Theses and Dissertations from UMD
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New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a give thesis/dissertation in DRUM
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Item Lab-on-a-Chip Integration of Size-based Separation Techniques for Isolation of Bacteria from Blood(2018) Han, Jung Yeon; DeVoe, Don L; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Clinical sample preparation is an essential process in modern diagnostics for maximizing sensitivity and specificity of detection and for ensuring reliability of assay readout. In general, sample preparation typically involves isolating and concentrating a population of target molecules, cells, or particles together with the removal of undesired components from specimen that could otherwise interfere with target detection. The identification of bacteria from complex clinical matrices such as blood presents a particular sample preparation challenge. Conventional culture-based methods typically require at least 24 h of incubation time, making this approach unsuitable for use in rapid diagnostics. Therefore, the development of sample preparation methods for bacteria with rapid processing time, high purification efficiency, and large volumetric throughput to enable analysis of low bacteria concentrations in blood remains a key challenge. This dissertation is focused on realizing a universal platform for preparing microbial sample from blood that is free lysis buffer, electric field, or affinity-based capture methods. First, we developed the porous silica monolith elements integrated into thermoplastic devices for isolation of intact bacteria from blood, enabling the application of emerging detection methods that supports bacterial identification from purified cell populations. Second, to support high throughput analysis of blood samples procured in resource-limited environments, microfluidics elements integrated directly into a syringe are demonstrated by utilizing the deterministic lateral displacement technique and the Dean flow focusing methods. Through these approaches blood cell reduction prior to bacteria isolation can be achieved, thereby increasing the overall sample volume that may be processed by the system. Additionally, a miniaturized hydrocylone capable of operating at tens of milliliters per minute feed rate is presented. Complex microstructures successfully realized at a hundred-micron scale by 3D printing technique presented a promising route to the unconventional microfluidic systems. Lastly, we demonstrated ancillary microfluidic components required to enable full operation of the system in a low-cost lab-on-a-chip format suitable for implementation in resource-limited environments and optimize overall operation of the platform to achieve throughput, sensitivity, and selectivity suitable for clinical application when coupling the platform with downstream detection methods designed for assay readout from intact bacteria.Item DYNAMICS OF CAPSULES IN COMPLEX MICROFLUIDIC DEVICES(2018) Koolivand, Abdollah; Dimitrakopoulos, Panagiotis; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The dynamics of micro-capsules has attracted a lot of attention in the last decade due to their vast applications in different industrial sectors such as cosmetic products, food industry, chemical processes, reaction systems, cell modeling, drug delivery, and medical processes. Additionally, biological cells such as red blood cells can be modeled as capsules. Understanding the rheological behavior of these cells provides great physical insight for early diagnosis of relevant diseases. The main objective of this research is to investigate the effects of physical and geometrical parameters on the hydrodynamics of simple and multiple capsules in complex mi- crofluidic devices. For this purpose, we have developed the mathematical formulation needed for modeling multiple capsules with or without complex internal structures. The developed framework provides an enormous flexibility in problem definition, and facilitates the investigation of the hydrodynamics of a wide class of capsules in microfluidic channels and vascular capillaries. We first study the deformation of a spherical capsule in a T-junction channel. It is shown that an initially spherical capsule develops a bean shape at low flow rates and an inverse kayak shape at high flow rates. Based on the non-trivial deformation of the capsule, a new methodology for the determination of membrane moduli is proposed. For an accurate determination of the membrane moduli, it is paramount to measure the capsule dimensions precisely, which is easier in the proposed device owning to the stagnation-point flow of the T-junction. To determine the membrane moduli, one needs to do a single experiment for different flow rates, and compare the experimental measurements of the capsule steady-state dimensions with the provided computational data. We then consider the flow dynamics of non-spherical capsules and investi- gate the effects spheroidity and initial orientation on the steady-state shape. It is found that a non-spherical capsule, placed with a non-zero initial orientation angle along the centerline of a microchannel, does not practically rotate during deforma- tion. Thus, precise instrumentation is required for proper alignment of the