No description available.

There is a clear need for testing targeted drug carrier systems in a more realistic microenvironment where both biochemical interactions and shear forces are present. This is critical both for understanding of the molecular mechanisms involved in this process and during the drug discovery process. Current in vitro models of the leukocyte adhesion cascade cannot be used for real-time studies of the entire leukocyte adhesion cascade including rolling, adhesion and migration in a single assay. In this study, we have developed and validated a novel bioinspired microfluidic device (bMFD) and used it to test the hypothesis that blocking of specific steps in the adhesion/migration cascade significantly affects other steps of the cascade. The bMFD consists of an endothelialized microvascular network in communication with a tissue compartment via a 3 µm porous barrier. Human neutrophils in bMFD preferentially adhered to activated human endothelial cells near bifurcations with rolling and adhesion patterns in close agreement with in vivo observations. Treating endothelial cells with monoclonal antibodies to E-selectin or ICAM-1 or treating neutrophils with wortmannin reduced rolling, adhesion, and migration of neutrophils to 60%, 20% and 18% of their respective control values. Antibody blocking of specific steps in the adhesion/migration cascade (e.g. mAb to E-selectin) significantly downregulated other steps of the cascade (e.g. migration). This novel in vitro assay provides a realistic human cell based model for basic science studies, identification of new treatment targets, selection of pathways to target validation, and rapid screening of candidate agents. / Mechanical Engineering

Nucleic-based acid technology (NAT) is a reliable and well-established method in molecular diagnosis for the detection of bacterial infection. Specifically, PCR (polymerase chain reaction) is the most popular technique to amplify the number of DNA or RNA copies in the sample. However, due to the thermal cycles in the PCR method, advanced equipment and technologies are required to precisely control the temperature during the cycles. To overcome this limitation, isothermal amplification methods have been developed which function at constant temperatures and help reduce the need for state-of-the-art machines to perform the amplification. Among isothermal amplification methods, LAMP (loop mediated isothermal amplification) has demonstrated robustness and sensitivity compared to PCR. Additionally, microfluidic lab-on-a-chip (LOC) technology can facilitate the intensive processes which have been used traditionally in laboratories by automating the required procedures, reducing the volume of the reagents and minimizing the cost and the time of experiments. Although many microfluidic LOC devices have been developed in order to be used in resource poor settings, there is still a need for a simple setup which is inexpensive, accurate and can be performed without the need for a trained technician. In this thesis, a disposable microfluidic device was developed which is capable of performing high-throughput DNA amplification by using a simple segmentation method in order to digitize the sample into multiple micro-wells. Moreover, design and fabrication of a disposable, inexpensive flexible heater which is an inevitable part of the setup using a direct write process was introduced in order to provide the required energy for the LAMP reaction. Parallel real-time DNA amplification with limit of detection down to few copies per micro-well in less than an hour was illustrated. Using E. coli 0157, it was demonstrated that the detection time of E.coli can be as quick as 11 to 55 minutes with sample concentrations varying from 700,000 copies/micro-well (11 minutes), 70,000 copies/micro-well (18 minutes), 700 copies/micro-well (31 minutes), 7 copies/micro-well (40 minutes) and 0.07 copies/micro-well (55 minutes). Finally, the capability of the device for on chip reagent storage up to 3 days without using any coating methods was illustrated. / Thesis / Master of Applied Science (MASc)

