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 ENABLING RAPID PHENOTYPIC DETECTION OF CEPHALOSPORIN RESISTANCE BEYOND THE CENTRAL LABORATORY(2019) Nguyen, Hieu Thuong; White, Ian; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The so-called bacterial “superbugs” are largely resistant to some of the most commonly prescribed antibiotics, including a drug class known as cephalosporins used to treat many hospital and community-acquired infections. This major public health threat has been acknowledged for decades by the Centers for Disease Control (CDC) as a major concern; yet, the detection of superbugs has not been made routine since standard testing practices have been limited to specialized “central” laboratories with sophisticated yet bulky and expensive equipment and highly trained personnel. As a result, the lack of simpler testing methods that can be used in everyday clinics and doctor’s offices can be viewed as a source of error contributing to incorrect antibiotic treatment and poorer patient outcomes, factors that drive even more advanced resistance, depleting our drugs or last resort. In this dissertation, we explore new strategies for simplified methods to test for cephalosporin resistance in order to give higher accessibility in the timely detection of superbugs to support the improvement of patient care. To do this, we take an organic chemistry and biochemical approach to develop new detection molecules that report resistance activity in bacteria expressing extended-spectrum β-lactamase (ESBL) enzymes, one of the most prolific resistance strategies used by superbugs. Next, we describe methods of integrating these detection molecules into practical testing methods, and detail the engineering of simpler assays that allow for rapid readout of ESBL phenotypes using commonplace laboratory plate readers, portable Raman devices, and even handheld personal glucose meters (used for diabetes monitoring) purchased from the drugstore.Item Dual Quorum Quenching Capsules: Disrupting two bacterial communication pathways that lead to virulence(2016) Rhoads, Melissa Katherine; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Healthcare Associated Infections (HAIs) in the United States, are estimated to cost nearly $10 billion annually. And, while device-related infections have decreased, the 60% attributed to pneumonia, gastrointestinal pathogens and surgical site infections (SSIs) remain prevalent. Furthermore, these are often complicated by antibacterial resistance that ultimately cause 2 million illnesses and 23,000 deaths in the US annually. Antibacterial resistance is an issue increasing in severity as existing antibiotics are losing effectiveness, and fewer new antibiotics are being developed. As a result, new methods of combating bacterial virulence are required. Modulating communications of bacteria can alter phenotype, such as biofilm formation and toxin production. Disrupting these communications provides a means of controlling virulence without directly interacting with the bacteria of interest, a strategy contrary to traditional antibiotics. Inter- and intra-species bacterial communication is commonly called quorum sensing because the communication molecules have been linked to phenotypic changes based on bacterial population dynamics. By disrupting the communication, a method called ‘quorum quenching’, bacterial phenotype can be altered. Virulence of bacteria is both population and species dependent; each species will secrete different toxic molecules, and total population will affect bacterial phenotype9. Here, the kinase LsrK and lactonase SsoPox were combined to simultaneously disrupt two different communication pathways with direct ties to virulence leading to SSIs, gastrointestinal infection and pneumonia. To deliver these enzymes for site-specific virulence prevention, two naturally occurring polymers were used, chitosan and alginate. Chitosan, from crustacean shells, and alginate, from seaweed, are frequently studied due to their biocompatibility, availability, self-assembly and biodegrading properties and have already been verified in vivo for wound-dressing. In this work, a novel functionalized capsule of quorum quenching enzymes and biocompatible polymers was constructed and demonstrated to have dual-quenching capability. This combination of immobilized enzymes has the potential for preventing biofilm formation and reducing bacterial toxicity in a wide variety of medical and non-medical applications.Item Interference Studies Using Multidimensional Mapping of Cross-Reactive Sensors: Applications in Blood Monitoring of Clozapine(2014) Chocron, Sheryl E.; Ghodssi, Reza; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Point-of-care sensors are used in clinical applications for diagnosing and monitoring health conditions. For example, a point-of-care sensor for therapeutic drug monitoring of the clozapine antipsychotic can reduce burdens from guidelines suggesting routine monitoring of this medication. However, when measuring chemical markers in complex fluids, there are challenges related to decreased sensor performance due to chemical interference. This work presents a methodology for identifying individual interfering species. A set of cross-reactive electrochemical sensors were developed, whose diversified responses provide a fingerprint-type pattern capable of differentiating various species. By mapping the multidimensional responses, patterns from complex solutions were discerned and matched to those of individual species. Applying this methodology to clozapine sensing in blood, a major source of chemical interference was identified. The understanding matrix components that cause interference can guide the design of reliable sensing systems and can be integrated with pattern recognition tools that can account for it.Item Evaluation of Optical Sensor Platforms for Multiplexed Detection of Proteins(2014) Spindel, Samantha; Sapsford-Medintz, Kim E; White, Ian; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This work investigated optical sensor platforms for protein multiplexing, the ability to analyze multiple analytes simultaneously. Multiplexing is becoming increasingly important for clinical needs because disease and therapeutic response often involve the interplay between a variety of complex biological networks involving multiple, rather than single, proteins. Moreover, one biomarker may be indicative of more than one disease, similar diseases can manifest with similar physical symptoms, and monitoring a disease requires the ability to detect subtle differences over time. Multiplexing is generally achieved through one of two routes, either through spatial separation on a surface (different wells or spots) or with the use of unique identifiers/labels (such as spectral separation - different colored dyes, or unique beads - size or color). We looked into combining both spatial separation and unique labels to further expand the multiplexing capabilities of surfaces. Our original research resulted in one of the few demonstrations of reactive semiconductor nanocrystal immunoassays for multiplexed analysis within a single well on a microtiter plate. Innovative planar surface fluorescent immunoassays were developed for both spatial and spectral multiplexing using Quantum Dots and prospective incorporation into a Point-of-Care (POC) device involving an evanescent wave scanner. These assays used standard microscope slides combined with flow cells and were designed to markedly reduce the amount of sample and reagents needed as compared to standard 96-well plate assays. The platform was optimized for detecting Chicken IgG and Staphylococcal Enterotoxin B (SEB); SEB is commonly used in the literature to characterize the performance of biosensor platforms. The planar surface fluorescent immunoassays were applied to a real-world public health need to detect renal injury. Two emerging novel biomarkers, Kidney Injury Marker-1 (KIM-1) and Neutrophil Gelatinase Associated Lipocalin (NGAL), were investigated for their potential to detect injury earlier and with more specificity than current methods using serum creatinine (SCr). Detecting these medically-relevant markers using planar surface fluorescence immunoassays could potentially allow for more rapid diagnosis of acute kidney injury (AKI), among other uses.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.