Rapid and label-free detection of Brucella melitensis in milk and milk products using an aptasensor

In this work, a novel quartz crystal microbalance (QCM) aptasensor is designed for the diagnosis of Brucella melitensis bacteria, which affects the Mediterranean fever (brucellosis) from the zoonotic diseases that are very common in the Middle East Countries. The method is based on the selection of B. melitensis bacterium from solutions using B. melitensis specific binding aptamer (Apt) attached magnetic nanoparticles. The surface of the magnetic nanoparticles (i.e.,Fe₃O₄) was modified by 3-aminopropyltriethoxysilane (APTES) and then grafted with a hydrophilic macromonomer poly(ethyleneglycol)-methacrylate (PEG-MA) as a first block polymer and glycidylmethacrylate (GMA) as a second block functional polymer via atom transfer radical polymerization (ATRP) method [Fe₃O₄ @SiO₂ @p(PEG-MA-GMA)], then, the specific binding aptamer was immobilized. The aptamer immobilized magnetic nanoparticles were used for the pre-concentration of the target bacterium, and the same aptamer sequence was also immobilized on the QCM chip and used for the quantitative detection of B. melitensis using QCM aptasensor. The detection limits of the QCM aptasensor were in the range 1.0²-1.0⁷ CFU mL^-1, with recoveries up to 79%. The synthesized [Fe₃O₄ @SiO₂ @p(PEGMA-GMA)] nanoparticles showed a good permanence and high isolation recoveries for the pull down of the target bacterium from food samples, after recycling eight times. The method was successfully applied to target bacterium determinations in milk and milk product samples.