capsule which influences the deformation and steady-state shape. This behavior may explain possible inconsistencies between measured (experimental) and calculated (compu- tational) shapes. We then study the lateral migration of capsules with different size in a mi- crofluidic channel with a trapezoidal cross-section. Owing to the emergence of 3D printing technology, fabrication of a channel with trapezoidal cross-section is fea- sible. Based on our computational data, we proposed an optimized geometry that could be utilized for separation of capsules or cells with different size. The main advantage of the proposed geometry is its inexpensive fabrication cost without the need for incorporating complicated inner structures, which automatically eliminates the risk of channel clogging. Moreover, the simple structure of the trapezoidal mi- crochannel allows an easy scale out through parallelization and reduction of the cell sorting time. In addition, we investigate the complex behavior of two (equal or unequal sized) capsules flowing in a square microfluidic channel. Capsules merging process controls the on-demand drug release and reaction. Thus, we identified the hydro- dynamic conditions that facilitates or hinders the merging of the capsules. The merging process is commonly accompanied by the drainage of existing liquid film between two particles. We observed that the capsules merging in most cases is ac- companied by the formation of dimple surfaces, and thus a simplified flat lubrication surface assumption which is widely-used in the theoretical studies might not be an ideal choice for modeling the film drainage time in merging process.Item THERMOPLASTIC MICROFLUIDIC PCR TECHNOLOGIES FOR NEAR-PATIENT DIAGNOSTICS(2017) Sposito, Alex J.; DeVoe, Don L; Mechanical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Microfluidic technologies have great potential to help create portable, scalable, and cost-effective devices for rapid polymerase chain reaction (PCR) diagnostics in near patient settings. Unfortunately, current PCR diagnostics have not reached ubiquitous use in such settings because of instrumentation requirements, operational complexity, and high cost. This dissertation demonstrates a novel platform that can provide reduced assay time, simple workflow, scalability, and integration in order to better meet these challenges. First, a disposable microfluidic chip with integrated Au thin film heating and sensing elements is described herein. The system employs capillary pumping for automated loading of sample into the reaction chamber, combined with an integrated hydrophilic valve for precise self-metering of sample volumes into the device. With extensive multiphysics modeling and empirical testing we were able to optimize the system and achieve cycle times of 14 seconds and completed 35 PCR cycles plus HRMA in a total of 15 minutes, for successful identification of a mutation in the G6PC gene indicative of von Gierke’s disease. Next, a scalable sample digitization method that exploits the controlled pinning of fluid at geometric discontinuities within an array of staggered microfluidic traps is described. A simple geometric model is developed to predict the impact of device geometry on sample filling and discretization, and validated experimentally using fabricated cyclic olefin polymer devices. Finally, a 768-element staggered trap array is demonstrated, with highly reliable passive loading and discretization achieved within 5 min. Finally, a technique for reagent integration by pin spotting affords simplified workflow, and the ability to perform multiplexed PCR. Reagent printing formulations were optimized for stability and volume consistency during spotting. Paraffin wax was demonstrated as a protective layer to prevent rehydration and reagent cross contamination during sample loading. Deposition was accomplished by a custom pin spotting tool. A staggered trap array device with integrated reagents successfully amplified and validated a 2-plex assay, showing the potential of the platform for a multiplexed antibiotic resistance screening panel.Item Integration of virus-like particle macromolecular bioreceptors in electrochemical biosensors(2016) Zang, Faheng; Ghodssi, Reza; Electrical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Rapid, sensitive and selective detection of chemical hazards and biological pathogens has shown growing importance in the fields of homeland security, public safety and personal health. In the past two decades, efforts have been focusing on performing point-of-care chemical and biological detections using miniaturized biosensors. These sensors convert target molecule binding events into measurable electrical signals for quantifying target molecule concentration. However, the low receptor density and the use of complex surface chemistry in receptors immobilization on transducers are common bottlenecks in the current biosensor development, adding to the cost, complexity and time. This dissertation presents the development of selective macromolecular Tobacco mosaic virus-like particle (TMV VLP) biosensing receptor, and the microsystem integration of VLPs in microfabricated electrochemical biosensors for rapid and performance-enhanced chemical and biological sensing. Two constructs of VLPs carrying different receptor peptides targeting at 