Master of Science / Department of Chemistry / Christopher T. Culbertson / Mixing is an essential process to many areas of science for example it is important in studying chemical reaction kinetics, chemical synthesis, DNA hybridization and PCR amplification. Mixing on the macroscale level is readily achieved through convection. Rapid mixing on microchips however, is problematic as the low Reynolds numbers and high Peclet numbers indicate that fluid flow is in the laminar regime and limits mixing on microchips to diffusion. Because of these limitations mixing on microchips is often relegated to diffusional mixing which requires long channels and long time periods. Several methods have been developed to increase the speed and efficiency of mixing on microfluidic devices. A variety of techniques have been employed to overcome these obstacles including for example 1) 3 dimensional channel designs to split up and recombine flows 2) employing sophisticated lithographic techniques to make grooves within a channel to generate transverse flows and 3) using lateral flow created by using spiral channels. Other groups have used outside energy sources to achieve mixing by changing of the zeta potential within the channel, using induced charge electroosmosis, and also by modifying the electrokinetic flow. We propose using dielectric elastomers (DEs) to modulate flow as a means to achieve rapid and active mixing on the microchip format. Electroactive polymers such as poly(dimethylsiloxane) function as DEs and are capable of converting electrical energy into mechanical energy. The application of an electrical potential across the PDMS results in a change in the dimensions of the PDMS dielectric layer between the two actuating electrodes creating an actuator. When employed in microfluidic devices this actuator can be used to change the volumes of the microfluidic channels on the PDMS. If the actuators are placed near a T-intersection where two components are entering the intersection the actuators can serve to improve mixing on microfluidic devices. Studies were conducted on how on the magnitude of the actuation, the frequency of actuation, the field strength, the electrode design and position relative to the T intersection, the channel dimensions and the overall channel design impacted mixing efficiency. Mixing results showed promise but further development of technology is necessary to achieve adequate mixing in microfluidic channels using DEs.

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Many applications for the analysis and processing of biological materials require the enrichment of cell subpopulations. Conventional cell sorting systems are large and expensive with complex equipment that necessitates specialized personnel for operation. Employing microfluidics technology for lab-on-a-chip adaptation of these devices provides several benefits: improved transport control, reduced sample volumes, simplicity of operation, portability, greater accessibility, and reduced cost. The designs of microfluidic cell sorting chips vary widely in literature; evaluation and optimization efforts are rarely reported. This study intends to investigate the primary components of the design to understand the effect of various parameters and to improve the performance of the microfluidic chip. Optimized individual elements are incorporated into a proposed cell sorter chip with the ability to dynamically sort target cells from a non-homogeneous solution using electrical driving forces. Numerical and experimental results are used to evaluate the sample focusing element for controlled cell dispensing, the sorting configuration for target cell collection, and the flow elements for reduced pressure effects and prevention of flow blockages. Compact models are adapted to solve the potential field and flow field in the chip and to predict the focused sample stream width. A commercial CFD package is used to perform 2-D simulations of the potential, velocity, and concentration fields. A fluorescence microscopy visualization system is implemented to conduct experiments on several generations of chip designs. The data from sample focusing experiments, performed with fluorescent dye samples, is analyzed using a Gaussian distribution model proposed in this work. A technique for real-time monitoring of fluorescent microspheres in the microfluidic chip enables the use of dynamic cell sorting to emulate fully autonomous operation. The performance values obtained from these experiments are used to characterize the various design configurations. Sample focusing is shown to depend largely on the relative size of the sheath fluid channel and the sample channel, but is virtually independent of the junction shape. Savings in the applied potential can be achieved by utilizing the size dependency. The focusing performance also provides information for optimizing the widths of the channels relative to the cell size. Successful sorting of desired cells is demonstrated for several designs. Key parameters that affect the sorting performance are discussed; a design employing the use of supplemental fluid streams to direct the particle during collection is chosen due to a high sorting evaluation and a low sensitivity to flow anomalies. The necessary reduction of pressure influences to achieve reliable flow conditions is accomplished by introducing channel constrictions to increase the hydrodynamic resistance. Also, prolonged operation is realized by including particle filters in the proposed design to prevent blockages caused by the accumulation of larger particles. A greater understanding of the behaviour of various components is demonstrated and a design is presented that incorporates the elements with the best performance. The capability of the microfluidic chip is summarized based on experimental results of the tested designs and theoretical cell sorting relationships. Adaptation of this chip to a stand-alone, autonomous device can be accomplished by integrating an optical detection system and further miniaturization of the critical components.

