis. To the best of our knowledge, no reports have yet been published on their role in immunoassays of small toxin molecules before submitted this study.

In this study, CdTe/CdS/ZnS QDs were synthesized and applied in immunoassays for the detection of mycotoxin ZEN. The artificial antigen-modified CdTe/CdS/ZnS QDs (QDs-OVA-ZEN) were employed as signal probes, and PG functionalized MNBs served as capture probes to seize the free IgG or QDs-OVA-ZEN-IgG (or ZEN-IgG) complex. Based on these probes, two formats of novel homogeneous immunoassays were engineered and investigated. One is based on the first capture last react (FCLR) format in which free IgG was firstly captured by capture probes as immunosensing carriers and then submitted for the immunoreaction, while the other is a first react last capture (FRLC) format in which PG modified MNBs were exploited to capture the immune complex (QDs-OVA-ZEN-IgG or ZEN-IgG) after an immunoreaction. The established methods were applied for monitoring ZEN in different kinds of cereal samples, and the assays’ performance was evaluated by ELISA with subsequently satisfactory results. The proposed assays have demonstrated huge potential for the routine detection of ZEN in food safety management.