Among all organs, lungs are the last ones to grow and develop fully. As a result, extreme premature neonates may suffer from respiratory failure due to their immature lungs and will require respiratory support in the form of mechanical ventilation or extracorporeal membrane oxygenation (ECMO). In addition, extreme prematurity is recognized as the primary cause of neonatal morbidity and mortality. The conventional standard of care for respiratory support of preterm neonates with respiratory failure are invasive and may lead to long-term morbidities and complications. Hence, a non-invasive respiratory support technique named “Artificial Placenta” has been developed to address the issues and challenges associated with the current technologies. An artificial placenta type device is one designed to provide required oxygenation in room air via non-invasive access to the umbilical vessels without the need of any external pump. In this thesis, microfluidic and microfabrication technologies have been employed in the development of a pumpless neonatal lung assist device (LAD) for preterm neonates in two approaches: 1) design and develop novel microfabrication techniques to fabricate advanced microfluidic blood oxygenators with high gas exchange capacity and reduced form factor and 2) design and construct several modular LADs based on the oxygenators that were developed to fulfill the required gas transfer needs for these babies. The new microfluidic blood oxygenators with double-sided gas transfer channels were found to enhance oxygenation up to 343 % in room air and be easily scaled-up to achieve higher gas exchange capacities without a noticeable increase in priming volume. Furthermore, this microfabrication method has been utilized to make the largest all PDMS ultra-thin double-sided blood oxygenator with higher gas exchange capabilities. Also, a novel composite material made of PDMS and PTFE was introduced that conferred high flexibility to the oxygenator to decrease the form factor of such devices. This device was one of the first microfluidic blood oxygenators with enough flexibility to be deformed, bent, or rolled without limitation and losing its functionality. In order to satisfy the gas transfer need of these preterm neonates, few microfluidic-based modular LADs were constructed to support different birth weights up to 2 kg. The main design criteria for such a LAD in this research was low pressure drops (capable of being operated by a baby’s heart), an oxygen transfer of 1.3 – 1.9 mL min-1 kg-1 of body weight (or an increase in oxygen saturation level from ~ 75 % to ~ 100 % and ideally in room air), and low priming volume (less than 10 % of the total blood volume of a baby). These LADs first were evaluated in vitro to measure their gas exchange capacities and those which could meet needed oxygenation would be tested in vivo. For the first time, it was shown that a pumpless microfluidic-based LAD could support a newborn piglet and provide adequate oxygenation in room air or the oxygen-rich environment. The application of these microfluidic blood oxygenators was not only limited to preterm neonates but also can be used to develop LADs for adult patients. / Thesis / Doctor of Philosophy (PhD)

Yes / We report the first application of a microfluidic device to observe chemotactic migration in multicellular spheroids. A microfluidic device was designed comprising a central microchamber and two lateral channels through which reagents can be introduced. Multicellular spheroids were embedded in collagen and introduced to the microchamber. A gradient of fetal bovine serum (FBS) was established across the central chamber by addition of growth media containing serum into one of the lateral channels. We observe that spheroids of oral squamous carcinoma cells OSC–19 invade collectively in the direction of the gradient of FBS. This invasion is more directional and aggressive than that observed for individual cells in the same experimental setup. In contrast to spheroids of OSC–19, U87-MG multicellular spheroids migrate as individual cells. A study of the exposure of spheroids to the chemoattractant shows that the rate of diffusion into the spheroid is slow and thus, the chemoattractant wave engulfs the spheroid before diffusing through it. / This work has been supported by National Research Program of Spain (DPI2011-28262-c04-01) and by the project "MICROANGIOTHECAN" (CIBERBBN, IMIBIC and SEOM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Biological systems of bacteria were investigated at the single-cell and single-molecule level. Additionally, aspects of the techniques employed were studied. A unifying theme in each project is the reliance on optical imaging techniques coupled to microfluidic devices. Hypo-osmotic shock experiments with an Escherichia coli mechanosensitive channel deletion mutant were carried out at the single-cell level. E. coli MJF465 cells in which the three major mechanosensitive channel genes are deleted (∆mscL, ∆mscS, ∆mscK) show only 10% cell viability upon hypo-osmotic shock (from LB + 0.5 M NaCl into distilled water), compared to 90% viability of the wild-type strain. Bacterial cells were trapped with optical tweezers in microfluidic devices, enabling the first direct observation of single-cell behaviour upon hypo-osmotic shock. Phase-contrast microscopy revealed intra-population diversity in the cells response: Different features of lysis included cells bursting rapidly and leakage of ribosomes, DNA and protein from the cytoplasm. Fluorescence microscopy of hypo-osmotically-shocked GFP-expressing MJF465 cells showed either bursting of cells, which was a rare event, or fast leakage of GFP, indicating cell membrane ruptures. Data were analysed in terms of their kinetic behaviour and showed that lysis occurs on a timescale of milliseconds to seconds. The implications of these findings for the bacterial cell wall and cell membranes are discussed. Enzymes involved in homologous recombination and repair of double-stranded DNA (dsDNA) breaks are essential for maintaining genomic integrity in both eukaryotes and prokaryotes. RecBCD of E. coli and AddAB, found widely in bacteria, are involved in these processes, carrying out the same function. Both enzymes were studied kinetically with single-molecule total internal reflection fluorescence microscopy (TIRFM). Surface-tethered, hydrodynamically stretched lambda-DNA molecules, stained with YOYO-1, were imaged with TIRFM in a microfluidic flowcell. The RecBCD enzyme is a well characterised DNA helicase and was introduced to this system for method validation purposes. The AddAB enzyme of Bacteroides fragilis was then characterised as a helicase acting on lambda-DNA. It was found that AddAB helicase unwinds dsDNA with high processivity of on average 14,000 bp and up to 40,000 bp for individual enzyme complexes at an ATP-dependent rate ranging from 50-250 bp s−1 (for Mg2+-ATP concentrations larger or equal than 0.1 mM). This activity was detected by DNA binding dye (YOYO-1) displacement from the dsDNA and studied for different Mg2+-ATP concentrations, flow (shear) rates and different YOYO-1 staining ratios of DNA. Aspects of this last experimental setup were investigated. A kinetic analysis of intercalation of YOYO-1 into lambda-DNA is presented, occurring on a timescale of minutes. Different flow rates and staining ratios that influence the apparent (stretched) DNA molecule length were also examined. Several image analysis techniques were employed to enhance the data quality in images showing stretched lambda-DNA molecules. The Singular Value Decomposition was found to be the most effective technique which strongly reduces the noise in the obtained kymograph images.

