Nanomaterial-based lateral flow immunoassays (LFIAs) have been widely used for the on-site detection of genetically modified components. However, the practical applications are often limited by the complex matrix, such as in red samples. In this study, a thionine (Thi) labeling-based LFIA was developed for the first time to detect CP4-EPSPS protein. The optimal labeling concentration of Thi was 0.5 mg/mL, and the antibody could be rapidly coupled to Thi in 10 min. The visual limit of detection (vLOD) levels for transgenic soybean, sugar beet, and cotton containing the CP4-EPSPS protein reached 0.05%, 0.1%, and 0.1%, respectively, and had no interference from other proteins. After storage at 4 °C for three months, the LFIA sensitivity remained unchanged and showed good stability. This method could be used to screen and detect a variety of transgenic crops containing the CP4-EPSPS protein, and the results were consistent with the current standard assay. This study pioneered the development of an immunochromatographic method using Thi as a marker and applied it to the detection of the CP4-EPSPS protein in herbicide-tolerant transgenic crops. This provides a new method for the rapid immunoassay of Thi as a dye and has good prospects for practical application.

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