Applications of nanoporous gold monoliths as substrates for the capture and release of lectins and glycoproteins

Nanoporous gold (np-Au) monoliths are a free-standing nanostructured material with typical pore dimensions in the tens of nanometers range. The microstructure of np-Au resembles those of macroporous monolithic materials being used in chromatographic separations. The surfaces of np-Au monoliths were modified via flow methods with different ligands to develop affinity substrates for separations. A carbohydrate-modified np-Au monolith was prepared by immobilizing thiolated saccharides and further used to separate lectins. The np-Au monolith surface was also functionalized with self-assembled monolayers (SAMs) of α-lipoic acid (LA) followed by activation of carboxyl terminal groups to create amine reactive esters. Concanavalin A (Con A) was then covalently immobilized to develop a substrate for extraction of glycoprotein from a mixture. Likewise, aminophenylboronic acid was immobilized to develop a substrate that was tested for pH-dependent capture and release of cis-diol containing molecules. Preservation of SAMs and immobilized ligands were possibly due to the in situ surface modification of np-Au monoliths that limited the possible damage and degradation of molecules on the surface. Selectivity of the developed substrates was enhanced by capping the unreacted functional groups or by incorporation of protein resistant spacers to limit the non-specific adsorption of unwanted molecules. The loading and surface coverage of molecules on np-Au monolith surface were determined by thermogravimetric analysis (TGA) and by an in situ solution depletion method. TGA was able to quantify the amount of loading based from the mass loss after the pyrolysis of modified np-Au monoliths. The in situ solution depletion method estimates the amount of loading by the difference in the initial and final concentration of a circulating solution monitored by a UV detector. This research aims to introduce np-Au monolith as an addition to the materials being used as substrates in chromatographic separation and extraction. The chemical stability, simple but reproducible preparation, high surface-to-volume ratio and availability of wide variety of Au surface functionalization are the features of np-Au monolith that could complement the limitations of the existing materials used in separations. The focus of this research is on the separation of lectins and glycoproteins, which is an important step towards an effective glycan analysis in glycomics

Application of label-free mass spectrometry-based proteomics to biomarker discovery

Mass spectrometry is an analytical technique which is used extensively in the fields of chemistry and physics. Developments in the field over the last two decades have permitted the analysis of a wide variety of biological molecules from a range of sources. The term proteomics relates to the study of the protein complement of a cell or organism with particular interest in the identification and quantification of these analytes. A biomarker is a characteristic that can be measured and evaluated to give an indication of normal, biological processes, or pharmacological responses to a therapeutic intervention. Bodily fluids are a rich source of potential biomarkers as they can be obtained in reasonable quantity and their extraction is generally minimally invasive. The plasma proteome, which contains many thousands of analytes spanning a dynamic range greater than 10 orders of magnitude, reflects the status of the many tissues and organs in the body serving as an ideal medium for potential biomarker discovery. The analytical challenges posed by the plasma proteome are significant. Depletion of the highly abundant proteins is usually a prerequisite of any biomarker study and no technique has the dynamic range to study all of the proteins present. Comprehensive characterisation of the plasma proteome requires significant experimental effort and cost. Use of pooled samples in biomarker studies is widespread and the majority of biomarkers, which have been identified in the discovery phase, have not passed clinical validation. A data independent, label-free quantitative approach has been evaluated for the study of depleted maternal plasma proteomes taken in the first trimester. Plasma was characterised from individual and groups of patients from three obstetric conditions using single and multi-dimensional chromatography. Potential biomarkers from each source were identified and evaluated. Multi-dimensional chromatography was used to simplify the complexity of the analytes introduced to the mass spectrometer and the benefits and limitations of the approach in terms of biomarker discovery have been demonstrated

DIGE Proteome Analysis Reveals Suitability of Ischemic Cardiac In Vitro Model for Studying Cellular Response to Acute Ischemia and Regeneration