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available via the Internet as viewed 10 July 2008.

Microfluidics deals with the behavior, control and manipulation of fluids which are confined at micrometer length scale. It has important application in lab-on-a chip technology, micro-propulsion, additive manufacturing, and micro-thermal technologies. Microfluidics has been widely used in detection, separation, transportation, and mixing of fluids and particles. The work carried out for the thesis to study the fluid-structure interaction in micro-channel involves an experimental part and a simulation part. In the experimental part the characterization of biofluid (RBC in BSA) is carried out based on the power law of fluid and flow behavior is studied. Also the dependence of fluid concentration on the viscosity in the channel is studied. The results are analyzed. Transition of fluid behavior from non-Newtonian shear thickening to non-Newtonian shear thinning is observed when the RBC concentration varies from 5.5×106 to 5.5×107 cells/ml in the channel. From the viscosity measurements of the biofluid it is observed that the average viscosity in the channel increases on increasing concentration of the fluid for shear thickening fluids. In the simulation part, interaction behavior of biomolecule DNA is studied in the channel containing biofluid which is characterized in the experimental part. Cell free DNAs are common problem in infectious disease detection. Based on the assumptions of the WLC model, DNA strand is assumed as a one dimensional elastic member with its one end fixed at the channel wall and the other end free to move in the fluid. Bent and straight DNAs are considered for the study. Multiple scales are involved in this problem which is not fully understood. DNA strands in the channel are exposed to different forces in the channel which are mainly due to the pressure and viscous effects. Numerical simulations are carried out for the multiphysics problem of DNA in the fluid using a coupled multiphysics finite element scheme and the results are obtained. Same procedure is carried out considering smaller channels and also for PBS solution as background fluid to obtain consistent results. It is found that when the channel width increases the tip displacement of DNA decreases. It was observed that DNA tip displacement is more in the channel when its end-to-end length is approximately half the width of the channel. Potential application of these modeling and simulation are in molecular screening processes to improve the performance of microfluidic DNA chips, and in design of micro-channel structures of microfluidic devices.

No description available.

No description available.