Design of an Antigen-Triggered Nanobody-Based Fluorescence Probe for PET Immunoassay to Detect Quinalphos in Food Samples.
Yi-Fan LiangJia-Dong LiRu-Yu FangZhen-Lin XuLin LuoZi-Jian ChenJin-Yi YangYu-Dong ShenHiroshi UedaBruce D HammockHong WangPublished in: Analytical chemistry (2023)
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.
Keyphrases
- liquid chromatography tandem mass spectrometry
- pet ct
- positron emission tomography
- label free
- computed tomography
- pet imaging
- ms ms
- single molecule
- sensitive detection
- energy transfer
- electron transfer
- simultaneous determination
- loop mediated isothermal amplification
- high resolution
- small molecule
- quantum dots
- real time pcr
- magnetic resonance
- magnetic resonance imaging
- crispr cas
- risk assessment
- photodynamic therapy
- tandem mass spectrometry