A Dual-T-Line Immunochromatographic Assay Based on Computational Chemistry-Guided Hapten Design for Rapid On-Site Detection of Chloroneb in Agricultural and Environmental Samples

The health risks posed by chloroneb (CHL), a representative fungicide, in agricultural products and the environment necessitate rapid detection methods. Here, computational chemistry-aided rational hapten design yielded a high-performance monoclonal antibody against CHL (IC50 = 2.63 ng/mL), guided by analysis of its spatial conformation and electronic properties. A dual-T-line gold nanoparticle-based immunochromatographic assay (AuNP-ICA) was established, achieving 2.6-fold higher sensitivity and a 2.9-fold higher upper detection limit compared with traditional single-T-line formats. The assay enables rapid (<20 min) on-site detection of CHL in lake water, soil, tea, soybeans, and mangoes, with limits of detection ranging from 2.51 to 4.54 μg/kg. Recovery and blind sample analyses demonstrated excellent agreement with instrumental methods. This work not only provides a practical tool for field detection of CHL but also establishes a generalizable technological framework that integrates rational hapten design with a dual-T-line configuration for developing sensitive immunoassays targeting other small-molecule contaminants.