**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].

High performance liquid chromatography (HPLC) [14], HPLC-tandem mass spectrometry (HPLC-MS) [15], and immunoassays [16,17] are used for detecting ZEN. Compared with the referenced HPLC and HPLC-MS methods, immunoassays have the characteristics of rapidity, being low cost, and being able to achieve high throughput screening on-site for a large number of samples [18–21]. Of the variety of immunoassays, the immunochromatographic assay (ICA) has attracted more attention. It is widely used for detecting contamination, because of its outstanding characteristics of simplicity, readability, and portability [22,23]. High performance liquid chromatography (HPLC) [14], HPLC‐tandem mass spectrometry (HPLC‐MS) [15], and immunoassays [16,17] are used for detecting ZEN. Compared with the referenced HPLC and HPLC‐MS methods, immunoassays have the characteristics of rapidity, being low cost, and being able to achieve high throughput screening on‐site for a large number of samples [18–21]. Of the variety of immunoassays, the immunochromatographic assay (ICA) has attracted more attention. It is widely used for detecting contamination, because of its outstanding characteristics of simplicity, readability, and portability [22,23].

*Toxins* **2020**, *x*, x FOR PEER REVIEW 2 of 12

ICA methods have been developed and applied for ZEN detection. An ICA strip has been used for the rapid detection of ZEN in wheat from Jiangsu, China, with a limit of detection (LOD) of 50 ng/mL, which was applied in 202 real wheat samples [24]. The ICA method based on the quantum dot nanobead and biotin-streptavidin system for the determination of aflatoxin B<sup>1</sup> (AFB1) and ZEN was developed, which improved the sensitivity of ZEN detection to 59.15 pg/mL [25]. A quantum dot microbead based fluorometric lateral flow ICA was developed for the simultaneous detection of AFB1, deoxynivalenol (DON), and ZEN, and the LOD reached 1.92 ng/g for ZEN in the real cereal samples [26]. Furthermore, amorphous carbon nanoparticles, aptamer, dyed latex microspheres, and other novel materials have been used to improve the performance of ICA for detecting ZEN [27–29]. The above-mentioned ICA methods for ZEN promoted detection sensitivity, for which it also showed the characteristics of convenience, rapidity, economy, visual detection on-site, and could even get accurate levels of ZEN contamination using the quantitative approach. ICA methods have been developed and applied for ZEN detection. An ICA strip has been used for the rapid detection of ZEN in wheat from Jiangsu, China, with a limit of detection (LOD) of 50 ng/mL, which was applied in 202 real wheat samples [24]. The ICA method based on the quantum dot nanobead and biotin‐streptavidin system for the determination of aflatoxin B1 (AFB1) and ZEN was developed, which improved the sensitivity of ZEN detection to 59.15 pg/mL [25]. A quantum dot microbead based fluorometric lateral flow ICA was developed for the simultaneous detection of AFB1, deoxynivalenol (DON), and ZEN, and the LOD reached 1.92 ng/g for ZEN in the real cereal samples [26]. Furthermore, amorphous carbon nanoparticles, aptamer, dyed latex microspheres, and other novel materials have been used to improve the performance of ICA for detecting ZEN [27–29]. The above‐mentioned ICA methods for ZEN promoted detection sensitivity, for which it also showed the characteristics of convenience, rapidity, economy, visual detection on‐site, and could even get accurate levels of ZEN contamination using the quantitative approach.

Given the high ZEN contamination rate and the large number of samples to be examined, a higher performance detection method for ZEN was developed and improved in this study. For this purpose, a practical ICA based on two judgment models for the semi-quantitative detection and quantitative detection of ZEN was developed and applied in authentic cereals and feeds (Figure 1). Combining the naked eye and strip reader, the ICA could quickly achieve the visualization and digitalization for ZEN detection, while improving the detection sensitivity and providing an alternative detection method for ZEN. The proposed ICA method was applied to detect the ZEN contamination levels in 187 samples of cereal and their products from China in 2019. Then, the referenced LC-MS/MS was used to verify the accuracy of the ICA method. Given the high ZEN contamination rate and the large number of samples to be examined, a higher performance detection method for ZEN was developed and improved in this study. For this purpose, a practical ICA based on two judgment models for the semi‐quantitative detection and quantitative detection of ZEN was developed and applied in authentic cereals and feeds (Figure 1). Combining the naked eye and strip reader, the ICA could quickly achieve the visualization and digitalization for ZEN detection, while improving the detection sensitivity and providing an alternative detection method for ZEN. The proposed ICA method was applied to detect the ZEN contamination levels in 187 samples of cereal and their products from China in 2019. Then, the referenced LC‐MS/MS was used to verify the accuracy of the ICA method.

**Figure 1.** Two models of result judgment for the immunochromatographic assay (ICA). **Figure 1.** Two models of result judgment for the immunochromatographic assay (ICA).
