*2.2. Key Parameters for the ICA 2.2. Key Parameters for the ICA*

It was a key procedure to optimize the parameters for the preparation of the ZEN‐McAb‐GNP probes and the ICA strips, which could be beneficial for improving the sensitivity of the ICA method. The evaluation criterion was based on using fewer ZEN in order to reduce the red color intensity in the T line and for maintaining the effectiveness of ICA. The optimal parameters for the ICA are shown in Table 1. For preparing the ZEN‐McAb‐GNP probes, 2.3 μL of 0.2 mol/L K2CO3 in an aqueous solution, 3.0 μL of a 1.0 mg/mL ZEN‐McAb in a 10 mmol/L PB buffer, and 100 μL of 10% bovine serum albumin (BSA) in purified water were used in 1 mL of GNPs. For spraying a 30 cm of nitrocellulose (NC) membrane, the 0.5 μL of 13.1 mg/mL goat anti‐mouse IgG (GAM‐IgG) and 1.0 μL of 8.8 mg/mL ZEN‐antigen in 30 μL of 10 mmol/L of phosphate buffered saline (PBS buffer) were immobilized on the C line and T line, respectively. It was a key procedure to optimize the parameters for the preparation of the ZEN-McAb-GNP probes and the ICA strips, which could be beneficial for improving the sensitivity of the ICA method. The evaluation criterion was based on using fewer ZEN in order to reduce the red color intensity in the T line and for maintaining the effectiveness of ICA. The optimal parameters for the ICA are shown in Table 1. For preparing the ZEN-McAb-GNP probes, 2.3 µL of 0.2 mol/L K2CO<sup>3</sup> in an aqueous solution, 3.0 µL of a 1.0 mg/mL ZEN-McAb in a 10 mmol/L PB buffer, and 100 µL of 10% bovine serum albumin (BSA) in purified water were used in 1 mL of GNPs. For spraying a 30 cm of nitrocellulose (NC) membrane, the 0.5 µL of 13.1 mg/mL goat anti-mouse IgG (GAM-IgG) and 1.0 µL of 8.8 mg/mL ZEN-antigen in 30 µL of 10 mmol/L of phosphate buffered saline (PBS buffer) were immobilized on the C line and T line, respectively.


**Table 1.** The optimization of key parameters for the preparation of ICA for ZEN. **Table 1.** The optimization of key parameters for the preparation of ICA for ZEN.

ZEN‐McAb 1.0 mg/mL 3.0 μL 10 mmol/L PB Note: The concentrations and volumes of ZEN-McAb and K2CO<sup>3</sup> in this table are used in 1 mL GNPs. Other parameters were used for preparing a 30 cm of NC membrane.

BSA 10% 100 μL Purified water
