Biofunctional interfaces capable of anchoring biomolecules and nanoparticles of interest onto a platform are the key components of many biomedical assays, clinical pathologies, as well as antibacterial and antiviral surfaces. In an ideal biofunctional surface, bio-entities and particles are covalently immobilized on a substrate in order to provide robustness and long-term stability. Nonetheless, most of the reported covalent immobilization strategies incorporate complex wet-chemical steps and long incubation times hindering their implementation for mass production and commercialization. Another essential factor in the biointerface preparation, specially with regard to biosensors and diagnostic applications, is utilization of an efficient and durable blocking agent that can inhibit non-specific adsorption of biomolecules thereby enhancing the sensitivity of sensors by diminishing the level of background noise. Many of the commonly used blocking agents lack proper prevention of non-specific adsorption in complex fluids. In addition, most of these agents are physically attached to surfaces making them unreliable for long-term usage in harsh environments (i.e. where shear stresses above 50 dyn/cm2 or strong washing buffers are involved). This thesis presents novel and versatile strategies to covalently immobilize nanoparticles and biomolecules on substrates. The new surface coating techniques are first implemented for robust attachment of TiO2 nanoparticles onto ceramic tiles providing self-cleaning properties. Further, we utilize similar strategies to covalently immobilize proteins and culture cells in microfluidic channels either as a full surface coating or as micropatterns of interest. The new strategies allow us to obtain adhesion of ~ 400 cells/mm2 in microfluidic channels after only 1-day incubation, which is not achievable by the conventional methods. Moreover, we show the possibility of covalently micropatterning of biomolecules on lubricant-infused surfaces (LISs) so as to attain a new class of biofunctional LISs. By integration of these surfaces into a biosensing platform, we are able to detect interleukin 6 (IL-6) in a complex biofluid of human whole plasma with a limit of detection (LOD) of 0.5 pg.mL-1. This LOD is significantly lower than the smallest reported IL-6 LOD in plasma, 23 pg mL-1, using a complex electrochemical system. The higher sensitivity of our developed biosensor can be attributed to the distinguish capability of biofunctional LISs in preventing non-specific adhesion of biomolecules compared to other blocking agents. / Thesis / Doctor of Philosophy (PhD) / The key goal of this thesis is to provide new strategies for preparation of robust and durable biointerfaces that could be employed for many biomedical devices such as self-cleaning coatings, microfluidics, point-of-care diagnostics, biomedical assays, and biosensors in order to enhance their efficiency, sensitivity, and precision. The introduced surface biofunctionalization methods are straightforward to use and do not require multiple wet-chemistry steps and incubation times, making them suitable for mass production and high throughput demands. Moreover, the introduced surface coating strategies allow for creation of antibody/protein micro-patterns covalently bound onto a biomolecule-repellent surface. The repellent property of the surfaces is resulted from infusion of an FDA-approved lubricant into the surface of a chemically modified substrate. While the surface repellency can effectively prevent non-specific adhesion of biomolecules, the patterned antibodies can locally capture the desired analyte, making them a great candidate for biosensing.

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Microfluidics is the science of processing microliters or less of fluid at a time in a channel with dimensions on the order of microns. The small size of the channels allows fluid properties to be studied in a world dominated by viscosity, surface tension, and diffusion rather than gravity and inertia. Microfluidic droplet generation is a well studied and understood phenomena, which has attracted attention due to its potential applications in biology, medicine, chemistry and a wide range of industries. This dissertation adds to the field of microfluidic droplet studies by studying individual droplet deformation and the process of scaling-up microfluidic devices for industrial use. The study of droplet deformation under extensional and mixed shear and extensional flows was performed within a microfluidic device. Droplets were generated using a flow-focusing device and then sent through a hyperbolic contraction downstream of the droplet generator. The hyperbolic contraction allowed the smallest droplets to be deformed by purely extensional flows and for the larger droplets to experience mixed extensional and shear flows. The shear resulted from the proximity of the droplet to the walls of the microfluidic channel. The continuous phase in all of these devices was oil and the dispersed phase was water, an aqueous surfactant solution, or an aqueous suspension of colloidal particles. Droplet deformation dynamics are affected by the use of surfactants and colloidal particles, which are commonly used to stabilize emulsion droplets again coalescence. Microfluidic droplet generating devices have many potential industrial applications, however, due to the low output of product from a single droplet generating device, their potential has not been realized. Using six parallel flow-focusing droplet generators on a single chip, the process of microfluidic droplet formation can be scaled up, thus resulting in a higher output of droplets. The tuning of droplet size and production frequency can be achieved on chip by varying the outlet tubing lengths, thus allowing for a single device to be used to generate a range of droplet sizes.

