Lab-on-a-Chip systems and the associated micro-fabrication technologies have been around for almost three decades. However, the rapidly shifting technological landscape and the multidisciplinary nature of the engineering know-how have made it extremely difficult for a majority of these technologies to materialize to find applications and find commercial products. In order to address this gap, researchers worldwide have attempted to implement design automation paradigms typically used for VLSI engineering and apply them to these Lab-on-a-Chip. However, almost all of these efforts have been disconnected, resulting in a delayed/stalled application of algorithmic advances on real-world device design. FluigiCAD will allow the rapid application and integration of innovative ideas into a single cohesive workflow. / 2024-06-13T00:00:00Z

With approximately 24,500 preterm children born annually in Canada and an estimated shortage of 6 million ounces of breast milk, the distribution of donor milk must be time-sensitive yet safe to efficiently meet this demand. Donor human milk banks take the greatest precautions to protect their users, but some of these microorganisms manage to circumvent the employed methods. The consumption of contaminated donor milk has the potential to be fatal particularly to the vulnerable, immunocompromised premature infants. The tools used by milk banks to ensure safe distribution rely heavily on the culture plate. It has been the gold standard in screening for microbiological specimens due to its wide availability, low cost, and simplicity. However, the procedural times for bacterial culture plates are tedious and long, lasting a minimum of 48 hours. Advances in microfluidics, particularly in combination with the concept of monitoring metabolites to indicate bacterial viability, hold much promise to significantly reducing the long processing times of culture plates. Combining the concept of compartmentalized culture and a chromogenic optical dye for the detection of metabolic changes as a diagnostic sensor would simplify the identification and quantification of microbial presence. The updated Rapid Culture Detection system is a nanowell device fabricated using polydimethylsiloxane (PDMS) that uses the oxygen-sensitive redox indicator Methylene Blue to determine the presence of bacteria. Preliminary studies have shown to detect bacteria in as little as 3.33 hours using these nanowells compared to the 24 hours required for microwell liquid culture (620%). Initial studies have also been conducted with human milk, indicating a slower detection than in LB media. The novel easy-to-use and low-cost Rapid Culture Detection system is a promising alternative detection tool for protecting infants from pathogenic illnesses caused by contaminated human milk and shortening the time required to access lifesaving nutrition. / Thesis / Master of Applied Science (MASc)

The epigenome carries dynamic information that controls gene expression and maintains cell identity during both disease and normal development. The inherent plasticity of the epigenome paves new avenues for developing diagnostic and therapeutic tools for human diseases. Microfluidic technology has improved the sensitivity and resolution of epigenomic analysis due to its outstanding ability to manipulate nanoliter-scale liquid volumes. In this thesis, I report three projects focusing on low-input, cell-type-specific and spatially resolved histone modification profiling on microfluidic platforms. First, I applied Microfluidic Oscillatory Washing-based Chromatin Immunoprecipitation followed by sequencing (MOWChIP-seq) to study the effect of culture dimensionality, hypoxia stress and bacterium infection on histone modification landscapes of brain tumor cells. I identified differentially marked regions between different culture conditions. Second, I adapted indexed ChIPmentation and introduced mu-CM, a low-input microfluidic device capable of performing 8 assays in parallel on different histone marks using as few as 20 cells in less than 7 hours. Last, I investigated the spatially resolved epigenome and transcriptome of neuronal and glial cells from coronal sections of adult mouse neocortex. I applied unsupervised clustering to identify distinct spatial patterns in neocortex epigenome and transcriptome that were associated with central nervous system development. I demonstrated that our method is well suited for scarce samples, such as biopsy samples from patients in the context of precision medicine. / Doctor of Philosophy / Epigenetic is the study of alternations in organisms not caused by alternation of the genetic codes. Epigenetic information plays pivotal role during growth, aging and disease. Epigenetic information is dynamic and modifiable, and thus serves as an ideal target for various diagnostic and therapeutic strategies of human diseases. Microfluidics is a technology that manipulates liquids with extremely small volumes in miniaturized devices. Microfluidics has improved the sensitivity and resolution of epigenetic analysis. In this thesis, I report three projects focusing on low-input, cell-type-specific and spatially resolved histone modification profiling on microfluidic platforms. Histone modification is one type of epigenetic information and regulates gene expression. First, we studied the influence of culture condition and bacterium infection on histone modification profile of brain tumor cells. Second, we introduced mu-CM, combining a low-input microfluidic device with indexed ChIPmentation and is capable of performing 8 assays in parallel using as few as 20 cells. Last, we investigated spatial variations in the epigenome and transcriptome across adult mouse neocortex, the outer layer of brain involving in higher-order function, such as cognition. I identified distinct spatial patterns responsible for central nervous system development using machine learning algorithm. Our method is well suited for studying scarce samples, such as cells populations isolated from patients in the context of precision medicine.

