A simple and sensitive fluoroimmunoassay based on the nanobody-alkaline phosphatase fusion protein for the rapid detection of fenitrothion

IF 3.7 2区 农林科学 Q2 FOOD SCIENCE & TECHNOLOGY Frontiers in Sustainable Food Systems Pub Date : 2023-12-21 DOI:10.3389/fsufs.2023.1320931
Zijian Chen, Ai-Jun Huang, Xiu-Xiu Dong, Yi-Feng Zhang, Lin Zhu, Lin Luo, Zhen-Lin Xu, Hongwu Wang
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Abstract

Immunoassay is a powerful tool for the rapid detection of small harmful organic molecules. In this study, a simple and sensitive fluoroimmunoassay (FIA) based on a nanobody-alkaline phosphatase fusion protein (VHHjd8-ALP) and blue-emissive carbon dots (bCDs) was developed for the rapid detection of fenitrothion. The bCDs were synthesized using the one-step hydrothermal method. Citric acid and urea were used as carbon and nitrogen sources, respectively. The synthesized bCDs were characterized by fluorescence spectrum, high-resolution transmission electron microscopy, x-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy. After one step of competitive immunoassay, the VHHjd8-ALP bound to the microplate and catalyzed the substrate p-nitrophenylphosphate (pNPP) into p-nitrophenol (pNP); the latter can quench the blue of bCDs due to an inner-filter effect. After condition optimization, an FIA calibration curve was finally created, which showed an IC50 value of 16.25 ng/mL and a limit of detection (LOD) of 0.19 ng/mL. Compared with the pNPP-based one-step conventional indirect competitive enzyme-linked immunoassay (icELISA), the developed FIA showed an 11-fold sensitivity improvement. Furthermore, the analysis period of FIA only takes approximately 55 min, which was obviously faster than that of the conventional icELISA. The recovery test showed recoveries from 81.8 to 119% with fruits and vegetable samples, which verified the practicability and accuracy of the developed FIA.
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基于纳米体-碱性磷酸酶融合蛋白的简单灵敏的荧光免疫测定,用于快速检测杀螟松
免疫测定是快速检测有害有机小分子的有力工具。本研究开发了一种基于纳米体-碱性磷酸酶融合蛋白(VHHjd8-ALP)和蓝色发光碳点(bCDs)的简单灵敏的荧光免疫分析法(FIA),用于快速检测杀螟松。bCDs 采用一步水热法合成。柠檬酸和尿素分别用作碳源和氮源。利用荧光光谱、高分辨透射电子显微镜、X 射线光电子能谱和傅立叶变换红外光谱对合成的 bCDs 进行了表征。经过一步竞争性免疫测定,VHHjd8-ALP 与微孔板结合,并将底物对硝基苯磷酸(pNPP)催化成对硝基苯酚(pNP),后者由于内滤效应可以淬灭 bCDs 的蓝色。经过条件优化,最终绘制出了一条 FIA 校准曲线,其 IC50 值为 16.25 纳克/毫升,检测限 (LOD) 为 0.19 纳克/毫升。与基于 pNPP 的一步式传统间接竞争酶联免疫分析法(icELISA)相比,所开发的 FIA 灵敏度提高了 11 倍。此外,FIA 的分析时间仅需约 55 分钟,明显快于传统的 icELISA。回收率测试显示,水果和蔬菜样品的回收率在 81.8% 至 119% 之间,这验证了所开发的 FIA 的实用性和准确性。
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来源期刊
Frontiers in Sustainable Food Systems
Frontiers in Sustainable Food Systems Agricultural and Biological Sciences-Horticulture
CiteScore
5.60
自引率
6.40%
发文量
575
审稿时长
14 weeks
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