Microfluidic devices are ideal for analysis of biological systems. The small dimensions result to controlled handling of the flow profile and the cells in suspension. Implementation of additional forces in the system, such as an electric field, promote further manipulation of the cells. In this dissertation, I show novel, unique microfluidic approaches for manipulation and analysis of mammalian cells by the aid of electrical methods or the architecture of the device. Specifically, for the first time, it is shown, that adoption of electrical methods, using surface electrodes, promotes cell concentration in a microchamber due to isoelectric focusing (IEF). In contrast to conventional IEF techniques for protein separation, a matrix is not required in our system, the presence of which would even block the movement of the bulky cells. Electric field is, also, used to breach the cell membrane and gain access to the cell interior by electroporation (irreversible and reversible). Irreversible electroporation is used in a unique, integrated microfluidic device for cell lysis and reagentless extraction of DNA. The genomic material is subsequently analyzed by on-chip PCR, demonstrating the possible elimination of the purification step. On the other hand, reversible electroporation is used for the delivery of exogenous molecules to cells. For the first time, the effect of shear stress on the electroporation efficiency of both attached and suspended cells is examined. On the second part of my dissertation, I explore the capabilities of the architecture of microfluidic devices for cell analysis. A simple, unique method for compartmentalization of a microchamber in an array of picochambers is presented. The main idea of the device lies on the fabrication of solid supports on the main layer of the device. These features may even hold a dual nature (e.g. for cell trapping, and chamber support), in which case, single cell analysis is possible (such as single cell PCR). On the final chapter of my dissertation, a computational analysis of the flow and concentration profiles of a device with hydrodynamic focusing is conducted. I anticipate, that all these novel techniques will be used on integrated microfluidic systems for cell analysis, towards point-of-care diagnostics. / Ph. D.

Instrument miniaturization is led by the desire to perform rapid diagnosis in remote areas with high throughput and low cost. In addition, miniaturized instruments hold the promise of consuming small sample volumes and are thus less prone to cross-contamination. Gas chromatography (GC) is the leading analytical instrument for the analysis of volatile organic compounds (VOCs). Due to its wide-ranging applications, it has received great attention both from industrial sectors and scientific communities. Recently, numerous research efforts have benefited from the advancements in micro-electromechanical system (MEMS) and nanotechnology based solutions to miniaturize the key components of GC instrument (pre-concentrator/injector, separation column, valves, pumps, and the detector). The purpose of this dissertation is to address the critical need of developing a micro GC system for various field- applications. The uniqueness of this work is to emphasize on the importance of integrating the basic components of μGC (including sampling/injection, separation and detection) on a single platform. This integration leads to overall improved performance as well as reducing the manufacturing cost of this technology. In this regard, the implementation of micro helium discharge photoionization detector (μDPID) in silicon-glass architecture served as a major accomplishment enabling its monolithic integration with the micro separation column (μSC). For the first time, the operation of a monolithic integrated module under temperature and flow programming conditions has been demonstrated to achieve rapid chromatographic analysis of a complex sample. Furthermore, an innovative sample injection mechanism has been incorporated in the integrated module to present the idea of a chip-scale μGC system. The possibility of using μGC technology in practical applications such as breath analysis and water monitoring is also demonstrated. Moreover, a nanotechnology based scheme for enhancing the adsorption capacity of the microfabricated pre-concentrator is also described. / Ph. D.

