Gold nanoparticle-based lateral flow immunoassay for the repaid and sensitive detection of pyraclostrobin in wheat, apple, Chinese cabbage, and soil samples

Pyraclostrobin is widely employed for controlling fungal pathogens. Given its potential adverse effects on aquatic organisms , beneficial insects, and mammals, there is an urgent need to develop on-site detection methods for monitoring pyraclostrobin residues. In this study, monoclonal antibodies (mAbs) targeting pyraclostrobin were developed, and a reliable structure of the mAb 4B4C3 was predicted using AlphaFold 3. The structure showed high predicted local distance difference test (pLDDT) values for both the complementarity-determining regions (CDRs) and framework regions (FRs). Molecular docking was employed to investigate its binding pattern with pyraclostrobin. Subsequently, a highly sensitive lateral flow immunoassay (LFIA) based on gold nanoparticles (AuNPs) for detecting pyraclostrobin was established. The AuNP-LFIA method exhibited a visual limit of detection (vLOD) of 1.25 ng/mL and a completely inhibitory concentration of 5 ng/mL, surpassing the sensitivity of previously described methods. The accuracy of the established method was confirmed through spiked-recovery tests, exhibiting a sensitivity of 30 ng/g in wheat, apple, and soil samples, and 60 ng/g in Chinese cabbage samples. The reliability of the method was validated using ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). This study presents a sensitive and cost-effective approach for rapidly detecting pyraclostrobin residues, demonstrating the significant potential of AlphaFold 3 in antibody engineering and small molecule on-site detection.