Integrated Quantum Dot Microsphere-Based Multiplex Lateral Flow Immunoassay for On-Site Hazard Screening of Azole Antifungal Residues in Ecological Environment and Aquatic Products

As widely applied azole antifungal agents, climbazole (CBZ), clotrimazole (CLZ), and isavuconazole (ISKZ) can persist in environmental media and bioaccumulate in aquatic organisms, thus bringing hidden risks to the ecological environment and aquatic products. Herein, a computer-assisted hapten design was adopted to prepare highly specific monoclonal antibodies, with the half-maximal inhibitory concentrations (IC50) of 1.825, 1.732, and 3.013 ng/mL for CBZ, CLZ, and ISKZ, respectively. Molecular recognition was driven by noncovalent interactions in antibody complementarity-determining regions. Based on this, a quantum dot microspheres-based multiplex lateral flow immunoassay was established for simultaneous detection of the three analytes, with calculated limits of detection in lake water, sludge, and fish of 0.198-1.205, 1.183-3.461, and 3.719-6.291 μg/kg, respectively. Satisfactory agreement with high-performance liquid chromatography-tandem mass spectrometry was validated via both spiked matrix recovery tests and real sample detection. This rapid, simple, and portable method provides a robust and convenient tool for large-scale on-site screening of azole antifungals, supporting efficient multicomponent pollutant monitoring.