Design of an Antigen-Triggered Nanobody-Based Fluorescence Probe for PET Immunoassay to Detect Quinalphos in Food Samples.
Design of an Antigen-Triggered Nanobody-Based Fluorescence Probe for PET Immunoassay to Detect Quinalphos in Food Samples.
复制标题
DOI：
10.1021/acs.analchem.3c01696
复制
发表时间：
2023-08
影响因子：
7.4
通讯作者：
Yifan Liang;Jiadong Li;Ru Fang;Zhenlin Xu;Lin Luo;Zijian Chen;Jinyi Yang;Yu-Dong Shen;
中科院分区：
文献类型：
--
作者：
Yifan Liang;Jiadong Li;Ru Fang;Zhenlin Xu;Lin Luo;Zijian Chen;Jinyi Yang;Yu-Dong Shen;
基于荧光位点特异性标记的纳米抗体（也称为微型淬灭体（Q-体））的光诱导电子转移（PET）免疫测定在均相非竞争模式下表现出非凡的灵敏度和节省大量时间，因此被认为是有价值的方法。然而，受限于与PET原理相关的抗原结合之前和之后荧光信号的猝灭和去猝灭的效率，并非所有原始纳米抗体都可以用作微型Q-抗体的候选物。本文以抗喹硫磷纳米抗体11 A（Nb-11 A）为模型，首次采用X射线结构分析与定点突变相结合的策略，设计并制备了突变体Nb-R29 W，并通过ATTO 520荧光素标记成功制备了微型Q体。在此基础上，建立了一种新的PET免疫分析方法，其检测限为0.007 μg/mL，检测时间仅为15 min，灵敏度提高25倍，比竞争性免疫分析方法快5倍。同时，蔬菜样品回收率试验和标准超高效液相色谱-串联质谱（UPLC-MS/MS）验证均表明，所建立的PET免疫分析方法是一种灵敏、准确的喹硫磷检测新方法。最终，这项工作的发现将为利用现有抗体资源开发触发PET荧光探针提供有价值的见解。
Photoinduced electron-transfer (PET) immunoassay based on a fluorescence site-specifically labeled nanobody, also called mini Quenchbody (Q-body), exhibits extraordinary sensitivity and saves much time in the homogeneous noncompetitive mode and is therefore regarded as a valuable method. However, limited by the efficiency of both quenching and dequenching of the fluorescence signal before and after antigen binding associated with the PET principle, not all original nanobodies can be used as candidates for mini Q-bodies. Herein, with the anti-quinalphos nanobody 11A (Nb-11A) as the model, we, for the first time, adopt a strategy by combining X-ray structural analysis with site-directed mutagenesis to design and produce a mutant Nb-R29W, and then successfully generate a mini Q-body by labeling with ATTO520 fluorescein. Based on this, a novel PET immunoassay is established, which exhibits a limit of detection of 0.007 μg/mL with a detection time of only 15 min, 25-fold improved sensitivity, and faster by 5-fold compared to the competitive immunoassay. Meanwhile, the recovery test of vegetable samples and validation by the standard ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) both demonstrated that the established PET immunoassay is a novel, sensitive, and accurate detection method for quinalphos. Ultimately, the findings of this work will provide valuable insights into the development of triggered PET fluorescence probes by using existing antibody resources.