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Structural and functional studies of membrane peptides : Glycophorin A transmembrane domain and melittin analoguesTakei, Jiro January 1998 (has links)
No description available.
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Development of a DNA extraction, amplification and storage microdeviceMarkey, Amelia Louise January 2013 (has links)
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.
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Single cell analysis on microfluidic devicesChen, Yanli January 1900 (has links)
Master of Science / Department of Chemistry / Christopher T. Culbertson / A microfluidic device integrated with valves and a peristaltic pump was fabricated using multilayer soft lithography to analyze single cells. Fluid flow was generated and mammalian cells were transported through the channel manifold using the peristaltic pump. A laser beam was focused at the cross-section of the channels so fluorescence of individual labeled intact cells could be detected. Triggered by the fluorescence signals of intact cells, valves could be actuated so fluid flow was stopped and a single cell was trapped at the intersection. The cell was then rapidly lysed through the application of large electric fields and injected into a separation channel. Various conditions such as channel geometry, pumping frequency, control channel size, and pump location were optimized for cell transport. A Labview program was developed to control the actuation of the trapping valves and a control device was fabricated for operation of the peristaltic pump. Cells were labeled with a cytosolic dye, Calcein AM or Oregon Green, and cell transport and lysis were visualized using epi-fluorescent microscope. The cells were transported at rates of [simular to] 1mm/s. This rate was optimized to obtain both high throughput and single cell trapping. An electric field of 850-900 V/cm was applied so cells could be efficiently lysed and cell lysate could be electrophoretically separated. Calcein AM and Oregon Green released from single cells were separated and detected by laser-induced fluorescence. The fluorescence signals were collected by PMT and sampled with a multi-function I/O card. This analyzing method using microchip may be applied to explore other cellular contents from single cells in the future.
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Lysis of 'Escherichia coli' for the Recovery of Pentamerised Single-Domain Antibody Used for the Gender Specific Separation of Bovine SpermO'Reilly, Jordan January 2016 (has links)
Gender of animal offspring is of great interest to farmers where gender selection is achieved via the separation of male-bearing from female-bearing sperms prior to performing artificial insemination. A start-up company (Ab Biotech Inc.) has developed a technique for gender selection based on the production of an intracellular single-domain antibody (sdAb) using the bacterium Escherichia coli capable of sexing bovine sperm. The purpose of this research was to provide a recommendation to Ab Biotech Inc. for the lysis of E. coli. An efficient lysis technique was required in order to release the intracellular sdAb. In the dairy industry, sexing for female calves is preferred since male calves are not useful for the purpose of milk production. Multiple lysis techniques were tested in order to provide a feasible recommendation for Ab Biotech Inc. These techniques included high pressure homogenization, sonication, bead milling and enzymatic/chemical lysis using lysozymes and Triton X-100. Required lysis time, extent of lysis and potential operating costs were contributing factors for determining an optimal technique. The extent of lysis was determined by quantifying the total amount of released protein using SDS-PAGE densitometry. It was recommended to choose bead milling for potential process upscaling since a large amount of fractional lysis (0.70) was obtained over a short amount of lysis time (3 min) with an inexpensive ($9.50/kg) 0.3 mm mixture of glass beads.
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Development of Cell Lysis Techniques in Lab on a chipShahini, Mehdi January 2013 (has links)
The recent breakthroughs in genomics and molecular diagnostics will not be reflected in health-care systems unless the biogenetic or other nucleic acid-based tests are transferred from the laboratory to clinical market. Developments in microfabrication techniques brought lab-on-a-chip (LOC) into being the best candidate for conducting sample preparation for such clinical devices, or point-of-care testing set-ups. Sample preparation procedure consists of several stages including cell transportation, separation, cell lysis and nucleic acid purification and detection. LOC, as a subset of Microelectromechanical systems (MEMS), refers to a tiny, compact, portable, automated and easy-to-use microchip capable of performing the sample-preparation stages together. Complexity in micro-fabrications and inconsistency of the stages oppose integration of them into one chip.
