Abstract: Covalent immobilization of avidin onto Fe₃O₄ magnetic nanoparticales(MNPs) via a facile solvethermal synthetic method was carried out using glutaraldehyde as a crosslinking agent. Immobilization process was monitored and avidin immobilized magnetic nanoparticles(Avi-MNPs) were characterized by TEM, XRD, UV-Vis, FTIR and fluorescence spectroscopy. Evaluation of Avi-MNPs activity and its application for the determination of the concentration of protein A were also performed. TEM images show the average particle diameters of the non-functional MNPs and Avi-MNPs are approximately 30 and 50 nm, respectively, and all the nanoparticles have a uniform distribution. XRD analysis indicates that the characteristic diffraction peaks of MNPs are the same as Fe₃O₄ and MNPs are high purity. Firstly glutaraldehyde was linked to MNPs and then avidin was immobilized onto the surface of the functional MNPs, which were verified by UV-Vis, FTIR and fluorescence spectroscopy. The activity evaluation of Avi-MNPs shows that the activity is 4.706 U/mg of D-biotin bound/mg Avi-MNPs and lower than 14.1 U/mg D-biotin bound/mg free avidin. The Avi-MNPs were applied to the determination of the concentration of protein A, the results indicated that this method has an advantage of saving time and effort compared to the traditional enzyme-linked immunosorbent assay and can be carried out with a simple UV-Vis spectrophotometer.

Key words: Magnetic nanoparticales, Avidin, Covalent immobilization, Activity evaluation