Digital microfluidics (DMF) is an emerging liquid-handling technique that facilitates manipulation of discrete droplets across an array of electrodes. Although the working principle of droplet movement is still under debate, it has gained significant interest as the technique has been applied to various applications in biology, chemistry and medicine. With recent advances in rapid prototyping and multilayer fabrication techniques using printed circuit boards, DMF has become an attractive and alternative solution to conventional macroscale fluidics techniques with additional capability of sample size reduction, faster analysis time, full automation, and multiplexing.
In this thesis, we explore the use of DMF for human plasma protein depletion due to its multiple advantages. The high abundance of human serum albumin (HSA) and immunoglobulins (Igs), which constitute 80% of total plasma proteins, is a major challenge in proteome studies. Unfortunately, conventional methods to deplete high abundant proteins (e.g. macro LC-columns) are labour-intensive, require dilution of sample, and run the risk of sample loss. Furthermore, most techniques lack the ability to process multiple samples simultaneously. Hence, we developed a new method of protein depletion using anti-HSA and Protein A/G immobilized paramagnetic beads manipulated by DMF to deplete HSA and IgG from human plasma.
Toward this goal, prototype DMF devices and electronic controller were designed, built and characterized (Chapter 2). Preliminary depletion experiments were first optimized in-tubes and then adapted for DMF manually (Chapter 3). At last, the entire depletion process was performed on DMF using an automated controller system (Chapter 4). Results showed that the protein depletion efficiency for immunoglobulin G (IgG) and HSA in 10 minutes for four samples simultaneously was as high as 98%, and an approximately 3-fold increase in signal-to-noise ratio after depletion was demonstrated by MALDI-MS analysis. The depletion process is sufficient for a tryptic digest to be performed on a model protein, cytochrome C, where 89% sequence coverage was obtained for a depleted sample. Although some improvements such as on-chip sample processing (e.g. digestion) need to be carried out as future work, we anticipate that the new technique is a significant alternative for applications involving protein depletion steps. / Thesis (Master, Chemistry) -- Queen's University, 2014-05-29 02:38:50.176
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OKQ.1974/12209 |
| Date | 29 May 2014 |
| Creators | Mei, NINGSI |
| Contributors | Queen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.)) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner. |
| Relation | Canadian theses |
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