Proteomic analysis of myocardial tissue from patient population is suited to yield insights into cellular and molecular mechanisms taking place in cardiovascular diseases. However, it has been limited by small sized biopsies and complicated by high variances between patients. Therefore, there is a high demand for suitable model systems with the capability to simulate ischemic and cardiotoxic effects in vitro, under defined conditions. In this context, we established an in vitro ischemia/reperfusion cardiac disease model based on the contractile HL-1 cell line. To identify pathways involved in the cellular alterations induced by ischemia and thereby defining disease-specific biomarkers and potential target structures for new drug candidates we used fluorescence 2D-difference gel electrophoresis. By comparing spot density changes in ischemic and reperfusion samples we detected several protein spots that were differentially abundant. Using MALDI-TOF/TOF-MS and ESI-MS the proteins were identified and subsequently grouped by functionality. Most prominent were changes in apoptosis signalling, cell structure and energy-metabolism. Alterations were confirmed by analysis of human biopsies from patients with ischemic cardiomyopathy

Multifunctional Natural Polysaccharides for Energy Storage Applications

Department of Energy Engineering(Battery Science and Technology)An agarose is a polysaccharide material, generally extracted from seaweeds. Agarose is a linear polymer made up of the repeating unit of agarobiose, which is a disaccharide made up of D-galactose and 3,6-anhydro-L-galactopyranose. Many applications of agarose are described in the literature from biology to energy field, such as gel electrophoresis, protein purification, solid culture media, motility assays, template for the fabrication of porous structures, binder, separator membrane, and carbon-coating material for lithium-ion batteries (LIBs). Silicon (Si) has attracted much attention as promising anode material due to its high theoretical capacity (3579 mA h g-1 with composition of Li15Si4 at room temperature), relatively low working potential ( 300% with composition of Li3.75Si at room temperature) during lithiation/delithiation leads to a serious aggregation of Si, the formation of unstable thick solid-electrolyte-interface (SEI) layers and depletion of electrolyte, which will make critical capacity fading. Furthermore, Si has low electrical conductivity and sluggish lithium-ion diffusivity. These fatal flaws prevent the commercialization of Si anode. There are several solutions to overcome these drawbacks, including Si composites with metal oxide, inactive metals, or carbon materials and Si nanostructuring (e.g., nanoparticles, nanowires, and nanotubes). Another attempt has been tried to develop functional polymeric binders which can alleviate severe volume change of Si anodes. Actually, overall quality of batteries depends on performance of binders, because polymeric binders give adhesion between electrode and current collector, allowing long-term cycling stability. Natural polysaccharide was used as polymeric binder for Si anodes, because it contains many functional groups, which are expected to generate strong adhesion between binder and active material. In chapter II, we demonstrate eco-friendly, abundant natural polysaccharide as a binder for Si-based anode, Si/C composite materials consisting of the Si foam dispersed in hard carbon (HC) synthesized by using agarose, and LiMn2O4 cathodes. Si foam@HC@C was successfully synthesized by a simple carbonization method. The nanostructured Si foam and agarose binder containing many functional groups enables to strong adhesion between Si foam and current collector, leading to enhanced electrochemical properties, including a high specific capacity of 1028 mAh g-1 (60% retention compared to 2nd cycle) and outstanding cycling performance after 200 cycles. Si foam@HC@C electrode showed first discharge capacity and discharge capacity were 654 and 513 mAh g-1 with enhanced initial coulombic efficiency of 78.4%, compared to HC@C with initial coulombic efficiency of 71.6%. LiMn2O4 cathode with agarose binder exhibited high initial coulombic efficiency of 96.2% and stable cycling performance with nearly 100% coulombic efficiency. These results indicate agarose binder can be used for both of anode and cathode due to good electrochemical stability in wide operating voltage. In chapter III, Si/Al2O3 foam particles were simply synthesized by the chemical etching of the Al?Si alloy and a subsequent selective thermal oxidation process. The Si/Al2O3 electrodes with tunable Al2O3 thickness exhibited highly stable cycling performance, excellent rate capability, and suppressed volume expansion. This strategy opens up an effective way to introduce various protecting layers on the surface of other inorganic materials.ope

Nanoscale Optical and Correlative Microscopies for Quantitative Characterization of DNA Nanostructures