Microfluidics has been the focal point of research in various disciplines due to its advantages of portability and cost effectiveness, and the ability to perform complex tasks with precision. In the past two decades microfluidic technology has been used to cool integrated circuits, for exoplanetary chemical analysis, for mimicking cellular environments, and in the design of specialized organ-on-a-chip devices. While there have been considerable advances in the complexity and miniaturization of microfluidic devices, particularly with the advent of microfluidic large-scale integration (mLSI) and microfluidic very-large-scale-integration (mVLSI), in which there are hundreds of thousands of flow channels per square centimeter on a microfluidic chip, there remains an actuation overhead problem: these small, complex microfluidic devices are tethered to extensive off-chip actuation machinery that limit their portability and efficiency. Insects, in contrast, actively and efficiently handle their respiratory air flows in complex networks consisting of thousands of microscale tracheal pathways. This work analytically and experimentally investigates the viability of incorporating some of the essential kinematics and actuation strategies of insect respiratory systems in microfluidic devices. Mathematical models of simplified individual tracheal pathways were derived and analyzed, and insect-mimetic PDMS-based valveless microfluidic devices were fabricated and tested. It was found that not only are these devices are capable of pumping fluids very efficiently using insect-mimetic actuation techniques, but also that the fluid flow direction and magnitude could be controlled via the actuation frequency alone, a feature never before realized in microfluidic devices. These results suggest that insect-mimicry may be a promising direction for designing more efficient microfluidic devices. / Ph. D. / Microfluidics or the study of fluids at the microscale has gained a lot of interest in the recent past due to its various applications starting from electronic chip cooling to biomedical diagnostic devices and exoplanetary chemical analysis. Though there has been a lot of advancements in the functionality and portability of microfluidic devices, little has been achieved in the improvement of the peripheral machinery needed to operate these devices. On the other hand insects can expertly manipulate fluids, in their body, at the microscale with the help of their efficient respiratory capabilities. In the present study we mimic some essential features of the insect respiratory system by incorporating them in microfluidic devices. The feasibility of practical application of these techniques have been tested, at first, analytically by mathematically modeling the fluid flow in insect respiratory tract mimetic microchannels and tubes and then by fabricating, testing and analyzing the functionality of microfluidic devices. The mathematical models, using slip boundary conditions, showed that the volumetric fluid flow through a trachea mimetic tube decreased with the increase in the amount of slip. Apart from that it also revealed a fundamental difference between shear and pressure driven flow at the microscale. The microfluidic devices exhibited some unique characteristic features never seen before in valveless microfluidic devices and have the potential in reducing the actuation overhead. These devices can be used to simplify the operating procedure and subsequently decrease the production cost of microfluidic devices for various applications.

The analysis of potentially harmful biological particles is imperative for the mitigation of disease. As a result, there is a growing need for tools which can characterize, detect, and separate biological particles for the alleviation of a multitude of disease. One powerful technique for the analysis of cells is the use of dielectrophoresis (DEP) forces for the manipulation of particle movement. DEP is a particle transport phenomenon, induced by the presence of non-uniform electric fields. The dependence on intrinsic electrical properties of cells, have enabled DEP force to be utilized for numerous biological analyses. This thesis presents the investigation of breast cancer, pathogen, neuronal and glial cells and their DEP profiles. The drug response of various breast cancer cell lines when exposed to a variety of chemical stimuli were analyzed using shifts in their DEP profiles in relation to control groups. These results were supplemented with gene expression analysis to identify biophysical changes which could contribute to the DEP shifts. Additional experiments were conducted for the monitoring of pathogens. Live/dead bacteria mixtures were evaluated using an integrated system with DEP enrichment and impedance spectroscopy. Another application of DEP which was investigated was the separation of heterogeneous mixtures. Through the use of a novel microfluidic channel design, the separation of simulated circulating tumor cells (CTCs) from diluted blood and neuron cells from glial cells was demonstrated. The wide range of applications examined in this thesis highlights the versatility of DEP and the flexibility of the reported devices. / MS / Microscale technology can be utilized for the identification, characterization and sorting of biological material in a plethora of biomedical applications. One promising technique which is capable of cell manipulation is dielectrophoresis (DEP). DEP is a microscale force which causes particles to be attracted or repelled by specific geometries in microchannels. The DEP force is produced by the application of electric fields and can be utilized to analysis biological cell populations. This is because biological particles have unique electrical properties based on their cell morphology. Distinctions in their external protrusions and internal structures contribute to their electrical properties and can be identified in their DEP profiles. Based on this concept a variety of biomedical applications of DEP was explored. Chapter 2 and 3 describe the investigation of cells when exposed to various drugs. Drug induced responses were characterized based on their shifts in their DEP profiles. Chapter 4 presents the a rapid and low-cost live/dead assay for bacteria in aqueous samples through DEP and impedance spectroscopy. In chapter 5, the development of a DEP platform for cell sorting is reported. The wide range of biomedical applications which were explored demonstrate the useful nature of the DEP phenomenon.

