There are situations in which it is very important to quickly and positively identify an individual. Examples include suspects detained in the neighborhood of a bombing or terrorist incident, individuals detained attempting to enter or leave the country, and victims of mass disasters. Systems utilized for these purposes must be fast, portable, and easy to maintain. The goal of this project was to develop an ultra fast, direct PCR method for forensic genotyping of oral swabs. The procedure developed eliminates the need for cellular digestion and extraction of the sample by performing those steps in the PCR tube itself. Then, special high-speed polymerases are added which are capable of amplifying a newly developed 7 loci multiplex in under 16 minutes. Following the amplification, a postage stamp sized microfluidic device equipped with specially designed entangled polymer separation matrix, yields a complete genotype in 80 seconds. The entire process is rapid and reliable, reducing the time from sample to genotype from 1-2 days to under 20 minutes. Operation requires minimal equipment and can be easily performed with a small high-speed thermal-cycler, reagents, and a microfluidic device with a laptop. The system was optimized and validated using a number of test parameters and a small test population. The overall precision was better than 0.17 bp and provided a power of discrimination greater than 1 in 106. The small footprint, and ease of use will permit this system to be an effective tool to quickly screen and identify individuals detained at ports of entry, police stations and remote locations. The system is robust, portable and demonstrates to the forensic community a simple solution to the problem of rapid determination of genetic identity.

abstract: Glioblastoma Multiforme (GBM) is a grade IV astrocytoma and the most aggressive form of cancer that begins within the brain. The two-year average survival rate of GBM in the United States of America is 25%, and it has a higher incidence in individuals within the ages of 45 - 60 years. GBM Tumor formation can either begin as normal brain cells or develop from an existing low-grade astrocytoma and are housed by the perivascular niche in the brain microenvironment. This niche allows for the persistence of a population of cells known as glioma stem cells (GSC) by supplying optimum growth conditions that build chemoresistance and cause recurrence of the tumor within two to five years of treatment. It has therefore become imperative to understand the role of the perivascular niche on GSCs through in vitro modelling in order to improve the efficiency of therapeutic treatment and increase the survival rate of patients with GBM. In this study, a unique three dimensional (3D) microfluidic platform that permitted the study of intercellular interactions between three different cell types in the perivascular niche of the brain was developed and utilized for the first time. Specifically, human endothelial cells were embedded in a fibrin matrix and introduced into the vascular layer of the microfluidic platform. After spontaneous formation of a vascular layer, Normal Human Astrocytes and Patient derived GSC were embedded in a Matrigel® matrix and incorporated in the stroma and tumor regions of the microfluidic device respectively. Using the established platform, migration, proliferation and stemness of GSCs studies were conducted. The findings obtained indicate that astrocytes in the perivascular niche significantly increase the migratory and proliferative properties of GSCs in the tumor microenvironment, consistent with previous in vivo findings. The novel GBM tumor microenvironment developed herein, could be utilized for further in-depth cellular and molecular level studies to dissect the influence of individual factors within the tumor niche on GSCs biology, and could serve as a model for developing targeted therapies. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2020

Neuropsychiatric disorders severely impact quality of life in millions of patients, contributing more Disease Affected Life Years (DALYs) than cancer or cardiovascular disease. The human brain is a complex system of 100 billion neurons connected by 100 trillion synapses, and human studies of neural disease focus on network-level circuit activity changes, rather than on cellular mechanisms. To probe for neural dynamics on the cellular level, animal models such as the nematode C. elegans have been used to investigate the biochemical and genetic factors contributing to neurological disease. C. elegans are ideal for neurophysiological studies due to their small nervous system, neurochemical homology to humans, and compatibility with non-invasive neural imaging. To better study the cellular mechanisms contributing to neurological disease, we developed automated analysis methods for characterizing the behaviors and associated neural activity during sleep and learning in C. elegans: two neural functions that involve a high degree of behavioral and neural plasticity. We developed two methods to study previously uncharacterized spontaneous adult sleep in C. elegans. A large microfluidic device facilitates population-wide assessment of long-term sleep behavior over 12 hours including effects of fluid flow, oxygen, feeding, odors, and genetic perturbations. Smaller devices allow simultaneous recording of sleep behavior and neuronal activity. Since the onset of adult sleep is stochastically timed, we developed a closed-loop sleep detection system that delivers chemical stimuli to individual animals during sleep and awake states to assess state-dependent changes to neural responses. Sleep increased the arousal threshold to aversive chemical stimulation, yet sensory neuron (ASH) and first-layer interneuron (AIB) responses were unchanged. This localizes adult sleep-dependent neuromodulation within interneurons presynaptic to the AVA premotor interneurons, rather than afferent sensory circuits. Traditionally, the study of learning in C. elegans observes taxis on agar plates which present variable environmental conditions that can lead to a reduction in test-to-test reproducibility. We also translated the butanone enhancement learning assay such that animals can be trained and tested all within the controlled environment of a microfluidic device. Using this system, we demonstrated that C. elegans are capable of associative learning by observing stimulus evoked behavioral responses, rather than taxis. This system allows for more reproducible results and can be used to seamlessly study stimulus-evoked neural plasticity associated with learning. Together, these systems provide platforms for studying the connections between behavioral plasticity and neural circuit modulation in sleep and learning. We can use these systems to further our understanding of the mechanisms underlying neural regulation, function, and disorder using human disease models in C. elegans.

