Acoustophoresis is a label free method where the acoustic radiation force is used to manipulate microparticles inside microfluidic channels. The magnitude of the force is dependent of several parameters, which include the density, speed of sound and size of the microparticles, as well as the amplitude of the pressure waves. Recently, acoustophoresis has been used in microfluidics to manipulate microparticles inside moving droplets. In this Master's thesis project, two microfluidic chip designs are used to enrich droplets with polystyrene beads (10 μm in diameter) using acoustophoresis. The microchips have been fabricated with two different fabrication methods; crystalline dependent wet etching and crystalline independent dry etching. In the microchips, water droplets in oil are generated with microparticles suspended in them. By using a channel width that is half a wavelength of the incoming acoustic waves, pressure nodal lines are created in the middle of the channel in which the microparticles align. The droplets then enters a droplet splitting feature, where they are divided into three daughter droplets. Since the majority of the incoming particles are recovered in the center daughter droplet while some of the droplet volume is removed, the center droplet is enriched with the microparticles. For the wet etched design stable droplet splitting was observed when the volumetric flow was 18 μL/min and the incoming droplets had a length-to-width ratio larger than 3. The maximum recovery for this design was 81.1% ± 13.8% with an applied voltage at 10 Vpp. Stable droplet splitting was observed for the dry etched chip at 10.5 μL/min and 18 μL/min at 10 and 20 Vpp, when the incoming droplet had a length-to-width ratio of 3. In this chip the maximum recovery was 93.2% ± 8.3% at the volumetric flow of 10.5 μL/min and an applied voltage of 20 Vpp.

Nanopores are apertures of nanometric dimensions in an insulating matrix. They are routinely used to sense and measure properties of single molecules such as DNA. This sensing technique relies on the process of translocation, whereby a molecule in aqueous solution moves through the pore under an applied electric field. The presence of the molecule modulates the ionic current through the pore, from which information can be obtained regarding the molecule's properties. Whereas the electrical properties of the nanopore are relatively well known, much less work has been done regarding their fluidic properties. In this thesis I investigate the effects of fluid flow within the nanopore system. In particular, the charged nature of the DNA and pore walls results in electrically-driven flows called electroosmosis. Using a setup which combines the nanopore with an optical trap to measure forces with piconewton sensitivity, we elucidate the electroosmotic coupling between multiple DNA molecules inside the confined environment of the pore. Outside the pore, these flows produce a nanofluidic jet, since the pore behaves like a small electroosmotic pump. We show that this jet is well-described by the low Reynolds number limit of the classical Landau-Squire solution of the Navier-Stokes equations. The properties of this jet vary in a complex way with changing conditions: the jet reverses direction as a function of salt concentration, and exhibits asymmetry with respect to voltage reversal. Using a combination of simulations and analytic modelling, we are able to account for all of these effects. The result of this work is a more complete understanding of the fluidic properties of the nanopore. These effects govern the translocation process, and thus have consequences for better control of single molecule sensing. Additionally, the phenomena we have uncovered could potentially be harnessed in novel microfluidic applications, whose technological implications range from lab-on-a-chip devices to personalised medicine.

Microfluidic technology is a rapidly progressing tool in biomedical engineering. Microfluidic devices are appreciated for their simplicity and production efficiency potential. Our research focuses on developing a microfluidic device capable of transfecting cells by applying shear stress to cause temporary membrane damage. The advantage of this physical method of transfection is the possibility of incorporating large molecules that cannot be inserted with more traditional chemical transfection methods, while avoiding the large fall in viability seen with other physical methods such as electroporation. Unlike previous groups, our device incorporates the use of microfluidic valves to allow tunability, and impedance sensing for cell membrane damage analysis. We achieve (95±5)% cell viability and up to (68±5)% efficiency in transfecting 3T3 cells with DNA-sized molecules. In future stages, we intend to add the device onto an existing cell-encapsulation device that is tasked with preparing therapeutic cells to be used in regenerative medicine applications.

The aim of this project was to work towards developing a droplet-based microfluidic device which can perform cell lysis, Whole Genome Amplification (WGA) and storage of the amplified DNA. This would provide an automated biobanking device capable of high-throughput sample processing whilst shielding the samples from the sample loss and contamination commonly experienced by conventional, isolated sample handling methods.WGA has been examined using two commercially available WGA kits (GenomiPhi V2 and HY) to produce a continuous flow device that is capable of amplifying both human genomic DNA (gDNA) and bacterial plasmid DNA samples in nanolitre volume droplets. A positive effect of reducing reaction volumes on the amplification of bacterial plasmid DNA was shown by obtaining an increase in yield with decreasing volumes. It was shown, however, that a reduction in the volume of the WGA reaction has a negative impact on the amplification of human gDNA, in terms of both reduced yield and copy number variation (CNV). Furthermore, a novel method for reducing this CNV has been achieved by pooling the products of multiple reaction volumes. Finally, a cell lysis device has been developed which can perform rapid lysis of a human neuroblastoma cell line in continuously flowing droplets through addition of an alkaline solution.These devices provide an advantage over previously developed methods, displaying cell lysis of a human cell line and amplification of both human gDNA and plasmid DNA, while the continuous flow design of the devices allows for both high-throughput processing of samples and the future integration of the devices to form a μTAS biobanking device.

