Computationally Guided Hapten Design Enables a Multicolor Immunochromatographic Assay for the Simultaneous Detection of Three Spirocyclic Tetronic/Tetramic Acid Derivative Insecticides in Environment and Agricultural Samples

The widespread application and environmental persistence of three spirocyclic tetronic/tetramic acid derivatives (STADs)─spirodiclofen (SDF), spiromesifen (SMF), and spirotetramat (STM)─have raised sustained concerns regarding their residue accumulation, potential ecotoxicity, and risks to human health, underscoring the urgent need for rapid detection methods. Herein, six haptens were rationally designed and screened using computational chemistry-assisted molecular modeling tools, followed by the successful generation of specific monoclonal antibodies against SDF, SMF, and STM via hybridoma technology, with half-maximal inhibitory concentrations of 4.91, 0.56, and 1.03 ng/mL, respectively. On this basis, a multiplex immunochromatographic assay was developed for the first time using red, purple, and blue polystyrene microspheres as tracer labels for the three antibodies. Coupled with a portable reader, it enables simultaneous detection of the three STADs in lake water, soil, and tomato samples within 15 min. This assay achieves visual limits of detection (LODs) of 2–10 ng/g and quantitative LODs of 0.48–2.29 ng/g across these matrices. Recovery experiments and blinded sample analyses yielded results consistent with those obtained by reference instrumental methods, confirming the assay’s accuracy and reliability-and thereby providing a sensitive, deployable, and practical tool for on-site, multiresidue monitoring of STADs in environmental and agricultural samples.