Recent development of super-resolution (SR) fluorescence microscopy techniques has provided a new tool for direct visualization of subcellular structures and their dynamics in cells. The homing of Hematopoietic stem/progenitor cells (HSPCs) to bone marrow is a multistep process that is initiated by tethering of HSPCs to endothelium and mediated by spatiotemporally organised ligand-receptor interactions of selectins expressed on endothelial cells to their ligands expressed on HSPCs which occurs against the shear stress exerted by blood flow. Although molecules and biological processes involved in this multi-step cellular interaction have been studied extensively, molecular mechanisms of the homing, in particular the nanoscale spatiotemporal behaviour of ligand-receptor interactions and their role in the cellular interaction, remain elusive. Using our new method of microfluidics-based super-resolution fluorescence imaging platform we can now characterize the correlation between both nanoscale ligand-receptor interactions and tethering/rolling of cells under external shear stress. We found that cell rolling on E-selectin caused significant reorganization of the nanoscale clustering behavior of CD44 and CD43, from a patchy clusters of ~ 200 nm in size to an elongated network-like structures where for PSGL-1 the clustering size did not change significantly as it was 85 nm and after cell rolling the PSGL-1 aggregated to one side or even exhibited an increase in the footprint. Furthermore, I have established the use of 3D SR images that indicated that the patchy clusters of CD44 localize to protruding structures of the cell surface. On the other hand, a significant amount of the network-like elongated CD44 clusters observed after the rolling were located in the close proximity to the E-selectin surface. The effect of the nanoscale reorganization of the clusters on the HSPC rolling over selectins is still an open question at this stage. Nevertheless, my results further demonstrate that this mechanical force-induced reorganisation is accompanied by a large structural reorganisation of actin cytoskeleton. Our microfluidics-based SR imaging also demonstrate an essential role of the nanoscale clustering of CD44 on stable rolling behaviours of cells. Our new experimental platform enhances understanding of the relationship between nanoscopic ligand-receptor interactions and macroscopic cellular interactions, providing a foundation for characterizing complicated HSPC homing

Abstract Application of Argon Plasma Technology to Hydrophobic and Hydrophilic Microdroplet Generation in PDMS Microfluidic Devices Brennan Graham Microfluidics has gained popularity over the last decade due to the ability to replace many large, expensive laboratory processes with small handheld chips with a higher throughput due to the small channel dimensions [1]. Droplet microfluidics is the field of fluid manipulation that takes advantage of two immiscible fluids to create droplets from the geometry of the microchannels. This project includes the design of a microfluidic device that applies the results of an argon plasma surface treatment to polydimethylsiloxane (PDMS) to successfully produce both hydrophobic and hydrophilic surfaces to create oil in water (O/W) and water in oil (W/O) microdroplets. If an argon plasma surface treatment renders the surface of PDMS hydrophilic, then O/W microdroplets can be created and integrated into a larger microdroplet emulsion device. The major aims of this project include: (1) validating previously established Cal Poly lab protocols to produce W/O droplets in hydrophobic PDMS microdroplet generators (2) creating hydrophilic PDMS microdroplet generators (3) making oil in water droplets in hydrophilic PDMS microdroplet generators (4) designing a multilayer microfluidic device to transfer W/O droplets to a second hydrophilic PDMS microdroplet generator v W/O droplets were successfully created and transferred to a second hydrophilic PDMS device. The hydrophilic PDMS device also produced O/W droplets in separate testing from the multilayered microfluidic PDMS device. The ultimate purpose of this project is to create a multilayer microdroplet generator that produces water in oil in water (W/O/W) microdroplet emulsions through a stacked device design that can be used in diagnostic microdroplet applications. Thesis Supervisor: Dave Clague Title: Professor of Biomedical Engineering

