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A passive microfluidic device for continuous buffer exchangeGedra, Olivia Rose 25 July 2024 (has links)
Generally, dielectrophoresis (DEP) analysis of biological cell samples relies on the differing electrical parameters between the cells and the surrounding fluid medium. To achieve effective positive DEP manipulation and sorting of mammalian cells in suspension, it is required to resuspend the cells into a low-conductivity fluid buffer. The use of a low conductivity buffer also aids in minimizing the effects of Joule heating, which can cause cell death and ineffective cell trapping. The common method to prepare the sample relies on centrifugation of sensitive cells, a time-consuming and tedious process that may result in decreased sample viability. Herein is presented a microfluidic device that passively moves cells from a high-conductivity growth media into a low-conductivity DEP buffer. It is comprised of con- verging rows of pillars and uses mechanical filtration to force cells into the new buffer while allowing for the old fluid to flow through the posts and out of separate outlets. Because this device is intended to be used upstream of a contactless dielectrophoresis (cDEP) device, the buffer exchange device must have an outlet flow rate that is within the range necessary for direct integration with the cDEP device, maintain a low shear stress that will not affect the integrity of the sample and achieve sufficiently high cell recovery. Methods of this project included optimizing the shape, size, and orientation of the posts, determining the flow rate for maintaining an ideal DEP buffer conductivity, numerical modeling of shear stress, and determining the cell recovery rate. It is anticipated that this device can be extended to physiological media sample processing such as for liquid biopsy. / Master of Science / In order to accomplish numerous biomedical experiments, cells must be transferred from their native fluid growth media into a different fluid solution, through a process referred to as buffer exchange. The current method for buffer exchange is time consuming, tedious, and affects the number of cells left alive for experimentation. In this work, we present a microfluidic device that can accomplish the buffer exchange process by simply flowing in the cells in their media in parallel with the new buffer solution. The results of this research work can be extended to aid in the process of buffer exchange for various biological experiments.
The proposed device utilizes mechanical filtration to force cells into the new buffer while allowing for the old fluid to flow through the posts and out of separate outlets. The design of the device was optimized through computational analysis of the concentration and fluid shear stress in conjunction with experimental tests of devices for outlet conductivity and cell retention.
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Novel Native Mass Spectrometry-based Fragmentation and Separation Approaches for the Interrogation of Protein ComplexesVanAernum, Zachary L. 29 September 2020 (has links)
No description available.
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Production and characterisation of a chlamydial antigen candidate for vaccine trialsKoivula, Therese January 2021 (has links)
The bacterium Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infection worldwide. When left untreated, chlamydial infections can lead to severe complications, such as infertility. Lack in current prevention and management due to its asymptomatic course of infection highlight the need for an effective vaccine against chlamydia. There is no vaccine at present to protect against chlamydia, but research is ongoing. A research group at Örebro University has developed a protein antigen candidate. This project focused on the production of the candidate, here called Protein X, for preclinical trials. This included optimising production in Escherichia coli to maximise formation of soluble protein, optimising purification, buffer exchange and removal of His-tag. It was found that formation of soluble protein was favoured in lower expression temperatures. Furthermore, purification was performed on soluble and insoluble protein fractions using immobilised metal affinity chromatography. However, issues with inefficient binding to the resin and purity could not be solved and further optimisation is needed. Buffers were tested to find a suitable buffer for preclinical experiments, but the protein precipitated in all buffers. It was however found that protein from the insoluble fraction dissolved in pure water. Lastly, removal of the His-tag was performed with a non-enzymatic method that utilises nickel ions instead of expensive proteases. Efficient removal was however not achieved and enzymatic methods may be considered instead. In conclusion, this project highlighted issues in the production of Protein X and may guide the research group towards improving this process for efficient preclinical preparations.
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