Among the variety of mechanisms utilized in LOC for cell lysis, electrical methods have the highest potential to be integrated with other microchip-based mechanisms. There are, however, major limitations in electrical cell lysis methods: the difficulty and high-cost fabrication of microfluidic chips and the high voltage requirements for cell lysis. Addressing these limitations, the focus of this thesis is on realization of cell lysis microchips suitable for LOC applications.
We have developed a new methodology of fabricating microfluidic chips with electrical functionality. Traditional lithography of microchannel with electrode, needed for making electro-microfluidic chips, is considerably complicated. We have combined several easy-to-implement techniques to realize electro-microchannel with laser-ablated polyimide. The current techniques for etching polyimide are by excimer lasers in bulky set-ups and with involvement of toxic gas. We present a method of ablating microfluidic channels in polyimide using a 30W CO2 laser. Although this technique has poorer resolution, this approach is more cost effective, safer and easier to handle. We have verified the performance of the fabricated electro-microfluidic chips on electroporation of mammalian cells.
Electrical cell lysis mechanisms need an operational voltage that is relatively high compared to other cell manipulation techniques, especially for lysing bacteria. Microelectro-devices have dealt with this limitation mostly by reducing the inter-distance of electrodes. The technique has been realized in tiny flow-through microchips with built-in electrodes in a distance of a few micrometers which is in the scale of cell size. In addition to the low throughput of such devices, high probability of blocking cells in such tiny channels is a serious challenge. We have developed a cell lysis device featured with aligned carbon nanotube (CNT) to reduce the high voltage requirement and to improve the throughput. The vertically aligned CNT on an electrode inside a MEMS device provides highly strengthened electric field near the tip. The concept of strengthened electric field by means of CNT has been applied in field electron emission but not in cell lysis. The results show that the incorporation of CNT in lysing bacteria reduces the required operational voltage and improves throughput. This achievement is a significant progress toward integration of cell lysis in a low-voltage, high-throughput LOC.
We further developed the proposed fabrication methodology of micro-electro-fluidic chips, described earlier, to perform electroporation of single mammalian cell. We have advanced the method of embedding CNT in microchannel so that on-chip fluorescent microscopy is also feasible. The results verify the enhancement of electroporation by incorporating CNT into electrical cell lysis. In addition, a novel methodology of making CNT-embedded microfluidic devices has been presented. The embedding methodology is an opening toward fabrication of a CNT-featured LOC for other applications.
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Microfluidic Particles / Cells Sorter Integrated with Concentration Friction Feeding Device for Biochemical Analysis ApplicationsLee, Chen-Yan 02 August 2006 (has links)
This study proposes a navel method for continuously particle sorting utilizing cascade squeeze jumping effect under microfluidic configuration. Microparticles with different sizes can be successfully separated at different stages of squeezing sheath flow. The method is based on that particles can not flow stably within a flow stream with the smaller stream width than their sizes. Big particles will jump from their original flow stream into the wider neighboring sheath flow. In this study, we have successfully designed and fabricated two kinds of particles/cells sorters using MEMS (Micro-electro-mechanical Systems) technology. The proposed microchip device includes a multi-stage sheath flow particles/cells sorter and an improved design of a cascade squeezed flow scheme. In the study, theoretical formulations, computer simulations and experimental operations are used to analyze the flow field in the microchip and evaluate the sorting performance of the devices. Results show the good sorting performance with cell recovery rate of 87.7% and yield rate of 94.1% can be obtained using the proposed micro particles/cells sorter.
Furthermore, it is also important to continiously prepare reagents for in-column bio-chemical reactions. Therefore, this study presents a sheath-flow based microfluidic device for concentration fraction delivery of liquid samples. The simple and novel structure proposed in this study is able to prepare reagent with different concentration and is also easy to be integrated with other multifunctional microfluidic device. In order to demonstrate the feasibility and performance of the proposed concentration fraction delivery device, this study designs an integrated microchip device for in-line preparation of lysin reagent for cell lysis and an integrated T-form microfluidic mixer for demonstration of RBC lysis in the same microchip. Reagents for cell lysis are firstly prepared by the concentration faction delivery part of the chip. The prepared reagent is mixed with RBC sample downstream in the reaction channel using the T-form mixer. Results show a high RBC lysing rate of upto 100% in 10 mm downstream the T-junction can be achieved utilizing the proposed chip.