The limited transport of conventional chemotherapy within the tumor microenvironment (TME) is due to irregular vascularization, increased tumor interstitial pressure, and a dense extracellular matrix (ECM). The lack of selectivity of anticancer drugs often leads to systemic toxicity and damage to healthy tissues. Bacteria-based cancer therapy (BBCT) is a promising alternative, as tumor-targeting bacteria have been shown to preferentially colonize primary and metastatic tumors and induce anti-tumor effects. In this dissertation, we focus on several aspects of bacteria-nanoparticle conjugates, wherein BBCT is synergistically combined with nanomedicine to augment the efficacy of both treatment modalities. We explore biofabrication of our bacteria-nanoparticle conjugates called NanoBEADS (Nanoscale Bacteria Enabled Autonomous Drug Delivery Systems) and their interaction with the TME. Specifically, (1) we investigate the effects of two bacteria-NP conjugation chemistry and assembly process parameters of mixing method, volume, and duration, on NP attachment density and repeatability. We evaluate the influence of linkage chemistry and NP size on NP attachment density, viability, growth rate, and motility of NanoBEADS. (2) We investigate the effect of dense stroma and ECM production on the intratumoral penetration of bacteria with a mathematical model of bacterial intratumoral transport and growth. (3) We develop a microfluidic device with multicellular tumor spheroids to study the transport of tumor-targeting bacteria and support real-time imaging and long-term experiments. (4) We develop a new type of bacteria-based bio-hybrid drug delivery system using engineered cell surface display for enhancing the attachment of nanoparticles. / Doctor of Philosophy / Chemotherapy faces challenges in effectively reaching tumors due to factors like irregular blood vessel distribution, increased tumor pressure, and the presence of dense structures such as the extracellular matrix (ECM). This often results in collateral damage to healthy tissues. Bacteria-based cancer therapy (BBCT) offers a promising alternative, utilizing tumor-targeting bacteria to selectively attack tumors. This dissertation focuses on optimizing NanoBEADS (Nanoscale Bacteria Enabled Autonomous Drug Delivery Systems), which are chemotherapy encapsulating nanoparticle-bacteria assemblies to overcome these challenges and characterizing its behavior in tumors. Firstly, we investigated the optimization of bacteria-nanoparticle attachment, exploring various linkage chemistries and assembly processes to enhance attachment density, viability, and motility. Secondly, we examine how dense stroma and ECM affect bacterial penetration providing insights into intratumoral transport dynamics. Thirdly, we develop a microfluidic device integrated with multicellular tumor spheroids to enable real-time imaging and long-term experimentation on bacteria and drug transport. Lastly, we explore the potential of engineered cell surface display to enhance nanoparticle attachment in NanoBEADS, paving the way for self-propelled and highly targeted drug delivery systems. This dissertation strives to contribute to the transformation of current approaches to cancer treatment by refining drug delivery precision and efficacy while minimizing systemic toxicity.

