Elsevier

Food Chemistry

Volume 336, 30 January 2021, 127713
Food Chemistry

Dual fluorescent immunochromatographic assay for simultaneous quantitative detection of citrinin and zearalenone in corn samples

https://doi.org/10.1016/j.foodchem.2020.127713Get rights and content

Highlights

  • Developed a fluorescent-labeled dual immunoassay for detecting citrin (CIT) and zearalenone (ZEN).

  • The europium nanoparticles were used as detection probes to label the anti-CIT and anti-ZEN antibodies.

  • The immunoassay resulted in a significant increase of the sensitivity.

  • The paper-based method shows excellent selectivity for simultaneous detection of CIT and ZEN.

Abstract

The presence of multiple mycotoxins in the agricultural products poses a serious threat to the health of humans and animals. Citrinin (CIT) causes slow growth in animals and damages the kidney function. Zearalenone (ZEN) causes chronic poisoning, abnormal functioning and even death in animals. Herein, a dual fluorescent immunochromatographic assay (DF-ICA) based on europium nanoparticles (EuNPs) was developed for the simultaneous detection of CIT and ZEN in the corn samples. After optimization, the limits of detection (LODs), IC50 and average recoveries for the simultaneous determination of CIT and ZEN were 0.06 and 0.11 ng/mL, 0.35 and 0.76 ng/mL, from 86.3% to 111.6% and from 86.6% to 114.4%, respectively. Moreover, the DF-ICA was validated by high performance liquid chromatography (HPLC) analyses, and a satisfactory consistency was obtained. In brief, this work demonstrates the feasibility of DF-ICA for simultaneous monitoring of CIT and ZEN in the corn samples.

Introduction

Mycotoxins are natural chemical substances produced by certain species of filamentous fungi (Stec et al., 2009, Santos et al., 2010); toxicity of mycotoxins can involve carcinogenic, teratogenic, nephrotoxic and mutagenic effects (Wang, Liu, Ning, Liu, & Li, 2012). Mycotoxin contamination has become the focus of food safety issues due to toxicity and universality (Duan, Li, Shao, Huang, & Xiong, 2019). Citrinin (CIT) and zearalenone (ZEN) are the common mycotoxins in agricultural products and are mainly produced by Aspergillus, Penicillium and Fusarium species (Ah et al., 2012). CIT and ZEN were reported to coexist in contaminated corn (Gott, Hendel, Mendoza, Hofstetter-Schahs, & Murugesan, 2017). Considering that mycotoxins are a potential huge threat to humans and animals, prevention and treatment of the mycotoxins contaminants is very important. Thus, a number of countries established legal limits on mycotoxin levels in various agricultural foods. Until now, no legal maximum limits of CIT in the corn samples were established (Zhang, Liu, Peng, Cui, Shi, & He, 2019). However, the limit of citrinin in the red fermented products in Japan is 200 μg/kg (Liao, Chen, Lin, Chiueh, & Shih, 2014), and the limit in the European Union is 100 μg/kg. For ZEN, the legal limits for corn and its products are 60 μg/kg in China (Luo et al., 2019).

In recent years, a large number of studies have developed methods for detection of mycotoxins, particularly multiple mycotoxins, to minimize the harm to humans and animals (Shao et al., 2018). Various chromatographic and mass spectrometric methods have been developed for detection of multiple mycotoxins in agricultural foods, such as liquid chromatography fluorescence (LC-FL) (Marley, Brown, Leeman, & Donnelly, 2016), high performance liquid chromatography (HPLC) (Vrabcheva, Usleber, Dietrich, & Märtlbauer, 2000), liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Bernhardt et al., 2016, Sun et al., 2017), and ultra-performance liquid chromatography with fluorescence detection (UHPLC-FL) (Wei et al., 2019). These techniques produce sensitive and accurate results; however, the complexity of operations, requirement of skilled personnel and expensive equipment and time-consuming procedures make them unsuitable for rapid screening and on-site analysis. The immunoassays have been widely applied for mycotoxins determination as screening methods because of their high sensitivity, low cost and specificity (Liu, Wang, Yao, & Zhu, 2018). Therefore, the presence of multiple mycotoxins in food justifies the need for rapid and cost-effective methods of simultaneous immunoassays (He et al., 2019). High-throughput immunoassays have significant advantages in the detection of multiple mycotoxins, such as enzyme-linked immunosorbent assay (Yirga, Ling, Yang, Yuan, & Wang, 2017), multiplex flow cellular immunoassays (Bienenmann-Ploum et al., 2013) and antibody microarray immunoassay (Zhang et al., 2018). However, these methods could not be widely used due to the demand for special instruments and skilled technicians. The molecularly imprinted biosensors (Atar et al., 2015, Eren et al., 2015, Ertan et al., 2016, Atar et al., 2016) are sensitive, fast and stable and are superior to the above methods and are widely used in biological detection. The simultaneous detection of multiple mycotoxins in agricultural products is a trend in the development of the testing methods.

