Eco-Friendly Fluorescent ELISA Based on Bifunctional Phage for Ultrasensitive Detection of Ochratoxin A in Corn.
Eco-Friendly Fluorescent ELISA Based on Bifunctional Phage for Ultrasensitive Detection of Ochratoxin A in Corn.
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基于双功能噬菌体的环保型荧光 ELISA 超灵敏检测玉米中赭曲霉毒素 A
DOI：
10.3390/foods10102429
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发表时间：
2021-10-13
期刊：
影响因子：
--
通讯作者：
Xiong Y
中科院分区：
文献类型：
--
作者：
Tong W;Fang H;Xiong H;Wei D;Leng Y;Hu X;Huang X;Xiong Y
传统的酶联免疫吸附试验（ELISA）是赭曲霉毒素A（OTA）检测的常用方法，但灵敏度低，酶-OTA结合物的竞争性抗原有害，限制了其应用。本文中，引入了具有融合在p3蛋白上的OTA模拟表位和在主要p8蛋白上修饰的生物素的双功能M13噬菌体作为生态友好的竞争抗原和酶容器以增强灵敏度。巯基丙酸修饰的量子点（MPA-QDs），这是非常敏感的过氧化氢，被选为荧光信号转换器，可以体现葡萄糖氧化酶诱导的荧光猝灭在葡萄糖的存在下。在此基础上，建立了一种灵敏度高、环境友好的OTA荧光免疫检测方法。在优化的条件下，该方法对OTA在4.8 ~ 625 pg/mL范围内具有良好的线性检测能力，检测限（LOD）为5.39 pg/mL。LOD比基于辣根过氧化物酶（HRP）的常规ELISA低约26倍，比基于GOx-OTA缀合物的荧光ELISA低6倍。所提出的方法也显示出很大的特异性和接受的准确度分析OTA在真实的玉米样品。检测结果与超高效液相色谱-荧光检测法检测结果高度一致，表明该方法检测OTA的可靠性高。总之，所提出的方法是一个很好的OTA筛选平台，在传统的ELISA，可以很容易地扩展到传感其他分析物通过改变特定的模拟肽序列在大肠杆菌中。
Conventional enzyme-linked immunosorbent assay (ELISA) is commonly used for Ochratoxin A (OTA) screening, but it is limited by low sensitivity and harmful competing antigens of enzyme-OTA conjugates. Herein, a bifunctional M13 bacteriophage with OTA mimotopes fused on the p3 protein and biotin modified on major p8 proteins was introduced as an eco-friendly competing antigen and enzyme container for enhanced sensitivity. Mercaptopropionic acid-modified quantum dots (MPA-QDs), which are extremely sensitive to hydrogen peroxide, were chosen as fluorescent signal transducers that could manifest glucose oxidase-induced fluorescence quenching in the presence of glucose. On these bases, a highly sensitive and eco-friendly fluorescent immunoassay for OTA sensing was developed. Under optimized conditions, the proposed method demonstrates a good linear detection of OTA from 4.8 to 625 pg/mL and a limit of detection (LOD) of 5.39 pg/mL. The LOD is approximately 26-fold lower than that of a conventional horse radish peroxidase (HRP) based ELISA and six-fold lower than that of a GOx-OTA conjugate-based fluorescent ELISA. The proposed method also shows great specificity and accepted accuracy for analyzing OTA in real corn samples. The detection results are highly consistent with those obtained using the ultra-performance liquid chromatography-fluorescence detection method, indicating the high reliability of the proposed method for OTA detection. In conclusion, the proposed method is an excellent OTA screening platform over a conventional ELISA and can be easily extended for sensing other analytes by altering specific mimic peptide sequences in phages.
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