Methods to engineer nanomaterials and devices with uniquely tailored properties are highly sought after in fields such as manufacturing, medicine, energy, and the environment. The macromolecule deoxyribonucleic acid (DNA) enables programmable self-assembly of nanostructures with near arbitrary shape and size and with unprecedented precision and accuracy. Additionally, DNA can be chemically modified to attach molecules and nanoparticles, providing a means to organize active materials into devices with unique or enhanced properties. One particularly powerful form of DNA-based self-assembly, DNA origami, provides robust structures with the potential for nanometer-scale resolution of addressable sites. DNA origami are assembled from one large DNA scaffold strand and many unique, short staple strands; each staple programmatically binds the scaffold at several distant domains, and the coordinated interactions of many staples with the scaffold act to fold the scaffold into a desired shape. The utility of DNA origami has been demonstrated through multiple applications, such as plasmonic and photonic devices, electronic device patterning, information storage, drug delivery, and biosensors. Despite the promise of DNA nanotechnology, few products have successfully translated from the laboratory to industry. Achieving high yield and high-precision synthesis of stable DNA nanostructures is one of the biggest challenges to applications of DNA nanostructures. For adoption in manufacturing, methods to measure and inspect assembled structures (i.e. metrology) are essential. Common high-resolution imaging techniques used to characterize DNA nanostructures, such as atomic force microscopy and transmission electron microscopy, cannot facilitate high-throughput characterization, and few studies have been directed towards the development of improved methods for nanoscale metrology. DNA-PAINT super-resolution microscopy enables high-resolution, multiplexed imaging of reactive sites on DNA nanostructures and offers the potential for inline optical metrology. In this work, nanoscale metrologies utilizing DNA-PAINT were developed for DNA nanostructures and applied to characterize DNA origami arrays and single site defects on DNA origami. For metrology of DNA origami arrays, an embedded, multiplexed optical super-resolution methodology was developed to characterize the periodic structure and defects of two-dimensional arrays. Images revealed the spatial arrangement of structures within the arrays, internal array defects, and grain boundaries between arrays, enabling the reconstruction of arrays from the images. The nature of the imaging technique is also highly compatible with statistical methods, enabling rapid statistical analysis of synthesis conditions. To obtain a greater understanding of DNA origami defects at the scale of individual strands, correlative super-resolution and atomic force microscopies were enabled through the development of a simple and flexible method to bind DNA origami directly to cover glass, simultaneously passivating the surface to single-stranded DNA. High-resolution, correlative microscopy was performed to characterize DNA origami, and spatial correlation in super-resolution optical and topographic images of 5 nm was achieved, validating correlative microscopy for single strand defect metrology. Investigations of single strand defects showed little correlation to structural defects on DNA origami, revealing that most site defects occur on strands that are present in the structure, contrary to prior reports. In addition, the results suggest that the structural stability of DNA origami was decreased by DNA-PAINT imaging. The presented work demonstrated the development and application of advanced characterization techniques for DNA nanostructures, which will accelerate fundamental research and applications of DNA nanotechnology

Analysis of the effects of BrdU on DLKP human lung cancer cells by two-dimensional difference gel electrophoresis and mass spectrometry

Bromodeoxyuridine (BrdU) is a thymidiie analogue that incorporates into DNA of dividing cells during the S-phase of the cell cycle. Previous work in laboratories reported that treatment with lOyM BrdU in the human lung carcinoma cell line (DLKP) resulted in increased expression of the cytoskeletal proteins Keratin 8 and 18 and the cell adhesion proteins a2 and b1 integrin. This study investigated protein expression changes in differentiating DLKP cells following exposure to 10yM BrdU. DLKP cells were grown in culture flasks and harvested after 7 days exposure to BrdU. Two-dimensional gel electrophoresis was used to investigate BrdU specific changes in the proteome of DLKP BrdU treated and control cells. Cy3-labeled DLKP control were combined with Cy-5 labeled BrdU DLKP treated proteins and separated on the same 2-D gel along with a Cy-2 labelled mixture of both samples as an internal standard. Using DIGE technology, the statistically significant comparisons of each protein abundance was made over three biological replicates. 43 protein spots were identified as differentially regulated. Among the 43 protein spots, 25 were found to be up-regulated and 18 were found to be downregulated

