Since its introduction in 1960's, the model organism Caenorhabditis elegans has played a crucial role towards scientific discoveries because of its relatively simple anatomy, conserved biological mechanisms, and mapped genome. The organism also has a rapid generation time and produces a large number of isogenic progeny, making C. elegans an excellent system for conducting forward genetic screens. Conventional screening methods, however, are labor intensive and introduce potential experimental bias; typically, large-scale screens can take months to years. Thus, automated screening and characterization platforms can provide an opportunity to overcome this bottleneck. The objective of this thesis is to develop tools to perform rapid phenotypical characterization of C. elegans to enable automated genetic screening systems for neural development. To achieve this goal, I developed methods to increase throughput of worm handling using microfluidic devices and demonstrate software modules to phenotype unknown mutants using quantitative and morphological image analysis methods. Microfluidic devices are constructed from PDMS using established soft-lithography techniques. The emphasis on the simplification of existing designs greatly facilitates the adoption of our developed systems by other scientists. This thesis also includes image processing modules using various techniques to determine animal phenotypes. For example, we adapted standard thresholding methods to detect animal motor neurons, developed a modified granulometry algorithm to rapidly characterize large numbers of lipid droplets in 3D, and developed a probability model to determine neuronal process morphology. This work is significant because it increases current capabilities of screening small animals with morphological phenotypes by enhancing throughput and reducing human bias. Genes or gene functions that can be discovered using these methods can further elucidate mechanisms relevant to neural development, degeneration, maintenance, and function; these discoveries in turn can facilitate discoveries of potential therapeutic strategies for human neurological diseases.

This thesis presents the development and optimization of a high-throughput fluorescence microbead based approach for multiplexed, large scale medical diagnostics of biological fluids. Specifically, different sizes of semiconductor nanocrystals, called quantum dots, are infused into polystyrene microspheres, yielding a set of spectrally unique optical barcodes. The surface of these barcodes are then used for sandwich assays with target molecules and fluorophore-conjugated detection antibodies, changing the optical spectra of beads that have associated with (or captured) biomolecular targets. These assayed microbeads are analyzed at a single bead level in a high-throughput manner using an electrokinetic microfluidic system and laser induced fluorescence. Optical signals collected by solid state photodetectors are then processed using novel signal processing algorithms. This document will discuss developments made in each area of the platform as well as optimization of the platform for improved future performance.

Lipidomics and metabolomics are powerful tools for the examination of cellular metabolism and physiology. Methods for lipid analysis need to be developed that begin with small samples and do not overly dilute or disperse the sample in the separation process. Microchips provide a platform for interfacing lysis of small cell populations with on-chip solid phase extraction for isolating lipid samples to generate high quality mass spectra from very small samples. Chapter 1 of this dissertation presents a novel method for small scale lipidomics of bacterial cells by microchip based extraction coupled with untargeted profiling of glycerophospholipids using nanoelectrospray ionization mass spectrometry. Chapter 2 and 3 focus on the development of LC-MS/MS methods to study biological pathways. In Chapter 2, I describe a method for analysis of the phospholipids metabolite, GroPIns, in the medium of the pathogenic yeast Candida albicans. This method was applied to aid in the characterization of the GroPIns transport protein, Git1, in C. albicans. Chapter 3 extends the studies of part two and describes an efficient method based on HILIC-MS/MS for the separation and quantification of five lipid-related extracellular metabolites in yeast Saccharomyces cerevisiae. This newly developed methodology was successfully applied to determine the extracellualr levels of glycerophosphoinositol, glycerophosphocholine, glycerol 3-phosphate, inositol and choline in wild type and mutant strains. / Bayer School of Natural and Environmental Sciences / Chemistry and Biochemistry / PhD / Dissertation

