Preparation Of Functional Nanoparticles And Their Interactions With Proteins

Nanoscience and nanotechnology are the top research areas nowadays. Functionalized nanoparticles have huge potential applications in the biological field, like protein separation, targeting drug delivery and drug controlled release. Interaction between nanoparticles and bioactive substances would be changed under different environment, and could be adjusted by functionalization of nanoparticles for further application.Two kinds of nanoparticles, gold nanoparticles and magnetic spherical polyelectrolyte brushes (MSPB), were synthesized in this thesis. Interactions between these two nanoparti-cles and two proteins, bovine serum albumin (BSA) andβ-lactoglobulin (BLG), were studied. Study of interaction between gold nanoparticle and proteins provided a scientific basis for mak-ing biosensors. While the study of interaction between spherical polyelectrolyte brushes and proteins was helpful for protein separation. Introduction of magnetic nanoparticles into spher-ical polyelectrolyte brushes improved protein separation velocity and efficiency. Furthermore, magnetic spherical polyelectrolyte brushes could be recycled and reused for cost-effective ap-plication. Details for this thesis are listed as follows:1. Gold nanoparticles were synthesized by a combination of Brust-Schiffrin phase transfer method and Murray place exchange method. Obtained gold nanoparticles were well dispersed in aqueous phase. Quaternary ammonium ligands with different hydrophobicity were attached to the gold core surface. TEM indicated the diameter of gold core was 2 nm, and DLS showed that the overall diameter of gold nanoparticle was 8-13 nm. Magnetic nanoparticles (Fe3O4) were prepared via coprecipitation, and the average diameter was around 10 nm. Further results showed that the best dispersion of magnetic nanoparticles in styrene could be obtained by using acetone as a cosolvent of oleic acid, adding oleic acid before ammonia. The optimal oleic acid content was near 25 wt%. Miniemulsion polymerization was applied to prepare magnetic polystyrene latex, and the magnetic spherical polyelectrolyte brushes were finally obtained by photoemulsion polymerization.2. Bovine serum albumin (BSA) andβ-lactoglobulin had a very similar isoelectric point (pI), and were chosen to study their selectivity by gold nanoparticles. Gold nanoparticle sur-face was modified by a ligand with an end group of tetra-glycol ethylene-trimethyl ammonium (TTMA) to eliminate the effect of hydrogen binding and hydrophobic group in the end group of gold nanoparticle ligand. Characterization contained high-resolution turbidimetry and dy-namic light scattering (DLS). Results showed that insoluble aggregates were formed immedi- ately when BSA bound to TTMA modified gold nanoparticle, while soluble complexes were formed as initial and then insoluble aggregates were formed for binding of BLG to TTMA modified gold nanoparticle. DelPhi was applied to simulate the surface potential distributions of BSA and BLG near their pHc. It was found that BSA owned discrete charge patch, and BLG owned dipole-like negative and positive charge patch. Different kind of charge patches derived from various surface charge distributions could be used to explain the difference of interactions between BSA/BLG and TTMA modified gold nanoparticle. To avoid the effect of hydrogen binding and hydrophobic group in protein structure, two BLG isoforms, BLGA and BLGB, were selected to study their interactions with TTMA modified gold nanoparticle.3. There were only two amino acids difference between BLGA and BLGB monomer. Namely, Asp64 (BLGA)→Gly64 (BLGB) and Val118 (BLGA)→Ala64 (BLGB). Results showed that TTMA modified gold nanoparticle had high selectivity to these two protein iso-forms. The pHc difference was 0.65 pH unit at ionic strength 5 mM, which was higher than 0.20 pH unit of selectivity for PDADMAC to BLGA/BLGB. We proposed a mechanism named "electrostatic selectivity" to explain what we had found, i.e., selectivity to different proteins was realized only by changing the electrostatic interactions. Isothermal titration calorimetry (ITC) was then used to get the thermodynamic parameters for BLGA/BLGB and TTMA mod-ified gold nanoparticle. Results indicated that both stoichiometry and binding constant for BLGA-TTMA modified gold nanoparticle were larger than that of BLGB-TTMA modified gold nanoparticle. Additionally, enthalpy for binding of BLGA/BLGB and TTMA modified gold nanoparticle was positive indicating that the binding process was entropy driven. Finally, ITC was applied to study the thermodynamic parameters for binding of BLGA/BLGB and gold nanoparticles modified by other quaternary ammonium ligands. Selectivity of gold nanoparti-cles to BLGA/BLGB was affected by introducing hydrophobic groups. When hydrophobicity was small, electrostatics dominated and the selectivity judged by the difference of binding constant was determined by the charge patch difference of BLGA/BLGB. When hydropho-bicity was large, hydrophobic interaction dominated leading to diminished selectivity for gold nanoparticle to BLGA/BLGB. While if hydrophobicity was moderate, highest selectivity was achieved due to synergistic effect of electrostatics and hydrophobic interactions.4. Using AIBN as the initiator, the highest magnetite content was limited to 15 wt%, and the real magnetite content was lower than that of theoretical values. Using KPS as the initiator, helped by introducing magnetite aggregates, the highest magnetite content reached 52.3 wt%, and the real magnetite content was higher than that of theoretical values. The reason was that existence of magnetic nanoparticles decreased the monomer conversion of styrene, increased the magnetite content. Using AIBN as the initiator, distribution of magnetic nanoparticles in the polystyrene matrix was inhomogeneous, and a part of magnetic nanoparticles exposed to the surface of polystyrene. When the initiator was changed to KPS, the distribution of magnetic nanoparticles in polystyrene was well improved and the magnetic nanoparticles were dispersed mainly in the center of the matrix. We proposed a mechanism to explain this difference. When AIBN was used, the polymerization started from the interior to the exterior of monomer droplet, excluding magnetic nanoparticles outside or even to surface of the polystyrene matrix. When KPS was used, polymerization started from the exterior to the interior and pushing magnetic nanoparticles to polystyrene matrix center. Magnetic spherical polyelectrolyte brushes were very stable compared to magnetic polystyrene latex due to electrostatic repulsion and steric hin-drance between poly (acrylic acid) chains. Magnetic spherical polyelectrolyte brushes could be separated rapidly by a strong magnet, and could be redispersed in water. Size of magnetic spher-ical polyelectrolyte brushes were not changed after several cycles of separation-redispersion operation, indicating a good redispersity. Velocity and efficiency of magnetic separation could be adjusted by changing magnetite content and the pH. At last, interaction between spherical polyelectrolyte brushes (SPB) and BSA/BLG was studied by turbidimetry and dynamic light scattering (DLS). Results indicated that separation could be realized at a pH range based on the different amount of protein adsorption.