Responses of ectomycorrhizal fungi to mineral substrates

Boreal forest soils are complex, heterogeneous growth substrates where organic and mineral components provide nutrient resources for soil organisms and plants. Mineral nutrients are cycled between living and dead organic components of the forest soil and weathering of soil minerals provides an important input of new resources, compensating for losses from the ecosystem. Predicting soil responses to changing climate and management practices is important to determine their effect on forest production. Models for this purpose are largely based on the concept of the soil solution as the interface controlling soil processes such as weathering and nutrient uptake by plants, whereas soil microbiology recognises microbial processes as the driving force in soil nutrient cycling. In boreal forests most tree root tips are colonised by ectomycorrhizal fungi. The mycelia of these symbiotic fungi mediate nutrient uptake by their tree hosts. These fungi are abundant in the organic layer of forest soils and ectomycorrhizal research has therefore largely focused on nutrient uptake from this horizon. Minerals in the soil may, however, also serve as nutrient resources for ectomycorrhizal fungi. Through combined chemical and physical processes fungi can affect nutrient availability by weathering minerals. This thesis describes a field experiment investigating the distribution of different ectomycorrhizal fungi in organic and mineral forest soil horizons, in vitro studies of fungal acidification of artificial substrates with different mineral element composition, microcosm studies of growth and carbon allocation in intact ectomycorrhizal systems colonising heterogeneous mineral substrates and a preliminary investigation of changes in surface micro-topography of minerals colonised by ectomycorrhizal hyphae. Half of the fungal species identified in the forest soil occurred exclusively in the mineral horizons. Mycelial growth, carbon allocation and substrate acidification by fungi colonising different mineral substrates in vitro and in microcosms appeared to be influenced by mineral element composition. Interpretation of possible modification of mineral surface micro-topography is more difficult but together the results obtained suggest that ectomycorrhizal fungi may contribute to the development of microenvironments on colonised mineral surfaces, where increased weathering can take place. Processes regulating nutrient availability in such microenvironments are different from those estimated from the bulk soil solution

High light stress in photosynthesis: the role of oxidative post-translational modifications in signaling and repair

Oxidative stress is a natural consequence of photosynthetic oxygen evolution and redox enzyme processes. Trp oxidation to N-formylkynurenine (NFK) is a specific, reactive oxygen species (ROS)-mediated reaction. This thesis work describes the identification and functional characterization of NFK in oxygen evolving Photosystem II (PSII). Although proteomics studies have confirmed NFK modifications in many types of proteins, limited knowledge on the biochemical significance exists. In vitro studies in thylakoids and PSII membranes were used to establish a correlation between oxidative stress, NFK formation, and photoinhibition. The in vivo effect of preventing Trp oxidation to NFK was assessed by site-directed mutation in the cyanobacteria Synechocystis sp. PCC 6803. This work provides insight into the role of NFK in photosynthetic oxygen evolution and photoinhibition. Based on the current knowledge of NFK, ROS, and repair, a new model is described. In this modified model for photoinhibition and repair, NFK plays a role in signaling for turnover of damaged proteins. NFK may play a similar role in replacement of damaged proteins in other systems.PhDCommittee Chair: Bridgette A. Barry; Committee Member: David Collard; Committee Member: Ingeborg Schmidt-Krey; Committee Member: Wendy Kelly; Committee Member: Yomi Oyeler

3D lithium ion batteries—from fundamentals to fabrication

3D microbatteries are proposed as a step change in the energy and power per footprint of surface mountable rechargeable batteries for microelectromechanical systems (MEMS) and other small electronic devices. Within a battery electrode, a 3D nanoarchitecture gives mesoporosity, increasing power by reducing the length of the diffusion path; in the separator region it can form the basis of a robust but porous solid, isolating the electrodes and immobilising an otherwise fluid electrolyte. 3D microarchitecture of the whole cell allows fabrication of interdigitated or interpenetrating networks that minimise the ionic path length between the electrodes in a thick cell. This article outlines the design principles for 3D microbatteries and estimates the geometrical and physical requirements of the materials. It then gives selected examples of recent progress in the techniques available for fabrication of 3D battery structures by successive deposition of electrodes, electrolytes and current collectors onto microstructured substrates by self-assembly methods