Ideally, every person in the world should have access to a safe and clean water supply; if not all sources of water are clean and safe, at the very least, an effective method to detect water contamination should be readily available. An effective detection method should not only be sensitive, rapid, robust, and affordable, but, ideally, it should also be equipment-free and easy to transport and deliver to the end-users. The main goal of this project is to develop a variety of bits and pieces of bioassay systems, with a particular focus on paper-based bioactive devices in order to provide portable and ready-to-use biosensors which can be useable by anyone anywhere around the world without requiring formal training. According to the World Health Organization (WHO), 76,000 people each year die in India alone because of pesticide poisoning. Long term exposure to organophosphate pesticides is known to have adverse effects on neurological function and can lead to Alzheimer's Disease, Attention Deficit Hyperactivity Disorder (ADHD), and reduced Intelligence Quotient (IQ). The likelihood of long term exposure to pesticides is heightened in developing countries, so a reliable and inexpensive pesticide sensor is a much-needed device in the developing world. To address this need, this project reports on the development of a fully-automated bioactive paper-based sensor for the detection of organophosphate pesticides. In the proposed biosensor, two innovations were implemented to achieve a full-automated format for the pesticide sensor: (I) First is a PUMP ON A PAPER (Jahanshahi-Anbuhi et al., LOC, 2012) that increases the flow rate of fluids within paper-based microfluidic analytical devices and sequentially brings two separate liquid streams to the enzyme test zone on the paper sensor, and (II) the second innovation is a PIPETTE ON A PAPER (Jahanshahi-Anbuhi et al., LOC, 2014) that involved the creation of a pullulan (a natural non-ionic polysaccharide) temporary bridge-system to transfer a known amount of solution to the sensing zone that, gives the enzyme zone a chance to dry and accept the substrate solution from the slow channel after a fixed period of time. This proposed format results in a simplified assay that detects the presence of pesticides automatically without any further manipulation from the user. However, the shelf life of this assay kit is challenging due to instability of both enzyme (AChE) and substrate (IDA) at room temperature. AChE loses its enzymatic activity when stored at room temperature and IDA becomes oxidized quickly. This problem is not unique to these two bio reagents, however; almost all bioassays which use bio-reagents (such as enzymes and small-molecular substrates) are unstable to varying degrees and require special shipping and storage. The instability of these molecules can arise from either thermal denaturation or chemical modification, such as oxidation or hydrolysis. Because of these issues, they often have to be shipped on dry ice with special packaging, which is costly. The cost of maintaining a cold chain for distributing bio-reagents accounts for up to 80% of the cost. Aside from the cost, these reagents also have to be stored in bulk in refrigerators or freezers to minimize the loss of activity, but they must be thawed and aliquoted for their intended tests. Repeated freezing and thawing can result in a significant loss of activity, which often leads to less reliable test results. These issues make running such assays in resource-limited settings a significant challenge. There is, therefore, an urgent need for an assay system with stable reagents that is easy to use, simple to read, inexpensive, and that includes a method for the long-term stabilization of enzymes and other unstable reagents in pre-measured quantities. To overcome to all these issues, pullulan is utilized for the development of pill-based-biosensors. Pullulan dissolves quickly in aqueous solutions and shows very high oxygen barrier properties in its film form. Considering the unique properties of pullulan, it is hypothesized that pullulan may be suitable for producing assay pills with encapsulated enzymes or other unstable molecules and may provide a simplified platform for carrying out bioassays in resource-limited settings. The application of these pill-based-biosensors is shown via the entrapment of AChE and IDA for the creation of an assay kit that can detect organophosphate pesticides (Jahanshahi-Anbuhi et al., Angew. Chem., 2014). Moreover, this thesis reports on the stabilization of highly unstable