In this study, we have successfully demonstrated three kinds of microfluidic device including a micro particles/cells sorter, a concentration fraction delivery device and a cell lysis reactor. Numerical analysis and experimental investigation confirm the proposed concepts and performance of the microfluidic devices. The contributions of the study are highly potential for developing a low-cost bioreactor system in the
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Microfluidic bases sample preparation for blood stream infectionsArdabili, Sahar January 2014 (has links)
Microfluidics promises to re-shape the current health-care system by transferring diagnostic tools from central laboratories to close vicinity of the patient (point-of-care). One of the most important operational steps in any diagnostic platform is sample preparation, which is the main subject in this thesis. The goal of sample preparation is to isolate targets of interest from their surroundings. The work in this thesis is based on three ways to isolate bacteria: immune-based isolation, selective cell lysis, size-based separation. The first sample-preparation approach uses antibodies against lipopolysaccharides (LPS), which are surface molecules found on all gram-negative bacteria. There are two characteristics that make this surface molecule interesting. First, it is highly abundant: one bacterium has approximately a million LPS molecules on its cell-wall. Second, the molecule has a conserved region within all gram-negative bacteria, so using one affinity molecule to isolate disease-causing gram-negative bacteria is an attractive option, particularly from the point of view of sample preparation. The main challenge, however, is antigen accessibility. To address this, we have developed a treatment protocol that improves the capturing efficiency. The strategy behind selective cell lysis takes advantage of the differences between the blood-cell membrane and the bacterial cell-wall. These fundamental differences make it possible to lyse (destroy) blood-cells selectively while keeping the target of interest, here the bacteria, intact and, what is more important alive. Viability plays an important role in determining antibiotic susceptibility. Difference in size is another well-used characteristic for sample- separation. Inertial microfluidics can focus size-dependent particle at high flow-rates. Thus, particles of 10 µm diameter were positioned in precise streamlines within a curved channel. The focused particles can then be collected at defined outlets. This approach was then used to isolate white blood cells, which account for approximately 1% of the whole blood. In such a device particles of 2µm diameter (size of bacteria) would not be focused and thereby present at every outlet. To separate bacteria from blood elasto-inertial microfluidics was used. Here, e blood components are diverted to center of the channels while smaller bacteria remain in the side streams and can subsequently be separated. / <p>QC 20141212</p>
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Development of Cell Lysis Techniques in Lab on a chipShahini, Mehdi January 2013 (has links)
The recent breakthroughs in genomics and molecular diagnostics will not be reflected in health-care systems unless the biogenetic or other nucleic acid-based tests are transferred from the laboratory to clinical market. Developments in microfabrication techniques brought lab-on-a-chip (LOC) into being the best candidate for conducting sample preparation for such clinical devices, or point-of-care testing set-ups. Sample preparation procedure consists of several stages including cell transportation, separation, cell lysis and nucleic acid purification and detection. LOC, as a subset of Microelectromechanical systems (MEMS), refers to a tiny, compact, portable, automated and easy-to-use microchip capable of performing the sample-preparation stages together. Complexity in micro-fabrications and inconsistency of the stages oppose integration of them into one chip.
Among the variety of mechanisms utilized in LOC for cell lysis, electrical methods have the highest potential to be integrated with other microchip-based mechanisms. There are, however, major limitations in electrical cell lysis methods: the difficulty and high-cost fabrication of microfluidic chips and the high voltage requirements for cell lysis. Addressing these limitations, the focus of this thesis is on realization of cell lysis microchips suitable for LOC applications.
