Nucleus Extraction from Cells by Performing Optically-Induced Cell Lysis on a Continuous-flow Platform
| Autor: | Huang, Shih Hsuan, 黃士軒 |
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| Rok vydání: | 2015 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 103 This study presents an innovative microfluidics-based approach for nucleus extraction and collection in a continuous format. Previously proposed micro-devices for cell lysis can be classified into several types, including thermal, electrical, chemical, acoustic, optical, and mechanical approaches, and can be combined with subsequent chemical treatments and analysis of extracted intracellular components. Comparing with previous micro-devices, a novel optically-induced cell lysis (OICL) device proposed in this study shows the advantages with significant flexibility, selectivity and efficiency. More importantly, once can precisely control the operating conditions of this new device to lyse cell membranes without disrupting nucleus. To facilitate this OICL module for continuous nucleus extraction, we further integrate an optically-induced dielectrophoresis (ODEP) module with OICL device into a microfluidic system. This novel approach avoids critical issues such as difficulty to extract intact nucleus rapidly and the requirement of professional skills to operate the traditional equipments, which are posed as common disadvantages to the traditional nucleus extraction approaches. Our new microfluidic system therefore provides a powerful method for automating four steps, including automatically focusing and transporting human embryonic kidney 293T cells, releasing the nucleus on the OICL module, nucleus isolation on the ODEP module and finally nucleus collection in the outlet chamber. The average throughput of nucleus extraction and collection was measured to be 1 cells/s. In order to prove the full function of this integrated microfluidic system, the nucleus was stained with green fluorescence dye (SYTO Green) and plasma membrane was stained with red fluorescence dye (CellMask Deep Red Plasma Membrane) for cell observation. The feasibility of this integrated system was demonstrated by observing fluorescent results showing no red fluorescence and maintenance of green fluorescence, indicating cell membrane lysis without disruption of the nucleus. The cell membrane lysis rate and ODEP nucleus separation rate were measured to be 78.04 ± 5.70 % and 80.90 ± 5.98 %, respectively. Therefore, the overall efficiency of nucleus extraction rate can be quantifiably up to 58.21 ± 2.21%. Furthermore, a flow control module was successfully integrated with whole micro-system, suggesting that the developed system is capable of transportation metered cells, releasing nucleus, and isolation of extracted nucleus for subsequent chemical treatments, applications and specific analysis of diseases. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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