Optical microlasers have been used in different engineering fields and for sensing various applications. They have been used in biomedical fields in applications such as for detecting protein biomarkers for cancer and for measuring biomechanical properties. The goal of this work is to propose a microfluidic-based fabrication method for fabricating optical polymer based microlasers, which has advantages, over current methods, such us the fabrication time, the contained cost, and the reproducibility of the microlaser's size. The microfluidic setup consisted of microfluidic pumps and a flow focusing droplet generator chip made of polydimethylsiloxane (PDMS). Parameters such as the flow rate (Q) and the pressure (P) of both continuous and dispersed phases are taken into account for determining the microlaser's size and a MATLAB imaging tool is used to reduce the microlaser's diameter estimation. In addition, two applications are discussed: i) electric field measurements via resonator doped with Di-Anepps-4 voltage sensitive dye, and ii) strain measurements in a 3D printed bone-like structure to mimic biomedical implantable sensors.

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The detection and identification of particles within fluid samples is key in the prevention of the spreading of disease. This has created a growing need for devices able to successfully separate and identify multiple particles for this purpose while operating at a high enough throughput to be applicable in the field. A well investigated method of manipulating particles in this way is Dielectrophoresis (DEP), which is the use of varied electric fields gradients to generate a force on small particles. The strength of DEP depends of the properties of the particle medium, the signal generating the electric field, and the properties of the particles themselves. This method and its interaction with all small particles, including biological particles such as blood and cancer cells, has allowed devices utilizing this idea to be investigated for various biological purposes. This thesis investigates methods to increase the throughput of these types of devices in order to increase their ability to process large amounts of samples in reasonable amounts of time. This is done in primarily two methods. One approach uses the application of chromatographic methods to DEP devices to separate particles by altering their individual transit time through a device, allowing identification during constant flow. Another method is through mass parallel channels which each individually operate as a standard DEP particle trapping device. This allows for the summation of the maximum flow through the device due to its design layout. / Master of Science / Micrometer scale devices are popular for the identification, separation, and characterization of micron scale particles. This includes uses in biological fields for the manipulation of particles such as blood cells, cancer cells, and bacteria. A common method of manipulating these particles is Dielectrophoresis, a force that causes particles to be repelled or attracted to geometric designs within the device generated by an applied electric field. The strength and direction of this force on the particles is dependent on the properties of the electrical signal applied to the device, the physical properties of the particles, such as size and shape, and the properties of the medium the particles are suspended in within the device. Biological devices utilizing this force have been tested before, allowing for particles to be separated out of mixed particle solutions. Most of these devices operate by moving through very little material at one time, somewhere in the microliter per hour range. This thesis explores attempts to increase the rate at which samples can be processed by these devices in multiple ways. Chapter 2 explores methods of DEP by applying Chromatography principles, which is to constantly move samples through the device at a high rate and slow the target particles, so they exit the device at a different time than other particles. Chapter 3 investigates increasing device throughput by replicating a standard DEP channel multiple times on one device so that several may operate all at once.

