Enteric pathogens form a large part of infectious diseases which contribute to a bulk of the healthcare costs. Enteric infections are usually contracted via the faecal-oral route or through contact with contaminated surfaces. Treatment by antibiotics is becoming increasingly ineffective due to the growing number of antibiotic resistant strains. Anti-microbial resistance poses a serious threat to the future of healthcare worldwide and necessitates the search for alternate forms of therapy. Bacteriophages (phages), are viruses which specifically infect and lyse bacteria. To introduce phages as a viable form of therapy, route of administration needs to be considered carefully. Model phages with broad host ranges are ideal for therapy however oral delivery to the lower gastro-intestinal (GI) poses several challenges. The acidic stomach environment can be detrimental to phages, rendering them inactive during passage. To overcome this challenge and improve the stability of phage during encapsulation and storage, this PhD research has been conducted. pH responsive polymers, Eudragit and alginate were used to develop composite microparticles which protected phage from acidic pH (pH 1-3). A novel method of acidifying oil was developed for crosslinking droplets in vitro to avoid the use of harsh solvent systems that can cause phage inactivation. Platform microfluidic technology was employed for phage encapsulation for the first time. Monodispersed droplets and particles were produced, offering fine-tuning of droplet diameter to tailor the release and pH protection of encapsulated phage. Process scale-up was attempted using membrane emulsification (ME) to produce larger volumes of encapsulated phage. In vitro and in-situ models investigated the efficacy of encapsulated phage-bacterial killing. Industrial scale method of spray drying, and electrospinning were also used to demonstrate the versatility of the formulation. Tableting dry powder phage, showed an effective method for producing solid dosage forms for therapy. Additionally, electrospun phage fibres also showed the potential use of pH responsive formulations in addressing wound infections. Improvement in encapsulated phage storage stability was observed with the addition of trehalose in the formulation. This research underpins the need for testing phage encapsulation for site-specific delivery and offers insight into the potential use of commercially available technologies.

In this thesis I explore how migratory properties of the model organism Dictyostelium discoideum are influenced by dimensionality and topology of the environment that surrounds the cell. Additionally, I sought to develop a microfluidic device able to measure mechanical properties of single cells with a sufficient throughput to account for the inherent heterogeneity of biological samples. Throughout this thesis I made use of microfabrication methods such as photo-lithography and soft-lithography, to develop ad hoc microstructured substrates. These tools enabled me to tackle different biological and biomedical questions related to cell migration and cell mechanics. Confining cells into channels with low dimensionality appeared to regulate the velocity of cellular locomotion, as well as the migration strategy adopted by the cell. Spatial confinement induced an altered arrangement of the acto-myosin cytoskeleton and microtubules. Moreover, the spatial constraint resulted in a simplified, mono-dimensional migration, characterised by constant average speed. Additionally, some cellular processes tended to occur in a periodic fashion, upon confinement. Interestingly, if Dictyostelium cells migrated through asymmetric bifurcating micro- channels, they appeared to be able to undergo a ’decision-making’ process leading to a directional bias. Although the biophysical mechanism underlying this response is yet to be understood, the data shown in this thesis suggest that Dictyostelium cells respond to differences in local concentrations of chemoattractants. The speed of a cell that crawls in a channel also depends on the cell’s stiffness, that in turn represents a measure of the density and structure of its cytoskeleton. To date, only a few methods have been developed to investigate cell mechanics with sufficient throughput. This motivated my interest in developing a microfluidic-based device that, exploiting the recording capabilities of a modern high speed camera, enabled me to assess the cellular mechanical properties at a rate greater than 10,000 cells per second, without the need for cell labelling. In this thesis I presented an example of how this method can be employed to detect differences between healthy and cancerous prostate cells, as well as to differentiate between prostate and bladder cancer cells based on their mechanical response. In conclusion, the work presented in this thesis highlights the interdisciplinarity required to investigate complex biological and biomedical problems. Specifically, the use of quantitative approaches that span from microtechnology, live imaging, computer vision and computational modelling enabled me to investigate novel biological processes as well as to explore new diagnostic technologies that aim to promote the improvement of the future healthcare.

