Fischell Department of Bioengineering Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/6628
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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 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 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 ENZYME INHIBITION IN MICROFLUIDICS FOR RE-ENGINEERING BACTERIAL SYNTHESIS PATHWAYS(2009) LARIOS BERLIN, DEAN EDWARD; RUBLOFF, GARY W; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Enzyme-functionalized biological microfluidic (EF-BioMEMS) systems are an emerging class of lab-on-chip devices that manipulate enzymatic pathways by localizing reaction sites in a microfluidic network. An EF-BioMEM system was fabricated to demonstrate biochemical enzyme inhibition. Further, design optimizations to the EF-BioMEM system have been proposed which improve system sensitivity and performance. The pfs enzyme is part of the quorum-sensing pathway that ultimately produces the bacterial signaling molecule AI-2. An EF-BioMEM system was fabricated to investigate the pfs conversion activity in the presence of a transition state analogue inhibitor. A reduction in enzyme conversion was measured in microfluidics for increasing inhibitor concentration that was comparable to the response expected on a larger scale. This EF-BioMEMS testbed is capable of investigating other compounds that inhibit quorum sensing. Design improvements were demonstrates that improve overall system responsiveness by minimizing unintended reactions from non-specifically bound enzyme. EF-BioMEMS signal-to-background performance increased from 0.72 to 2.43.