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Contamination of Zearalenone from China in 2019 by a Visual and Digitized Immunochromatographic Assay

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Contamination of Zearalenone from China in 2019 by a Visual and Digitized Immunochromatographic Assay toxins Article Contamination of Zearalenone from China in 2019 by a Visual and Digitized Immunochromatographic Assay Xia Hong y, Yuhao Mao y, Chuqin Yang, Zhenjiang Liu, Ming Li * and Daolin Du * Institute of Environmental Health and Ecological Security, School of the Environment and Safety Engineering, Jiangsu University, Xuefu Road 301, Zhenjiang 212013, China; [email protected] (X.H.); [email protected] (Y.M.); [email protected] (C.Y.); [email protected] (Z.L.) * Correspondence: [email protected] (M.L.); [email protected] (D.D.); Tel.: +86-511-8879-0955 (M.L. & D.D.) These authors contributed equally to this work. y Received: 10 July 2020; Accepted: 12 August 2020; Published: 14 August 2020 Abstract: Zearalenone (ZEN) is a prevalent mycotoxin that needs intensive monitoring. A semi-quantitative and quantitative immunochromatographic assay (ICA) was assembled for investigating ZEN contamination in 187 samples of cereal and their products from China in 2019. The semi-quantitative detection model had a limit of detection (LOD) of 0.50 ng/mL with visual judgment and could be completely inhibited within 5 min at 3.0 ng/mL ZEN. The quantitative detection model had a lower LOD of 0.25 ng/mL, and ZEN could be accurately and digitally detected from 0.25–4.0 ng/mL. The ICA method had a high sensitivity, specificity, and accuracy for on-site ZEN detection. For investigation of the authentic samples, the ZEN-positive rate was 62.6%, and the ZEN-positive levels ranged from 2.7 to 867.0 ng/g, with an average ZEN-positive level being 85.0 ng/g. Of the ZEN-positive samples, 6.0% exceeded the values of the limit levels. The ZEN-positive samples were confirmed to be highly correlated using LC-MS/MS (R2 = 0.9794). This study could provide an efficiency and accuracy approach for ZEN in order to achieve visual and digitized on-site investigation. This significant information about the ZEN contamination levels might contribute to monitoring mycotoxin occurrence and for ensuring food safety. Keywords: zearalenone; immunochromatographic assay; semi-quantification; quantification Key Contribution: A semi-quantitative and quantitative test strip had been developed and applied for investigating zearalenone contamination in 187 authentic samples in China in 2019. 1. Introduction Zearalenone (ZEN) is a common secondary metabolite from the Fusarium species, which has become one of the most widespread mycotoxins and has caused substantial economic losses to grains around the world [1,2]. ZEN exposure could cause the genotoxic, hepatoxic, immunotoxic, and even estrogenic effects [3–6]. Thus, ZEN is categorized as a class III carcinogen [7]. Recent investigations into mycotoxin contamination from all over the world found that a high percentage of ZEN contamination exists in cereals and animal feed [8–10]. To better monitor ZEN contamination and maintain human health, the maximum limits (MLs) of ZEN in unprocessed cereals and unprocessed maize have been regulated by the European Commission (EC) to be no more than 100 ng/g and 350 ng/g, respectively [11]. The Expert Committee of both the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) had set a provisional maximal tolerable daily intake of 0.5 ng/g of body weight for ZEN [12]. The China National Standard regulates that ZEN should be no more than 60 ng/g in wheat, wheat flour, corn, and corn flour [13]. Toxins 2020, 12, 521; doi:10.3390/toxins12080521 www.mdpi.com/journal/toxins Toxins 2020, 12, 521 2 of 12 Toxins 2020, x, x FOR PEER REVIEW 2 of 12 High performance liquid chromatography (HPLC) [[14],14], HPLC-tandemHPLC‐tandem mass spectrometry (HPLC-MS)(HPLC‐MS) [15[15],], and and immunoassays immunoassays [16 ,17[16,17]] are usedare forused detecting for detecting ZEN. Compared ZEN. Compared with the referenced with the referencedHPLC and HPLC HPLC-MS and HPLC methods,‐MS immunoassaysmethods, immunoassays have the characteristics have the characteristics of rapidity, of beingrapidity, low being cost, lowand cost, being and able being to achieve able to high achieve throughput high throughput screening screening on-site for on a‐site large for number a large number of samples of samples [18–21]. [18–21].Of the variety Of the of variety immunoassays, of immunoassays, the immunochromatographic the immunochromatographic assay (ICA) has assay attracted (ICA) more has attention.attracted moreIt is widely attention. used forIt detectingis widely contamination, used for detecting because contamination, of its outstanding because characteristics of its ofoutstanding simplicity, characteristicsreadability, and of portability simplicity, [ 22readability,,23]. and