Double-imprinted polymers for selective HSA depletion from human serum

Human Serum Albumin (HSA) is the most abundant protein in human blood plasma ̶ it constitutes about half of serum proteins. As in the all proteomic studies of the blood plasma, high-abundant proteins, i.e. albumin, hemoglobin, interfere with the analysis of low-abundant and disease-related proteins, so removal or depletion of these proteins is vital for proteomic studies. In this thesis study, it was aimed to deplete HSA from human serum in one step using double-imprinted cryogels, prior to use in proteomic analysis. For this purpose, HSA-imprinted cryogels were synthesized with 8% monomer concentration and a second imprinting step with 16% monomer concentration in presence of HSA was achieved on the first gel. Thereby, HSA double-imprinted cryogel columns (HSA-DIP) were obtained. Cryogel column synthesized by double-layer imprinting technique but not containing template molecule (DL-NIP) and monolayer HSA-specific polymers with monomer concentrations of 8% and 16% (called HSA-MIP8% and HSA-MIP16%, respectively) were synthesized as the control group. Synthesized columns were characterized by swelling tests, Scanning Electron Microscopy (SEM) and Micro Computed Tomography (Micro-CT) analysis. The water uptake ratios were calculated as 84% for HSA-DIP, 82% for DL-NIP, 89% for HSA-MIP8% and 88% for HSA-MIP16%. HSA adsorption capacity was investigated, and selectivity studies were performed against Human Hemoglobin (Hb) and Immunoglobulin G (IgG) molecules which occupy a significant portion of human serum. Obtaining results were investigated by kinetic analyses to determine adsorption properties of the system. Finally, depletion of HSA directly from human serum was studied. In addition, HSA-DIP columns were tested in terms of reusability, and it was demonstrated that they can be used up to 10 times without significant decrease in selective HSA adsorption capacity.