K2CO3 0.2 mol/L 2.3 μL Purified water *2.3. Semi-Quantitative Detection of the ICA*

Note: The concentrations and volumes of ZEN‐McAb and K2CO3 in this table are used in 1 mL GNPs. Other parameters were used for preparing a 30 cm of NC membrane. *2.3. Semi‐Quantitative Detection of the ICA* A series of ZEN standards (0, 0.25, 0.5, 0.7, 1.0, 1.5, 2.0, 3.0, 4.0, and 5.0 ng/mL in 20% methanol of 10 mmol/L of PBS buffer) were detected by the developed ICA. With the increasing in ZEN standards, the red color intensities were in a gradually decreasing process, and eventually disappeared for the T lines (Figure 3). The red color intensities for the C lines always existed to prove the effectivity of the ICA. It was observed that a significant reduction in the red color in the T line was shown in 0.50 ng/mL of ZEN. Thus, the semi‐quantitative LOD of the ICA could be established A series of ZEN standards (0, 0.25, 0.5, 0.7, 1.0, 1.5, 2.0, 3.0, 4.0, and 5.0 ng/mL in 20% methanol of 10 mmol/L of PBS buffer) were detected by the developed ICA. With the increasing in ZEN standards, the red color intensities were in a gradually decreasing process, and eventually disappeared for the T lines (Figure 3). The red color intensities for the C lines always existed to prove the effectivity of the ICA. It was observed that a significant reduction in the red color in the T line was shown in 0.50 ng/mL of ZEN. Thus, the semi-quantitative LOD of the ICA could be established as 0.50 ng/mL. The red color intensities eventually disappeared in the T line when the concentration of ZEN exceeded 3.0 ng/mL. These results of the ICA judged by the naked eye indicated that the visual detection of the ZEN levels could be in three intervals: <0.50 ng/mL (−, negative), 0.50 ng/mL ≤ZEN concentration <3.0 ng/mL (±, weakly positive), and ≥3.0 ng/mL (+, positive).

≤ZEN concentration <3.0 ng/mL (±, weakly positive), and ≥3.0 ng/mL (+, positive).

*Toxins* **2020**, *x*, x FOR PEER REVIEW 4 of 12

*Toxins* **2020**, *x*, x FOR PEER REVIEW 4 of 12

**Figure 3.** The semi‐quantitative detection of ICA for the series of ZEN standards. **Figure 3.** The semi-quantitative detection of ICA for the series of ZEN standards. **Figure 3.** The semi‐quantitative detection of ICA for the series of ZEN standards.

### *2.4. Quantitative Detection of the ICA 2.4. Quantitative Detection of the ICA 2.4. Quantitative Detection of the ICA*

*2.5. Specificity of the ICA*

*2.5. Specificity of the ICA*

The strips of ICA were digitally detected by the strip reader, and the gray values of the T lines were plotted against the concentrations of ZEN in order to get a reduction curve (Figure 4A). This showed a good linear relationship between the inhibition ratios of the gray values in the T lines and the logarithm of the ZEN concentrations (Figure 4B; Y = 81.88X + 37.86, R2 = 0.9977). In this case, ZEN could achieve linear detection in the range of 0.25 to 4.0 ng/mL, and the quantitative LOD could be defined as 0.25 ng/mL (Table 1). With the semi‐quantitative and quantitative LOD of the ICA all below the MLs for ZEN, the sensitivity of the developed ICA was satisfied for detecting ZEN. The strips of ICA were digitally detected by the strip reader, and the gray values of the T lines were plotted against the concentrations of ZEN in order to get a reduction curve (Figure 4A). This showed a good linear relationship between the inhibition ratios of the gray values in the T lines and the logarithm of the ZEN concentrations (Figure 4B; Y = 81.88X + 37.86, R<sup>2</sup> = 0.9977). In this case, ZEN could achieve linear detection in the range of 0.25 to 4.0 ng/mL, and the quantitative LOD could be defined as 0.25 ng/mL (Table 1). With the semi-quantitative and quantitative LOD of the ICA all below the MLs for ZEN, the sensitivity of the developed ICA was satisfied for detecting ZEN. The strips of ICA were digitally detected by the strip reader, and the gray values of the T lines were plotted against the concentrations of ZEN in order to get a reduction curve (Figure 4A). This showed a good linear relationship between the inhibition ratios of the gray values in the T lines and the logarithm of the ZEN concentrations (Figure 4B; Y = 81.88X + 37.86, R2 = 0.9977). In this case, ZEN could achieve linear detection in the range of 0.25 to 4.0 ng/mL, and the quantitative LOD could be defined as 0.25 ng/mL (Table 1). With the semi‐quantitative and quantitative LOD of the ICA all below the MLs for ZEN, the sensitivity of the developed ICA was satisfied for detecting ZEN.

