Novel magnetic nanobeads-based fluoroimmunoassays for zearalenone detection in cereals using protein G as the recognition linker
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.
Section snippets
Apparatus and reagents
Ovalbumin (OVA), 4-morpholineethanesulfonic acid (MES), Tween-20, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysulfosuccinimide (NHS), standards of ZEN and other used toxins were purchased from Sigma-Aldrich (St. Louis, MO, USA). MNBs were obtained from Baseline Sciences (Tianjin, China). Recombinant protein G was acquired from ProSpec-Tany TechnoGene Ltd (Ness-Ziona, Israel). Tellurium powder, NaBH4, CdCl2·2.5H2O and thioglycolic acid were purchased from Sinopharm Chemical
Principle of the proposed fluoroimmunoassays
Two types of MNBs-based fluoroimmunoassays were established here. The schematic illustration of the proposed methodology is shown in Scheme 1. For the FCLR format, the MNBs-PG-IgG complexes were self-assembled by adding anti-ZEN IgG into the as-prepared MNBs-PG probes. Sequentially, samples containing ZEN and QDs-OVA-ZEN probes were simultaneously competitively reacted with the MNBs-PG-IgG complex. After removing the uncoupled components under the magnetic field, the sediment was re-dispersed
Conclusions
In summary, both FCLR and FRLC formats of fluoroimmunoassays were assembled in this study for the sensitive detection of ZEN. For these two assays, CdTe/CdS/ZnS QDs were synthesized to which the OVA-ZEN antigen was conjugated, and used as novel signal probes. The PG modified MNBs served as capture probes to specifically recognize the Fc fragment of IgG antibody, ensuring the binding sites of the IgG antibody could be exposed to the antigen signal probes or target ZEN with a certain orientation
Acknowledgments
The authors are grateful for the financial support from the National Key R&D Program of China (No. 2017YFE0110800), the EU H2020 (No. EU-China-Safe 727564) and International Science and Technology Cooperation Program of China (No. 2014DFR30350).
Fuyuan Zhang is currently a Cotutelle Ph.D. student between Tianjin University of Science and Technology in China and Macquarie University in Australia. Meanwhile, he is working as a Visiting Academic in the University of New South Wales (UNSW). He also received the Postgraduate Research Scholarship (PGRA) funded by Australian Institute of Nuclear Science and Engineering (AINSE). His researches mainly focus on the nanoparticle sensors in Food Safety and Biomedical Research.
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2021, Sensors and Actuators, B: ChemicalCitation Excerpt :The working mechanism of the resulting sensor was recognition of protein G binder: MNBs functionalized with G attached to the crystallizable region of immunoglobulin G (IgG) at a particular orientation. At the same time, the antibody binding sites were not damaged during this chemical coupling [120]. CDs encapsulated into MIP with quenchable fluorescence were also used as active materials to determine ZEN cereal [121].
Fuyuan Zhang is currently a Cotutelle Ph.D. student between Tianjin University of Science and Technology in China and Macquarie University in Australia. Meanwhile, he is working as a Visiting Academic in the University of New South Wales (UNSW). He also received the Postgraduate Research Scholarship (PGRA) funded by Australian Institute of Nuclear Science and Engineering (AINSE). His researches mainly focus on the nanoparticle sensors in Food Safety and Biomedical Research.
Bing Liu received her Ph.D. in 2007 from Nankai University, now she is serving as a professor in Tianjin University of Science and Technology. Her researches mainly focus on Food Safety and Simulation Chemistry.
Guozhen Liu received her Ph.D. degree (2006) in Chemistry from Prof Justin Gooding's group at the University of New South Wales (UNSW). Dr. Liu conducted her postdoctoral research at CSIRO (2006–2008) and UNSW (2008–2010), respectively before she accepted a faculty position as Associate Professor at the Central China Normal University. Meanwhile, Dr. Liu achieved her industrial experience as the R&D Manager, China (2011–2015) on developing medical devices at AgaMatrix Inc., a US-based company for making glucose test strips and glucose meters. Before Liu was awarded the ARC Future Fellowship, Liu worked as the Research Fellow of ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP) (2015–2016). Liu’s research covers the area of nanoscale analysis, active nanoparticles, bionanofabrication, and advanced sensing technologies, with the focus on developing multifunctional theranostic tools for biomedical science.
Wei Sheng received her Ph.D. in 2008 from China Agricultural University, now she is serving as a professor in Tianjin University of Science and Technology. Her researches mainly focus on Food Safety.
Yan Zhang received her Ph.D. degree in 2005 from Tianjin University of Science and Technology, Tianjin, China. Now she is serving as a professor in Tianjin University of Science and Technology. Her researches mainly focus on the Food Science.
Qi Liu is a master student at Tianjin University of Science and Technology. Her research topic is the preparation of monoclonal antibody against zearalenone.
Shuo Wang received his B.S. from Nankai University in 1991 and obtained his Ph.D. from University of Sydney in 1999. He was a research fellow at University of Sydney and CSIRO Plant Industry from 2001 to 2003. He has been a professor at Tianjin University of Science and Technology since 2003 and currently work in Nankai University. His research is mainly focused on the fundamental theories and applied technology on rapid analysis of trace-level hazardous chemicals in food, and has made a number of pioneering achievements in related academic research frontiers, including the immunoassay for micromolecule, the enrichment and separation technology of trace-level contaminants in food, the synthesis and reaction mechanism of “biomimetic antibody” and the development on the immunoassay theory and technology of substitutable biological antibody. Prof. Wang has over 150 SCI publications. He is awarded Yangtze River Scholars Distinguished Professor, Distinguished Young Scholar of the National Science Foundation of China.