Cancer is a major health issue that has been associated with over 80 million deaths worldwide in the last decade. Recently, significant improvements have been made in terms of treatment and diagnosis. However, despite these advancements there is still a demand for low-cost, high-accuracy, and easy-to-use technologies capable of classifying cells. Analysis of cell behavior in microfluidic deformability assays provides a label-free method of observing cell response to physical and chemical stimuli. This body of work shows advancements made toward reaching our goal of a robust and cost-effective biosensing device that allows for the identification of normal and cancer cells. These devices can also monitor cell responses to physical and chemical stimuli in the form of mechanical deformation and chemotherapeutic drugs, respectively. Our initial design was a microfluidic device that consisted of three channels with varying deformation and relaxation regions. Cell velocities from the deformations regions allowed us to distinguish between normal and cancer cells at the single-cell level. The next design used a singular deformation channel that was embedded with an array of electrodes in order to measure entry time, transit time and velocities as a single cell passes through the channel. These factors were found to reveal information about the biomechanical properties of single cells. Embedded electrodes were implemented in order to reduce post processing times of the data analysis and provide more insight into the bioelectrical information of cells. Finally, we report a microfluidic device with parallel deformation channels and a single electrode pair to improve throughput and automate data collection of deformability assays. This thesis demonstrates how microfluidic deformability assays, with and without embedded electrodes, show promising capabilities to classify different cells based on their biophysical traits which can be utilized as a valuable tool for testing responses to physical and chemical stimuli. / MS / Cancer is a worldwide health issue with approximately 1.7 million new cases each year in the United State alone. Although a great amount of research has been conducted in this field, the numerous uncertainties and heterogeneity among tumors, which is amplified by the large diversity between patients, has limit progress in both diagnostics and therapy. Traditionally, cancer studies have primarily focused on biological and chemical techniques. However, more recently, researchers have begun to leverage engineering techniques to acquire a new perspective on cancer to better understand the underlying biophysical attributes. Thus far, various engineering methodologies have produced meaningful results, but these techniques are costly and tend to be laborious. As a result, there is a need for low-cost, high-accuracy, and easy-to-use technologies to aid with cancer research, diagnostics, and treatment. An emerging field to alleviate these concerns is microfluidics, which is a science involving the flow of fluids in micro-scale channels. The field of microfluidics shows a great deal of promise for the development of clinically ready devices for analyzing cancer cells at both the population and single cell levels. Investigating the behavior of cancer cells at a single cell level can provide valuable information to help better understand the responsiveness of tumors to physical or chemical stimuli, such as chemotherapeutic drugs. This thesis reports multiple robust and cost-effective biomedical micro-devices that are used to analyze normal and cancerous cells. These devices consist of a microfluidic channel with sensors and are created using micro-fabrication techniques. The unique designs have enabled the evaluation of cells based on their mechanical and electrical properties. Specifically, the mechanical properties can be measured by forcing a cell into a microfluidic channel that is smaller than the diameter of the cell and recording its response to this physical stimulus. Electrical properties are measured simultaneously as the cells are probed for their mechanical properties. In general, the mechanical and electrical properties of cells can be altered when they undergo internal change (i.e. diseased cells) or experience external stimuli. Thus, these properties can be utilized as indicators of cancer progression and can be used to distinguish tumorigenic from non-tumorigenic cells. Data collection from these devices is automated, allowing for the rapid acquisition of mechanical and electrical properties of cells with minimal post-processing. Results from these devices have been promising in their ability to indicate significant differences among various normal and cancer populations based on their mechanical and electrical attributes.

Recent attempts to investigate living systems from a biophysical point of view has opened new windows for development of new diagnostic methods and therapies. Pulsed electric fields (PEFs) are a new class of therapies that take advantage of biophysical properties and have proven to be effective in drug delivery and treating several disorders including tumors. While animal models are commonly being used for development of new therapies, the high cost and complexity of these models along with the difficulties to control the electric field in the animal tissue are some of the obstacles toward the development of PEFs-based therapies. Microengineered models of organs or Organs-on-Chip have been recently introduced to overcome the hurdles of animal models and provide a flexible and cost-effective platform for early investigation of a variety of new therapies. In this study microfluidic platforms with integrated micro-sensors were designed, fabricated and employed to study the consequences of PEFs at the cellular level. These platforms were specifically used to study the effects of PEFs on the permeabilization of the blood-brain barrier for enhanced drug delivery to the brain. Different techniques such as fluorescent microscopy and electrical impedance spectroscopy were used to monitor the response of the cell monolayers under investigation. Irreversible electroporation is a new focal ablation therapy based on PEFs that has enabled ablation of tumors in a non-thermal, minimally invasive procedure. Despite promising achievements and treatment of more than 5500 human patients by this technique, real-time monitoring of the treatment progress in terms of the size of the ablated region is still needed. To address that necessity we have developed micro-sensor arrays that can be implemented on the ablation probe and give real-time feedback about the size of the ablated region by measuring the electrical impedance spectrum of the tissue. / Ph. D.

