Smart Platform Development with Biomolecules for Biotechnological and Biomedical Applications

Abstract

The main objective of this dissertation is the synthesis and study of modified surface systems for the development of bioactive platforms and their use in specific biotechnological and biomedical applications. This work has led to various biological template development projects; all in attempts to provide new surfaces and probes in nanotechnology. These projects focus mainly on protein modified surface platforms, liposome based spherical platforms, and carbon nanotubes based magnetic platforms. The planar platforms include gold, silicon and aluminum oxide surfaces. Spherical surfaces such as liposomes and nanoparticles were also studied, and finally, surface modification was extended to carbon nanotubes and magnetic nanoparticles. In this dissertation, the planar surface work focuses on demonstrating the behavior of proteins at interfaces in terms of conformation, stability and activity (e.g., of avidin, trypsin and antibodies) using fluorescence microscopy. Different ligands were attached chemically on the surfaces to incorporate hydrophobic hydrophilic and charged characteristics. A chelating agent (iminodiacetic acid, IDA), an affinity ligand (biotin), and reactive groups (amino and carboxylic groups) were covalently incorporated onto the surfaces. Proteins including myoglobin, cytochrome C, avidin, trypsin and immunoglobulin G (IgG) were used in this study. The results show that proteins and ligands were successfully attached to different surfaces. Protein adsorption studies illustrate activity decrease by using fluorescence intensity. After attachment on hydrophobic functionalized surfaces. Along the same line, experiments were conducted on the comparison of silicon dioxide and gold-coated surfaces with immobilized enzymes, small molecules, and polymers for potential use as biosensors. Silicon dioxide wafers were prepared via silanization with 3-aminopropyl triethoxysilane (APTES) followed by glutaraldehyde activation and, finally, protein and/or small ligand attachment. Gold-coated surfaces were utilized for immobilizations using 16-mercaptohexadecanoic acid (MHA) which forms self-assembled monolayers (SAMs) on gold surfaces followed by covalently attachment of proteins. The activity of trypsin immobilized onto these surfaces was also measured. The silicon dioxide wafers when modified first with NH₂-PEG-NH₂ allowed for trypsin a relatively higher activity with about 11% greater activity than when attached on gold surfaces and 84% higher activity than on bare silicon surfaces. Furthermore, the bimolecular silicon dioxide surfaces were shown to be much more stable than the gold surfaces. The silicon dioxide surfaces with an immobilized reversible inhibitor, p-aminobenzamidine (PAB), show to very effectively bind proteins from solution compared to gold surfaces. Liposome were studied because their versatility and vast implications in bio-sensing and drug-delivery potential. In this work, liposomes were prepared with the phospholipids 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol. The amino groups of DMPE were then modified with ligands that included iminodiacetic acid (IDA), and PEG. These functionalized liposomes were used to prepare dispersed gold “nano-dots” on their surface. These novel functional liposomes, with chelating ligands and polymers can be used to bind biomolecules and active compounds (nanoparticles of gold, quantum dots, drugs) with long stability. The results show that we can successfully manufacture functional liposomes and form gold nanoshells on their external surface. These two types of systems can be used as drug delivery, and as imaging systems. Their characterization and potential use in biomedical applications as contrast agents seems quite promising once complexity and stability of these gold nanoshells is elucidated. The modification and preparation of functional-carbon nanotubes was investigated with the chemical hetero-junction analysis between magnetic nanoparticles coated poly-acrylic acid (PAA) and multi-wall carbon nanotubes (MWCNTs). Magnetic nanoparticles were covalently attached to open-ended nanotubes. Initial evidence suggests that short functionalized multi-wall nanotubes can be continuously connected at their terminal ends for build-up of relatively large nanostructures based on serial configurations. It is shown that magnetic carbon nanotubes systems exhibit defined arrangements due to the influence of magnetic fields. Indeed, linear arrays of carbon nanotubes inter-connected through magnetic nanoparticles were prone to be manipulated in the presence of a magnet device. A potential application of these magnetic nanostructures was shown by successfully manipulating agarose beads in buffer solution as a model system. These results suggest that the use of continuously connected magnetic nanostructures with non-modified sidewall surfaces will find potential applications in the areas of bio-sensing, force transduction and cancer screening-manipulation among others.