Les anticorps sont des protéines en forme de Y, trouvées comme composant du sérum circulant, qui aident le système immunitaire à cibler et à répondre aux agents pathogènes et aux molécules étrangères, mais peuvent aussi contribuer à la maladie en réagissant aux protéines constitutives. Les anticorps sont produits par des Plasmocytes, qui les sécrètent dans la circulation. Parce qu'il n'y a pas de lien physique entre les plasmocytes et leurs anticorps sécrétés, la détection d'anticorps spécifiques d’un antigène est problématique. Dans cette thèse, j'explore l'utilisation de la microfluidique en gouttelettes pour générer et manipuler des compartiments aqueux homogènes dans lesquels des cellules sécrétant anticorps peuvent être isolées et analysées à haut débit a'échelle d'une seule cellule. Pour caractériser les cellules sécrétant des anticorps à l'intérieur des gouttelettes, j'utilise un nouveau test qui permet d'interroger les cellules en fonction de la spécificité de leur sécrétion. Ces compartiments de gouttelettes peuvent être triés pour le séquençage d'anticorps, ou analysés au cours du temps pour obtenir des informations cinétiques de l'interaction anticorps-antigène à l'intérieur de chaque gouttelette. En utilisant une nouvelle technologie, j'ai pu obtenir le répertoire d'anticorps de souris immunisées contre deux antigènes différents à partir de cellules sécrétant des anticorps spécifiques d’un antigène, avec des capacités égales ou supérieures aux technologies disponibles actuelles. Aussi, j'ai pu suivre le processus de maturation d'affinité des anticorps à l'échelle de la cellule unique, à la fois dans l'immunisation et la maladie auto-immune. Avec ces outils, je démontre comment la microfluidique peut être utilisée pour caractériser les réponses immunitaires et auto-immunes à travers l'évaluation de cellules sécrétant des anticorps. / Antibodies are Y shaped proteins, found as a component of circulating serum, that help the immune system target and respond to pathogens and foreign molecules, but can also contribute to disease when reacting to constitutive self-proteins. Antibodies are produced Plasma Cells, which secrete them into circulation. Because there’s no physical link between Plasma Cells and their secreted antibodies, the detection of antigen-specific antibodies is problematic. In this thesis I explore the use of droplet microfluidics to generate and manipulate homogeneous aqueous compartments in which single antibody secreting cells can be isolated and analyzed in a high-throughput manner. To characterize single antibody secreting cells inside the droplets I use a novel assay that allows to interrogate cells based on the specificity of their secretion. These droplet compartments can be sorted for single cell antibody sequencing, or analyzed over time to obtain kinetic information of the antibody-antigen interaction inside each droplet. Using new established technology I was able to obtain the antibody repertoire of mice immunized against two different antigens from single antigen-specific antibody secreting cells, with equal or better capacities than current available technologies. Also, I was able to follow the affinity maturation process of antibodies at the single-cell level, both in immunization and autoimmune disease. With these tools I demonstrate how microfluidics can be used to characterize the immune and the autoimmune responses through the evaluation of single antibody secreting cells.

Conventional healthcare and the practice of medicine largely relies on the ineffective concept of one size fits all. Personalized medicine is an emerging therapeutic approach that aims to develop an advanced therapeutic technique that provides tailor-made therapy based on every individuals’ needs by delivering the right drug at the right time with the right amount of dosage. The advancement in technologies such as flexible electronics, microfluidics, biosensors, and advanced artificial intelligence can enable the realization of a truly effective personalized therapy. However, currently, there is a lack for a personalized minimally-invasive wearable closed-loop drug delivery system that is continuous, automated, conformal to the skin and cost-effective. Thus, this thesis focuses on the design, fabrication, optimization, and application of an automated personalized microfluidics drug delivery platform augmented with flexible biosensors, heaters, and expandable polymeric actuator. The platform provides precise drug delivery with spatiotemporal control over the administered dose as a response to real-time physiological changes of the individual. The system is flexible enough to be conformal to the skin and drug is transdermally administered through biocompatible microneedles. The platform includes a flexible multi-reservoir microfluidics layer, flexible and conformal heating elements, skin sensors and processing units which are powered by a lightweight battery integrated into the platform. The developed platform was fabricated using rapid, cost-effective techniques that are independent of advanced microfabrication facilities to expand its applications to low-resource setting and environments.