A novel fabrication process is presented which allows for three-dimensionally independent features to be etched in silicon using SF6 gas in a deep reactive ion etcher (DRIE) after a single etch step. The mechanism allowing for different feature depths and widths to be produced over a wafer is reactive ion etch lag, where etch rate scales with the exposed feature size in the mask. A modified Langmuir model has been developed relating the geometry of the exposed areas in a specific mask pattern as well as the etch duration to the final depth and width of a channel that is produced after isotropic silicon etching. This fabrication process is tailored for microfluidic network design, but the capabilities of the process can be applied elsewhere. A characterization of an Alcatel DRIE tool is also presented in order to enhance RIE lag by varying etch process parameters, increasing the variety of channel sizes that can be fabricated. High values of flow rate, coil power, and pressure were found to produce this effect. The capability of the modeled process for creating a microchip cooling device for high-heat flux applications was also investigated. Using meander channels, heat flux in excess of 100W/cm2 were cooled using 750µL/s flow rate of water through the chip. This single-mask process reduces risk of damage to the chip and provides the capability to cool high-heat-flux microprocessors for the next 10 years, and for an even longer time once the geometry of the channels is optimized. / Master of Science

2025 / The role of the lymphatic vasculature in disease has been gaining appreciation in recent years. For example, lymphatic vessels can serve as a route for tumor metastasis to secondary organs. Additionally, obesity is often associated with lymphatic dysfunction. In vitro tissue-engineered models that incorporate lymphatic vessels can be a useful tool to study lymphatics in disease. These models offer several advantages, including the use of human cells, the ability to replicate 3D environments, the incorporation of interstitial fluid flow, and precise control over tissue geometry. Here, I develop multiple tissue engineered models to study lymphatics in the context of breast cancer and obesity. Using a co-culture model with a breast microtumor adjacent to an engineered lymphatic, I investigate the role of the lymphatic endothelium in tumor invasion and vascular escape. Cancer cells that escape into the vessel can present inside, in line with, or outside the lymphatic endothelium and can displace endothelial cells on the vessel wall during this process. The presence of the lymphatic endothelium has an inhibitory effect on breast cancer invasion and escape through both physical and nonphysical mechanisms. Next, to examine how obesity can affect lymphatic solute drainage, I simulate the obesity-associated microenvironment in multiple lymphatic models using tumor necrosis factor-α, cobalt chloride, and oleic acid to model inflammation, hypoxia, and hyperlipidemia, respectively. Simulated obesity directly impairs lymphatic solute drainage rates of engineered lymphatics, likely by damaging endothelial junctions and promoting solute leakage from lymphatics. In contrast, conditioned medium from obesity-treated adipocytes does not affect lymphatic drainage. Surprisingly, co-culturing adipocytes with the lymphatics prevents the harmful effects of simulated obesity on the lymphatics, revealing a potential protective role of adipocytes in obesity-related lymphatic dysfunction. Overall, these model systems elucidate the role of lymphatic vessels in breast cancer and obesity and provide a platform for studying both tumor vascular escape and lymphatic drainage function in disease.

Today many cell biological techniques study large cell populations where an average estimate of individual cells’ behavior is observed. On the other hand, single cell analysis is required for studying functional heterogeneities between cells within populations. This thesis presents work that combines the use of microfluidic devices, optical microscopy and automated image analysis to design various cell biological assays with single cell resolution including cell proliferation, clonal expansion, cell migration, cell-cell interaction and cell viability tracking. In fact, automated high throughput single cell techniques enable new studies in cell biology which are not possible with conventional techniques. In order to automatically track dynamic behavior of single cells, we developed a microwell based device as well as a droplet microfluidic platform. These high throughput microfluidic assays allow automated time-lapse imaging of encapsulated single cells in micro droplets or confined cells inside microwells. Algorithms for automatic quantification of cells in individual microwells and micro droplets are developed and used for the analysis of cell viability and clonal expansion. The automatic counting protocols include several image analysis steps, e.g. segmentation, feature extraction and classification. The automatic quantification results were evaluated by comparing with manual counting and revealed a high success rate. In combination these automatic cell counting protocols and our microfluidic platforms can provide statistical information to better understand behavior of cells at the individual level under various conditions or treatments in vitro exemplified by the analysis of function and regulation of immune cells. Thus, together these tools can be used for developing new cellular imaging assays with resolution at the single cell level. To automatically characterize transient migration behavior of natural killer (NK) cells compartmentalized in microwells, we developed a method for single cell tracking. Time-lapse imaging showed that the NK cells often exhibited periods of high motility, interrupted with periods of slow migration or complete arrest. These transient migration arrest periods (TMAPs) often overlapped with periods of conjugations between NK cells and target cells. Such conjugation periods sometimes led to cell-mediated killing of target cells. Analysis of cytotoxic response of NK cells revealed that a small sub-class of NK cells called serial killers was able to kill several target cells. In order to determine a starting time point for cell-cell interaction, a novel technique based on ultrasound was developed to aggregate NK and target cells into the center of the microwells. Therefore, these assays can be used to automatically and rapidly assess functional and migration behavior of cells to detect differences between health and disease or the influence of drugs. The work presented in this thesis gives good examples of how microfluidic devices combined with automated imaging and image analysis can be helpful to address cell biological questions where single cell resolution is necessary. / <p>QC 20130927</p>