Host-microbe interactions at the gastrointestinal interface have emerged as a key component in the governance of human health and disease. Advances in micro-physiological systems are providing researchers with unprecedented access and insights into this complex relationship. These systems combine the benefits of microengineering, microfluidics, and cell culture in a bid to recreate the environmental conditions prevalent in the human gut. Here we present the human-microbial cross talk (HuMiX) platform, one such system that leverages this multidisciplinary approach to provide a representative in vitro model of the human gastrointestinal interface. HuMiX presents a novel and robust means to study the molecular interactions at the host-microbe interface. We summarize our proof-of-concept results obtained using the platform and highlight its potential to greatly enhance our understanding of host-microbe interactions with a potential to greatly impact the pharmaceutical, food, nutrition, and healthcare industries in the future. A number of key questions and challenges facing these technologies are also discussed. (C) 2017 THE AUTHORS. Published by Elsevier LTD on behalf of the Chinese Academy of Engineering and Higher Education Press Limited Company. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

The continuing threats from viral infectious diseases highlight the need for new tools to study viral interactions with host cells. Understanding how these viruses interact and respond to their environment can help predict outbreaks, shed insight on the most likely strains to emerge, and determine which viruses have the potential to cause significant human illness. Animal studies provide a wealth of information, but the interpretation of results is confounded by the large number of uncontrolled or unknown variables in complex living systems. In contrast, traditional tissue culture approaches have provided investigators a valuable platform with a high degree of experimental control and flexibility, but the static nature of flask-based cell culture makes it difficult to study viral evolution. Serial passaging introduces un-physiological perturbations to cell and virus populations by drastically reducing the number of species with each passage. Low copy, high fitness viral variants maybe eliminated, while in vivo these variants would be essential in determining the virus’ evolutionary fate. Bridging technologies are urgently needed to mitigate the unrealistic dynamics in static flask-based cultures, and the complexity and expense of in vivo experiments. This thesis details the development of a continuous perfusion platform capable of more closely mimicking in vivo cell-virus dynamics, while surpassing the experimental control and flexibility of standard cell culture. First, a microfluidic flow through acoustic device is optimized to enable efficient and controllable separation of cells and viruses. Repeatable isolation of cell and virus species is demonstrated with both a well-characterized virus, Dengue Virus (DENV), and the novel Golden Gate Virus. Next, a platform is built around this device to enable controllable, automated, continuous cell culture. Beads are used to assess system performance and optimize operation. Subsequently, the platform is used to culture both murine hybridoma (4G2) and human monocyte (THP-1) cell lines for over one month, and demonstrate the ability to manipulate population dynamics. Finally, we use the platform to establish a multispecies culture with THP-1 cells and Sindbis Virus (SINV). This work integrates distinct engineering feats to create a platform capable of enhancing existing cell virus studies and opening the door to a variety of high-impact investigations.