Optical feedback interferometry is a sensing technique with relative recent implementation for the interrogation of fluidic systems. The sensing principle is based on the perturbation of the laser emission parameters induced by the reinjection in the laser cavity of light back-scattered from a distant target. The technique allows for the development of compact and noninvasive sensors that measure various parameters related to the motion of moving targets. In particular, optical feedback interferometers take advantage of the Doppler effect to measure the velocity of tracers in flowing liquids. These important features of the optical feedback interferometry technique make it wellsuited for a variety of applications in chemical engineering and biomedical fields, where accurate monitoring of the flows is needed. This thesis presents the implementation of optical feedback interferometry based sensors in multiple fluidic systems where local velocity or flow rate are directly measured. We present an application-centered study of the optical feedback sensing technique used for flow measurement at the microscale with focus on the reliability of the signal processing methods for flows in the single and the multiple scattering regimes. Further, we present experimental results of ex vivo measurements where the optical feedback sensor is proposed as an alternative system for myography. In addition we present a real-time implementation for the assessment of non-steady flows in a millifluidic configuration. A semi-automatized system for single particle detection in a microchannel is proposed and demonstrated. Finally, an optical feedback based laser sensor is implemented for the characterization of the interactions between two immiscible liquid-liquid flowing at the microscale, and the measurement is compared to a theoretical model developed to describe the hydrodynamics of both fluids in a chemical microreactor. The present manuscript describes an important contribution to the implementation of optical feedback sensors for fluidic and microfluidic applications. It also presents remarkable experimental results that open new horizons to the optical feedback interferometry.

Li, Yuefang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 46-48). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.ii / Acknowledgement --- p.iii / Table of contents --- p.iv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Introduction to protein crystallization --- p.1 / Chapter 1.1.1 --- Principles of protein crystallization --- p.2 / Chapter 1.1.2 --- Classical methods to crystallize protein --- p.4 / Chapter 1.2 --- Crystal growth in unique environments: the pursuit of better crystals --- p.6 / Chapter 1.2.1 --- Protein crystallization in space --- p.6 / Chapter 1.2.2 --- Crystallization in gel and capillary --- p.7 / Chapter 1.3 --- Microfluidic methods for protein crystallization: high through-put screenings --- p.9 / Chapter 1.3.1 --- Valve-controlled methods --- p.10 / Chapter 1.3.2 --- Droplet-based methods --- p.11 / Chapter 1.3.3 --- Microwell-based methods --- p.11 / Chapter 1.4 --- Objective of the project --- p.13 / Chapter Chapter 2 --- Rehydratable hydrogel in nanoliter microwells --- p.15 / Chapter 2.1 --- Introduction --- p.15 / Chapter 2.2 --- Experimental --- p.17 / Chapter 2.2.1 --- Fabrication of SU-8 mould --- p.17 / Chapter 2.2.2 --- Fabrication of the PDMS device --- p.19 / Chapter 2.2.3 --- Liquid dispensing in PDMS device --- p.20 / Chapter 2.2.4 --- Polymerization of PA gel --- p.21 / Chapter 2.2.5 --- Drying and Rehydration of PA gel --- p.22 / Chapter 2.3 --- Results and discussions --- p.23 / Chapter 2.3.1 --- Preparation of PA gel in PDMS device --- p.23 / Chapter 2.3.2 --- Immobilization of PA gel in microwells --- p.25 / Chapter 2.3.3 --- Dehydration and Rehydration of PA gel --- p.25 / Chapter 2.3.4 --- Liquid dispensing in the gel-preloaded microwells --- p.29 / Chapter 2.4 --- Conclusion --- p.31 / Chapter Chapter 3 --- Protein crystallization by gel-based FID --- p.32 / Chapter 3.1 --- Introduction --- p.32 / Chapter 3.2 --- Experimental --- p.34 / Chapter 3.2.1 --- Conditions used for crystallize proteins --- p.34 / Chapter 3.2.2 --- Protein crystallization by microbatch method --- p.34 / Chapter 3.2.3 --- Protein crystallization in microchip --- p.35 / Chapter 3.3 --- Results and discussions --- p.35 / Chapter 3.3.1 --- Crystallization in microplate --- p.36 / Chapter 3.3.2 --- Crystallization in microwells --- p.38 / Chapter 3.4 --- Conclusion --- p.41 / Chapter Chapter 4 --- Conclusions --- p.43 / Chapter 4.1 --- Summary of the work --- p.43 / Chapter 4.2 --- Future perspectives --- p.44 / Reference --- p.46

