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

Abstract

<p>Strains of <em><a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/penicillium" title="Learn more about Penicillium from ScienceDirect's AI-generated Topic Pages">Penicillium</a></em> spp. are used for fungi-ripened cheeses and <em>Aspergillus</em> spp. routinely contaminate maize and other crops. Some of these strains can produce toxic secondary metabolites (mycotoxins), including the <a href="https://www.sciencedirect.com/topics/chemistry/neurotoxin" title="Learn more about neurotoxin from ScienceDirect's AI-generated Topic Pages">neurotoxin</a> α-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 epitope mimicking peptide, or <a href="https://www.sciencedirect.com/topics/chemistry/mimotope" title="Learn more about mimotope from ScienceDirect's AI-generated Topic Pages">mimotope</a>, selected by <a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/phage-display" title="Learn more about phage display from ScienceDirect's AI-generated Topic Pages">phage display</a>. CPA-specific antibody was used to isolate mimotopes from a cyclic 7-mer <a href="https://www.sciencedirect.com/topics/chemistry/peptide-library" title="Learn more about peptide library from ScienceDirect's AI-generated Topic Pages">peptide library</a> in consecutive selection rounds. Enrichment of <a href="https://www.sciencedirect.com/topics/engineering/binding-antibody" title="Learn more about antibody binding from ScienceDirect's AI-generated Topic Pages">antibody binding</a> phages was achieved, and the analysis of individual phage clones revealed four different mimotope <a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/peptide-sequence" title="Learn more about peptide sequences from ScienceDirect's AI-generated Topic Pages">peptide sequences</a>. The mimotope sequence, ACNWWDLTLC, performed best in phage-based immunoassays, <a href="https://www.sciencedirect.com/topics/chemistry/surface-plasmon-resonance" title="Learn more about surface plasmon resonance from ScienceDirect's AI-generated Topic Pages">surface plasmon resonance</a> binding analyses, and UC-RET-based immunoassays. To develop a homogeneous assay, upconversion <a href="https://www.sciencedirect.com/topics/chemistry/nanoparticle" title="Learn more about nanoparticles from ScienceDirect's AI-generated Topic Pages">nanoparticles</a> (UCNP, type NaYF₄:Yb³⁺, Er³⁺) were used as energy donors and coated with <a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/streptavidin" title="Learn more about streptavidin from ScienceDirect's AI-generated Topic Pages">streptavidin</a> to anchor the synthetic biotinylated mimotope. <a href="https://www.sciencedirect.com/topics/chemistry/alexa-fluor-555" title="Learn more about Alexa Fluor 555 from ScienceDirect's AI-generated Topic Pages">Alexa Fluor 555</a>, used as an energy acceptor, was conjugated to the anti-CPA <a href="https://www.sciencedirect.com/topics/engineering/antibody-fragment" title="Learn more about antibody fragment from ScienceDirect's AI-generated Topic Pages">antibody fragment</a>. The homogeneous single-step immunoassay could detect CPA in just 5 min and enabled a limit of detection (LOD) of 30 pg mL⁻¹ (1.5 μg kg⁻¹) and an IC₅₀ value of 0.36 ng mL⁻¹. No significant cross-reactivity was observed with other co-produced <a href="https://www.sciencedirect.com/topics/chemistry/mycotoxin" title="Learn more about mycotoxins from ScienceDirect's AI-generated Topic Pages">mycotoxins</a>. Finally, we applied the novel method for the detection of CPA in spiked maize samples using high-performance liquid chromatography coupled to a <a href="https://www.sciencedirect.com/topics/engineering/diode-array" title="Learn more about diode array from ScienceDirect's AI-generated Topic Pages">diode array</a> detector (HPLC-DAD) as a reference method.</p>