Fischell Department of Bioengineering Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/6628
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Item Enhanced Throughput Single-Cell Capillary Electrophoresis Mass Spectrometry(2023) Mendis, John Udara; Nemes, Peter; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Mass spectrometry (MS) has allowed for the analysis of small molecules and metabolites with high specificity and sensitivity. Capillary Electrophoresis mass spectrometry (CE-MS) is an ultrasensitive analytical technique to process amount-limited samples. Robust high-throughput ultrasensitive CE-MS methods and technologies are needed to be developed to comprehensively study the metabolome or proteome of a sample with a limited amount of material. In this study, we developed an enhanced-throughput multi field amplified sample stacking (M-FASS) method. The resulting approach has a sample processing throughput of 5–10 times that of conventional CE methods. FASS voltage duration and strength were optimized for peak area and peak resolution. The M-FASS CE-MS method was then applied for single cell analysis (SCA) of metabolic differences and gradients in the developing embryo of Xenopus Laevis. The statistical analysis: PCA and Fuzzy-c means clustering analysis revealed cell-to-cell differences among D11, V11, D12, and V12 cells and uncovered 6 distinct metabolite gradients between the four cells in X. Laevis 16-cell embryos. The findings showcase inherent metabolic gradients in the developing embryo.Item THE APPLICATION OF MICRODEVICES FOR INVESTIGATING BIOLOGICAL SYSTEMS(2018) Shang, Wu; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The gastrointestinal (GI) tract is a complex ecosystem with cells from different kingdoms organized within dynamically-changing structures and engaged in complex communication through a network of molecular signaling pathways. One challenge for researchers is that the GI tract is largely inaccessible to experimental investigation. Even animal models have limited capabilities for revealing the rich spatiotemporal variation in the intestine and fail to predict human responses due to genetic variation. Exciting recent advances in in vitro organ model (i.e., organ-on- chips (OOC)) based on microfluidics are offering new hope that these experimental systems may be capable of recapitulating the complexities in structure and context inherent to the intestine. A current limitation to OOC systems is that while they can recapitulate structure and context, they do not yet offer capabilities to observe or engage in the molecular based signaling integral to the functioning of this complex biological system. This dissertation focuses on developing microfluidic tools that provide access to interrogating signaling events amongst populations in the GI tract (e.g., microbes and enterocytes). First, a membrane-based gradient generator is built to establish linear and stable chemical gradients for investigating gradient-mediated behaviors of bacteria. Specifically, this platform enables the study of bacterial chemotaxis and potentially facilitates the development of genetically rewired lesion-targeted probiotics. Second, “electrobiofabrication” is coupled with microelectronics, for the first time, to create molecular-to-electronic (i.e., “molectronic”) sensors to observe and report the dynamic exchange of biochemical information in OOC systems. Last, to address the issue of poor compatibility between OOCs and sensors, we assemble OOCs with molectronic sensors in a modular format. The concept of modularity greatly reduces the system complexity and enables sensors to be built immediately before applications, avoiding functional decay of active biorecognition components after long-term device storage and use. We envision this work will “open” OOC systems for molecular measurement and interrogation, which, in turn, will expand the in vitro toolbox that researchers can use to design, build and test for the investigation of GI disease and drug discovery.Item Low-Cost Paper-Based Assays for Multiplexed Genetic Analysis using Surface Enhanced Raman Spectroscopy(2013) Hoppmann, Eric Peter; White, Ian M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In order to improve human health it is critical to develop low-cost sensors for chemical detection and healthcare applications. Low-cost chemical detectors can enable pervasive monitoring to identify health threats. Rapid yet accessible infectious disease diagnostics have the potential to improve patient quality of care, reduce healthcare costs and speed recovery. In both cases, when multiple targets can be detected with a single test (multiplexing), accessibility is improved through lowered costs and simplicity of operation. In this work we have investigated the practical considerations and applications of ink-jet printed paper surface enhanced Raman spectroscopy (SERS) devices. SERS enables specific simultaneous detection of numerous analytes using a single excitation source and detector. Sensitive detection is demonstrated in several real-world applications. We use a low-cost portable spectrometer for detection, further emphasizing the potential for on-site detection. These ink-jet printed devices are then used to develop a novel DNA detection assay, in which the multiplexing capabilities of SERS are combined with DNA amplification through polymerase chain reaction (PCR). In this assay, the chromatographic properties of paper are leveraged to perform discrimination within the substrate itself. As a test case, this assay is then used to perform duplex detection of the Methicillin-resistant Staphylococcus aureus (MRSA) genes mecA and femB, two genes which confer antibiotic resistance on MRSA. Finally, we explore statistical multiplexing methods to enable this assay to be applied to perform highly-multiplexed detection gene targets (5+), and demonstrate the differentiation of these samples using partial least-squares regression (PLS). By averaging the signal over a region of the SERS substrate, substrate variability was mitigated allowing effective identification and differentiation, even for the complex spectra from highly multiplexed samples which were impossible to visually analyze.Item INKJET PRINTED PAPER SURFACE ENHANCED RAMAN SPECTROSCOPY DEVICES FOR TRACE CHEMICAL ANALYSIS(2013) Yu, Wei Wen; White, Ian M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The needs of an ever growing human population are fueling demands for better and cheaper sensors for the early detection of harmful chemicals, pathogens and diseases markers from a variety of sources such as food, water, bodily fluids and contaminated surfaces. To address this, recent innovations utilize Microelectromechanical Systems (MEMS) technology to integrate multiple laboratory functions onto millimeter-sized chips to form Micro Total Analysis Systems (µTAS) or Lab-on-chip (LOC) devices. While sophisticated and powerful, the use of these devices for chemical and biological sensing is limited by complicated fabrication processes, high cost and robustness of the sensors. In this work we have developed a simple and inexpensive but exceptionally sensitive portable chemical and biological sensing platform through the innovative use of paper combined with Surface Enhanced Raman spectroscopy (SERS). Paper is functionalized with plasmonic nanostructures to transform it into a SERS substrate, while the natural properties of paper are leveraged for sample collection, cleanup, and analyte concentration in user-friendly formats such as wipes, dipsticks, and filters. The use of simple deposition methods such as inkjet printing for sensor fabrication combined with paper as the construction material means that sensors can be made at a very low cost. Additionally, the ability to be printed on demand eliminates issues with sensor shelf-life, while the absence of mechanical components makes these paper sensors much more robust than conventional sensors. In this work, practical applications of paper SERS sensors for the detection of food contaminants, narcotics, pesticides and other chemicals at trace levels are presented. Paper SERS sensors, by virtue of their low cost, simplicity of fabrication, high sensitivity and ease of use, promises to make chemical and biological sensing more accessible to the common user.