L’objet de ce travail expérimental est l’étude de la mécanique de films de mousses dont la structure est confinée par leur faible épaisseur. A partir d’une méthode de moussage de dispersion de particules de polyuréthane, nous sommes parvenus à contrôler, de façon indépendante, la fraction d’air (ou la densité) de ces films, leur épaisseur e et la distribution des tailles de leurs pores (de diamètre moyen D ̅_b). Pour ce dernier paramètre, nous nous sommes intéressés au cas de distributions monodisperse, bidisperse et polydisperses. Les différentes structures obtenues ont été étudiées par tomographie aux rayons X, de manière à quantifier l’ordre induit par le confinement (profils de densité, position des centres de pores, tailles des éléments structuraux,…). Nous nous sommes intéressés à la mécanique de ces films de mousse en tension (dans le plan du film) et en compression (dans le plan orthogonal au film). Un travail spécifique mené sur la matrice de polymère nous a permis de déterminer des grandeurs adimensionnées (modules et contraintes caractéristiques) permettant une comparaison avec les données et les modèles de la littérature. Nous montrons qu’en plus de l’effet classique de la densité, le nombre moyen de bulles à travers l’épaisseur, i.e. e⁄D ̅_b , est un paramètre déterminant pour les deux directions de sollicitation. En élongation, les couches pariétales contribuent à augmenter le module élastique des films par rapport à une mousse non-confinée. Ce renforcement est d’autant plus important (jusqu’à deux fois) lorsque e⁄D ̅_b est faible, quel que soit le type de distribution des tailles de pores. En compression, les couches pariétales ne contribuent pas directement, mais le confinement joue également un rôle important, avec cette fois-ci un impact déterminant de la distribution des tailles de pores. Ainsi, un film de mousse monodisperse est ordonné comme un polycristal et présente des caractéristiques mécaniques nettement plus élevées qu’un film de mousse polydisperse, comportant de nombreuses zones de faiblesse mécanique engendrées par les défauts d’empilement des bulles initiales. Les films de mousse bidisperses ont une réponse mécanique qui se rapproche soit des mousses monodisperses, pour de relativement grands rapports e⁄D ̅_b , soit des mousses polydisperses, pour de relativement petits rapports( e)⁄D ̅_b / The subject of this experimental work is the study of foam films mechanics with a confined structure because of their low thickness. With our foaming method using polyurethane particles dispersion, we generated foam films with independent control over the gas fraction (or the density), the thickness e or the pore size distribution (with a mean diameter D ̅_b). For this last parameter, we focused on monodisperse, bidisperse and polydisperse distributions. Obtained structures were studied using X-ray tomography to quantify confinement-induced order (density profiles, pore center spatial position, structural elements size,…). The mechanics of such foam films was studied in both uniaxial tension (in-plane) and compression (orthogonal plan). A particular work was done on the polymer matrix in order to determine reduced values (moduli and characteristic stress) to compare our results with models in the literature. We show that in addition to the classic density effect, the mean number of bubbles across the thickness, i.e. e⁄D ̅_b is a determinant parameter for both stress directions. In tension, parietal walls contribute to increase the elastic modulus of films with respect to non-confined foam. This effect on the mechanical strength is even more important (up to two times) when e⁄D ̅_b is small without any effect regarding the pore size distribution. In compression, parietal walls do not contribute directly to the measured values but the confinement still has an important role, this time depending on the pore size distribution. Indeed, monodisperse foam films are organized in polycrystals with much better mechanical characteristics compared to polydisperse foam films that present numerous mechanical weak spots caused by initial bubble packing defects. Bidisperse foam films mechanical behavior is either close to monodisperse or polydisperse foam films respectively for high or small e⁄D ̅_b ratio

