Determination of monensin in milk samples by front-surface long-wavelength fluoroimmunoassay using nile blue-doped silica nanoparticles as labels

A heterogeneous immunoassay for monensin determination in milk samples using a tracer formed by anti-monensin antibodies bound to nile blue (NB)-doped silica nanoparticles (NPs), 96-well microplates as solid supports and long-wavelength fluorescence measurements is described for the first time. The assay relies on the competition of the monensin present in the samples with a monensin–bovine serum albumin conjugate, which was immobilized onto the well surface, for the active sites of anti-monensin antibodies. After subsequent incubation and washing steps, the fluorescence of the bound tracer fraction is measured onto the dry surface of the well. An antigen capture format was also assayed by immobilizing anti-sheep IgG previously to the incubation of sheep anti-monensin antibodies and using a tracer formed by monensin bound to nile blue-doped silica NPs, which competes with the analyte for binding the immobilized antibody. Although the fluorescence signal obtained in both formats can be correlated to the analyte concentration, better results were obtained using the antibody capture format. After the optimization of the system using this format, the method features a detection limit of 0.015 μg L−1 and a dynamic range from 0.05 to 5 μg L−1. The precision, assayed at two different analyte concentrations, 0.2 and 1 μg L−1, and expressed as relative standard deviation, gave values of 5.9% and 4.0%, respectively. The method was satisfactorily applied to the analysis of milk samples, which only required a simple extraction step in order to remove the proteins from samples, giving recoveries in the range 83.3–107.5%.

► A long-wavelength luminescent immunoassay for monensin using nanoparticles as labels is described for the first time.
► The fluorophor nile blue is encapsulated into silica matrix to obtain the label.
► Front-surface fluorescence is used as the detection system.
► Monensin residues in milk can be directly monitored owing to the relatively low LOD of the method.
► The sample treatment consists in protein removal and further dilution.