Outstanding characteristics of the immunochromatographic assay (ICA) promote the method as a popular diagnostic tool due to simplicity, speed, low cost, user-friendliness and suitability for high sample throughput (Wang et al., 2016, Safenkova et al., 2019, Zhang et al., 2019). In fact, the dual ICA has been widely applied in food safety, clinical diagnosis and environmental monitoring (Kong et al., 2016, Taranova et al., 2015). However, traditional ICA was hampered by inherent shortcomings, such as relatively poor reproducibility and low sensitivity, which limits its development (Tripathi, Upadhyay, & Nara, 2017).

The signal label is one of the main factors influencing the sensitivity of ICA. Colloidal gold (CG) nanoparticles are the most widely used labels, which can be rapidly synthesized, easily bound to antibodies and visually observed. Due to insufficient sensitivity, this label has not been applied to the detection of compounds at low concentrations (Li et al., 2013). The development of fluorescent signal had attracted widespread attention because it enables enhances to enhance the detection signal and increase the sensitivity of ICA, including europium nanoparticles (EuNPs) (Tang et al., 2015), dye-doped nanoparticles (Xu et al., 2013), and quantum dots (Chmangui et al., 2019). EuNPs are one of the ideal fluorescent labels. EuNPs offer high fluorescence, long fluorescence lifetime and nontoxic effect on the extracted sample. After a short-lived background fluorescence attenuation during sample detection, the specific fluorescence of the EuNPs was analysed enabling to eliminate the background fluorescence interference by extending the measurement time (Zhou et al., 2012). It had been reported that the new format of competitive immunochromatographic assay using EuNPs for ultrasensitive and quantitative determination of ochratoxin A improved the LODs up to 100-fold compared to that in the case of colloidal gold-based traditional ICA (Majdinasab, Zareian, Zhang, & Li, 2019). Therefore, EuNPs can be used as excellent tool to improve the sensitivity of the ICA.

In this study, the dual fluorescent immunochromatographic assay (DF-ICA) has been established by using EuNPs conjugate with two specific antibodies. The competitive EuNPs-mAb-based immunochromatographic assay enables simultaneous quantitative and sensitive detection of two mycotoxins under optimal conditions in a corn sample. Experimental results demonstrated that DF-ICA is a reliable method for simultaneous screening of dual targets within a single sample. Thus, our study furthers the application of DF-ICA in the field of food safety and quality monitoring.

Section snippets

Reagents and chemicals

CIT, ZEN, ochratoxin A (OTA), fumonisin B1 (FB1), and aflatoxin B1 (AFB1) standards were purchased from the National Institute of Metrology, P. R. China (Beijing, China). In this work, the formaldehyde and carbodiimide methods were used to couple CIT and ZEN to carrier proteins, respectively (Li et al., 2013, Chen et al., 2019). Monoclonal antibodies against citrinin and zearalenone were prepared in our laboratory (Li et al., 2013, Chen et al., 2019). 2-(N-Morpholino) ethanesulfonic acid (MES),

Detection principle

As shown in Fig. 1, the principle of DF-ICA is based on the competition between mycotoxins in the sample and the mycotoxin-BSA conjugates immobilized onto the nitrocellulose membrane for binding to the EuNPs-labelled antibodies (the emission spectrum of carboxylic acid-functional EuNP and the appearance of the strip under 365 nm radiation are shown in Fig. 1b and c, respectively). Initially, the detection reagents were prepared by mixing the EuNPs-mAb probes and sample solutions containing

Conclusions

In an early stage in our laboratory, two antigens were prepared, and specific monoclonal antibodies were screened. By using two specific monoclonal antibodies and a fluorescent substance to form a probe, the DF-ICA with reaction time of 25 min was established. The main objective of DF-ICA was ultrasensitive detection of CIT and ZEN contamination to be used in on-site screening of corn samples. For the development of sensitive DF-ICA, various parameters were optimized including immunoreaction

CRediT authorship contribution statement

Ying Xu: Investigation, Software, Writing - original draft, Formal analysis. Biao Ma: Writing - review & editing. Erjing Chen: Validation, Visualization, Data curation. Xiaoping Yu: Supervision, Funding acquisition. Zihong Ye: Data curation, Writing - review & editing. Chuanxin Sun: Conceptualization, Methodology. Mingzhou Zhang: Conceptualization, Methodology, Resources, Funding acquisition.

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.

Acknowledgements

The work was supported by the National Key Research and Development Program of China (2018YFF0215205, 2017YFF0210200) and the Important Science and Technology Specific Program of Zhejiang Province (2020C02024).

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