**Figure 4.** The quantitative detection of ICA for the series of ZEN standards. (**A**) The corresponding relationship between the gray value (T/C) and the concentration of ZEN (ng/mL). (**B**) The linear relationship between the inhibition (%) and the logarithm of the ZEN concentrations (ng/mL). **Figure 4.** The quantitative detection of ICA for the series of ZEN standards. (**A**) The corresponding relationship between the gray value (T/C) and the concentration of ZEN (ng/mL). (**B**) The linear relationship between the inhibition (%) and the logarithm of the ZEN concentrations (ng/mL). **Figure 4.** The quantitative detection of ICA for the series of ZEN standards. (**A**) The corresponding relationship between the gray value (T/C) and the concentration of ZEN (ng/mL); (**B**) The linear relationship between the inhibition (%) and the logarithm of the ZEN concentrations (ng/mL).

Comparing the developed ICA with the enzyme‐linked immunosorbent assay (ELISA) for ZEN, ICA showed more convenient and rapid operation steps for on‐site detection, which could achieve ZEN detection within 5 min using one step. Two models of judgment using the naked eye and strip reader could allow for the ICA detection to be more flexible. The digitized detection of ICA showed a lower LOD and a wider detection range than the visual detection. It was close to the sensitivity of the enzyme‐linked immunosorbent assay (ELISA; Table S1 and Figure S1). Moreover, the digitized detection of ICA could obtain accurate ZEN levels in order to realize quantitative detection. It was suggested that the visual and digitized detection of ICA for ZEN contamination could be selected as needed or used simultaneously in order to achieve semi‐quantitative and quantitative detection for on‐site testing. Comparing the developed ICA with the enzyme‐linked immunosorbent assay (ELISA) for ZEN, ICA showed more convenient and rapid operation steps for on‐site detection, which could achieve ZEN detection within 5 min using one step. Two models of judgment using the naked eye and strip reader could allow for the ICA detection to be more flexible. The digitized detection of ICA showed a lower LOD and a wider detection range than the visual detection. It was close to the sensitivity of the enzyme‐linked immunosorbent assay (ELISA; Table S1 and Figure S1). Moreover, the digitized detection of ICA could obtain accurate ZEN levels in order to realize quantitative detection. It was suggested that the visual and digitized detection of ICA for ZEN contamination could be selected as needed or used simultaneously in order to achieve semi‐quantitative and quantitative detection for on‐site testing. Comparing the developed ICA with the enzyme-linked immunosorbent assay (ELISA) for ZEN, ICA showed more convenient and rapid operation steps for on-site detection, which could achieve ZEN detection within 5 min using one step. Two models of judgment using the naked eye and strip reader could allow for the ICA detection to be more flexible. The digitized detection of ICA showed a lower LOD and a wider detection range than the visual detection. It was close to the sensitivity of the enzyme-linked immunosorbent assay (ELISA; Table S1 and Figure S1). Moreover, the digitized detection of ICA could obtain accurate ZEN levels in order to realize quantitative detection. It was suggested that the visual and digitized detection of ICA for ZEN contamination could be selected as needed or used simultaneously in order to achieve semi-quantitative and quantitative detection for on-site testing.

The cross‐reactivity tests (CR; %) of the ICA for the analogs of ZEN and the common mycotoxins

The cross‐reactivity tests (CR; %) of the ICA for the analogs of ZEN and the common mycotoxins
