Three-color triple platform for point-of-care detection of three antifungal azoles in livestock wastewater and sludge.

Due to the environmental persistence of antifungal azoles (AFZs), and their potential ecotoxicity, and ability to induce resistance, their occurrence in the environment has become a concern, with livestock wastewater and sludge serving as important reservoirs for their residues. Hence, it is crucial to devise an efficient, precise, and comprehensive screening approach for these compounds. In this study, computer-aided simulation technology was employed to analyze the spatial structures and electronic properties of miconazole (MCZ), ketoconazole (KCZ), fluconazole (FCZ) and haptens, ensuring their structural similarity for the preparation of highly sensitive monoclonal antibodies (mAbs). The results showed that the half maximal inhibitory concentration values of the three mAbs were 5.46, 0.54, and 18.01 ng/mL, respectively. Based on this foundation, we established a multiplex-latex nanoparticles immunochromatography assay (multi-LNPs-ICA) capable of detecting three AFZs simultaneously within livestock-sourced wastewater and sludge. In wastewater, the limits of detection (LODs) were determined to be 1.35 ng/g for MCZ, 0.54 ng/g for KCZ, and 4.54 ng/g for FCZ, with corresponding visual limits of detection (vLODs) of 5, 2, and 20 ng/g, respectively. Similarly, in sludge samples, the LODs of 3.77 ng/g (MCZ), 1.51 ng/g (KCZ), and 17.69 ng/g (FCZ), with vLODs reaching 10, 5, and 50 ng/g for these analytes, respectively. Through validation against high-performance liquid chromatography-tandem mass spectrometry and analysis of actual samples, our multi-LNPs-ICA demonstrated the accuracy and reliability, demonstrating its utility for rapid field screening applications.