Upconversion-linked immunosorbent assay for the biomimetic detection of the mycotoxin cyclopiazonic acid

Authors: PRADANAS-GONZALEZ, FERNANDO - Complutense University Of Madrid (UCM); PELTOMAA, RIIKKA - University Of Turku; LAHTINEN, SATU - University Of Turku; LUQUE-URIA, ALVARO - Complutense University Of Madrid (UCM); RODRIGUEZ, YOEL - Mount Sinai School Of Medicine; NAVARRO-VILLOSLADA, FERNANDO - Complutense University Of Madrid (UCM); Maragos, Chris; SOUKKA, TERO - University Of Turku; MORENO-BONDI, MARIA - Complutense University Of Madrid (UCM); BENITO-PENA, ELENA - Complutense University Of Madrid (UCM)

Submitted to: Analytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/28/2024
Publication Date: 12/7/2024
Citation: Pradanas-Gonzalez, F., Peltomaa, R., Lahtinen, S., Luque-Uria, A., Rodriguez, Y., Navarro-Villoslada, F., Maragos, C.M., Soukka, T., Moreno-Bondi, M.C., Benito-Pena, E. 2024. Upconversion-linked immunosorbent assay for the biomimetic detection of the mycotoxin cyclopiazonic acid. Analytical Chemistry. https://doi.org/10.1021/acs.analchem.4c05168.
DOI: https://doi.org/10.1021/acs.analchem.4c05168

Interpretive Summary: Cyclopiazonic acid (CPA) is a neurotoxin that can be produced by certain fungi commonly found on maize and in certain cheeses. Researchers at the University of Madrid-Complutense, the University of Turku, and the Mycotoxin Prevention and Applied Microbiology Unit of USDA-ARS (Peoria, IL) developed a rapid antibody-based test for CPA in maize. Many rapid tests use enzymes to amplify signal and improve sensitivity. In this report an alternative approach was used that involved a highly sensitive fluorescent nanomaterial (upconversion nanoparticles). Doing so reduced the number of steps required and improved the sensitivity relative to a traditional enzyme-based assay by 60-fold. These improvements will help to ensure food safety by facilitating the rapid, low cost, monitoring for this toxin in maize.

Technical Abstract: The neurotoxin a-cyclopiazonic acid (CPA) is an emerging mycotoxin produced as a secondary metabolite by several fungi species (i.e. Penicillium spp. and Aspergillus spp.). It commonly contaminates maize, crops, cheese, and wine. In this work, CPA detection in foodstuff is accomplished by combining two innovative strategies, upconversion nanoparticles (UCNPs) and epitope-mimicking peptides, to develop a competitive upconversion-linked immunosorbent assay (ULISA). On the one hand, we have applied UCNPs (type NaYF4:Yb3+, Er3+) as background-free optical labels due to their anti-Stokes shift with excitation in the near-infrared region and emission in the ultraviolet-visible region. On the other hand, a CPA epitope-mimicking cyclic peptide (A2) was used as a substitute for the toxin-conjugates traditionally applied to competitive assays. UCNPs were decorated with an anti-CPA fragment antigen-binding antibody (UCNP-Fab), and CPA detection was accomplished through competition with a biotinylated CPA epitope-mimicking cyclic peptide (A2, ACNWWDLTLC-GGGSK (Biotin)-NH2), anchored to a streptavidin-coated microtiter plate, for antibody binding. ULISA platform offers ultrasensitive detection of CPA (limit of detection of 1.3 pg mL-1 and IC50 value of 15 pg mL-1), and no cross-reactivity was observed with other coproduced mycotoxins. These results substantially outperformed the analytical features of conventional heterogeneous immunoassays based on enzymatic detection. The proposed ULISA was applied to detect CPA in spiked maize samples, and the results were validated by high-performance liquid chromatography coupled to a tandem mass spectrometry detector (HPLC-MS/MS).