The ability to generate a range of concentrations of various solutions rapidly and conveniently is an ongoing need in biotechnology. In this thesis we demonstrate how we took advantage of the full process control afforded by our recent custom high resolution 3D printer and resin advances to realize highly integrated and miniaturized microfluidic components for simultaneous on-chip serial dilution for dose-response assays. With judicious selection of mixed layer thicknesses and pixel-by-pixel dose control, we show that the diameter of 3D printed membrane valves can be reduced from 300 µm to 46 µm. We further introduce an entirely new kind of 3D printed valve, termed a squeeze valve, in which the active area is reduced still further to 15 µm x 15 µm. We demonstrate and characterize pumps based on each type of valve and introduce a short (<1 mm long) high aspect ratio channel that enables rapid diffusion-based mixing. We show that combining two pumps with this diffusion mixing channel results in a highly compact 1:1 mixer component. Connecting 10 of these components in series yields a miniature 10 stage 2-fold microfluidic serial dilution module that from two solution inputs simultaneously generates 10 output concentrations that cover three orders of magnitude. We show the efficacy of our serial dilution approach by demonstrating an assay for dose-dependent permeabilization of A549 cells in different concentrations of digitonin integrated into a single device. Our demonstration of component miniaturization in conjunction with a high degree of integration illustrates the promise of 3D printing to enable highly functional and compact microfluidic devices for a variety of biomolecular applications.

Three-dimensional spheroid arrays represent in vivo activity better than conventional 2D cell culturing. A high-throughput microfluidic chip may be capable of depositing cells into spheroid arrays, but it is difficult to regulate the path of individual cells for deposition. Droplets that encapsulate cells may aid in facilitating cell delivery and deposition in the return bend of a microfluidic chip. In this study, a low-cost method for fabricating polymer-cast microfluidic chips has been developed for rapid device prototyping. Computational fluid dynamic (CFD) simulations were conducted to quantify how a change in geometry or fluid properties affects the dynamics of a droplet. These simulations have shown that the deformation, velocity, and trajectory of a droplet are altered when varying the geometry and fluid properties of a multiphase microfluidic system. This quantitative data will be beneficial for the future design of a microfluidic chip for cell deposition into 3D spheroid arrays.

In this diploma thesis we will study the luminescence properties of nanocrystals. We will summarize the basic division according to size and standard method of theoretical description of semiconductor and metal nanocrystals. We will describe the luminescence properties of nanocrystals and the influence of the surrounding environment. In the se- cond part of the thesis we will follow up the construction of an apparatus for imaging luminescence spectroscopy intended for the measurement of individual nanoobjects in a liquid environment. Finally, we will study luminescence properties of organixally passi- vated metal clusters in a liquid environment with changes in temperature and excitation intensity. We compare the luminescence of gold nanocrystals with and without long po- lyethylene glycol chains bound on the surface. 1