portability [22,23]. ICA methods have been developed and applied for ZEN detection. An ICA strip has been used for the the rapid rapid detection detection of of ZEN ZEN in in wheat wheat from from Jiangsu, Jiangsu, China, China, with with a limit a limit of ofdetection detection (LOD) (LOD) of 50 of ng/mL,50 ng/mL, which which was was applied applied in in202 202 real real wheat wheat samples samples [24]. [24]. The The ICA ICA method method based based on on the the quantum dot nanobead and biotin-streptavidinbiotin‐streptavidin system for the determinationdetermination of aflatoxinaflatoxin BB11 (AFB(AFB1)) and ZEN was developed, which improved the sensitivity of ZEN detection to 59.15 pgpg/mL/mL [[25].25]. A quantum dot microbead based fluorometric fluorometric lateral flowflow ICA was developed for the simultaneous detection of AFB1,, deoxynivalenol deoxynivalenol (DON), (DON), and and ZEN, ZEN, and and the the LOD LOD reached reached 1.92 1.92 ng/g ng/g for ZEN in the real cereal samples [26]. [26]. Furthermore, Furthermore, amorphous amorphous carbon carbon nanoparticles, nanoparticles, aptamer, aptamer, dyed dyed latex latex microspheres, microspheres, and otherand other novel novel materials materials have have been been used used to improve to improve the the performance performance of of ICA ICA for for detecting detecting ZEN ZEN [27–29]. [27–29]. The above above-mentioned‐mentioned ICA methods for ZEN promoted detection sensitivity, for which it also showed the characteristics of convenience, rapidity, rapidity, economy, visual detection on-site,on‐site, and could even get accurate levels of ZEN contamination using the quantitative approach. Given thethe high high ZEN ZEN contamination contamination rate rate and and the the large large number number of samples of samples to be examined,to be examined, a higher a performancehigher performance detection detection method method for ZEN for was ZEN developed was developed and improved and improved in this study. in this For study. this purpose, For this purpose,a practical a ICApractical based ICA on two based judgment on two models judgment for themodels semi-quantitative for the semi‐quantitative detection and detection quantitative and quantitativedetection of ZENdetection was developed of ZEN was and developed applied in and authentic applied cereals in authentic and feeds cereals (Figure and1). feeds Combining (Figure the 1). Combiningnaked eye and the strip naked reader, eye theand ICA strip could reader, quickly the achieveICA could the visualizationquickly achieve and the digitalization visualization for ZENand digitalizationdetection, while for improving ZEN detection, the detection while sensitivity improving and the providing detection an alternativesensitivity detection and providing method foran alternativeZEN. The proposed detection ICA method method for was ZEN. applied The proposed to detect the ICA ZEN method contamination was applied levels to in detect 187 samples the ZEN of contaminationcereal and their levels products in 187 from samples China in of 2019. cereal Then, and the their referenced products LC-MS from /MSChina was in used 2019. to Then, verify the referencedaccuracy of LC the‐MS/MS ICA method. was used to verify the accuracy of the ICA method. Figure 1. TwoTwo models of result judgment for the immunochromatographic assay (ICA). 2. Results and Discussion 2. Results and Discussion 2.1. Identification of the Gold Nanoparticles 2.1. Identification of the Gold Nanoparticles The image of the transmission electron micrograph showed that the prepared colloidal gold The image of the transmission electron micrograph showed that the prepared colloidal gold nanoparticles (GNPs) were about 17 nm and the particles exhibited a uniform distribution (Figure2A). nanoparticles (GNPs) were about 17 nm and the particles exhibited a uniform distribution (Figure The ultraviolet–visible spectra (UV/vis) for the GNPs solution had a smooth protruding peak at 518 nm. 2A). The ultraviolet–visible spectra (UV/vis) for the GNPs solution had a smooth protruding peak at Qualitative differences were found between the UV/vis spectra of the ZEN-monoclonal antibody 518 nm. Qualitative differences were found between the UV/vis spectra of the ZEN‐monoclonal (McAb)-GNP probes and GNPs (Figure2B). The ZEN-McAb-GNP probes showed a smooth protruding antibody (McAb)‐GNP probes and GNPs (Figure 2B). The ZEN‐McAb‐GNP probes showed a smooth peak at 527 nm, indicating that the GNPs had been successfully coupled in ZEN-McAb to produce protruding peak at 527 nm, indicating that the GNPs had been successfully coupled in ZEN‐McAb to produce probes. Further studies of the ICA evaluation and application also demonstrated the availability and excellent performance of the prepared GNPs. Toxins 2020, 12, 521 3 of 12 probes. Further studies of the ICA evaluation and application also demonstrated
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