Highly Specific Monoclonal Antibodies Combined with A Multiplex Quantum Dot Microsphere-based Fluorescence Lateral Flow Immunochromatographic Assay for Simultaneous Detection of Vanillin, Methyl Vanillin, and Ethyl Vanillin in Food Samples

Vanillin (VAN), methyl vanillin (VER), and ethyl vanillin (EVA) are widely used as flavor enhancers in processed foods. Monitoring their levels is essential to prevent overuse or adulteration, especially in infant foods. Herein, a computational chemistry-aided molecular modeling technique was employed to design specific haptens for VAN, VER, and EVA. High-affinity monoclonal antibodies were generated via hybridoma technology with matrix effect-enhanced screening, yielding half-maximal inhibitory concentrations of 22.10, 49.94, and 40.84 ng/mL. A multiplex quantum dot microsphere-based fluorescence lateral flow immunoassay was developed for the simultaneous detection of VAN, VER, and EVA in infant formula and rice. The quantitative detection ranges in infant formula were 6.31–142.59, 24.07–119.88, and 23.34–214.77 μg/kg; in rice, the detection ranges were 6.71–143.63, 18.51–183.75, and 21.42–167.65 μg/kg. The method was validated through spiked recovery tests and real sample analysis, demonstrating its accuracy and reliability for rapid, high-throughput screening applications.