firefly luciferase for the detection of microorganisms and, more particularly, ATP. Through the use of pullulan, this thesis demonstrates that both the enzyme and the substrate can be protected, immobilized, and stabilized at room temperature, instead of the existing storage methods, which require temperatures <-20˚C. This innovation allows for a more convenient method of shipping the bioassay kits around the world without any extra care. Furthermore, pullulan-based films are utilized for the development of a method for controlled multidirectional flow within paper-based biosensors. This method provides the possibility of trapping labile and volatile reagents and stabilizing them by forming thin films with pullulan. The trapped reagents within pullulan films can be strategically stacked and assembled on a paper strip in different directions. Furthermore, should the need arise, these reagents can be released and delivered sequentially or simultaneously in both vertical and lateral directions through the paper. The application of this method is shown for: (I) creation of "ready-to-use" assay kit for the detection of Escherichia coli (E. Coli). This assay kit has the step of cell lysing and proceeds automatically to the step in which enzymes react. The second application (II) shows the trapping of Simon’s reagents, which is widely used for methamphetamine detection. Overall, these unique fabrication techniques can be widely used for the preparation of highly stable, ready-to-use, and user-friendly biosensors. We are currently working on the detection of other contaminants such as heavy metals, and we are starting on vaccine stabilization and delivery, which would have a tremendous impact for society. / Dissertation / Doctor of Engineering (DEng)

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Integration and miniaturisation in electronics has undoubtedly revolutionised the modern world. In biotechnology, emerging lab-on-a-chip (LOC) methodologies promise all-integrated laboratory processes, to perform complete biochemical or medical synthesis and analysis encapsulated on small microchips. The integration of electrical, optical and physical sensors, and control devices, with fluid handling, is creating a new class of functional chip-based systems. Scaled down onto a chip, reagent and sample consumption is reduced, point-of-care or in-the-field usage is enabled through portability, costs are reduced, automation increases the ease of use, and favourable scaling laws can be exploited, such as improved fluid control. The capacity to manipulate single cells on-chip has applications across the life sciences, in biotechnology, pharmacology, medical diagnostics and drug discovery. This thesis explores multiple applications of optical manipulation within microfluidic chips. Used in combination with microfluidic systems, optics adds powerful functionalities to emerging LOC technologies. These include particle management such as immobilising, sorting, concentrating, and transportation of cell-sized objects, along with sensing, spectroscopic interrogation, and cell treatment. The work in this thesis brings several key applications of optical techniques for manipulating and porating cell-sized microscopic particles to within microfluidic chips. The fields of optical trapping, optical tweezers and optical sorting are reviewed in the context of lab-on-a-chip application, and the physics of the laminar fluid flow exhibited at this size scale is detailed. Microfluidic chip fabrication methods are presented, including a robust method for the introduction of optical fibres for laser beam delivery, which is demonstrated in a dual-beam optical trap chip and in optical chromatography using photonic crystal fibre. The use of a total internal reflection microscope objective lens is utilised in a novel demonstration of propelling particles within fluid flow. The size and refractive index dependency is modelled and experimentally characterised, before presenting continuous passive optical sorting of microparticles based on these intrinsic optical properties, in a microfluidic chip. Finally, a microfluidic system is utilised in the delivery of mammalian cells to a focused femtosecond laser beam for continuous, high throughput photoporation. The optical injection efficiency of inserting a fluorescent dye is determined and the cell viability is evaluated. This could form the basis for ultra-high throughput, efficient transfection of cells, with the advantages of single cell treatment and unrivalled viability using this optical technique.