In Chapter 1, we briefly review the thermodynamics of polymer solutions, including the ideal solution based on the Flory-Huggins lattice chain model. The entropy change in the mixing of macromolecules with small solvent molecules is much smaller than that in the mixing of two kinds of small molecules. Therefore, the effect of the solution temperature is also smaller. / In Chapter 2, we describe the basic difference between the complicate polymeric and simple Newtonian fluid and list some dimensionless numbers relevant to various physical phenomena, including the Reynolds number (Re) and the capillary number (Ca), respectively related to the inertial effect and the interfacial tension. As a fluid goes down to the micro scale, the inertial effect is usually negligible so that the flow becomes laminar. However, the interfacial tension starts to play a significant role, which leads to the development of some droplet-based (or digital) microfluidic systems. Using small droplets leads to the following advantages: (1) the reagents are limited within a small boundary of each droplet; and (2) no complex microfluidic device is required. / In Chapter 3, we use PNIPAM in water as a model system to detail how to construct a polymer phase diagram by using a microfluidic device, including the choice of the carrier fluid, the principle and experimental procedure of forming concentration-controllable PNIPAM droplets, the determination of PNIPAM concentration in each droplet by using a fluorescein probe, the effect of fluorescein on the phase transition, and the detection of the phase transition by dark field viewing. For comparison, we also did the normal LLS measurement of the phase transition of PNIPAM in water. / In Chapter 4, PS in cyclohexane is used as a model system to illustrate how to handle organic solvents because cyclohexane swells the PDMS channels. The swelling is much eliminated by directly loading the PS solution into the junction via glass capillaries. Since the addition of a fluorescence concentration probe dramatically influences the PS phase transition, we have to use a so-called parallel experimental method to produce concentration-controllable PS droplets. In this method, several PDMS chips from the same batch are used in the formation of small PS droplets. When the numbers of small PS droplets produced in the same procedure are similar to each other, the PS concentrations in different corresponding droplets are comparable. Therefore we are able to index the PS concentration in each droplet by comparing it with the droplets prepared by the same procedure, but with some added fluorescence probes. / In Chapter 5, on the basis of numerous experiments, we find inorganic salts play a significant role on the droplet forming. Thus we propose that droplet formation is a kinetics governed process when two immiscible liquids meet each other in microchannels. / In this thesis we have proposed and established a new method of constructing polymer phase diagrams. By employing the droplets-based microfluidic system, we are able to form an array of droplets of polymer solutions with several nanoliters in size. Each droplet has a controllable composition. The array of polymer droplets can be transferred and stored in a glass capillary; there the turbidity of each droplet due to the difference of scatted light immediately after the phase transition can be monitored under a microscope via dark field viewing, when the solution temperature changes. Therefore, we are able to construct a polymer phase diagram by simply combing each phase transition temperature with its corresponding compositions of polymer solution droplets. / This method has two distinguished advantages; namely, minimal sample consumption and much reduced experimental time required for the phase transition to reach its equilibrium at each given temperature. This is because the greatly increased surface-to-volume ratio allows rapid diffusion and fast heat transfer. To demonstrate the principle, we have chosen PS in cyclohexane with an upper critical solution temperature (UCST) and Poly(N-isopropylacrylamide) (PNIPAM) in water with a lower critical solution temperature (LCST) as two model systems. Primarily established phase diagrams of these two polymer solutions have demonstrated the feasibility of using droplets-based microfluidic system to construct polymer phase diagrams. / Shi, Feng. / Advisers: Chi Wu; Bo Zheng. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3532. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Pre-eclampsia, fetal growth restriction and stillbirth are major pregnancy disorders throughout the world. The underlying pathogenesis of these diseases is defective placentation characterised by inadequate invasion of extravillous trophoblast (EVT) cells into the uterine decidua. This invasion is necessary to transform the uterine arteries, ensuring an adequate maternal blood supply into the intervillous space for normal fetal growth and development. The mechanisms that regulate trophoblast invasion remains poorly understood, but it is known to be influenced by a number of factors in the uterine environment. These include interactions with maternal immune cells as well as cytokines and the products from the uterine glands. In this thesis, physical factors, specifically, tissue stiffness and oxygen are studied as regulators of trophoblast invasion. The mechanical environment is known to regulate cell fate and the migratory behaviour of cells. Despite invasion of EVT cells through decidual tissue rich in extracellular matrix (ECM) proteins, there has been no study investigating how tissue stiffness might regulate EVT invasion. Oxygen has also long been investigated as a regulator for trophoblast invasion, but evidence is conflicting on whether low oxygen promotes or inhibits invasion. This is in part because of the wide variation in methods used and the over-reliance on trophoblast cell lines. To examine the effects of tissue stiffness and oxygen tension, a robust in vitro method to examine the motility and migration of primary EVT cells in three-dimensions (3D) was first established. This system offers significant benefits compared with two-dimensional (2D) systems used previously. Importantly, only cells expressing the HLAG antigen, a marker for the extravillous phenotype are analysed. The stiffness of decidual tissues at the maternal-fetal interface was determined using atomic force microscopy. In patient matched samples, a 3-4 fold increase in stiffness was found where the placenta implants into the decidua, compared to where there is no implantation. Migration of single EVT cells under different matrix stiffness and oxygen concentrations in 3D were investigated. To determine whether EVT migration is directed, a microfluidic system was established, which models the oxygen gradient at the maternal-fetal interface in the first trimester of pregnancy. This system is simple and economical to setup, and permits analysis of the migration dynamics of trophoblast cells in 3D and in real-time under different oxygen concentrations. In conclusion, the change in stiffness at the site of implantation, is further evidence of the dramatic change that occurs in the uterine wall during pregnancy. A microfluidic system to study whether primary EVT cell invasion is directed under oxygen gradients was developed.