In the cancer research, it is important to understand protein dynamics which are involved in cell signaling. Therefore, particular protein detection and analysis of target protein behavior are indispensable for current basic cancer research. However, it usually performed by conventional biochemical approaches, which require long process time and a large amount of samples. We have been developed the new applications based on microfluidics and Raster image Correlation spectroscopy (RICS) techniques. A simple microfluidic 3D hydrodynamic flow focusing device has been developed for quantitative determinations of target protein concentrations. The analyte stream was pinched not only horizontally, but also vertically by two sheath streams by introducing step depth cross junction structure. As a result, a triangular cross-sectional flow profile was formed and the laser was focused on the top of the triangular shaped analyte stream. Through this approach, the target protein concentration was successfully determined in cell lysate samples. The RICS technique has been applied to characterize the dynamics of protein 53 (p53) in living cells before and after the treatment with DNA damaging agents. P53 tagged with Green Fluores-cent Protein (GFP) were incubated with and without DNA damaging agents, cisplatin or eptoposide. Then, the diffusion coefficient of GFP-p53 was determined by RICS and it was significantly reduced after the drug treatment while that of the one without drug treatment was not. It is suggested that the drugs induced the interaction of p53 with either other proteins or DNA. This result demonstrates that RICS is able to detect protein-protein or protein-DNA interactions in living cells and it may be useful for the drug screening. As another application of microfluidics, an integrated microfluidic platform was developed for generating collagen microspheres with encapsulation of viable cells. The platform integrated four automated functions on a microfluidic chip, (1) collagen solution cooling system, (2) cell-in-collagen microdroplet generation, (3) collagen microdroplet polymerization, and (4) incubation and extraction of the microspheres. This platform provided a high throughput and easy way to generate uniform dimensions of collagen microspheres encapsulating viable cells that were able to proliferate for more than 1 week.

Emulsions and foams are thermodynamically unstable dispersions that will eventually succumb to coalescence, leading to phase separation. However the kinetic stability of emulsions and foams can vary from transiently stable systems with lifetimes of seconds to indefinitely stable systems with lifetimes of many years. Understanding and controlling emulsion and foam stability is fundamental to their widespread application in consumer products and industrial processes. Designed stimuliresponsive peptide surfactants that allow the stability of emulsions and foams to be controlled by changes in solution conditions have recently been developed at the University of Queensland. The research objective of this thesis was to establish the mechanism by which these switchable biosurfactants control emulsion and foam stability and hence contribute design rules for future generations of peptide surfactants. In particular, research focused on the control of emulsion coalescence kinetics and the fundamental insights that these peptide-based emulsions provide into the coalescence phenomena. It was proposed that these switchable peptide surfactants allow the mechanical strength of the viscoelastic surfactant layer to be decoupled from other contributions to emulsion stability. It was found that the established Derjaguin– Landau–Vervey–Overbeek (DLVO) theory, which is frequently used as the basis for predicting emulsion stability, was not able to describe the stability switching observed in the peptide-based emulsions. Different designs of peptide surfactant were used to demonstrate that the kinetics of emulsion coalescence could be shifted by changing the interfacial elasticity, clearly illustrating the critical role of the surfactant layer’s mechanical properties in the coalescence mechanism. Where the peptide-surfactant-based emulsions enabled triggering a rapid transition to coalescence from a flocculation stable system it was shown that both the electrostatic repulsion (flocculation barrier) and the interfacial elasticity (coalescence barrier) were switched. This work made use of a number of experimental techniques to study the coalescence mechanism, including the observation of droplet interactions in microfluidic channels. The switchable peptide surfactants were shown to enable triggered coalescence in droplet based microfluidics, something that had hereto with proved an intractable challenge for surfactant containing oil-in-water systems. Having established the importance of the mechanical properties of the adsorbed peptide layer in enabling control over coalescence kinetics, it was of interest to study the effect of adding other surfactant species. Mixed surfactant systems are likely to be encountered in industrial applications or commercial products. The peptide surfactant AM1 was mixed with the common anionic surfactant sodium dodecyl sulfate (SDS) and synergistic behaviour was identified, including enhanced interfacial adsorption and reversible association of structures in the bulk solution. Furthermore the interfacial layers formed by AM1-SDS retained the switchable mechanical behaviour despite considerable increases in the absolute mechanical strength.