Drugs of Abuse (DOAs) are among the single largest contributor to crime in the United States and present a high cost to society in terms of financial costs and physical/mental well-being of individuals. The forensic community requires a variety of validated methods to detect and analyze DOAs in a variety of different sample types, and most developed methods utilize a liquid or gas chromatography (GC or LC) separation system paired to a mass spectrometer (MS) detection detector. Capillary Electrophoresis (CE) based separation techniques have also been experimented with due to this technique’s high efficiency and speed, high resolving power, low sample consumption, and potentially lower cost when compared to GC or LC based techniques, even though the sensitivity of these systems is perceived to be weaker. The goal of this research to develop a CE-MS/MS method utilizing the ZipChipTM to demonstrate it can accurately and reliably detect and quantify DOAs. The DOAs analyzed for this method were opioids and benzodiazepines, and these were 6-monacetylmorphine, 7-aminoclonazepam, codeine, diazepam, dihydrocodeine, 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine fentanyl, heroin, hydrocodone, hydromorphone, meperidine, methadone, morphine, norfentanyl, oxycodone, and oxymorphone. Standard Practices for Method Validation in Forensic Toxicology guidelines from the Academy Standards Board (ASB) of Toxicology were used as the template for this validation; samples were prepared and analyzed as neat standards in diluent, blood and urine were assessed for interferences, ionization suppression/enhancement, and extraction recovery. The total runtime for the method was 3.5 minutes, with the retention time range being 1.4 to 2.9 minutes. All samples were prepared using compound standards diluted in metabolite diluent, which consisted of methanol, ammonium acetate, and water prior to injection. The calibration curves consisted of eight calibrator samples that ranged from 0.5 ng/ml to 200 ng/ml for all analytes, and a linear model was used for each compound. The minimum acceptable 𝑅2 value was set to >0.98, and each curve had a weighing factor of 1𝑥2. Each curve for most of the compounds achieved the minimum requirement apart from two Codeine curves (0.9781 and 0.9785) and 7-aminoclonazepam (0.9791). Bias and precision were assessed at three concentrations- 5, 100, and 150 ng/ml. The minimum requirement for bias and precision for a compound was if the percent bias or coefficient of variation was within +/- 20%. Most compounds in this method exhibit acceptable levels of bias (except for Dihydrocodeine which had a bias of 24.58% at 100 ng/ml), and the only compounds to meet the minimum requirement for precision were 6-MAM, 7-aminoclonazepam, diazepam, fentanyl, methadone, and morphine. The limit of detection and limit of quantitation were both set at the lowest calibrator level of 0.5 ng/ml, and no carryover was observed in this method. No interferences occurred due to both deuterated internal standards and from common compounds such as benzylecogine, cocaine, and lidocaine, but blood cause signal interference with fentanyl and urine caused signal interference with methadone and norfentanyl. Ionization suppression and enhancement was observed for a majority of the compounds, and this observation will need to be assessed as to the effect it has on validation parameters in the future. The results collected suggest that accurate, reliable, and sensitive data may be collected if a compound has a specifically paired deuterated internal standard included in the sample. The speed of the suggested method and the minimal sample preparation could be desirable for forensic use. Further testing will need to be conducted to fully validate this method for blood and urine.

Neuropsychiatric disorders severely impact quality of life in millions of patients, contributing more Disease Affected Life Years (DALYs) than cancer or cardiovascular disease. The human brain is a complex system of 100 billion neurons connected by 100 trillion synapses, and human studies of neural disease focus on network-level circuit activity changes, rather than on cellular mechanisms. To probe for neural dynamics on the cellular level, animal models such as the nematode C. elegans have been used to investigate the biochemical and genetic factors contributing to neurological disease. C. elegans are ideal for neurophysiological studies due to their small nervous system, neurochemical homology to humans, and compatibility with non-invasive neural imaging. To better study the cellular mechanisms contributing to neurological disease, we developed automated analysis methods for characterizing the behaviors and associated neural activity during sleep and learning in C. elegans: two neural functions that involve a high degree of behavioral and neural plasticity. We developed two methods to study previously uncharacterized spontaneous adult sleep in C. elegans. A large microfluidic device facilitates population-wide assessment of long-term sleep behavior over 12 hours including effects of fluid flow, oxygen, feeding, odors, and genetic perturbations. Smaller devices allow simultaneous recording of sleep behavior and neuronal activity. Since the onset of adult sleep is stochastically timed, we developed a closed-loop sleep detection system that delivers chemical stimuli to individual animals during sleep and awake states to assess state-dependent changes to neural responses. Sleep increased the arousal threshold to aversive chemical stimulation, yet sensory neuron (ASH) and first-layer interneuron (AIB) responses were unchanged. This localizes adult sleep-dependent neuromodulation within interneurons presynaptic to the AVA premotor interneurons, rather than afferent sensory circuits. Traditionally, the study of learning in C. elegans observes taxis on agar plates which present variable environmental conditions that can lead to a reduction in test-to-test reproducibility. We also translated the butanone enhancement learning assay such that animals can be trained and tested all within the controlled environment of a microfluidic device. Using this system, we demonstrated that C. elegans are capable of associative learning by observing stimulus evoked behavioral responses, rather than taxis. This system allows for more reproducible results and can be used to seamlessly study stimulus-evoked neural plasticity associated with learning. Together, these systems provide platforms for studying the connections between behavioral plasticity and neural circuit modulation in sleep and learning. We can use these systems to further our understanding of the mechanisms underlying neural regulation, function, and disorder using human disease models in C. elegans.