A microfluidic device was created for single-cell capture and analysis using polydimethylsiloxane (PDMS) channels and a glass substrate to develop a microfluidic single-cell impedance sensor for cell diagnostics. The device was fabricated using photolithography to create a master mold which in turn will use soft lithography to create the PDMS components for constant device production. The commercial software, COMSOLTM Multiphysics, was used to quantify the fluid dynamics in shallow micro-channels. The device will be able to capture a cell and sequester it long enough to enable measurement of the impedance spectra that can characterize cell. The proposed device will be designed to capture a single cell and permit back-flow to flush out excess cells in the chamber. The device will be designed to use syringe pumps and the syringe-controlled channel will also be used to capture and release the cell to ensure cell control and device reusability. We hypothesize that these characteristics along with other proposed design factors will result in a unique microfluidic cell-capture device that will enable single-cell impedance sensing and characterization.

Silk fibroin from silkworm cocoons is found in numerous applications ranging from textiles to medical implants. Its recent adoption as a biomaterial is due to the material's strength, biocompatibility, self-assembling behavior, programmable degradability, optical clarity, and its ability to be functionalized with antibodies and proteins. In the field of bioengineering it has been utilized as a tissue scaffolding, drug delivery system, biosensor, and implantable electrode. This work suggests a new application for porous silk in a microscale chromatography column. We demonstrate in situ cryotropic polymerization of highly porous structures in microscale geometries by freezing aqueous silk with a solvent. The resulting cryogels are experimentally characterized using flow parameters common in chromatography design; tortuosity, global pressure drop, pore diameter, and porosity. These empirical parameters are put into porous flow models to calculate an order-of-magnitude increase in functional surface area over the blank capillaries and packed-sphere columns used in traditional designs. Additionally, the pressure requirements to produce relevant flow rates in these structures are found not to threaten the integrity of microfluidic seals or connectors.

When confined within containers or conduits, drops and bubbles migrate to regions of minimum energy by the combined effects of surface tension, surface wetting, system geometry, and initial conditions. Such capillary phenomena are exploited for passive phase separation operations in micro-fluidic devices on earth and macro-fluidic devices aboard spacecraft. Our study focuses on the migration and ejection of large inertial-capillary drops confined between tilted planar hydrophobic substrates. In our experiments, the brief nearly weightless environment of a drop tower allows for the study of such capillary dominated behavior for up to 10 mL water drops with migration velocities up to 12 cm/s. We control ejection velocities as a function of drop volume, substrate tilt angle, initial confinement, and fluid properties. We then demonstrate how such geometries may be employed as passive no-moving-parts droplet generators for very large drop dynamics investigations. The method is ideal for hand-held non-oscillatory drop generation for fun, educational, and insightful astronaut demonstrations aboard the International Space Station.