According to the third international consensus definition (sepsis-3), sepsis is characterized as life-threatening organ dysfunction resulting from an uncontrolled host response to infection. Sepsis stands as a prominent contributor to worldwide mortality. A study revealed approximately 50 million reported cases of sepsis and 11 million associated deaths worldwide, constituting nearly 20% of all global fatalities. Various biomarkers have been investigated for sepsis prognosis including Procalcitonin (PCT), C-reactive protein (CRP), interleukin-1β (IL-1β), interleukin-6 (IL-6), and protein C. In addition to proteomic markers genomic biomarkers have also been investigated for sepsis. For instance, research indicates a substantial rise in plasma cell-free DNA (cfDNA) and total circulating histones levels during sepsis, correlating with its severity and mortality. The complexity arises in creating a measurement tool for sepsis, given the diverse nature of these biomarkers, each requiring distinct detection methods. The objective of this doctoral thesis is to develop a low-cost fully integrated microfluidic device for detecting a genomic biomarker (cfDNA) and a proteomic biomarker (total circulating histones) using a new method for integration of hydrogels inside microfluidic devices during the fabrication process. This method involves using porous and fibrous membranes as scaffolds to support gels. The scaffold facilitates the drying and reconstitution of these gels without any loss of shape or leakage, making it advantageous in various applications, especially in point-of-care (POC) devices where long-term storage of gels inside the device is required. This hydrogel integration method was applied to demonstrate gel electrophoretic concentration and isoelectric trapping of cfDNA and histones respectively in rehydrated agarose gates with proper pH embedded in a porous membrane in a microfluidic device. Then, these two detections were performed in a single fully integrated microfluidic device. Additionally, nonspecific fluorescent dyes were incorporated within the device, eliminating the necessity for off-chip sample preparation. This enables direct testing of plasma samples without the need to label DNA and histones with fluorescent dyes beforehand. In all the fabrication steps of the microfluidic device, xurography, a cost-effective and rapid fabrication method, was utilized. This device demonstrated the effective separation of cfDNA and histones in the agarose gates in a total time of 20 minutes, employing 10 and 30 Volts for cfDNA and histone accumulation, respectively. This device could be further developed to create a POC device for the quantification of cfDNA and histones simultaneously in severe sepsis patients plasma sample. / Thesis / Doctor of Philosophy (PhD)

Neutrophils are the innate immune system's first line of defense in response to an infection. During an infection in the tissue, chemical cues called chemoattractants are released, which signal neutrophils to exit circulation and enter the tissue. Once in the tissue, neutrophils directionally migrate in response to the chemoattractant and toward the site of infection in a process called chemotaxis. At the site of infection, they initiate antimicrobial responses to clear the infection and resolve inflammation, restoring homeostasis. However, neutrophils are exposed to multiple chemoattractants and must prioritize these signals in order to correctly migrate to the appropriate site. The ability of neutrophils to properly undergo chemotaxis in the presence of infection and inflammation is crucial for resolution of inflammation and pathogen clearance. It has been recently shown that when pre-conditioned with bacterial endotoxin (LPS), innate immune function can become dysregulated. Neutrophils start to display altered antimicrobial response as well as dysfunctional migration patterns. This behavior has been seen in patients with sepsis, where a person's immune system overreacts to an infection, leading to systemic inflammation throughout the body, causing tissue damage, multiple organ failure, and in many cases, death. We explore the effects of inflammation on neutrophil migratory patterns and decision-making within chemotaxis. Additionally, to understand how inflammation within disease impacts chemotaxis, we measure the difference between neutrophils from healthy individuals and those from septic patients. We approached this using a combination of experimental and computational techniques. We developed a microfluidic assay to measure neutrophil decision-making in a competitive chemoattractant environment between an end-target (fMLP) and intermediary (LTB4) chemoattractant. Additionally, we probed for the expression level of molecules related to neutrophil chemotaxis. We also built a system of ordinary differential equations to model the dynamics of the molecular interactions underlying neutrophil chemotaxis. Our results showed that when neutrophils were induced into a highly inflammatory state, they prioritized pro-inflammatory signals over pro-resolution signals and displayed dysfunctional migration patterns. Similarly, neutrophils from patients with sepsis also displayed dysregulated migration patterns. This aberrant neutrophil chemotaxis may be implicated in the pathogenesis of sepsis, where accumulation of neutrophils in off-target organs is often seen. These results shed light onto the directional migratory decision-making of neutrophils exposed to inflammatory signals. Understanding these mechanisms may lead to the development of pro-resolution therapies that correct the neutrophil compass and reduce off-target organ damage. / Doctor of Philosophy / Neutrophils are innate immune cells that act as the first line of defense toward an infection. During an infection, chemical signals are released, stimulating neutrophils to migrate toward that specific site of infection. Once the cells are in the tissue, they can clear the pathogen and resolve inflammation. However, when neutrophils are migrating in the tissue, they are overwhelmed with multiple signals, directing them toward different sites. These signals must be prioritized by the cell so they can properly migrate toward the correct location. It has been recently shown that neutrophils that have been preconditioned into inflammatory states will display dysfunctional migration patterns. They are unable to migrate to the site of infection and instead migrate to healthy tissue, where they can cause damage. This has been shown in patients with sepsis, which is a condition where a person's immune system overreacts to an infection, causing inflammation throughout the body, leading to tissue damage and multiple organ failure. Our work explores the impact of inflammation on neutrophil migration patterns and the ability of the cell to properly prioritize when stimulated by multiple chemical signals. Additionally, we look at how neutrophils from healthy individuals differ from neutrophils from patients with sepsis, to understand how inflammation within disease impacts cellular migration. We approach this both experimentally and computationally. We designed a microfluidic assay to measure neutrophil migration in the presence of two competing chemical signals. We also measured the expression levels of molecules relevant to cell migration. We also built a mathematical model to investigate the molecular interactions underlying these processes. These results shed light on how inflammation impacts neutrophil migration and its role in inflammatory diseases.

