Preclinical testing is necessary to investigate the safety and efficacy of novel therapeutics before moving to clinical trials, yet approximately 90% of these therapies fail once tested in humans. This has led to increased interest in developing robust preclinical models that accurately mimic the complex human in vivo physiology. Microfluidic devices that can introduce dynamic conditions to 3D cell/organoid cultures, also known as tissue-on-a-chip, have emerged as physiologically relevant in vitro preclinical models that can achieve high throughput screening of therapeutics. The research presented here aimed to develop an angiogenic environment within a novel microfluidic device to stimulate formation of endothelial networks that will eventually be integrated into a vascularized tumor model for screening chemotherapeutics. The novel microfluidic devices were fabricated using photolithography to create a patterned mold, casting polydimethylsiloxane (PDMS) over the mold, and bonding patterned PDMS to a glass slide. Three sets of experiments were then conducted, with each introducing different angiogenic stimuli to human umbilical vein endothelial cells (HUVECs) co-cultured with human dermal fibroblasts (HDFs) within the devices. The first set of experiments sought to develop a standard protocol for plating human cells in the novel microfluidic device and to investigate if the mechanism of nutrient transport and interstitial flow would induce an angiogenic response resulting in endothelial network formation. A working protocol was developed but it was determined that further development of an angiogenic environment within the device was necessary to stimulate endothelial network formation. The second set of experiments investigated if seeding HUVECs in a peripheral channel of the device and introducing a concentration gradient of vascular endothelial growth factor (VEGF) would stimulate endothelial network formation directed by a growth factor gradient, similar to angiogenesis in vivo. This was repeated under hypoxic conditions to more accurately mimic the in vivo angiogenic environment, but significant endothelial network formation was not observed and seeding of HUVECs in the peripheral channel presented no perceptible improvements. The final set of experiments investigated if v returning HUVECs to the center chamber in local co-culture with HDFs and exposing devices to hypoxic conditions would provide the necessary angiogenic environment to stimulate endothelial network formation within the microfluidic device. Lack of quantifiable endothelial network formation in the final set of experiments led to an analysis of 3D HUVEC colony formation, however, no statistically significant trends were discovered. Even though no significant differences were found, these experiments succeeded in developing a protocol for plating human cells in the novel microfluidic device that can be translated to the tumor side of the Microphysiological Systems lab. From these experiments we can also conclude that co-cultures of HUVECs and HDFs can survive and form into colonies within the novel microfluidic device but additional angiogenic stimuli are necessary to develop robust endothelial networks. Based on the current literature and knowledge gained throughout the experiments presented here, several suggestions are presented to potentially stimulate angiogenesis and develop endothelial networks in the device such as increasing cell densities, varying length of incubation, introducing mediators of angiogenesis like nitric oxide, and addition of tumor cells.

Colorectal cancer is the second most common cause of cancer-related deaths in the United States, with the relative 5-year survival rate for distant stage cancer being only 14%. The most common treatment for colorectal cancer is with chemotherapeutic drugs; however, the discovery of these drugs is costly, time-consuming, and often requires the use of animal models that do not yield results that translate to clinical trials. Due to these shortcomings, researchers seek to develop physiologically relevant in vitro tumor models that more accurately mimic the tumor microenvironment for cheaper and faster high-throughput drug screening. The aim of this research was to develop a colorectal cancer tumor model co-cultured with endothelial and stromal cells, followed by validation with clinically relevant chemotherapeutic agents within microfluidic devices. The first experiment consisted of a lipofection of fibroblasts to yield fluorescently tagged cells that could be later imaged using a fluorescence microscope. The next experiment consisted of a co-culture of tumor, endothelial, and fibroblast cells at varying densities in a twodimensional (2D) culture to determine the optimal plating densities that would yield quantifiable tumor and endothelial network formation. The following experiment used these optimal densities to test the effects of the chemotherapeutic agents oxaliplatin and SN38 on the tumor and endothelial cells in 2D. After the various densities and drug concentrations were tested in 2D, the model was introduced into microfluidic devices. The first experiment in the devices was similar to the first experiment plated in 2D, as it involved the establishment of optimal plating densities of all three cell types within the devices. Similarly, the goal of this experiment was to yield quantifiable tumor and endothelial network formation within the devices. The final experiment performed in this research was the introduction of oxaliplatin and SN38 to the optimized densities v of cells determined from the previous experiment, with the aim of evaluating the effects of these chemotherapeutic agents on the tumor and endothelial cells within microfluidic devices. The two experiments plated in 2D established plating densities to be tested in the devices. These experiments also showed that increasing drug concentrations resulted in reduced tumor count and size and revealed no disruption in the endothelial networks when exposed to oxaliplatin concentrations as high as 50 µM. The final two experiments in microfluidic devices revealed that endothelial network formation is not yet possible within the devices with the current protocols, but that tumor cells still showed dose-dependent responses to drug exposure as they did in 2D. Due to the lack of network formation in this device model, future work is required to allow for endothelial cell organization into networks, to further increase the physiological relevancy of this model to in vivo tumor conditions.