2,4,6-trinitrotoluene (TNT) explosive or anti-FLAG antibody are successfully bioengineered. The VLP-based TNT electrochemical sensor utilizes unique diffusion modulation method enabled by biological binding between target TNT and receptor VLP. The method avoids the influence from any interfering species and environmental background signals, making it extremely suitable for directly quantifying the TNT level in a sample. It is also a rapid method that does not need any sensor surface functionalization process. For antibody sensing, the VLPs carrying both antibody binding peptides and cysteine residues are assembled onto the gold electrodes of an impedance microsensor. With two-phase immunoassays, the VLP-based impedance sensor is able to quantify antibody concentrations down to 9.1 ng/mL. A capillary microfluidics and impedance sensor integrated microsystem is developed to further accelerate the process of VLP assembly on sensors and improve the sensitivity. Open channel capillary micropumps and stop-valves facilitate localized and evaporation-assisted VLP assembly on sensor electrodes within 6 minutes. The VLP-functionalized impedance sensor is capable of label-free sensing of antibodies with the detection limit of 8.8 ng/mL within 5 minutes after sensor functionalization, demonstrating great potential of VLP-based sensors for rapid and on-demand chemical and biological sensing.Item Image-Guided Precision Manipulation of Cells and Nanoparticles in Microfluidics(2016) Cummins, Zachary; Shapiro, Benjamin; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Manipulation of single cells and particles is important to biology and nanotechnology. Our electrokinetic (EK) tweezers manipulate objects in simple microfluidic devices using gentle fluid and electric forces under vision-based feedback control. In this dissertation, I detail a user-friendly implementation of EK tweezers that allows users to select, position, and assemble cells and nanoparticles. This EK system was used to measure attachment forces between living breast cancer cells, trap single quantum dots with 45 nm accuracy, build nanophotonic circuits, and scan optical properties of nanowires. With a novel multi-layer microfluidic device, EK was also used to guide single microspheres along complex 3D trajectories. The schemes, software, and methods developed here can be used in many settings to precisely manipulate most visible objects, assemble objects into useful structures, and improve the function of lab-on-a-chip microfluidic systems.Item INTERFACIAL CONSIDERATIONS FOR DROPLET PCR LAB-ON-CHIP DEVICES(2015) Pandit, Kunal; White, Ian M; Raghavan, Srinivasa R; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Lab-on-chip devices have the potential to decentralize the current model of diagnostics to point-of-care diagnostics. Easy to use, low cost, rapid infectious disease diagnostic tools could especially impact and improve healthcare in low resource areas. Micro-total-analytical-systems could also enable smarter medical decisions, quicker patient recoveries, and cheaper healthcare costs in fully developed settings. Significant innovations to standard technologies used today will help realize the promise of lab-on-chip devices. In this work, innovative technologies compatible with current lab-on-chip devices were investigated to simplify their operation, decrease their complexity, and reduce their cost. The interfacial aspects that dominate microfluidic systems, and in particular droplet polymerase chain reaction (PCR) devices, are emphasized. Droplet PCR utilizing microfluidic technology has largely been automated, but sample preparation methods prior to amplification remains a laborious process. We have developed particles that condense the many steps of sample preparation into a single buffer protocol. The particles were made by crosslinking chitosan, a pH responsive biopolymer. DNA was electrostatically and sterically adsorbed to the beads at pH 8.5. Furthermore, amplification of DNA directly off the beads was demonstrated eliminating the need to desorb DNA into solution. Implementation of these particles will drastically simplify droplet PCR lab-on-chip devices. We also characterized the adsorption of polymerase at the oil-water interfaces of droplets and identified a surfactant to prevent the loss of polymerase in solution. The pendant drop technique was used to observe the change in interfacial tension due to adsorption of Taq Pol and/or surfactants to the interface. PCR performance of two surfactants, Brij L4 and ABIL EM90, were predicted from equilibrium interfacial tension measurements. Brij L4, a surfactant that had never been used with PCR, prevented polymerase adsorption and enabled more efficient PCR than ABIL EM90, a popular PCR surfactant. Lastly, we ambitiously designed a system to conduct droplet PCR without oil or surfactants. Droplets were generated on-chip by adapting a co-flow droplet generating device previously developed in our group. Then droplets were immobilized on-chip in hydrodynamic traps. Two different modes of trapping were demonstrated, indirect and direct. Also, all aspects of an air continuous phase droplet PCR device were considered such as protein adsorption to channel walls and droplet evaporation during thermal cycling.Item New Sensing Modalities for Bacterial and Environmental