We have developed a new methodology of fabricating microfluidic chips with electrical functionality. Traditional lithography of microchannel with electrode, needed for making electro-microfluidic chips, is considerably complicated. We have combined several easy-to-implement techniques to realize electro-microchannel with laser-ablated polyimide. The current techniques for etching polyimide are by excimer lasers in bulky set-ups and with involvement of toxic gas. We present a method of ablating microfluidic channels in polyimide using a 30W CO2 laser. Although this technique has poorer resolution, this approach is more cost effective, safer and easier to handle. We have verified the performance of the fabricated electro-microfluidic chips on electroporation of mammalian cells.
Electrical cell lysis mechanisms need an operational voltage that is relatively high compared to other cell manipulation techniques, especially for lysing bacteria. Microelectro-devices have dealt with this limitation mostly by reducing the inter-distance of electrodes. The technique has been realized in tiny flow-through microchips with built-in electrodes in a distance of a few micrometers which is in the scale of cell size. In addition to the low throughput of such devices, high probability of blocking cells in such tiny channels is a serious challenge. We have developed a cell lysis device featured with aligned carbon nanotube (CNT) to reduce the high voltage requirement and to improve the throughput. The vertically aligned CNT on an electrode inside a MEMS device provides highly strengthened electric field near the tip. The concept of strengthened electric field by means of CNT has been applied in field electron emission but not in cell lysis. The results show that the incorporation of CNT in lysing bacteria reduces the required operational voltage and improves throughput. This achievement is a significant progress toward integration of cell lysis in a low-voltage, high-throughput LOC.
We further developed the proposed fabrication methodology of micro-electro-fluidic chips, described earlier, to perform electroporation of single mammalian cell. We have advanced the method of embedding CNT in microchannel so that on-chip fluorescent microscopy is also feasible. The results verify the enhancement of electroporation by incorporating CNT into electrical cell lysis. In addition, a novel methodology of making CNT-embedded microfluidic devices has been presented. The embedding methodology is an opening toward fabrication of a CNT-featured LOC for other applications.
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Bridging Inductively Coupled Plasma - Mass Spectrometry (ICP-MS) from Metalloproteomics to the Undergraduate CurriculumDonnell, Anna M. 30 October 2017 (has links)
No description available.
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Microfluidics for Cell Manipulation and AnalysisLoufakis, Despina Nelie 21 October 2014 (has links)
Microfluidic devices are ideal for analysis of biological systems. The small dimensions result to controlled handling of the flow profile and the cells in suspension. Implementation of additional forces in the system, such as an electric field, promote further manipulation of the cells. In this dissertation, I show novel, unique microfluidic approaches for manipulation and analysis of mammalian cells by the aid of electrical methods or the architecture of the device. Specifically, for the first time, it is shown, that adoption of electrical methods, using surface electrodes, promotes cell concentration in a microchamber due to isoelectric focusing (IEF). In contrast to conventional IEF techniques for protein separation, a matrix is not required in our system, the presence of which would even block the movement of the bulky cells. Electric field is, also, used to breach the cell membrane and gain access to the cell interior by electroporation (irreversible and reversible). Irreversible electroporation is used in a unique, integrated microfluidic device for cell lysis and reagentless extraction of DNA. The genomic material is subsequently analyzed by on-chip PCR, demonstrating the possible elimination of the purification step. On the other hand, reversible electroporation is used for the delivery of exogenous molecules to cells. For the first time, the effect of shear stress on the electroporation efficiency of both attached and suspended cells is examined. On the second part of my dissertation, I explore the capabilities of the architecture of microfluidic devices for cell analysis. A simple, unique method for compartmentalization of a microchamber in an array of picochambers is presented. The main idea of the device lies on the fabrication of solid supports on the main layer of the device. These features may even hold a dual nature (e.g. for cell trapping, and chamber support), in which case, single cell analysis is possible (such as single cell PCR). On the final chapter of my dissertation, a computational analysis of the flow and concentration profiles of a device with hydrodynamic focusing is conducted. I anticipate, that all these novel techniques will be used on integrated microfluidic systems for cell analysis, towards point-of-care diagnostics. / Ph. D.
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