The quorum sensing signaling system based on intercellular exchange of N-acyl-homoserine lactones is used by many proteobacteria to regulate the transcription of essential genes in a signal density-dependent manner. It is involved in a number of processes including the development of highly organized bacterial communities, e.g., biofilms, the regulation of expression of virulence factors, production of antibiotics, and bioluminescence. The extensive genetic and biochemical data available on the quorum sensing system in Vibrio fischeri allows the development of a systems biology approach to undertake a spatial and dynamical analysis of the regulation throughout the population. The quorum sensing regulated lux genes are organized in two divergent transcriptional units: luxR and luxICDABEG. The latter contains the genes required for luminescence and the luxI gene necessary for synthesis of an N-acyl-homoserine lactone commonly called autoinducer (AI). The luxR gene codes for a transcriptional regulatory protein that activates the transcription of both operons at a threshold concentration of AI. The positive feedback loop induces a rapid increase of transcription level of the lux genes when a critical population density is reached (reflected by the concentration of AI in the environment). With a combination of molecular biology tools, physiological analysis, and mathematical modeling we identified critical characteristics of the system and expect to assign parameter values in order to achieve a comprehensive understanding of the dynamics. An ordinary differential equation mathematical model is used to investigate the dynamics of the system and derive parameter values. In parallel a novel microfluidic cell culture experimental set-up is used to carefully control environmental parameters as well as to achieve chemostatic conditions for high-density cell populations. An unstable variant of the green fluorescent protein was used as a reporter to follow the time response at a single cell level. Thus spatial organization and noise across the population can be analyzed. Plasmids carrying different genetic constructs were transformed in a recombinant Escherichia coli strain to specifically identify genetic and biochemical elements involved in the regulation of the lux genes under diverse conditions. Then the quantitative data extracted from batch culture and microfluidic assays were used to assign parameter values in the models. The particular question being investigated first is the nature of the regulation to increasing concentration of the signal. The hypothesis tested is that the regulation of the production of the signal by individual cells is biphasic and, therefore, quorum sensing should be robust to global and local variations in cell density. / Master of Science

Bio-hybrid approaches have recently provided a possible solution to address the challenge of on-board actuation, control and communication modules for micro/nanoscale cargo-carrying vehicles by integrating live prokaryotic or eukaryotic cells with synthetic objects. More specifically, because micro/nanoparticles are able to transport cargos efficiently and bacteria can play the role of targeted and selective delivery agents, a hybrid of these two can advance the current strategies for environmental monitoring, drug delivery and medical imaging. The main goal of this dissertation was to fabricate, assemble, and characterize different components of a mobile network of bacteria-based bio-hybrid systems for long-term applications in drug delivery and biosensing. First, a new library of bacteria-enabled delivery systems was developed by coupling live engineered bacteria with non-spherical particles and the transport of these bacteria-based systems was investigated in the absence and presence of chemical cues using microfluidic platforms. Next, a quorum-sensing (QS) based bacterial cell-cell communication network was characterized in a high-throughput manner in order to understand the coordinated behavior of the bacterial species ferrying the cargoes. Lastly, the QS behavior of a chemotactic population of the bacterial species in response to the endogenously produced signaling molecules was studied. The work presented in this dissertation lays the foundation for a well-characterized generation of bacteria-assisted cargo delivery devices with enhanced transport properties and capable of executing pre-programmed multi-agent coordinated tasks upon their arrival at the target site. / Ph. D.

The lack of efficacy of existing chemotherapeutic treatments of solid tumors is partially attributed to the limited diffusion distance of therapeutics and the low selectivity of anti-cancer drugs with respect to cancerous tissue, which also leads to high levels of systemic toxicity in patients. Thus, chemotherapy can be enhanced through improving anti-cancer drug carrier selectivity and transport properties. Several strains of gram positive (e.g. Clostridium and Bifidobacterium) and gram-negative (e.g. Salmonella Typhimurium and Escherichia coli) bacteria have been shown to possess the innate ability to preferentially colonize tumor tissues. The overall goal of this dissertation is to characterize the transport and sensing of Bacteria-Enabled Drug Delivery Systems (BEADS) in select relevant environments and to investigate the associated underlying principles. BEADS consist of an engineered abiotic load (i.e. drug-laden micro or nano-particles) and a living component (i.e. bacteria) for sensing and actuation purposes. Findings of this dissertation work are culminated in experimental demonstration of deeper penetration of the NanoBEADS within tumor tissue when compared to passively diffusing chemotherapeutic nanoparticles. Lastly, the transport mechanisms that Salmonella Typhimurium VNP20009 utilize to preferentially colonize solid tumors are also examined with the ultimate goal of engineering intelligent and more efficacious drug delivery vehicles for cancer therapy. / Ph. D.