Among various sources for biofuels, microalgae provide at least three-orders-of-magnitude higher production rate of biodiesel at a given land area than conventional crop-based methods. However, microalgal biodiesel still suffers from significantly lower harvesting efficiency, making such a fuel less competitive. To increase the separation efficiency of microalgae from cultivation solution, an orbital microchannel was utilized that enabled the isolation of biofuel-algae particles from the effluent. The results obtained showed that the separation efficiency in the microfluidic centrifugal separator can be as high as 76% within a quick separation time of 30 seconds. Multiple parameters of algae behaviors and separation techniques such as initial concentration, pH and temperature were studied and manipulated to achieve better efficiencies. It was found that changing these factors altered the separation efficiency by increasing or decreasing flocculation, or “clumping” of the microalgae within the microchannels. The results suggested that an acidic condition would enhance the separation efficiency since in a basic environment, large flocs of microalgae would block and hinder the separation process. Furthermore, a hot temperature solution (around 33 °C) yielded to a higher separation efficiency. The important characteristics of the separator geometry and the infusion rate on algae separation were also very effective in the separation process. This study revealed that there is an opportunity to improve the currently low efficiency of algae separation in centrifugal systems using much smaller designs in size, ensuring a much more efficient algae harvesting. / Includes bibliography. / Thesis (MS)--Florida Atlantic University, 2021. / FAU Electronic Theses and Dissertations Collection

This diploma thesis deals with optimization of analysis process and measuring antibiotics induced changes in E. coli cells via Raman spectroscopy, LTRS and microfluidic systems. Optical micromanipulation by a laser beam allows noncontact and noninvasive manipulation of objects on scale 10^-5–10^-8 m, for example bacterial cells. Microfluidic device consists of microchannels and microchambers in transparent polymer and it is used for isolation, observation and cultivation of bacterial cells. Combination of these methods gives an effective tool for observation, manipulation and analysis of microorganisms. E. coli is a microorganism potentially pathogenic for humans and faster detection of its sensitivity to antibiotic treatment would make the whole process of diagnostics and treatment easier. We performed laser tweezer-Raman spectroscopy and conventional Raman spectroscopy of bacterial cells and cells under antibiotic stress and collected Raman spectra and characteristic areas were compared with literature to establish the reliability and usefulness of this method.

The demand for novel and robust microbial biocatalysts for industrial and pharmaceutical applications continue to grow at a fast pace.This warrants a continuous need for advanced tools and technologies to exploit the vast metabolic potential of microorganisms in different environments. Unlike culture-based studies that can only reveal the metabolic potential of cultivable microorganisms, functional metagenomics charts the enzymatic potential of the entire microbial communities in a given environment. This method has substantially contributed to the effective discovery of unique microbial genes for industrial and medical applications. Functional metagenomics involves the extraction of microbial DNA directly from environmental samples,construction of an expression library containing the entire microbial genome, and screening the libraries for the presence of desired phenotypes. Therefore, development of a pipeline for analyzing and screening metagenomic libraries is essential for rapid detection of the desired features from thousands of clones of a single library. Here, we developed a pipeline for high-throughput screening of the lipolytic genes from the Red Sea.Further, a high-throughput single cell microfluidics platform combined with a laser-based fluorescent screening bioassay was deployed to discover new lipolytic genes. Our analysis led to the identification of 24 microbial genes for lipases and esterase from a metagenomic library of the Red Sea water. The results further showed that the constructed pipeline is robust in conducting functional metagenomics and for the discovery of new genes. It also implies that the Red Sea is a rich under- investigated source of natural resources of new genes and gene products.

Microencapsulation of stem cells in hydrogel for use in therapeutic applications has been shown to improve cell retention at the site of injuries due to their mechanical and immunoprotective properties. These microscale droplets (cocoons) can be produced at high throughputs within microfluidic channels. Currently, the ability for cells to egress hydrogel cocoons is under investigation. This egress can correlate with therapeutic efficacy, and so promoting or inhibiting the egress of cells can be a vital component of viable treatments. Previously, a second hydrogel layer was shown to reduce egress, but issues involving cell proliferation were unchanged. We propose a microfluidic process to encapsulate cells in two layers of thermoresponsive hydrogels, in which the inner core melts at physiological temperatures to form hollow cocoons that allow cells free motion inside the immunoprotective shell. We hypothesize that the open volume would increase cell viability and proliferation, without increasing cell egress due to the uninterrupted hydrogel shell. In this project the encapsulation of NIH 3T3 cells in hollow agarose cocoons was achieved. 3T3 cells were first encapsulated in thermoreversible gelatin which were then re-encapsulated in agarose through the use of a flow-focusing microfluidic channel with on-chip mixing of two inlet flows to produce hollow cocoons. The production of these cocoons showed the potential of high throughput, monodisperse samples with future investment. Preliminary investigation in the behavior of the encapsulated cells showed that the cells maintain high viability over the course of 48 hours. There are early indications that the hollow nature of correctly formed cocoons can limit cell egress, and may allow for proliferation in the cocoon.