Homogeneous immunoassays represent an attractive alternative to traditional heterogeneous assays due to their simplicity, sensitivity, and speed. On the basis of a previously identified epitope-mimicking peptide, or mimotope, we developed a homogeneous fluorescence quenching immunoassay based on gold nanoparticles (AuNPs) and a recombinant epitope-mimicking fusion protein for the detection of mycotoxin fumonisin B₁ (FB₁). The fumonisin mimotope was cloned as a fusion protein with a yellow fluorescent protein that could be used directly as the tracer for FB₁ detection without the need of labeling or a secondary antibody. Furthermore, owing to the fluorescence quenching ability of AuNPs, a homogeneous immunoassay could be performed in a single step without washing steps to separate the unbound tracer. The homogeneous quenching assay showed negligible matrix effects in 5% wheat extract and high sensitivity for FB₁ detection, with a dynamic range from 7.3 to 22.6 ng mL^–1, a detection limit of 1.1 ng mL^–1, and IC₅₀ value of 12.9 ng mL^–1, which was significantly lower than the IC₅₀ value of the previously reported assay using the synthetic counterpart of the same mimotope in a microarray format. The homogeneous assay was demonstrated to be specific for fumonisins B₁ and B₂, as no significant cross-reactivity with other mycotoxins was observed, and acceptable recoveries (86% for FB₁ 2000 μg kg^–1 and 103% for FB₁ 4000 μg kg^–1), with relative standard deviation less than 6.5%, were reported from spiked wheat samples, proving that the method could provide a valuable tool for simple analysis of mycotoxin-contaminated food samples.

中文翻译：

---

基于金纳米粒子和模拟表位的黄色荧光蛋白的伏马菌素B _1的均相猝灭免疫分析

均质免疫测定法由于其简单性，灵敏性和快速性，代表了传统异质测定法的一种有吸引力的替代方法。基于先前鉴定的表位模拟肽或模拟表位，我们开发了基于金纳米颗粒（AuNPs）和重组表位模拟融合蛋白的均相荧光猝灭免疫测定法，用于检测霉菌毒素伏马菌素B ₁（FB ₁）。伏马菌素模拟表位被克隆为带有黄色荧光蛋白的融合蛋白，可直接用作FB ₁的示踪剂无需标记或二抗即可进行检测。此外，由于AuNPs的荧光淬灭能力，可以在一个步骤中进行均相免疫测定，而无需洗涤步骤以分离未结合的示踪剂。均相淬灭测定显示，在5％小麦提取物中的基质效应可忽略不计，并且对FB _1的检测具有很高的灵敏度，动态范围为7.3至22.6 ng mL ^–1，检测限为1.1 ng mL ^–1，IC ₅₀值为12.9 ng mL ^–1，显着低于IC ₅₀使用相同模拟表位的合成对应物以微阵列形式进行的先前报道的测定的值。同质性试验被证明是特异于伏马毒素乙₁和B ₂，如观察到与其他真菌毒素无显著交叉反应性，和可接受的回收率（86％为FB ₁ 2000微克千克^-1和用于FB 103％₁ 4000微克从加标的小麦样品中报告了相对标准偏差小于6.5％的kg ^–1），证明该方法可以为简单分析受霉菌毒素污染的食品样品提供有价值的工具。