Faculty of Applied Sciences
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Item Interaction studies of nanomaterials with plasma protein using experimental and computational methods(2018) Sabela, Myalowenkosi Innocent; Bisetty, Krishna; Sagrado, Salvador; Mwakikunga, B.Nanomaterials have received considerable attention due to their unique physicochemical properties and various applications. The present study attempts to fill in the knowledge gaps by investigating the synthesis, stability, antimicrobial, and antioxidant properties, along with their sensing capabilities. For this purpose, chemical and green methods were used to synthesise silver nanoparticles, titanium dioxide nanomaterials and zinc oxide nanoparticles. For the chemical synthesis, bio-discrimination of macromolecules were investigated by calorimetric methods. Whereas for green synthesis, silver nanoparticles and zinc oxide nanoparticles were synthesized using plant extracts and tested for cytotoxicity and antioxidant properties followed by the electrochemical sensing of silymarin. The synthesized nanomaterials were analyzed by high-resolution transmission electron microscopy, X-ray diffraction, photoluminescence, fourier transform infrared spectroscopy, thermogravimetric analyzer, dynamic light scattering and zeta potential analyzer to better understand the morphology and capping. In addition, computational modelling techniques were implemented to assess the adsorption and interaction of nanomaterial with biomolecules through docking. The results arising from this work are presented in a case-study fashion ranging from colorimetry, protein- nanomaterial interactions through to biosynthesis. For the colorimetric discrimination an extensive literature survey revealed that silver nanoparticles and gold nanoparticles based colorimetric assays, are widely used for screening in biosciences and metal analysis. A detailed review of colorimetric-based assays and identification of the fundamental parameters that influences such strategies are presented in this work. Accordingly, colorimetric discrimination of bovine serum albumin, lysozyme, single-stranded deoxyribonucleic acid, double-stranded deoxyribonucleic acid, and polynucleotides using silver nanoparticles was performed. This work demonstrates the importance of the starting material for colorimetric measurements preferably, with particles that are active in a wider absorbance region. Furthermore, an in-depth statistical analysis of the colorimetric data using principal component analysis provided a framework for sensing of the selected proteins, deoxyribonucleic acid, and polynucleotides by simultaneously varying the concentration of a salt and biomacromolecules from 10 to 100 mM and from 0.1 to 10 µg mL-1 respectively. In the case of the protein-nanomaterial interaction, the bioconjugate formation was studied between human serum albumin and TiO2 nanostructures. The TiO2 nanostructures were prepared by microwave assisted calcination at 200, 400, 700, and 900 °C. The results revealed that calcination above 400 °C had a significant impact on the surface of the nanomaterial, resulting in a lower surface adsorption of the protein confirmed by characterization techniques mentioned above. To further elucidate interactions between human serum albumin and titanium dioxide nanostructures, computational molecular docking studies were performed, resulting in the characteristic shapes of TiO2 nanostructures correlating well with the experimental observations. The nanowires and nanotubes showed the much greater binding energy of –2.33 Kcal mol-1 and -2.85 Kcal mol-1, respectively. In contrast to the nanotubes, the docked conformation of the nanowires showed a greater number of interactions. In regard to the biosynthesis of silver nanoparticles prepared from aqueous plant extracts of Iboza riparia leaf and Ilex Mitis root bark, the formation of nanoparticles were due to the reduction of silver ions caused by electron transfer from the electron rich atoms of the extracts such as saponins and diterpenes. Therefore, relevant cytotoxicity studies were conducted with the MTT assay on HEK293T followed by an antioxidant activity with ABTS. Antimicrobial activity analysis depicted susceptibility of microorganisms in the order of nanoparticles by diterpenes (156 nm) followed by nanoparticles by saponins (50 nm). Another biosynthesis of zinc oxide nanoparticles was performed using the extracts from jacaranda mimosifolia flowers and the carica papaya seeds. The composition of both extracts was analyzed by gas chromatography–mass spectrometry. Oleic acid was found to be the major component in the extract of jacaranda mimosifolia flowers. The microwave assisted biosynthesis of nanoparticles was successfully completed within 5 min resulting in 2–4 nm particle size. Further characterization was performed using the aforementioned instruments. The zinc oxide nanoparticles from jacaranda mimosifolia flowers were used to test the viability of gram-negative Escherichia coli and gram-positive Enterococcus faecium bacteria, which was found to be 48% and 43% respectively. In order to better understand the influence of oleic acid on the size and shape of nanoparticles, its adsorption on the different facets was explored computationally. The preferential passivation of the (101) facet was evident from the concentration profiles with an average distance of 1.4 Å for oleic acid. On the other hand, facets (100) and (002) were located at varying distances of 4 and 9 Å respectively. Overall the results indicated that the presence of oleic acid could drive the preferential growth of zinc oxide nanomaterials to nanorod array and other structures. Furthermore, the composition of the zinc oxide nanoparticles from the carica papaya seed extracts was also identified using the aforementioned techniques. The results highlighted the role of oleic acid as a capping agent for these nanoparticles. In relation to their electrochemical applications, they were also tested for sensing activities of silymarin by incorporating them with multiwalled carbon nanotubes immobilized on the surface of a glassy carbon electrode. The electrochemical signals obtained from the modified electrode was 2-fold higher than that of a bare glassy carbon electrode. The electrode based on zinc oxide nanoparticle was able to detect silymarin in the commercial Milk Thistle tablet.Item Study of interaction between polyphenolic compounds and protein using computational and capillary electrophoresis techniques(2013-07-30) Sabela, Myalowenkosi Innocent; Bisetty, KrishnaThe present work involves the interaction studies of chiral compounds with the Human Serum Albumin (HSA) protein using computational and experimental methods. The HSA protein has multiple binding sites that forms the basis for its exceptional ability to interact with many organic and inorganic molecules, which makes this protein an important regulator of intercellular fluxes and the pharmacokinetic behaviour of many drugs. This study was undertaken to evaluate the related pharmacokinetic and enantioselective binding parameters of the racemic catechin enantiomers with the HSA. Accordingly, this work involved a method development for the chiral separation of a racemic compound, by capillary electrophoresis-electrokinetic chromatography (CE-EKC) with a highly sulphated beta-cyclodextrin (HS--CD) as a chiral selector. The experimental work was supported by two molecular docking studies. The first included the mimicking of the host-guest interactions between a chiral selector and an enantiomeric compound. The second study included the estimation of the pseudo enantioselective (ES) binding of catechin to HSA. Overall, it was found that CE-EKC is the preferred method for the(±)-catechin binding to HSA protein evaluation. Moreover, the technique used in this work is not restricted to HSA or polyphenols, but can also be applied to other proteins and ligands that possess chirality. Furthermore, the molecular docking approaches also proved to be very useful for the evaluation of chiral recognition systems and for elucidation of the ligand-protein interactions.