Delivery of a desired ‘active’ compound (for example, starch (as an energy substrate)) to the gastrointestinal (GI) tract is most easily achieved by oral administration. Unfortunately, the efficacy of most actives is greatly reduced due to the aggressive nature of digestive enzymes and processes which occur in this environment. A commonly applied strategy to prevent deactivation of the active prior to absorption at the target site is to encapsulate the active in another ‘sacrificial’ or non-degradable polymer matrix. Traditionally, the active and matrix is processed into a microparticle format for easy oral delivery (dispersed in a liquid or paste). However, established encapsulation methods which rely on bulk-phase processing to produce these microparticles (e.g. emulsification) are far from ideal as they lack control over the final microparticle size, size distribution, composition and shape. The lack of control in the physical properties of the resultant microparticles in turn results in an inherent lack of control over the kinetics of release of the active at the target site. In contrast, recent advances in microfluidic device fabrication and methodology development have firmly proven that these new generation devices can produce monodisperse droplets and microparticles in a continuous, controllable and predictable manner. Their potential as a processing tool for the production of highly tailored microparticles for targeted delivery, however, remains to be fully explored. Both the physical and chemical (physicochemical) properties of microparticles made from a single polymer system may be altered by the deposition of one or more additional polymer layers onto the microparticle surface (for example, alternating layers of oppositely charged polyelectrolytes to produce core-shell like particles), and this method has proven to be favorable with regards to retarding the release of active compounds. However, this addition of alternate layers of oppositely charged polyelectrolytes (so called Layer-by-Layer (LbL) deposition or assembly) does increase the number of processing steps the particles must undergo prior to storage or delivery. Further, the overall effectiveness of this additional processing is still highly dependent on the properties of the original (core) microparticles. In this thesis, a microfluidic technique was developed to encapsulate starch granules in alginate-based microparticles. Using this continuous technique, the size of the microparticles produced were shown to be monodisperse and reproducible. The developed microfluidic device included a drop formation section, followed by a gelation region and a transfer section, where the particles made on-chip are transferred from the carrier oil phase to an aqueous phase prior to collection. The microparticles collected from this microfluidic device were found to be stable for several weeks and in stark contrast to particles produced via a standard bulk emulsification routes, no aggregation was observed over this time frame. The release profile of glucose (as a result of starch hydrolysation) from microparticles produced using both a standard bulk emulsification method and the developed microfluidic-based method were compared. It was found that the monodisperse particles produced using the microfluidic method showed significantly more retardation to release compared to the glucose release profile from bulk-processed particles. This retardation effect was more pronounced when a thin layer of an oppositely charged polyelectrolyte (chitosan) was adsorbed onto the negatively charged surface (alginate is an anionic polyelectrolyte) of the microfluidic-processed microparticle. The microfluidic device developed within this thesis and the resulting tailored microparticles thus show significant potential with regards to offering a new generation of microparticle delivery systems with highly deterministic delivery over extended lifetimes.

Delivery of a desired ‘active’ compound (for example, starch (as an energy substrate)) to the gastrointestinal (GI) tract is most easily achieved by oral administration. Unfortunately, the efficacy of most actives is greatly reduced due to the aggressive nature of digestive enzymes and processes which occur in this environment. A commonly applied strategy to prevent deactivation of the active prior to absorption at the target site is to encapsulate the active in another ‘sacrificial’ or non-degradable polymer matrix. Traditionally, the active and matrix is processed into a microparticle format for easy oral delivery (dispersed in a liquid or paste). However, established encapsulation methods which rely on bulk-phase processing to produce these microparticles (e.g. emulsification) are far from ideal as they lack control over the final microparticle size, size distribution, composition and shape. The lack of control in the physical properties of the resultant microparticles in turn results in an inherent lack of control over the kinetics of release of the active at the target site. In contrast, recent advances in microfluidic device fabrication and methodology development have firmly proven that these new generation devices can produce monodisperse droplets and microparticles in a continuous, controllable and predictable manner. Their potential as a processing tool for the production of highly tailored microparticles for targeted delivery, however, remains to be fully explored. Both the physical and chemical (physicochemical) properties of microparticles made from a single polymer system may be altered by the deposition of one or more additional polymer layers onto the microparticle surface (for example, alternating layers of oppositely charged polyelectrolytes to produce core-shell like particles), and this method has proven to be favorable with regards to retarding the release of active compounds. However, this addition of alternate layers of oppositely charged polyelectrolytes (so called Layer-by-Layer (LbL) deposition or assembly) does increase the number of processing steps the particles must undergo prior to storage or delivery. Further, the overall effectiveness of this additional processing is still highly dependent on the properties of the original (core) microparticles. In this thesis, a microfluidic technique was developed to encapsulate starch granules in alginate-based microparticles. Using this continuous technique, the size of the microparticles produced were shown to be monodisperse and reproducible. The developed microfluidic device included a drop formation section, followed by a gelation region and a transfer section, where the particles made on-chip are transferred from the carrier oil phase to an aqueous phase prior to collection. The microparticles collected from this microfluidic device were found to be stable for several weeks and in stark contrast to particles produced via a standard bulk emulsification routes, no aggregation was observed over this time frame. The release profile of glucose (as a result of starch hydrolysation) from microparticles produced using both a standard bulk emulsification method and the developed microfluidic-based method were compared. It was found that the monodisperse particles produced using the microfluidic method showed significantly more retardation to release compared to the glucose release profile from bulk-processed particles. This retardation effect was more pronounced when a thin layer of an oppositely charged polyelectrolyte (chitosan) was adsorbed onto the negatively charged surface (alginate is an anionic polyelectrolyte) of the microfluidic-processed microparticle. The microfluidic device developed within this thesis and the resulting tailored microparticles thus show significant potential with regards to offering a new generation of microparticle delivery systems with highly deterministic delivery over extended lifetimes.

