Conjugates between food proteins and magnetic nanoparticles: production and properties

Advances in processes for producing nanostructured materials have made possible the production of magnetic nanosupports with potential applications in the biochemical and biotechnological fields. This kind of support is generally synthesized by encapsulating magnetic materials within a polymer layer. The interest for magnetic nanosupports is not only limited to the obvious ease of their separation under micro- and nanofluidic conditions. Most relevant is the fact that nanostructures can be conjugated to biologically active molecules, including hormones, antibodies, drugs, and various peptides, taken up by cells, and circulated among tissues expressing their cognate receptors. Given their intrinsic magnetism, magnetic nanoparticles may be used as tracers in NMR and MRI experiments, and are easily detectable in standard transmission electron microscopy. This work is aimed at producing and characterizing conjugates between magnetic nanoparticles and bioactive proteins, and at assessing their use as biological tracers (for instance in monitoring the intracellular and/or intratissutal path of bioactive molecules of food origin), and their suitability for controlling enzyme activity in a number of applications. Dextran-coated iron oxide nanoparticles were modified in order to obtain an activated coating which allowed the covalent binding of different bioactive proteins through simple chemical procedures. Proteins considered in our studies included food allergens, enzymes, and antibodies to food proteins. Dot blotting with specific antibodies, followed by immunoenzymatic detection of the bound antibodies, demonstrated the actual presence of these allergenic proteins on the conjugated nanoparticles. As a further test of protein immobilization, immunoprecipitation experiments were performed to demonstrate the coupling of the proteins to the functionalized dextran-coated nanoparticles. Both immunological approaches confirmed the suitability of our immobilization strategy. We also prepared conjugates between analytical-grade trypsin and dextran-coated nanoparticles, and assessed the immobilized enzyme activity on synthetic substrates. These experiments led us to estimate the coupling yield of our coupling approach, which compares with those reported in the literature for equivalent procedures. Preliminary tests aimed at assessing the citotoxicity of unmodified dextran-coated nanoparticles showed that the viability of differentiated HT-29 cells, from a human adenocarcinoma cell line, after various times of incubation with the nanoparticles was close to that of control untreated cells, suggesting full biocompatibility of the unmodified particles with this peculiar cell line.