Grâce à leurs propriétés physiques/chimiques uniques, les nanoparticules colloïdales sont au cœur de nombreuses applications innovantes. Afin de faciliter leur caractérisation ou de les intégrer dans des dispositifs fonctionnels, il est nécessaire de les assembler de manière dirigée sur des surfaces solides. Dans ce contexte, l’objectif de cette thèse est de mieux comprendre et d’optimiser la technique de nanoxérographie, méthode d’assemblage dirigé où des nanoparticules sont piégées sur des motifs de charges électrostatiques. Après un premier travail consistant à améliorer le procédé de nanoxérographie, trois problématiques spécifiques ont été adressées : (i) l’assemblage de particules micrométriques. Le couplage de simulations numériques et de manipulations expérimentales a permis d’identifier les paramètres clés de l’assemblage de telles particules colloïdales et d’élargir (facteur 100) la gamme de tailles de particules assemblables par nanoxérographie. (ii) l’analyse de l’assemblage multicouche. Par le biais de nanoparticules modèles luminescentes et par la mise en place d’un nouveau protocole d’assemblage, les critères clés génériques pour l’assemblage 3D de colloïdes par nanoxérographie ont été dégagés. (ii) l’assemblage dirigé de nanogels sensibles à un stimulus environnemental extérieur. L’utilisation d’un protocole d’assemblage optimisé a permis d’élaborer des assemblages de nanogels interactifs avec leur environnement et du faire du tri sélectif de ces nanoparticules sur une même surface. / Owing to their unique physico-chemical properties, colloidal nanoparticles are building blocks for the creation of plentiful innovative devices. In order to make easier their characterization and to incorporate them into functional nano-devices, it is necessary to perfectly control their directed assemblies onto solid surfaces. In this context, this thesis’ purpose is to simultaneously better understand and optimize the nanoxerography method, which allows electrostatic and selective directing assemblies of nanoparticles onto charged patterns. After an optimization of the nanoxerography process, three specific problematics have been addressed: (1) micron-sized particles assembly. The combined use of numerical simulations and experiments enabled to unveil the key parameters involved in micron-sized particles assembly and to expend the particle size range foreseeable for an assembly by nanoxerography (factor 100). (2) the 3D assembly analysis. The influence of diverse parameters on the 3D assembly of luminescent model nanoparticles was quantified by using a new assembly protocol. The results gave the generic key criterions for the 3D assembly of colloids by nanoxerography. (3) directed assembly of nanogels sensitive to an external environmental stimulus. The use of an optimized protocol allowed elaborating nanogels assemblies interactive with their environment and to sort these nanoparticles onto the same surface.

Indiana University-Purdue University Indianapolis (IUPUI) / The purpose of this thesis is to use experimental methods to seek deeper understanding and better performance in the self-circulating self-regulating microfluidic gas generator initially developed in Dr. Zhu’s group, by changing the major features and dimensions in the reaction channel of the device. In order to effectively conduct experiments described above, a microfabrication method that is capable of making new microfluidic devices with low cost, short time period, as well as relatively high accuracy was needed first. Developing such a fabrication method is the major part of this thesis. We initially used patterned polymer films and glass slide, and bonded them together by sequentially aligning and stacking them into a microfluidic device with patterned double-sided tapes. Later we developed a more advanced microfabrication method that used only patterned polystyrene (PS) films. The patterned PS films were obtained from a digital cutter and they were bonded into a microfluidic device by thermopress bonding method that required no heterogeneous bonding agents. This new method did not need manual assembly which greatly improved its precision (~ 100 µm), and it used only PS as device material that has favorable surface wetting property for microfluidics applications. In order to find the optimized microfluidic channel design to improve gas generating performance, we've designed and fabricated microfluidic devices with different channel dimensions using the PS fabrication method. Based on the gas generation testing results of those devices, we were able to come up with the optimal dimensions for the reaction channel that had the best gas generation performance. To obtain a more fundamental understanding about the working mechanism of our device and its bubble dynamics, we have conducted ultrafast X-ray imaging test at Advanced Photon Source (APS), Argonne National Laboratory. High speed (100 KHz) phase contrast images were captured that allowed us to observe directly inside the reaction channel on the cross section view during the self-circulating catalytic reaction. The images provided us with lots of insightful information that in turn helped the dimensional improvement for the microchannel design. The 100 KHz high speed images also gave us useful information about the dynamics of bubble development on a catalyst bed, such as growth and merging of the bubbles.