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Cancer is still among those diseases that prominently contribute to the numerous deaths that are caused each year. But as technology and research is reaching new zeniths in the present times, cure or early detection of cancer is possible. The detection of rare cells can help understand the origin of many diseases. The current study deals with one such technology that is used for the capture or effective separation of these rare cells called Lab-on-a-chip microchip technology. The isolation and capture of rare cells is a problem uniquely suited to microfluidic devices, in which geometries on the cellular length scale can be engineered and a wide range of chemical functionalizations can be implemented. The performance of such devices is primarily affected by the chemical interaction between the cell and the capture surface and the mechanics of cell-surface collision and adhesion. This study focuses on the fundamental adhesion and transport mechanisms in rare cell-capture microdevices, and explores modern device design strategies in a transport context. The biorheology and engineering parameters of cell adhesion are defined; chip geometries are reviewed. Transport at the microscale, cell-wall interactions that result in cell motion across streamlines, is discussed. We have concentrated majorly on the fluid dynamics design of the chip. A simplified description of the device would be to say that the chip is at micro scale. There are posts arranged on the chip such that the arrangement will lead to a higher capture of rare cells. Blood consisting of rare cells will be passed through the chip and the posts will pose as an obstruction so that the interception and capture efficiency of the rare cells increases. The captured cells can be observed by fluorescence microscopy. As compared to previous studies of using solid microposts, we will be incorporating a new concept of cylindrical shell micropost. This type of micropost consists of a solid inner core and the annulus area is covered with a forest of silicon nanopillars. Utilization of such a design helps in increasing the interception and capture efficiency and reducing the hydrodynamic resistance between the cells and the posts. Computational analysis is done for different designs of the posts. Drag on the microposts due to fluid flow has a great significance on the capture efficiency of the chip. Also, the arrangement of the posts is important to contributing to the increase in the interception efficiency. The effects of these parameters on the efficiency in junction with other factors have been studied and quantified. The study is concluded by discussing design strategies with a focus on leveraging the underlying transport phenomena to maximize device performance.

Indiana University-Purdue University Indianapolis (IUPUI) / The purpose of this thesis is to use experimental methods to seek deeper understanding and better performance in the self-circulating self-regulating microfluidic gas generator initially developed in Dr. Zhu’s group, by changing the major features and dimensions in the reaction channel of the device. In order to effectively conduct experiments described above, a microfabrication method that is capable of making new microfluidic devices with low cost, short time period, as well as relatively high accuracy was needed first. Developing such a fabrication method is the major part of this thesis. We initially used patterned polymer films and glass slide, and bonded them together by sequentially aligning and stacking them into a microfluidic device with patterned double-sided tapes. Later we developed a more advanced microfabrication method that used only patterned polystyrene (PS) films. The patterned PS films were obtained from a digital cutter and they were bonded into a microfluidic device by thermopress bonding method that required no heterogeneous bonding agents. This new method did not need manual assembly which greatly improved its precision (~ 100 µm), and it used only PS as device material that has favorable surface wetting property for microfluidics applications. In order to find the optimized microfluidic channel design to improve gas generating performance, we've designed and fabricated microfluidic devices with different channel dimensions using the PS fabrication method. Based on the gas generation testing results of those devices, we were able to come up with the optimal dimensions for the reaction channel that had the best gas generation performance. To obtain a more fundamental understanding about the working mechanism of our device and its bubble dynamics, we have conducted ultrafast X-ray imaging test at Advanced Photon Source (APS), Argonne National Laboratory. High speed (100 KHz) phase contrast images were captured that allowed us to observe directly inside the reaction channel on the cross section view during the self-circulating catalytic reaction. The images provided us with lots of insightful information that in turn helped the dimensional improvement for the microchannel design. The 100 KHz high speed images also gave us useful information about the dynamics of bubble development on a catalyst bed, such as growth and merging of the bubbles.