No description available.

A novel laser desorption system, with improved signal stability and extraordinary long lifetime, is presented for the study of jet cooled biomolecules in the gas phase using vibrationally resolved photoionisation spectroscopy. Tryptophan (Trp) is used as the test substance to characterize this desorption source. Here, the surface of a moving and rotating rod (graphite/Trp, 3mm diameter and 6 mm length) is exposed to a pulsed desorption beam from a Nd:YAG (1064 nm) laser running at 20Hz (Continuum Minilite).The characteristics of the source developed here and its properties with respect to cooling and stability have been investigated. Good control over the rod movement and the delivery of the IR beam result in a highly stabilized source with no noticeable fragmentation products.The combination of premixing within the source and using a pellet has made it possible to produce a stable jet-cooled beam of Trp, which lasts for several weeks without changing the sample. Additionally, the stability and signal to noise ratio has been improved by averaging the signal over the entire sample pellet by synchronizing the data acquisition with the rotation of the sample rod. The results demonstrate how a combination of the above helps to produce stable time of flight (TOF) signal and good quality one- and two-colour resonant two-photon ionisation (R2PI), photoionisation efficiency (PIE) and mass analyzed threshold ionization (MATI) spectra of Trp. The existence of six low-lying conformers of Trp in the gas phase has been confirmed. The first MATI spectrum of an isolated biomolecule (Trp) via R2PI for the determination of IE with high accuracy as ± 3 cm-1 has been recorded.

The molecular components of life (i.e., biomolecules such as DNA, proteins, lipids) have the potential to preserve in animals that have been extinct for millions of years, offering a scale of analysis previously inaccessible from the fossil record. As new technology (e.g., high resolution mass spectrometry) has been incorporated into fossil analyses, researchers have begun to detect biomolecules in terrestrial vertebrates dating back to the Triassic Period (~230 Ma). However, these biomolecules have not been demonstrated to be the biological remains of these ancient animals and may instead be exogenous organic contaminants. Here, I developed a series of analytical techniques to detect and interpret the preservation of the degraded remains of the most common protein in bone, collagen, in terrestrial vertebrates from two time slices that represent the two ends of the preservation spectrum: a "shallow time" study of fossils <150,000 years old from different burial environments (i.e., permafrost, fluvial and hot springs) and a deep time study of dinosaurs (~212 - 66 Ma) from the same burial environment (i.e., fluvial), representing the current limit of the reported protein preservation in the fossil record. Unlike previous studies that have focused on organic extractions to detect biomolecules, I studied intact fossil bones and the rocks they were found in, to understand more about the effect of burial conditions on preservation and potential alternative sources of organic compounds. I found endogenous amino acids (the degradation products of proteins) and lipids in the mammoth bones, although they were already heavily degraded in fluvial environments, even on such short timescales. I also found that there were amino acids and lipids preserved in the dinosaur bones, however tests on the age of the amino acids and the types of lipids present, demonstrate that they are not original to the animals in this study. Therefore, fluvial environments, one of the most common depositional environments preserved in the geologic record, are not conducive to the preservation of proteins on long timescales and researchers should be cautious when using these biomolecules to make interpretations about the biology of ancient animals. / Doctor of Philosophy / An outstanding challenge in the geosciences is understanding how living tissues are altered and preserved when an organism enters the fossil record. Studying the information encapsulated in fossils holds the key to an organism’s journey from death to discovery. Over the last few decades, studies of the taphonomy (i.e, how an organism decays and fossilizes) of extinct organisms have shifted their focus from how animals are preserved to what of the original tissues remain. The preservation of organic molecules (e.g., nucleic acids) over long time scales has raised a number of interesting questions (e.g., the preservation potential of DNA) and has been met with equal shares of optimism and apprehension. But ultimately, the preservation of molecular information has the potential to expand what is currently known about the biology of ancient animals and lead to a better understanding of the processes of fossilization, goals that require an understanding of how organic molecules (biomolecules) are altered over short-term and long-term scales and what organic compounds have persisted over the organism’s journey from death to discovery. Considering burial context is critical in determining if the biomolecules (i.e., DNA, proteins and lipids) being detected in fossils are the biological remains of ancient animals or organic contaminants from other sources. Therefore, I studied terrestrial vertebrates from two different periods of time: the “shallow time” dataset consists of mammoth bones from different burial environments (i.e., permafrost, fluvial, hot springs) that are all less than 150,000 years old and the deep time dataset consists of dinosaur bones from the same burial environments (i.e., fluvial) and range from ~212 to 66 million years old. Focusing on the influence of fluvial environments, where the majority of terrestrial vertebrate fossils are found, is key to understanding the long term preservation potential of the most common organic biomolecule in bone, collagen. Researchers have detected biomolecules like amino acids (as far back as the Triassic Period, ~230 million years), that they have linked to collagen preservation, however, no definitive evidence has been found to determine that the biomolecules detected belong to the animal preserved. I studied intact fossil bone to determine what biomolecules are present and if they can be definitively linked to the animal in which they were found. Mammoth bones are preserved on a timeline that is conducive to collagen preservation (<150,000 years) and preserve original amino acids (the degradation products of collagen) and lipids. However, degradation of these biomolecules is already apparent in the bones found in fluvial environments. The dinosaur bones have both amino acids and lipids (as well as other organics, like lignin, which is found in plants) present in the bones that are not present in the rocks where the bones were found. However, tests on the ages of the amino acids indicate that the amino acids are not old enough to be original. Therefore, I have found no evidence of original biomolecules in the dinosaur bones and suggest researchers proceed with caution when attempting to make biological interpretations about ancient animals from biomolecules discovered in fluvial environments, particularly on long (i.e., millions of years) timescales.

