A Highly Sensitive Multiplex Immunosensor for Detecting Three Sulfonylurea Herbicides: From Hapten Design to Molecular Recognition Mechanism

Sulfonylurea herbicides (SUs) have the characteristic of persistent residues in the environment and crops, and their threat to environmental safety and human health is becoming increasingly prominent. In this study, three new haptens were designed targeting chlorimuron-ethyl (CRM), chlorsulfuron (CSF), and cinosulfuron (CIN), respectively, and their rationality was evaluated by computer simulation technology. Three monoclonal antibodies (mAbs) were obtained through mouse immunization and cell fusion, and their half-maximal inhibitory concentrations (IC₅₀) were 2.42 ng/mL for CRM, 2.00 ng/mL for CSF, and 1.48 ng/mL for CIN, respectively. Molecular docking revealed that the specific recognition ability of CRM-mAb was mainly mediated by the key amino acid residues (PHE-B36, ALA-B34, ARG-A73, ARG-A73, and ARG-B96) in its binding pocket. Similarly, the key amino acid residues (TYR-A32, PRO-B58, and THR-A105) in the CSF-mAb pocket and the key amino acid residues (ARG-B56, ALA-B57, ALA-A105, ASP-B52, and ASN-B55) in the CIN-mAb pocket ensured their specificity and sensitivity. A multiplex colloidal gold immunochromatographic assay (multi-GNPs-ICA) was constructed based on this, which can simultaneously detect the aforementioned three SUs in paddy water, soil, and rice. Among them, the visual limit of detection (vLOD) is 1-50 ng/mL, the cutoff value is 5-200 ng/mL, and the calculated limit of detection (cLOD) is 0.25-4.29 ng/mL. The detection results of positive samples are highly consistent with those of HPLC-MS, validating the reliability of the multi-GNPs-ICA and demonstrating its significant application potential in rapid on-site screening.