This research developed methods for culturing self-assembling capillaries in an injectable gel as a potential method for vascularizing tissue-on-a-chip models to mimic physiological drug delivery. Additionally, a mathematical model was developed as a tool for understanding nutrient delivery and comparison of potential delivery systems. Organs-on-a-chip provide novel platforms for studying biology and physiology in 3D, allow exploration of tissue engineering on a manageable scale, and serve as models for drug screening and drug-delivery testing. Methods were first developed for co-culture of endothelial cells and fibroblasts (3T3s or HDFs) in 2D, evaluating culture time, seeding density and ratio of HUVECs and fibroblasts, and immunostaining with a HUVEC-specific marker. Cells formed large sheets with no signs of vessel formation in 2D; therefore, the setup was translated to 3D culture to further induce stress and release of angiogenetic factors, using fibrin gel to suspend cells in 3D. After 9 days of culture, HUVECs had extensive network formation with a high degree of complexity in the experimental cell ratios (especially with 5:1 HUVECs:HDFs). Therefore, these parameters can be used as a starting point for further development of vascularized tissue constructs. A mathematical model was also successfully developed to assess the impact of cell concentration, consumption, and mode of nutrient delivery on 3D cellular constructs which can be used to predict the spatial distribution of glucose over time. Although the model shows flow introduced through a device is sufficient to maintain nutrient levels for cell growth, developing perfusable capillaries is still a critical part of creating physiologically representative tissues.

Surface enhanced Raman spectroscopy (SERS) is highly promising analytical technique for trace detection of analytes. It is particularly well suited for environmental analyses due to its high sensitivity, specificity, ease of operation and rapidity. The detection and characterization of environmental contaminants, using SERS is highly related to the uniformity, activity and reproducibility of the SERS substrate. In this thesis, SERS substrates were produced by gold nanoparticle formation on wax patterned chromatography paper. In situ reduction of hydrogen tetrachloroaurate (gold precursor) by trisodium citrate dihydrate (reducing agent) was used to produce gold nanoparticles within a paper matrix. These gold nanoparticle based SERS substrates were analyzed by FE-SEM, UV-Vis and Raman spectroscopy. This work discusses the SERS signal enhancements for Raman active MGITC dye for a series of substrates prepared by in situ reduction of gold salt and pre-produced gold nanoparticles. UV-Vis analysis was performed to understand the effect of different molar ratio (reducing agent to gold precursor) and reaction time on the size and shape of the localized surface plasmon resonance (LSPR) band that dictates the SERS enhancements. It was concluded that lower molar ratio (1:1 and 2:1) of citrate-to gold produced better SERS signal enhancements and broader LSPR band. Therefore, use of lower molar ratio (MR) was recommended for paper-based substrates using in situ-based reduction approach. / Master of Science

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Existing methods for pathogen/pollutant detection or wound infection monitoring employ high-cost instruments that could only be operated by trained personnel, and costly device-based detection requires a time-consuming field-to-lab process. This expensive process with multiple prerequisites prolongs the time that patients must wait for a diagnosis. Therefore, improved methods for point-of-care biosensing are necessary. In this study, we aimed to develop a direct, easy-to-use, portable, low cost, highly sensitive and selective sensor platform with the goal of pollutant detection and wound infection/cancer migration monitoring. This study has two main parts, including microfluidic, electrical, and optical sensing platforms. The first part, including chapters 2, 3, and 4, focuses on Bisphenol A (BPA) lateral flow assay (LFA) detection; the second part, including chapter 5 focuses on the electrical sensing platform fabrication for one of the markers of inflammation, matrix metalloproteinases-9 (MMP-9), monitoring/detection. In chapters 2, 3, and 4, we found that the few lateral flow assays (LFAs) established for detecting the endocrine-disrupting chemical BPA have employed citrate-stabilized gold nanoparticles (GNPs), which have inevitable limitations and instability issues. To address these limitations, in chapter 2, a more stable and more sensitive biosensor is developed by designing strategies for modifying the surfaces of GNPs with polyethylene glycol and then testing their effectiveness and sensitivity toward BPA in an LFA. In chapter 3, we describe the development of a new range-extended bisphenol A (BPA) detection method that uses a surface enhanced Raman scattering lateral flow assay (SERS-LFA) binary system. In chapter 4, we examine advanced bisphenol A (BPA) lateral flow assays (LFAs) that use multiple nanosystems. The assays include three nanosystems, namely, gold nanostars, gold nanocubes, and gold nanorods, which are rarely applied in LFAs, compared with general gold nanoparticles. The developed LFAs show different performances in the detection of BPA. In chapter 5, a stable electrical sensing platform is developed for MMP-9 detection.