This thesis describes the use of mass spectrometric methods based upon electrospray ionization, ESI, and (matrix-assisted) laser desorption/ionization, (MA)LDI, for the quantitative analysis of small biomolecules. Structure analysis when required, was obtained through tandem mass spectrometry (MS/MS). The Girard type reagent, 4-hydrazino-4-oxobutyl tris(2,4,6 trimethoxy)phenyl phosphonium bromide, in combination with the solid phase derivatization technique, is used to selectively prepare a pre-ionized malondialdehyde derivative to be analyzed by MALDI or LDI. The in situ derivatization and isolation minimize interferences from other components in biological samples. The combination of pre-ionization and aromatic functionalities allows for laser induced ionization without the need of matrix. The combination of these techniques provides an avenue for development of automation to produce a high throughput method of analysis. Chapter 3 involves the study of the complexation of diols to the oxovanadium ion. The oxovanadium (IV) complex of ethylene glycol is used as a reference to study the complexation of other diols and amines with the vanadyl ion. The ES spectra of various diols studied produce intense signals for the mixed and the analyte complexes, indicating effective complexation of the analytes with the vanadyl ion. Oxovanadium (IV) is observed to be more selective for complexation to diols than amines. This eliminates the possibility of interference from N-containing ligands to the detection of diols by the reference complex. The electrospray spectrum is used for quantitation and the tandem mass spectrometry spectrum for structure confirmation. The MS/MS spectrum also assists the identification of the diols by the structural differences within their isomers. The equilibrium constant of a set of diols was determined and its calibration curve were constructed. This study produces an alternative method to detect and quantify diols in aqueous solutions and blood samples. / Thesis / Master of Science (MSc)

In this dissertation, I present electronic spectroscopy of a few biologically relevant species and their complexes with solvent molecules in the gas phase using a variety of techniques, including resonantly enhanced multiphoton ionization (REMPI), laser induced fluorescence (LIF), and zero kinetic energy (ZEKE) photoelectron spectroscopy. My work on several methylated uracils and thymines and thymine-water complexes alludes to a new interpretation with regard to the origin of the photostability of our genetic code. I believe that it is the water solvent that stabilizes the photophysical and photochemical behavior of these bases under UV irradiation. For systems that demonstrate vibrational resolution in the first electronically excited state (S₁) and the cationic state, I performed vibrational analysis of both states with the aid of ab initio and density functional calculations. These observations are explained in terms of the structural changes from the ground state to S₁ and further to the cation. To bridge results from the gas phase to the solution phase, I also report studies of supersonically cooled water complexes of the three isomers of aminobenzoic acid. Density functional theory calculations are carried out to identify structural minima of water complexes in the ground state. The solvation mechanism is investigated based on vibrational analysis of the S₁ state of the neutral complex and the shift of ionization thresholds with increasing water content. / Graduation date: 2005

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

No description available.

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.