Novel cation-based hapten design strategy and development of a monoclonal antibody-based immunochromatographic assay strip for highly specific detection of imazalil: Theoretical chemical insights

Imazalil is an imidazole fungicide that controls fruit diseases by inhibiting ergosterol synthesis but has resulted in residues in food. There is a need to develop rapid immunoassays suitable for food safety monitoring and primary screening of large samples. The development of high-performance antibodies represents a pivotal front-end process in the establishment of immuno-rapid assays for harmful small molecules. This study innovatively adopted a hapten preparation strategy by introducing a quaternary ammonium cationic arm to enhance specific immune responses without exposing the common fragment imidazole ring. Theoretical chemistry techniques, including electrostatic potential, lipophilic potential, molecular structure superposition, and frontier molecular orbital energy gap-based molecular softness, were used to analyze the structural features of the quaternary ammonium cationic hapten IMZ-A. The results showed that IMZ-A retained imazalil’s microelectronic and macroscopic three-dimensional spatial superposition structures and showed increased softness for higher bioreactivity. The obtained imazalil monoclonal antibody demonstrates high specificity and sensitivity, with an IC of 1.96 ng mL. A colloidal gold immunochromatographic assay was developed, detecting imazalil residues in fruit samples within 7 min, with a qLOD of 0.57–2.05 ng mL and an IC of 3.67–12.41 ng mL in citrus, apple, and banana samples. This work offers a convenient and highly sensitive immunoassay for large-scale detection of trace imazalil residues. More significantly, it provides a novel and general design idea for hapten, namely, introducing cations into hapten molecules to improve their specific immune potential.