Phenomena(2013) Betz, Jordan; Rubloff, Gary W; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Intercellular communication is a ubiquitous phenomenon across all domains of life, ranging from archaea to bacteria to eukarya. In bacteria, this is often achieved using small molecules that allow bacteria to sense and respond to environmental cues about the presence, identity, and number of neighboring bacteria. This confers survival and competitive advantages to bacteria by providing a coordinated, population-scale response to a given stimulus in the environment. This dissertation describes the development of a microfluidic system for immobilizing and culturing of cells that also enables control over the genetic composition of the bacteria and their subsequent response to environmental stimuli via a new nonviral nucleic acid delivery mechanism. This nonviral nucleic acid delivery occurs outside the parameter space of traditional nonviral nucleic acid delivery methods such as electroporation and chemical transformation. The bacteria are immobilized in an optically clear alginate hydrogel which simulates the physical and chemical environment normally experienced by bacteria in a biofilm. Complementing the microfluidic cell culture work, surface enhanced Raman spectroscopy (SERS), a label-free vibrational spectroscopic technique that lends itself well to use in aqueous systems, was used to detect bacterial signaling molecules. SERS was performed with three different examples of bacterial communication molecules: the universal quorum sensing molecule autoinducer-2 (AI-2), the species-specific Pseudomonas Quinolone Signal (PQS), and the stationary phase indicator molecule indole. SERS substrates were formed by galvanic displacement, a substrate fabrication method that can be adapted to many SERS applications. Taken together, these new sensing modalities represent a step toward developing systems that allow researchers to investigate, understand, and ultimately control a cell's response to its environment.Item Microfluidic Planar Phospholipids Membrane System Advancing Dynamics Studies of Ion Channels and Membrane Physics(2012) Shao, Chenren; DeVoe, Donald L; Mechanical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The interrogation of lipid membrane and biological ion channels supported within bilayer phospholipid membranes has greatly expanded our understanding of the roles membrane and ion channels play in a host of biological functions. Several key drawbacks of traditional electrophysiology systems used in these studies have long limited our effort to study the ion channels. Firstly, the large volume buffer in this system typically only allows single or multiple additions of reagents, while complete removal either is impossible or requires tedious effort to ensure the stability of membrane. Thus, it has been highly desirable to be able to rapidly and dynamically modulate the (bio)chemical conditions at the membrane site. Second, it is difficult to change temperature effectively with large thermal mass in macro device. Third, traditional PPM device host vertical membranes, therefore incompatible with confocal microscopy techniques. The miniaturization of bilayer phospholipid membrane has shown potential solution to the drawbacks stated above. A simple microfluidic design is developed to enable effective and robust dynamic perfusion of reagents directly to an on-chip planar phospholipid membrane (PPM). It allows ion channel conductance to be readily monitored under different dynamic reagent conditions, with perfusion rates up to 20 µL/min feasible without compromising the membrane integrity. It is estimated that the lower limit of time constant of kinetics that can be resolved by our system is 1 minute. Using this platform, the time-dependent responses of membrane-bound ceramide ion channels to treatments with La3+ and a Bcl-xL mutant were studied and the results were interpreted with a novel elastic biconcave distortion model. Another engineering challenge this dissertation takes on is the integration of fluorescence studies to micro-PPM system. The resulting novel microfluidic system enables high resolution, high magnification and real-time confocal microscope imaging with precise top and bottom (bio)chemical boundary conditions defined by perfusion, by integrating in situ PPM formation method, perfusion capability and microscopy compatibility. To demonstrate such electro-optical chip, lipid micro domains were imaged and quantitatively studied for their movements and responses to different physical parameters. As an extension to this platform, a double PPM system has been developed with the aim to study interactions between two membranes. Potential application in biophysics and biochemistry using those two platforms were discussed. Another important advantage of microfluidics is its lower thermal mass and compatibility with various microfabrication methods which enables potential integration of local temperature controller and sensor. A prototype thermal PPM chip is also discussed together with some preliminary results and their implication on ceramide channel assembly and disassembly mechanism.Item Oxygen Measurement During Cell Culture: From Multiwell Plates to Microfluidic Devices(2011) Thomas, Peter Chung; Forry, Samuel P; Raghavan, Srinivasa R; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Oxygen