Prostate cancer presents a significant paradox: it is very common, yet rarely fatal. To wit, the prostate is the most common non-skin tissue for cancer diagnosis in men in the United States. Despite its high incidence, fatal malignancy occurs in only a small fraction of diagnosed men. The fatal cases are characteristically defined by distant spread in the body, also known as metastasis. In order to metastasize a cancer cell must complete several sequential steps. These include degradation of and invasion through the epithelial basement membrane, typically through the loss of static intracellular adhesions with fellow epithelial cells; entrance into the blood stream (intravasation); survival within circulation; exit from the blood stream upon arrival at a new tissue (extravasation); and survival and colonization at the secondary site. At the time of diagnosis, it is not currently possible to accurately predict future metastasis and thereby clinicians cannot delineate those men at high risk for fatal disease from the vast majority of men who are likely to experience an indolent disease course. Consequently, we examined the behavior of cancer cells in several steps of the metastatic cascade. In doing so, we uncovered both molecular and biophysical characteristics of cancer cells that may facilitate successful metastatic dissemination and tumor outgrowth. Epithelial-to-mesenchymal transition (EMT) is physiological process of transdifferentiation that is normally initiated during vertebrate development, but has recently been implicated in tumor development, progression, and metastases. The EMT program results in dramatic changes, including the exchange of epithelial for mesenchymal markers, altered cellular morphology, and gain of motility. EMT-like cellular alterations have been implicated most strongly in the metastasis steps of invasion and survival of cells at primary tumors sites. How EMT-like changes may facilitate survival and growth in the microenvironment of a micrometastatic niche has been less clearly elucidated. Consequently, we evaluated how EMT-like changes may affect the survival and subsequent outgrowth of prostate cancer cell lines following restrictive growth conditions. We observed that EMT-like cells as compared to their more epithelial counterparts displayed enhanced maintenance of their proliferative potential following extended culture in nutrient restriction. This phenotype depended on an EMT-associated increase in autophagy. Notably, the post-stress outgrowth phenotype could be conferred through a paracrine signaling mechanism that may involve autophagy-derived exosome-like extracellular vesicles. These studies demonstrated that EMT-like cells have a resistance to nutrient restriction through enhanced autophagy and may have uncovered a novel autophagy-dependent exosomal secretion pathway. Metastatic efficiency is thought to be strongly limited by the destruction of circulating tumor cells by the hemodynamic shear forces within the vasculature. However, such a persistent belief has little appropriate published experimental evidence. We developed an in vitro assay to expose cells to fluid shear stress (FSS). By monitoring the viability of the cells, we determined that transformed cells had a highly conserved ability to resist even very high FSS. The mechanism depended on the capacity to patch membrane defects, extracellular calcium, and a dynamic cytoskeleton. We also observed a stiffening of cancer cell membranes after exposure to FSS. Taken together, these studies expand the understanding of how cancer cells survive in circulation and indicate that metastatic efficiency is less limited by hemodynamic forces than previously thought. The steps of hematogenous metastasis between intravasation and extravasation necessitate the existence of circulating tumor cells (CTCs). Collection, enumeration, and study of CTCs have the potential to serve as a "liquid biopsy" of the metastatic cascade. In prostate cancer, the enumeration of CTCs by detection of the expression of epithelial markers has displayed limited clinical utility. We hypothesized that the prognostic value of CTC number may be enhanced by detection of cells which have undergone the pro-metastatic EMT-like program. We developed a flow cytometry-based experimental assay for enumeration of CTCs using epithelial (EpCAM) and mesenchymal-like (N-cadherin) surface proteins. We detected from prostatectomy patients before and after surgery events expressing EpCAM, N-cadherin, and both. However, the detection of background events from healthy control subjects was unacceptably high. These studies support the idea of mesenchymal-like tumor cells in circulation, but will require further assay development for reliable conclusions to be drawn. In sum, the work described above has provided descriptive and mechanistic insight to molecular and biophysical properties that may facilitate prostate cancer metastasis. It is our hope that these data will result in the development of relevant preventative, diagnostic, and therapeutic clinical strategies for prostate cancer.

This research utilizes Electrical Impedance Spectroscopy, a technique classically used for electrochemical analysis and material characterization, as the basis for a non-destructive, label-free assay platform for three dimensional (3D) cellular spheroids. In this work, a linear array of microelectrodes is optimized to rapidly respond to changes located within a 3D multicellular model. In addition, this technique is coupled with an on chip micro-pH sensor for monitoring the environment around the cells. Finally, the responses of both impedance and pH are correlated with physical changes within the cellular model. The impedance analysis system realized through this work provides a foundation for the development of high-throughput drug screening systems that utilize multiple parallel sensing modalities including pH and impedance sensing in order to quickly assess the efficacy of specific drug candidates. The slow development of new drugs is mainly attributed to poor predictability of current chemosensitivity and resistivity assays, as well as genetic differences between the animal models used for tests and humans. In addition, monolayer cultures used in early experimentation are fundamentally different from the complex structure of organs in vivo. This requires the study of smaller 3D models (spheroids) that more efficiently replicate the conditions within the body. The main objective of this research was to develop a microfluidic system on a chip that is capable of deducing viability and morphology of 3D tumor spheroids by monitoring both the impedance of the cellular model and the pH of their local environment. This would provide a fast and reliable method for screening pharmaceutical compounds in a high-throughput system.