摘要
蛋白質分析是醫學研究領域最重要的技術之一，生物技術主要有兩大基礎，一是由核酸建構的分子生物學，二是蛋白質技術。核酸所調控生理現象的最後產物依舊是蛋白質，由此可知生理運作的表現終究是受蛋白質調控的，因此了解蛋白質與生理現象相對應的關係可以幫助我們了解疾病生成的原因，或準確地檢測疾病。在我們的研究中，我們希望設計出一種玻璃微流體晶片，希望透過少量的樣本即可完成精準的疾病檢測。在蛋白質分析的整個過程中，蛋白質消化過程需耗費大量時間，因此往往是整個研究的瓶頸，於是我們提出了一種用於蛋白質分析的晶片，它具有產生精準的蛋白質消化片段的能力、分解時間短、高通量蛋白質消化這些優勢。我們微流體晶片的流道容量尺度為μL，我們的目標是應用於mL到μL規模的樣品採集分析。由於樣品採集量充足，我們能夠使用超靈敏的LC-MS（液相層析質譜儀）系統來檢測樣品，我們設計的奈米流道的深度和寬度分別為300nm和1mm，裝置的總體積為0.15nL，我們也應用了胰蛋白酶原固定方法來避免酵素自我消化的狀況，以提升酵素存活分解蛋白質的效率，總結來說在這個研究之中，我們避免了傳統蛋白質消化方法的缺點同時又保持了足量蛋白質消化樣本收集檢測的體積。

Protein analysis is one of the most important technologies in the field of medical research. There are two main foundations of biological technology, one is Molecular biology which is built of nucleic acids, and the other one is Protein technology. Protein analysis is the basis of protein technology. The expression of the physiological operation is regulated by proteins. Thus, to realize the expression profile of protein can help us to understand the cause of disease or to detect the disease precisely. In our Topic, we hope to design a device that is able to be used in disease detection by a small amount of specimens. In the whole process of protein analysis, protein digestion is often the bottleneck of the whole research due to its time-consuming. However, we present a device that is used for high digestion precision, low incubation time, and high-throughput protein digestion. The scale of our device is in uL scale. We aim to use the device in the mL to μL scale of sample collection. Due to the sufficient amount of sample collection, we are able to detect the sample by using ultrasensitive LC-MS (Liquid Chromatography Mass Spectrometry) system. Depth and width of nanochannel reactor we design is 300nm and 1mm respectively. The total volume of the device is 0.15nL. Also we applied trypsinogen immobilization method to prevent self-digestion of trypsin. Finally, the device can conquer the disadvantage of traditional bulk digestion but also maintain the bulk volume of protein digestion.

Abstract…………………………………………………………………..……………2
Abstract (中文)………………………………………………………………………...3
Table of Content………………………………………………………....…………….4
Chapter 1 Introduction……………………………………………….………………..6
1.1 Protein analysis…………………………………………………….……..…….6
1.2 Protein digestion methods………………………….……………………..……7
1.2.1 Bulk-scale method…………………………………………...…………..8
1.2.2 Micro-scale and Nano-scale method…………………………….………9
1.3 Issue of conventional protein digestion method………………………...….…10
1.3.1 Digestion time……………………………………………………....….10
1.3.2 Digestion volume………………………………………………..….…..11
1.4 Goal: Thin-layer nanofluidic protein digestion with uL volume…….….…….13
1.4.1 Experiment design……………………………….……………………..13
1.4.2 Fabrication of the thin-layer nanofluidic digestion device………..……14
1.4.3 Verification of digestion by LC-MS analysis……………………...…...15

Chapter 2 Design…………………………………………….……………………….17
2.1 protein digestion process………………………………………....…….…..…17
2.2 Shorten digestion time……………………………..……………………...…..18
2.3 Channel size and flow rate…….…………………….……………….…..……18
2.4. Flow situation in 1mm width nanochannel…………………….…….….……20

Chapter 3 Device fabrication………………………………………..……………..…23
3.1 Glass device fabrication………………………………………..….…………..23
3.1.1 Cr deposition………………………………………………………...…24
3.1.2 EVG-610 Exposure………………………………………….…………24
3.1.3 Dry etching Nano/Microchannel fabrication………………………..…26
3.2 Protein digestion device fabrication………………………………….……….28
3.2.1 APTES coating………………………………………………..………..28
3.2.2 VUV patterning………………………………………………...………29
3.2.3 Substrates bonding…………………………………………………..…31
3.2.4 Trypsin immobilization……………………………...…………………33

Chapter 4 Protein digestion experiment…………………………….……………….36
4.1 Cytochrome C digestion……………………………………..……..…………36
4.1.1 Traditional bulk cytochrome C digestion……………………..………..36
4.1.2 Nanochannel device protein digestion……………..………..…………37
4.2 Analysis with LC-MS………………………………………………..….……39
4.2.1 LC-MS analysis condition……………………..……………..………..39
4.2.2 MS results of cytochrome C digestion………………………..…..……39
4.2.3 Digestion rate……………………………….………….…..……..……45

Chapter 5 Conclusion and Future Work………………………………….……….....49
5.1 Conclusion………………………….…............………….……….………….49
5.2 Future Work………………………..……..….……………………………….50

References……………………………………………………………………………52

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