Delivery of a desired ‘active’ compound (for example, starch (as an energy substrate)) to the gastrointestinal (GI) tract is most easily achieved by oral administration. Unfortunately, the efficacy of most actives is greatly reduced due to the aggressive nature of digestive enzymes and processes which occur in this environment. A commonly applied strategy to prevent deactivation of the active prior to absorption at the target site is to encapsulate the active in another ‘sacrificial’ or non-degradable polymer matrix. Traditionally, the active and matrix is processed into a microparticle format for easy oral delivery (dispersed in a liquid or paste). However, established encapsulation methods which rely on bulk-phase processing to produce these microparticles (e.g. emulsification) are far from ideal as they lack control over the final microparticle size, size distribution, composition and shape. The lack of control in the physical properties of the resultant microparticles in turn results in an inherent lack of control over the kinetics of release of the active at the target site. In contrast, recent advances in microfluidic device fabrication and methodology development have firmly proven that these new generation devices can produce monodisperse droplets and microparticles in a continuous, controllable and predictable manner. Their potential as a processing tool for the production of highly tailored microparticles for targeted delivery, however, remains to be fully explored. Both the physical and chemical (physicochemical) properties of microparticles made from a single polymer system may be altered by the deposition of one or more additional polymer layers onto the microparticle surface (for example, alternating layers of oppositely charged polyelectrolytes to produce core-shell like particles), and this method has proven to be favorable with regards to retarding the release of active compounds. However, this addition of alternate layers of oppositely charged polyelectrolytes (so called Layer-by-Layer (LbL) deposition or assembly) does increase the number of processing steps the particles must undergo prior to storage or delivery. Further, the overall effectiveness of this additional processing is still highly dependent on the properties of the original (core) microparticles. In this thesis, a microfluidic technique was developed to encapsulate starch granules in alginate-based microparticles. Using this continuous technique, the size of the microparticles produced were shown to be monodisperse and reproducible. The developed microfluidic device included a drop formation section, followed by a gelation region and a transfer section, where the particles made on-chip are transferred from the carrier oil phase to an aqueous phase prior to collection. The microparticles collected from this microfluidic device were found to be stable for several weeks and in stark contrast to particles produced via a standard bulk emulsification routes, no aggregation was observed over this time frame. The release profile of glucose (as a result of starch hydrolysation) from microparticles produced using both a standard bulk emulsification method and the developed microfluidic-based method were compared. It was found that the monodisperse particles produced using the microfluidic method showed significantly more retardation to release compared to the glucose release profile from bulk-processed particles. This retardation effect was more pronounced when a thin layer of an oppositely charged polyelectrolyte (chitosan) was adsorbed onto the negatively charged surface (alginate is an anionic polyelectrolyte) of the microfluidic-processed microparticle. The microfluidic device developed within this thesis and the resulting tailored microparticles thus show significant potential with regards to offering a new generation of microparticle delivery systems with highly deterministic delivery over extended lifetimes.

Thesis (M.S.M.E.)--State University of New York at Binghamton, Watson School of Engineering and Applied Science, 2007. / Includes bibliographical references.

Thesis (M.S.)--Oregon State University, . / Printout. Includes bibliographical references (leaves 99-103). Also available on the World Wide Web.