The mechanisms behind cardiovascular disease (CVD) initiation and progression are not fully elucidated. It is hypothesized that blood flow patterns regulate endothelial cell (EC) function to affect the progression of CVDs. A system that subjects ECs to physiologically-relevant shear stress waveforms within microfluidic devices has not yet been demonstrated, despite the advantages associated with the use of these devices. In this work, a bioreactor was designed to fulfill this need. Waveforms from regions commonly affected by CVDs including were derived. Pump motion and fluid flow profiles were validated by actuator motion tracking, particle image velocimetry, and flowmeters. While several relevant waveforms were successfully replicated, physiological waveforms could not be produced at physiological frequencies owing to actuator velocity and accuracy limitations, as well as dampening effects in the system. Overall, this work lays the foundation for designing a system that provides insight into the role of shear stress in CVD pathogenesis.

The mechanisms behind cardiovascular disease (CVD) initiation and progression are not fully elucidated. It is hypothesized that blood flow patterns regulate endothelial cell (EC) function to affect the progression of CVDs. A system that subjects ECs to physiologically-relevant shear stress waveforms within microfluidic devices has not yet been demonstrated, despite the advantages associated with the use of these devices. In this work, a bioreactor was designed to fulfill this need. Waveforms from regions commonly affected by CVDs including were derived. Pump motion and fluid flow profiles were validated by actuator motion tracking, particle image velocimetry, and flowmeters. While several relevant waveforms were successfully replicated, physiological waveforms could not be produced at physiological frequencies owing to actuator velocity and accuracy limitations, as well as dampening effects in the system. Overall, this work lays the foundation for designing a system that provides insight into the role of shear stress in CVD pathogenesis.

<p dir="ltr">The necessity for on-farm, point-of-need (PON) nucleic acid amplification tests (NAATs) arises from the prolonged turnaround times and high costs associated with traditional laboratory equipment. This thesis aims to address these challenges by developing devices and a user-interface application designed for the efficient, accurate, and rapid detection of <i>Bacteroidales</i> as an indicator of fecal contamination on fresh produce farms.</p><p dir="ltr">In pursuit of this, I collaborated with lab members to engineer a Field-Applicable Rapid Microbial Loop-mediated isothermal Amplification Platform, FARM-LAMP. This device is portable (164 x 135 x 193 mm), energy-efficient (operating under 20 W), achieves the target 65°C with ± 0.2°C fluctuations, and is compatible with paper-based biosensors for loop-mediated isothermal amplification (LAMP). Subsequently, I led the fabrication of the microfluidic Field-Applicable Sampling Tool, FAST, designed to deliver high-throughput (10 samples per device), equal flow-splitting of fluids to paper-based biosensors, eliminating the need for a laboratory or extensive training. FARM-LAMP achieved 100% concordance with standard lab-based tests when deployed on a commercial lettuce farm and FAST achieved an average accuracy of 89% in equal flow-splitting and 70% in volume hydration.</p><p dir="ltr">A crucial aspect of device development is ensuring that results are easily interpretable by users. To this end, I developed a Python-based image analysis codebase to quantify sample positivity for fecal contamination, ranging from 0% (no contamination) to nearly 100% (definite contamination) and the concentration of field samples. It utilizes calculus-based mathematics, such as first and second derivative analysis, and incorporates image analysis techniques, including hue, saturation, and value (HSV) binning to a sigmoid function, along with contrast limited adaptive histogram equalization (CLAHE). Additionally, I developed a preliminary graphical user interface in Python that defines a prediction model for the concentration of <i>Bacteroidales</i> based on local weather patterns.</p><p dir="ltr">This thesis encompasses hardware development for on-field quantification and the creation of a preliminary user-interface application to assess fecal contamination risk on fresh produce farms. Integrating these devices with a user-interface application allows for rapid interpretation of results on-farm, aiding in the effective development of strategies to ensure safety in fresh produce operations.</p>