Effects Of Ion Exchange Ligand Density On Protein Adsorption

As one of the most effective ways to separate and purify the protein, ion exchange chromatography show lots advantages, such as the mild separation conditions and high purity of the target protein. The chromatographic separation is based on protein adsorption and desorption on interfaces. Because of the complexity of protein structure and interaction between media and proteins, the space structure is easy to be destroyed by surrounding environments. For ion exchange chromatography, the ligand density is a key factor affecting the denaturation of proteins. In this study, the sensor chips of dual-polarization interferometry(DPI) were modified with aminosiliane of different densities. The behaviors of protein absorption on the modified chips were characterized by DPI with five kinds of proteins as model proteins. The main results are as follows:(1) The silicon chips were modified by(N,N-Diethyl-3-aminopropyl)- trimethoxysilane(DAPTMS) in different concentrations. X-ray photoelectron spectroscopy(XPS), and Atom force microscope(AFM) were used to characterization of the surface composition and surface morphology. The results showed that the elements in modified silicon surfaces contain nitrogen. And the data show that silicon chips’ surface which were modified contain C-C bond, C-N bond and Si-O bond. The nitrogen percentage on silicon chips were determined as 1.08~2.38%. The AFM shows the results that modified silicon chips have lots of differences with the original chips in morphology, thickness and roughness. The nitrogen percentage on the silicon chips can be controlled by selection of DAPTM concentration(10-5~10-3mol/L) and reaction time(10-60min).(2) Five proteins(Lysozyme, Cytochrome C, Chymotrypsin, BSA and IgG) were used as model proteins. The adsorption and elution behaviors of model proteins on different silicon chips surface with different ligand density were determined using DPI. In this study, not only the different proteins adsorb in the same silicon chip were researched, but also the same protein adsorb in different silicon chip. The maximum adsorption thickness of Lysozyme, Cytochrome C, Chymotrypsin, BSA and IgG are 2.21 nm, 2.22 nm, 3.00 nm, 2.99 nm and 6.35 nm respectively. All of them are smaller than hydrodynamic diameters of the proteins. The result shows that the adsorption and elution behaviors have connection with protein types and sizes. The ligand density affects the behavior of protein adsorption. When the ligand distance is less than the protein diameter, the structure of adsorbed protein prefer to form deformed state.The research have both theoretic and practical values for the study of new chromatography medium and a deep understanding of protein inactivation during the absorption on the surface.