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標題: | 利用微流道系統進行奈米級生物微粒濃縮 On-chip Nano-scale Bio-particles Concentration Utilizing Microfluidics |
作者: | Zih-Hua Chen 陳姿樺 |
指導教授: | 郭柏齡 |
關鍵字: | 胞外泌體,微流道系統,微量濃縮,奈?級?物粒?,樣品製備, exosome,microfluidics,micro-concentrator,nano-bioparticles,sample preparation, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 近年來關於細胞胞外泌體的研究備受矚⽬,原因是胞外泌體被研究證實在細胞與細胞間的溝通和某些疾病發展中扮演重要的⾓⾊。例如癌症轉移、阿茲海默症的發展。然⽽,由於胞外泌體體積通常介於30 奈⽶到150 奈⽶間,樣品製備有兩⼤困難。其⼀,⽬前標準製備流程是以超⾼速離⼼法分離胞外泌體,不僅耗時耗⼒,也需要昂貴的儀器和專業操作⼈員。其⼆,由於胞外泌體在樣品中濃度偏低,必須經過濃縮步驟才能進⾏後續分析。
在這份研究中,我們運⽤微流道技術實現⼀可⽤於濃縮胞外泌體等奈⽶級⽣物微粒的薄膜式微流道系統,提供作為⼀種可替代超⾼速離⼼機的胞外泌體樣本製備⽅法。此裝置每分鐘可去除25μl 的⽔分。屬於獨⽴式系統,可以很容易與現有的實驗⽅法、流程結合。 此微流道裝置包含三層結構,依序為:淺層的樣品流道、多孔性薄膜、深層的氣流流道。加熱系統會將裝置控制在接近⼈體的溫度,保持樣品中的蛋⽩質不被破壞。多孔性薄膜⽤於隔離乾燥氣流和液體,通⼊的微氣流會產⽣強制對流,在短時間內交換⼤量熱能,促使⽔分蒸發,藉此達到濃縮樣品的⽬的。即使在運作⼗分鐘後,多孔性薄膜會稍微被阻塞,但此裝置還是能穩定的以25μl/min的速率去除溶劑。在細胞培養液的實驗中,我們能在⼆⼗分鐘內將1ml 的樣品濃度提⾼為1.5 倍,且回收率達到94%。 In recent years, exosomes have aroused people’s attention for it’s important role in cell-cell communication and transmission of disease states. However, since exosomes’diameter is generally between 30 nm and 150 nm, there are two main difficulties of preparing exosomal samples. First, the isolation process is time-consuming and costly. Ultracentrifugation is the most common and standard protocol which takes about five hours of the entire process. High demand of lab skills is also required. Second, the concentration of exosomes in conditional medium or plasma is too low to be detected. A condensing process is necessary prior to subsequent analysis. In this research, we demonstrate a membrane-base microfluidic chip that replace the ultracentrifugation process during exosomal sample preparation. Remove water by forced convection at the average rate of 25μl/min. This stand-alone system is suitable and easy for integrating with other existing protocols. In our approach, the microfluidic chip is composed of two layers, a shallow sample layer and a deep gas flow layer, with a porous membrane separating liquid sample from drying gas flow. The porous membrane creates an air-liquid interface. Dry air-flow with positive pressure promotes forced convection at the air-liquid interface. Water would evaporate and diffuse through the porous membrane. The drying gas flow would bring moisture out of the chip, facilitating the efficiency of evaporation and hence concentrate the exosome sample. As to peripheral devices, there are gas pump, humidity detective sensor and thermal control module. Since our device is a stand-alone microfluidic system, it is suitable and easy for integrating with other microfluidic device or existing protocols. The operating temperature is controlled to be around human body temperature. Even though there is a clogging effect after infuse for 10 minutes, our device performs a steady water loss rate at about 25 μl /min. In the conditional medium experiment, we raised the concentration of 1 ml sample to 1.5 times in 20 minutes while the recovery was 94%. In sum, we reveal a microfluidic concentrator for nano-scale bioparticles with high recovery rate, and provide the optimized operating parameters of peripheral devices when the operating temperature is equals to human body temperature. This device would advance the process of preparing exosome sample for analysis and would do benefit to fundamental research of exosomes and clinical liquid biopsy. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72430 |
DOI: | 10.6342/NTU201803409 |
全文授權: | 有償授權 |
顯示於系所單位: | 生醫電子與資訊學研究所 |
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