Competitive upconversion-linked immunoassay using peptide mimetics for the detection of the mycotoxin zearalenone

Due to increasing food safety standards, the analysis of mycotoxins has become essential in the food industry. In this work, we have developed a competitive upconversion-linked immunosorbent assay (ULISA) for the analysis of zearalenone (ZEA), one of the most frequently encountered mycotoxins in food worldwide. Instead of a toxin-conjugate conventionally used in competitive immunoassays, we designed a ZEA mimicking peptide extended by a biotin-linker and confirmed its excellent suitability to mimic ZEA by nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) analysis. Upconversion nanoparticles (UCNP, type NaYF₄:Yb,Tm) served as background-free optical label for the detection of the peptide mimetic in the competitive ULISA. Streptavidin-conjugated UCNPs were prepared by click reaction using an alkyne-PEG-neridronate linker. The UCNP conjugate clearly outperformed conventional labels such as enzymes or fluorescent dyes. With a limit of detection of 20 pg mL^-1 (63 pM), the competitive ULISA is well applicable to the detection of ZEA at the levels set by the European legislation. Moreover, the ULISA is specific for ZEA and its metabolites (α- and β-zearalenol) without significant cross-reactivity with other related mycotoxins. We detected ZEA in spiked and naturally contaminated maize samples using liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) as a reference method to demonstrate food analysis in real samples.