Magnetic Nanoparticles in Medical Diagnostic Applications: Synthesis, Characterization and Proteins Conjugation

Background: Magnetic nanoparticles (NPs) used in biomedical applications should be discrete with small particle sizes, narrow size distribution and superparamagnetic. NPs can be tailored to target, through chemical bonds, specific organs, cells, or even molecular markers of different diseases in vivo, with suitable surface chemistry modification. Methods: Nanoparticles are synthesized by a low cost coprecipitation reaction of ferrous and ferric salts with alkaline solution. The characteristics of the NPs are modified by varying the addition rate of the alkaline solution. NPs surface is silica coated using a modified Stöbe method. The conversion of the surface hydroxyl groups into amino-groups is performed by two different alkoxysilanes and the silanization reaction is conducted either in Methanol – Glycerol environment at elevated temperature, or in water at room temperature. The surface amine groups of the NPs are further converted, either to aldehyde groups by glutaraldehyde, or to carboxyl groups using glutaric anhydride. Bovine Serum Albumin and Vena human natural immunoglobulin are used in order to study the protein conjugation capacity of the functionalized NPs. The amount of protein attached to the nanoparticles is determined by UV–Vis spectroscopy of the supernatant. Conjugation of synthesized nanoparticles to protein BSA is examined by FTIR spectroscopy. SDS-PAGE electrophoresis followed by protein immunoblotting is used to test the effect of nano-conjugation to the antibodies. Results: Superparamagnetic Fe3O4 nanoparticles with saturation magnetization 60emu/g, a mean diameter 8-12 nm and BET surface areas between 100-250 m2/gr are obtained with total time of addition of the base between 1-5 minutes. They are coated with a thin and nearly uniform silica (SiO2) layer with thickness 1-2 nm. The most appropriate source for surface functionalization with amino groups is 3-aminopropyltriethoxysilane (APTES), while the two silanization methods used, proved to be equally efficient. NPs with surface aldehyde groups display better conjugation capacity than NPs functionalized with carboxyl groups. The FTIR spectra of the protein conjugated NPs samples, contain the two main peaks, at 1529 cm-1 and 1661 cm-1, attributed to the amide bond of the protein, which confirms the conjugation of the protein to the NPs. During a SDS-PAGE electrophoresis - protein immunoblotting experiment, the antibodies, after being conjugated to the nanoparticles, are selectively attached to their antigen, which indicates of lack of significant conformation changes secondary to the conjugation process. Conclusion: The conjugation capacity of the optimized nanoparticles is higher for Ig antibody than for BSA protein, under similar reaction conditions. The conjugational efficacy and conformational stability and the effect on electrophoretic mobility of the antibodies conjugated to the nanoparticles are verified by protein immunoblotting. Keywords: Conjugation, magnetite, nanoparticles, proteins, silica coating, superparamagnetic.