Ultra-sensitive determination of cyclopiazonic acid in complex food samples via competitive time-resolved luminescence immunoassays coupled to green microextraction

In this study, an ultra-sensitive competitive luminescence immunoassay for detecting and quantifying the mycotoxin cyclopiazonic acid (CPA) in food was developed. Analyses were conducted in 96-well plates using magnetic beads as support for a CPA-mimetic peptide, enhancing the surface area available for competition. Light emission from an anti-CPA antibody fragment linked to a long-lifetime luminescent Tb (III) chelate, serving as optical label, was measured in time-resolved luminescence (TRL) mode upon binding to the mimetic peptide. In the presence of the target toxin, emission intensity decreased proportionally to analyte concentration. The TRL readout provided an improved measurement of CPA concentrations when compared to two other optical detection methods, namely fluorescence and colorimetry, through effective background reduction and noise suppression. The broad applicability of the method was tested by examining two highly dissimilar matrices for the toxin: corn and blue cheese. The latter is especially relevant due to the use of Penicillium fungi, one of the main CPA producers alongside Aspergillus , in the fermentation process. Two different strategies for analyte extraction were applied, i.e., a traditional solvent mixture (methanol and water) and a sustainable alternative: a choline chloride and ethylene glycol natural deep eutectic solvent (NADES). The use of the NADES improved the removal of the lipophilic toxin from the fatty matrix as well as its handling during the sample preparation stage. The immunoassay exhibited remarkable selectivity and sensitivity, with limits of detection of 0.022 μg/kg and 4.0 μg/kg, and limits of quantification of 0.040 μg/kg and 8 μg/kg, for corn and cheese, respectively. This integrated approach offers a robust and environmentally sustainable platform for monitoring CPA contamination in food, contributing to improved food safety and public health protection.