abstract: Microfluidic systems have gained popularity in the last two decades for their potential applications in manipulating micro- and nano- particulates of interest. Several different microfluidics devices have been built capable of rapidly probing, sorting, and trapping analytes of interest. Microfluidics can be combined with separation science to address challenges of obtaining a concentrated and pure distinct analyte from mixtures of increasingly similar entities. Many of these techniques have been developed to assess biological analytes of interest; one of which is dielectrophoresis (DEP), a force which acts on polarizable analytes in the presence of a non-uniform electric fields. This method can achieve high resolution separations with the unique attribute of concentrating, rather than diluting, analytes upon separation. Studies utilizing DEP have manipulated a wide range of analytes including various cell types, proteins, DNA, and viruses. These analytes range from approximately 50 nm to 1 µm in size. Many of the currently-utilized techniques for assessing these analytes are time intensive, cost prohibitive, and require specialized equipment and technical skills. The work presented in this dissertation focuses on developing and utilizing insulator-based dielectrophoresis (iDEP) to probe a wide range of analytes; where the intrinsic properties of an analyte will determine its behavior in a microchannel. This is based on the analyte’s interactions with the electrokinetic and dielectrophoretic forces present. Novel applications of this technique to probe the biophysical difference(s) between serovars of the foodborne pathogen, Listeria monocytogenes, and surface modified Escherichia coli, are investigated. Both of these applications demonstrate the capabilities of iDEP to achieve high resolution separations and probe slight changes in the biophysical properties of an analyte of interest. To improve upon existing iDEP strategies a novel insulator design which streamlines analytes in an iDEP device while still achieving the desirable forces for separation is developed, fabricated, and tested. Finally, pioneering work to develop an iDEP device capable of manipulating larger analytes, which range in size 10-250 µm, is presented. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2018

Micro-fluid mixing is an important aspect of many of the various micro-fluidic systems used in biochemical production, biomedical industries, micro-energy systems and some electronic devices. Typically, because of size constraints and laminar flow conditions, different fluids may only have the opportunity to mix by diffusion, which is extremely rate limited. Therefore, active or highly effective passive mixing techniques are often required. In this study, two pulsed injectors are used to actively enhance mixing in a high aspect ratio microchannel (125 ��m deep and 1 mm wide). The main channel has two adjacent flowing streams with 100% dye and 0% dye concentrations, respectively. Two injectors (125 ��m deep and 250 ��m wide) are located on separate sides of the channel, with one downstream 2 mm (equivalent to two main channel widths or eight injector widths) from the other. This results in an asymmetric mixing as the flow proceeds downstream. A dye solution is used to map local mixing throughout the channel by measuring concentration variations as a function of both space and time. The primary flow rates are varied from 0.01 to 0.20 ml/min (Reynolds numbers of 0.3 to 26.6), the injector flow rate ratios are varied from 0.125 to 2, and the pulsing frequencies are varied from 5 to 15 Hz. Images of the concentration variations within the channel are used to quantify mixing by calibrating the intensity of the image with the concentration of the dye solution. The degree of mixing (DoM) is used as a measure of quality and is defined based on the integration across the channel of the difference between the local concentration and the 50% concentration values. DoM is normalized by the 50% concentration value and subtracted from one to yield a parameter that varies from 0 (no mixing) to 1 (perfect mixing). It is shown that there is a high degree of repeatability of concentration distribution as a function of phase of the pulsing cycle. A mixing map is constructed over the range of variables tested which indicates an optimum set of flow and pulsing conditions needed to achieve maximum mixing in the main channel flow. The flow rate ratio between the injectors and main channel is found to be the most influential parameter on overall mixing. The highest DoM in the main channel was found to be 0.89. It is also noticed that improved mixing can occur at very low flow ratios under a unique set of primary flow and low frequency pulsing conditions. In general, there is an inverse relationship between primary flow rate and pulsing frequency to achieve better overall mixing. / Graduation date: 2003

The research presented in this work explores the application of microfluidics to the field of proteomics through the design of a multi-channel microfluidic platform and the investigation of individual components of the system. The design of this microfluidic device allows the integration of several protein sample preparation steps for automated electrospray ionization mass spectrometric (ESI-MS) analysis, including protein separation, fractionation and collection, preconcentration and cleanup, and protein digestion. In order for the multi-channel system to function properly, I first evaluated each individual component of the device. Several areas were explored: (i) optimization of polymer monolith for solid-phase extraction (SPE) preconcentration; (ii) investigation of cationic coatings for microchannel surface modification to facilitate positive electrospray of peptides and proteins for chip-MS coupling; (iii) combination of the hydrophobic monolith and the PolyE-323 coating in a single channel device for on-chip SPE and on-bed tryptic digestion with on-line coupling to ESI-MS. Multiplexed microfluidic devices for protein analysis, which integrate a series of microfluidic features, were then designed, built and tested. The multiplexed microfluidic architecture employed a separation channel, a fractionator, an array of microchambers to accommodate monolithic polymer for SPE preconcentration, and an elution channel for the detection of eluted sample using fluorescence detector or mass spectrometer. The performance of the multiplexed devices for integration of multiple analytical steps was explored with sequential fractionation, collection, and elution of fluorescent sample, evaluating the ability to trap and release individual fractions without cross-contamination. Thorough analysis of each of the individual components on the multiplexed microfluidic platform provides valuable insights into the design of such systems, which brings us closer to our final goal of a proteomic processing microchip.