Waterborne or foodborne diseases are caused by consuming contaminated fluids or foods. The presence of pathogenic microorganisms can contaminate food or drinking water. These microorganisms can cause sickness even if they are present in minimal concentrations. The World Health Organization (WHO) has defined the standards for clean drinking water as the absence of E. coli in a 100 mL collected volume. Contaminated water or food can cause many diseases, and diarrhea is one of a prominent disease. Early detection of contamination in food or drinking water is critical. Conventional culture-based methods are time-consuming, labour intensive, and not suitable for on-site testing. Nucleic acid-based tests are sensitive and can rapidly detect pathogens. Microfluidic technology can play a significant role to develop low-cost, rapid, integrated, and portable nucleic acid-based detection devices. Microfluidic systems for isothermal amplification assays can be classified into two groups such as droplet-based and chamber-based systems. In this thesis, both droplet-based and chamber-based approaches were used to build the microfluidic methods for isothermal amplification assays. First, a simple electromechanical probe (tweezers) was developed that can manipulate a small aqueous droplet in a bi-layer oil phase. The tweezer consisted of two needles positioned close to each other and used polarization of the aqueous droplet in an applied electrical field to confine the droplet between the needles with minimal solid contact. AC electric potential was applied to the two metal electrodes. Droplet acquired a charge from the high voltage electrode and consequently performed an oscillatory motion with the same electrode. This droplet motion was controlled using two parameters of electric potential and frequency of the applied signal. Initially, electrically actuated droplet (0.3 µL) motion was investigated for a range of applied potential (400-960 Volts) and frequencies (0.1-1000 Hz). The droplet motion with high voltage electrode was characterized into three modes such as detachment, oscillation, and attachment. Mechanical motion of tweezer was used to transport droplet to various positions. Consequently, operations such as transportation, extraction, and merging were demonstrated. First, droplet (5 µL) transportation was characterized under the applied potential of 2000 Volts at various frequencies (5 to 1000 Hz). The droplet was successfully transported to the speed of 15 mm/s at higher frequencies (100 or 1000 Hz). Droplets of various volumes (12-80 µL) were extracted by increasing applied electric potential, from 0 to 6000 Volts at 5 Hz. Then, the operation of droplets merging was demonstrated using operational conditions for electrical tweezer. Finally, electrical tweezer was used to prepare samples for isothermal amplification assays. Two droplets consisted of various reagents of isothermal amplification assays, were transported and merged using the electrical tweezer. Then, a merged droplet (25 µL) was transported and immobilized in the amplification zone. The temperature of the amplification zone (~65°C) was maintained using an in-situ heater. DNA amplification was verified by measuring the off-chip end-point fluorescence intensity of isothermal assays. Second, an integrated microfluidic device has been developed to prepare a sample for isothermal amplification assays. And in-situ real-time amplification assays were performed to detect bacteria. The device consisted of two chambers (lysis and amplification) connected through a microchannel. A low-cost fabrication method was introduced to embed two resistive wire heaters around both chambers. Initially, bacteria cells were thermally lysed in the lysis chamber at 92°C for 5 min. Then, DNA was electrophoretically transported from lysis to the amplification chamber. The electric potential of 10 Volts was applied for 10 min for DNA transportation. Next, transported DNA was amplified at 65°C and DNA amplification was detected by measuring in-situ fluorescence intensity in the real-time format. The operation of the integrated microfluidic device was demonstrated in three steps. 1) Operation of individual components. 2) Operation of two components in a coupled format. 3) Integrated operation of three components with measurement of fluorescent intensity in a real-time format. The bacteria samples with the concentration of 100 CFU/mL were detected in less than one hour. / Thesis / Doctor of Philosophy (PhD)