Competitive cytometry-based immunoassay for patulin determination in apple juice

• A cytometry-based assay using monoclonal antibodies to detect a stable patulin derivative was developed. • The assay achieves a detection limit of 0.03 µg L −1 in buffer. • Sample preparation requires only a simple dilution step and enables rapid detection without calibration. • Successful validation and compliance with EU regulations for apple juice was achieved. Patulin is a mycotoxin that is frequently found in apples and apple-derived products. Given the potential harm it can cause to humans, maximum levels for patulin in food have been set worldwide. Conventional methods for the detection of patulin are often time-consuming or lack sensitivity. In this study, a novel cytometry approach based on specific monoclonal antibodies is presented. These high-affinity binders do not target patulin itself, but a stable derivative (adduct) that is rapidly obtained in an aqueous medium at room temperature. To develop the assay, a specific fluorescent competitor was designed and synthesized. After optimizing the assay conditions, including the concentration of the fluorescent competitor and of the antibody bound to polystyrene-silica core–shell microparticles, a detection limit of 0.03 µg L −1 in buffer was achieved. Finally, validation according to Commission Regulation (EU) 2023/2782 demonstrated that apple juice samples spiked with patulin at 25 or 50 µg L −1 (permissible limits set by the EU) were properly scored as non-compliant without any additional treatment other than a simple dilution step in buffer. The developed assay offers several key advantages, including rapid analysis, high sensitivity and specificity, and the potential for multiplexing, making it a promising analytical tool for routine monitoring of patulin contamination in food.