Cell-level and tissue-level mechanical properties are key to healthy biological functions, and many diseases and disorder arise or progress due to altered cell and tissue mechanics. Pulse electric field (PEFs), which employ intense external electric fields to cause electroporation, a phenomenon characterized by increased cell membrane permeability, also can cause significant changes to cell and tissue mechanics. Here, we investigate the mechanics of brain and brain cancer cells, specifically focusing on how PEFs impact cell mechanics and PEF-induced blood-brain barrier disruption. In our first study, we investigate single-cell mechanical disruption of glioblastoma cells after reversible electroporation using Nanonet Force Microscopy (NFM). A precise network of extracellular-matrix mimicking nanofibers enabled cell attachment and contraction, resulting in measurable fiber deflections. Cell contractile forces were shown to be temporarily disrupted after reversible electroporation, in an orientation and field-dependent manner. Furthermore, we found that cell response is often a multi-stage process involving a cell-rounding stage, biphasic stage, and a cell re-spreading stage. Additionally, cell viability post-PEFs was orientation-dependent. In another study, we investigated the mechanical properties of brain cancer for various-grade glioma cells (healthy astrocytes, grade II, grade III, and grade IV (glioblastoma) cells). A microfluidic constriction channel caused cell deformation as cells, driven by hydrostatic pressure, entered a narrow constriction. Finite element models of cell deformation and a neural network were used to convert experimental results (cell entry time and cell elongation within the channel) into elastic modulus values (kPa). We found that the that low-grade glioma cells showed higher stiffnesses compared to healthy and grade IV glioma cells, which both showed similar values. These results warrant future studies to investigate these trends further. PEFs can induce Blood-brain barrier (BBB) disruption, an effect we studied using a multiplexed, PDMS microdevice. A monolayer of human cerebral endothelial cells on a semi-permeable membrane was used to model the BBB, and permeability was assessed by the diffusion of a fluorescent dye from an upper to lower channel. A custom tapered channel and branching channel design created a linear gradient in the electric field within the device that enabled six electric field strengths to be tested at once against two unexposed (control) channels. Normalization of permeability by the control channels significantly removed experimental noise. We found that after high-frequency bipolar irreversible electroporation (HFIRE) electric pulses, permeability transiently increased within the first hour after electroporation, in a voltage- and pulse-number dependent manner. However, we found significant electrofusion events after pulsing at high voltages, which reduced monolayer permeability below baseline values. This device enables efficient exploration of a wide range of electroporation parameters to identify the optimal conditions for blood-brain barrier disruption. In another blood-brain barrier study, we incorporate dense, polystyrene nanofiber networks to create ultra-thin, ultra-porous basement-membrane-mimics for In vitro blood-brain barrier models. Fiber networks are fabricated using the non-electrospinning Spinneret-based Tunable Engineered Parameters (STEP) technique. Endothelial cells cultured on one side of the fiber network are in close contact with supporting cell types (pericytes) cultured on the backside of the fibers. Contact-orientation co-cultures have been shown to increase blood-brain barrier integrity, and our nanofiber networks increase the physiological realism of basement-membrane mimics for improve modeling. Finally, we investigate how cell viability post-electroporation is impacted by cell morphology. The impact of cell morphology (shape and cytoskeletal structure) on cell survival after electroporation is not well understood. Linking specific morphological characteristics with cell susceptibility to electroporation will enhance fundamental knowledge and will be widely useful for improving electroporation techniques where cell viability is desirable (gene transfection, electrofusion, electrochemotherapy) or where cell viability is undesirable (tumor ablation, cardiac ablation). Precise control of cell shape and orientation enabled by nanofiber scaffolds provides a convenient and expedient platform for investigating a wide variety of factors (morphological and experimental) on cell viability. Altogether, these investigations shed new light on cell mechanical changes due to disease and pulsed electric fields, and suggest opportunities for improving brain cancer therapies. / Doctor of Philosophy / In biology, structure and function are interrelated. Cells and tissue have structures that enable them to perform their proper function. In the case of disease, cell and tissue properties are altered, leading to dysfunction. Alternatively, healthy structures sometime hinder effective treatments, and therefore can be therapeutically disrupted to improve treatments. In this study, we investigate single-cell and multi-cellular mechanical change due to disease or after pulsed electric fields (PEFs), with a specific focus on the brain. Pulsed electric fields (PEFs) use electrodes to deliver short, intense pulses of electrical energy to disrupt cell membranes and change cell mechanics. We studied as single-cell contractility, cancer cell stiffness, and blood-brain barrier (BBB) disruption by PEFs. We found that PEFs cause significant change to cell shape and mechanics, and can disrupt the BBB. By studying several grades of brain cancers, we found that low-grade brain cancer (gliomas) showed increased stiffness compared to healthy and highly diseased (grade IV) cells. To mimic the BBB, we used microfluidic devices to grow specialized brain cells (endothelial cells) on permeable membranes and nanofibers networks and showed that these devices can mimic structures found in animals/humans. Finally, we studied how cell properties (such as shape) determine whether cells will survive PEFs. Taken together, our investigations improve the understanding of brain mechanics during disease and after PEFs, and suggest the usefulness of PEFs for improved brain cancer therapies.