Design of an Antigen-Triggered Nanobody-Based Fluorescence Probe for PET Immunoassay to Detect Quinalphos in Food Samples

Photoinduced electron-transfer (PET)immunoassay basedon a fluorescencesite-specifically labeled nanobody, also called mini Quenchbody (Q-body),exhibits extraordinary sensitivity and saves much time in the homogeneousnoncompetitive mode and is therefore regarded as a valuable method.However, limited by the efficiency of both quenching and dequenchingof the fluorescence signal before and after antigen binding associatedwith the PET principle, not all original nanobodies can be used ascandidates for mini Q-bodies. Herein, with the anti-quinalphos nanobody11A (Nb-11A) as the model, we, for the first time, adopt a strategyby combining X-ray structural analysis with site-directed mutagenesisto design and produce a mutant Nb-R29W, and then successfully generatea mini Q-body by labeling with ATTO520 fluorescein. Based on this,a novel PET immunoassay is established, which exhibits a limit ofdetection of 0.007 & mu;g/mL with a detection time of only 15 min,25-fold improved sensitivity, and faster by 5-fold compared to thecompetitive immunoassay. Meanwhile, the recovery test of vegetablesamples and validation by the standard ultraperformance liquid chromatography-tandemmass spectrometry (UPLC-MS/MS) both demonstrated that the establishedPET immunoassay is a novel, sensitive, and accurate detection methodfor quinalphos. Ultimately, the findings of this work will providevaluable insights into the development of triggered PET fluorescenceprobes by using existing antibody resources.