Homogeneous immunoassay for cyclopiazonic acid based upon mimotopes and upconversion-resonance energy transfer

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); MAS, VICENTE - Instituto De Salud Carlos Iii; BARDERAS, RADRIGO - Instituto De Salud Carlos Iii; Maragos, Chris; CANALES, ANGELES - Complutense University Of Madrid (UCM); SOUKKA, TERO - University Of Turku; BENITO-PENA, ELENA - Complutense University Of Madrid (UCM); MORENO-BONDI, MARIA - Complutense University Of Madrid (UCM)

Submitted to: Biosensors and Bioelectronics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/17/2023
Publication Date: 4/29/2023
Citation: Pradanas-Gonzalez, F., Peltomaa, R., Lahtinen, S., Luque-Uria, A., Mas, V., Barderas, R., Maragos, C.M., Canales, A., Soukka, T., Benito-Pena, E., Moreno-Bondi, M.C. 2023. Homogeneous immunoassay for cyclopiazonic acid based upon mimotopes and upconversion-resonance energy transfer. Biosensors and Bioelectronics. 233. Article 115339. https://doi.org/10.1016/j.bios.2023.115339.
DOI: https://doi.org/10.1016/j.bios.2023.115339

Interpretive Summary: Some fungi commonly found in food can produce toxins. One such toxin is cyclopiazonic acid (CPA), a neurotoxin produced by some species of Penicillia. CPA can contaminant food, so it is important to monitor for its presence. Current detection methods require multiple steps including a time-consuming separation step. Researchers at the University of Madrid-Complutense, the University of Turku (Finland), and the Mycotoxin Prevention and Applied Microbiology Unit of USDA-ARS (Peoria, IL) developed a rapid detection method for CPA in corn that does not require the separation step. In addition to being faster the new method does not need a CPA standard, reducing the possible exposure of the person conducting the test to the toxin. Increasing the speed and improving the safety of testing helps with monitoring for the toxin to ensure food safety.

Technical Abstract: Strains of Penicillium spp. are used as fungi-ripened cheeses but also routinely contaminate maize. Some of these strains can produce toxic secondary metabolites (mycotoxins), including the neurotoxin a-cyclopiazonic acid (CPA). In this work, we developed a homogeneous upconversion-resonance energy transfer (UC-RET) immunoassay for the detection of CPA using a novel peptide mimicking probe (mimotope) selected by phage display. CPA-specific antibody was used to isolate mimotopes from a cyclic 7-mer peptide library in consecutive selection rounds. Enrichment of antibody binding phages was achieved and the analysis of randomly selected monoclonal phages revealed four different conserved peptide sequences. Clone A2, with peptide sequence ACNWWDLTLC, showed the best sensitivity and reproducibility in phage-based enzyme-linked immunosorbent assay (phage-ELISA), surface plasmon resonance (SPR) binding analysis and UC-RET-based immunoassays. UCNPs (NaYF4:Yb3+, Er3+) were used as energy donors, and they were bioconjugated with streptavidin to anchor the biotinylated mimotope A2. Alexa Fluor 555 (AF555), used as an energy acceptor, was conjugated to the monoclonal anti-CPA antibody fragment (Fab). The single-step immunoassay could detect CPA in 5 min and enabled a limit of detection (LOD) of 30 pg mL-1 (1.5 µg kg-1) with an IC50 value of 0.36 ± 0.03 ng mL-1. No significant cross-reactivity was observed with other co-produced mycotoxins. We detected CPA in spiked contaminated maize samples using liquid chromatography-tandem mass spectrometry and diode array detection (UPLC-MS/MS and HPLC-DAD) as a reference method to demonstrate food analysis in real samples.