An Ultrasensitive Fluorescence Immunoassay Based on Magnetic Separation and Upconversion Nanoparticles as Labels for the Detection of Chloramphenicol in Animal-Derived Foods

Abstract

In this work, an ultrasensitive fluorescence immunoassay has been proposed for the detection of chloramphenicol. The proposed assay is based on using the carboxyl-functionalized NaYF4: Yb/Tm upconversion nanoparticles with maximum emission at 482 nm under excitation at 980 nm conjugated with anti-chloramphenicol (CAP) antibody as the signal probe and the magnetic polystyrene microspheres conjugated with coating antigen as the sensing probe. The coating antigens on the sensing probes compete with CAPs to bind with the antibodies on the signal probes and the immune complexes form. Via magnetic action, these complexes can be separated to measure their fluorescence intensity. The limit of detection (LOD) of this assay for CAP in PBS is 0.01 pg mL−1, and the linear range extends from 0.05 to 100 pg mL−1 with a linear equation of y = 387.64 lgx + 1153.93 (R2 = 0.9911). The recoveries of CAP in spiked animal-derived foods range from 76.85 to 105.18%. Trace CAP levels have been measured in the real sample by the proposed fluorescence immunoassay, and the results have been verified by commercial ELISA test kit and liquid chromatography-tandem mass spectrometry. The quantitation limits of this immunoassay for CAP in the muscle tissue, milk, and honey samples are 0.25 pg g−1, 0.4 pg g−1, and 0.4 pg g−1, respectively. The fluorescence immunoassay proposed in this study can be used to highly sensitively, accurately, and rapidly detect CAP residues in animal-derived foods.