Fischell Department of Bioengineering Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/6628
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Item SEROTONIN SENSOR-INTEGRATED IN VITRO SYSTEMS AS RESEARCH TOOLS TO ADDRESS THE GUT BRAIN AXIS(2022) Chapin, Ashley Augustiny; Ghodssi, Reza; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The gut-brain-axis (GBA) is a bi-directional communication system between the gastrointestinal (GI) enteric nervous system and the central nervous system, capable of complex crosstalk between the gut and the brain to maintain GI homeostasis and influence mood and higher cognitive functions. Under healthy conditions, this communication is beneficial for regulating immune function, proper peristaltic motion, and hormone release related to hunger and feeding behaviors. However, GBA communication can cause co-morbid occurrence of both GI and neural disorders. For instance, chronic inflammatory conditions of the gut, such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), often present with symptoms of depression and anxiety. Clinical studies, animal models, and molecular research techniques have implicated serotonin (5-HT) as a key signaling molecule to both regulate GI functions and stimulate enteric nerves. These studies are limited by the inability to study sub-mucosal 5-HT on the basolateral side of the epithelium, wheremost of the 5-HT is released and acts on nerves endings. The ability to measure 5-HT release patterns in this area, at native spatial and temporal scales, within an in vitro culture of the gut epithelium, would allow researchers to distinguish 5-HT release patterns stimulated by different GI luminal conditions associated with health and disease, to better understand how these stimuli affect the brain. In this dissertation, electrochemical sensors are fabricated within two types of in vitro platforms to measure 5-HT at physiological scales (sub-micromolar concentrations). The goal of this design is to facilitate the direct detection of 5-HT released from cells cultured in the platform to improve both spatial and temporal access to basolaterally-secreted molecules and provide continuous, automated measurements over experimental time scales. 5-HT sensors fabricated on both porous and smooth cell culture substrates are demonstrated, achieving sensitivities of ~1 – 10 μA/μM and limits of detection of ~100 nM. Electrochemical characterization allow understanding of 5-HT adsorption kinetics, which was modeled to track and predict sensor fouling over continuous measurements. These sensor-integrated substrates were packaged in 3D printed structures, which allowed rapid fabrication of custom designs and were shown to be biocompatible and support growth of RIN14B cells, a model 5-HT-secreting cell line. Finally, cell-secreted 5-HT was detected at ~100 – 500 nM, corresponding to ~4 pmol 5-HT / 105 cells. Ultimately, slow adsorption kinetics prevented direct detection of 5-HT from cells cultured directly on top of the sensors, but the thorough characterization of the platform demonstrated here lays significant groundwork for future optimization of the sensing protocol.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.