is an important regulator of normal cell behavior. Proper supply of oxygen is required to maintain ATP production, while perturbation of oxygen supply alters cell behavior and leads to tissue damage and cell death. In vivo, cells are exposed to a mean partial pressure of oxygen between 0.03 to 0.09 atm that is tissue specific. In contrast, conventional cell cultures are routinely performed at an atmospheric oxygen level of 0.21 atm. The disparity between in vivo and in vitro oxygen levels have been shown to affect cell viability, growth and differentiation. Continuous measurements and control of oxygen levels are thus critical to maintaining proper cell behavior. Current methods of oxygen measurement are invasive, difficult to integrate with microscopy and lack imaging capabilities. To improve the current state of measurements, we have developed a new non-invasive oxygen sensor for in vitro cell culture. The sensor was prepared by incorporating a porphyrin dye, Pt(II) meso-Tetra(pentafluoro-phenyl)porphine (PtTFPP), into gas permeable poly(dimethylsiloxane) (PDMS) thin films. The response of the sensor to oxygen followed the linear Stern-Volmer equation and demonstrated an order of magnitude higher sensitivity compared to other sensors (KSV = 548 ± 71 atm-1). A multilayer design created by sandwiching the PtTFPP-PDMS with a thin film of Teflon AF followed by a second layer of PDMS effectively mitigated against cytotoxicity effects and provided a suitable substrate for cell attachment. To demonstrate the utility of the sensor, oxygen measurements were made continuously with NIH 3T3 mouse fibroblast cells. The oxygen levels were found to decrease as a result of oxygen consumption by the cells. Using Fick's law, the data was analyzed and a per-cell oxygen consumption rate for the 3T3 fibroblasts was calculated. In addition, cells were clearly visualized on the sensor demonstrating the ability to integrate with phase-contrast and fluorescence microscopy. Next, human hepatocellular carcinoma HepG2 were cultured on the oxygen sensor and continuous oxygen measurements showed a drastic decrease in oxygen level such that the cells were exposed to hypoxic conditions within 24 h. The per-cell oxygen consumption rate for HepG2 was determined to be 30 times higher than the 3T3 fibroblasts, confirming the high metabolic nature of these cells. At high densities, oxygen flux measurements showed an asymptotic behavior reaching the theoretical maximum of the culture condition. When the oxygen diffusion barrier was reduced, the oxygen flux increased, demonstrating insufficient oxygenation for HepG2 at these densities. In routine culture, HepG2 adhere to their neighboring cells which results in formation of cell clusters. Oxygen measurement confirmed the presence of oxygen gradient across the cell clusters with the lowest oxygen levels observed in the middle. Finally, we successfully integrated the oxygen sensor into microfluidic systems. The sensor provided real-time non-invasive measurements of oxygen levels on-chip. To regulate the oxygen levels in the device, water with different dissolved oxygen concentrations was used instead of gas. This method successfully mitigated the problems of pervaporation associated with previous devices. Physiologically relevant oxygen levels and oxygen gradients were easily generated on the device and the results showed excellent agreement with numerical simulations.Item Precise steering of particles in electroosmotically actuated microfluidic devices(2010) Chaudhary, Satej; Shapiro, Benjamin; Aerospace Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In this thesis, we show how to combine microfluidics and feedback control to independently steer multiple particles with micrometer accuracy in two dimensions. The particles are steered by creating a fluid flow that carries all the particles from where they are to where they should be at each time step. Our control loop comprises sensing, computation, and actuation to steer particles along user-input trajectories. Particle positions are identified in real-time by an optical system and transferred to a control algorithm that then determines the electrode voltages necessary to create a flow field to carry all the particles to their next desired locations. The process repeats at the next time instant. Our method achieves inexpensive steering of particles by using conventional electroosmotic actuation in microfluidic channels. This type of particle steering has significant advantages over other particle steering methods, such as laser tweezers. (Laser tweezers cannot steer reflective particles, or particles where the index of refraction is lower than (or for more sophisticated optical vortex holographic tweezers does not differ substantially from) that of the surrounding medium.). In this thesis, we address three specific aspects of this technology. First, we develop the control algorithms for steering multiple particles independently and validate our control techniques using simulations with realistic sources of initial position errors and system uncertainties. Second, we develop optimal path planning methods to efficiently steer particles between given initial and final positions. Third, we design high performance microfluidic devices that are capable of simultaneously steering five particles in experiment.