跳到主要內容

臺灣博碩士論文加值系統

(34.204.169.230) 您好!臺灣時間:2024/02/21 22:12
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:宋旺洲
研究生(外文):Wang-Chou Sung
論文名稱:研發以高分子材料為基礎之實驗室晶片系統於生醫分析上的應用
論文名稱(外文):Development of Polymer-based Lab-on-Chip System for Bioanalysis
指導教授:陳淑慧陳淑慧引用關係
指導教授(外文):Shu-Hui Chen
學位類別:博士
校院名稱:國立成功大學
系所名稱:化學系碩博士班
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:101
中文關鍵詞:電噴灑游離化質譜實驗室晶片蛋白質學染色體基因
外文關鍵詞:Electrospray IonizationMass SpectrometryLab-on-ChipProteomicsChromosome DNA
相關次數:
  • 被引用被引用:5
  • 點閱點閱:294
  • 評分評分:
  • 下載下載:39
  • 收藏至我的研究室書目清單書目收藏:0
  系統的整合在微小化的分析系統上是非常重要的,因為它具有減少資源浪費, 操作自動化以及改善分析效率的優點。如今微小化的裝置已經被廣泛的應用於生化分析、藥物篩檢、化學或藥品檢驗上。利用微機電製程技術,許多傳統的分析技術可以被微小化並整合在玻璃或高分子材質的晶片上,這就是所謂的程序晶片(Lab-on-chip)。然而,分析系統的整合仍具有一些困難,比如說當晶片利用質譜進行偵測時緩衝溶液間的搭配的問題便會顯現出來。其他如晶片材料的選擇,偵測模式的建立以及不同反應步驟的串聯都是整合型晶片所需克服的問題。在此篇論文中,我們將利用兩種高分子材料; PDMS與PMMA,來製作分析DNA與蛋白質的分析晶片。由所得到的結果顯示,此兩種整合型分析晶片可得到極佳的分析效率。

  對於非正常染色體DNA的臨床檢測所需要的是能對DNA片段進行快速的分離與偵測。在第一個研究中,利用高分子電泳晶片針對易脆性X染色體的DNA來建立一個可快速篩選的分析工具。利用6公分長的微晶片電泳管道,可在三分鐘內將PCR放大之後的產物中,差異只有6個 CGG重複單位的DNA進行分離與偵測。實驗中, 總共針對12個男生及女生的檢體做測試。由結果顯示,不僅高分子微電泳晶片能在短時間完成分析,所得到的數據與經由一維平板膠電泳得到的結果完全相符。這證明了高分子微電泳晶片是可以當作快速分析與篩檢DNA的工具。

  在蛋白質學的研究中,質譜儀在蛋白質鑑定上扮演了關鍵的角色。藉由質譜的偵測,可免去需要染料標定的步驟,除此之外,更可以經由質譜儀得到層析圖譜中每一根訊號所代表的意義,如分析物種類、分子的質量數據等等。相對於傳統的光學偵測而言,微流體晶片與質譜偵測模式的結合在蛋白質學的研究上顯然更為有利。因此在此研究中, 將一拉尖的毛細管置於PMMA 晶片的微流體管道的末端當作的電噴灑介面,使晶片具有氣態游離化的能力。整個裝置可被設置在市售的質譜儀上且訊號品質與市售的微游離化噴頭相當。由結果顯示,經由此高分子材質的微電灑晶片只需少量的蛋白質樣品就可進行蛋白質鑑定的任務。

  對於蛋白質分析而言,一個好的分離工具更能提高蛋白質的鑑定效率。然而此外,在傳統分析方法中所用到的酵素水解、去鹽等步驟,可增加分析的準確度。 在此研究中,利用表面修飾後PDMS微流體晶片與具有酵素水解與去鹽功能的注射器做一整合,來建立一個蛋白質鑑定的模組,以進行快速的蛋白質分析。由結果顯示,4個胜呔的混合物不但可在2分鐘內達到完全的分離,而且藉由二次質譜的分析,可將個別的胺基酸序列完整解析出來。針對更複雜的蛋白質樣品,經由整個蛋白質鑑定微流體模組(PIMM)的分析,兩個原生的蛋白質可在兩個小時內將進行連續分析達到快速分析的目的。
  Integration of analytical miniaturized systems is important because it has the potential for the cost reduction, automation and analysis efficiency. The microdevices have been widely applied in the field of bioanalysis, drug screening and chemical/medical detection. By making use of the microelectronic mechanical system (MEMS) technologies, we can miniaturize conventional analytical techniques and integrate them to make total analysis systems in a glass or polymer based microchip, this is what we called lab-on-chip. However, for example, some difficulties like buffer systems compatibility with the MS detection always arise when interfacing chip with mass instrument. Other problems like chip substrate choosing, detection system development and the connection of different reaction system also have to be overcomed. In this dissertation, we built two kinds of polymer-based; poly(dimetyl siloxane) (PDMS) and poly (methyl methacrylate) (PMMA) for integrated analysis of DNA and protein samples. From the results obtained, the integration on the microchip is successful and proceeds high analysis efficiency in both analysis.

  Fast clinical screening of abnormal chromosomes demands a high-throughput method including DNA sizing and detection of the amplified products. The first study is to explore the use of polymer microchip electrophoresis for the analysis of PCR products of fragile X (CGG)n alleles to facilitate a fast exclusion test of fragile X syndrome (FXS). The PCR bands with more than six CGG-repeats in difference could be clearly distinguished in less 3 mins by microchip electrophoresis with a separation length of 6 cm. Twelve samples from males and females were tested in this study. From the results shown that not only the samples could be finished in a short time, but show correlation between the microchip electrophoresis and the traditional one dimensional (1D) gel electrophoresis. This proves the feasibility of PMMA chip to be a high throughput and fast exclusion DNA analysis tool.

  For proteomics research, mass analyzer plays an important role in protein identification. Because MS detection could eliminate the dye-labelling procedure and get some information of each chromatographic peak, like analyzes specices and molecule mass. This makes the coupling of microfluidic chip and MS detection more attractive and useful than traditional optical detection in proteomics research. In th other part of the dissteration, an pulled capillary tip was inserted into the end of the PMMA microfluidic channel to be an electrospary interface. This is an easy method was developed to build an ionization interface on the polymer substrate microchip. The whole device could be easily mounted on the commercial MS instrument and offer a high signal quality compared with the commercially available nano electrospray tips. In this research, this polymer-based microdevice would like to generate MS signals for protein identification from the small amounts of protein samples.

  In the proteomics research, a good separation tool could improve to identify the proteins. Beside, in the convetional protein analysis, the procedures like enzyme digestion and desalting were also used to increase the protein identification precision. In this study, a microfabricated PDMS electrospray microfluidic that integrated AMPS modified separation channel, an injector with trypsin enzyme cartridge and desalting cartride was developed to build the protein identification microfluidic module (PIMM) to do high-speed proteins analysis. Results show that a four peptide mixture could be separated in 2 mins and identified the amino acid sequence by tandem MS. In order to prove the high efficiency of PIMM, two native proteins could be sequential analysis in two hours. This proposed a protein identification microfluidic module (PIMM) can successfully operated in a high efficiency.
Table of Contents
Abstract I
中文摘要 IV
Table of Contents VI
List of Figures IX
List of Tables XIII
Abbreviations XIV

Chapter 1: Introduction

1.1 Lab-on-chip……………………………………………………… 2
1.2 Polymer-based Microdevice………………………………… 3
1.2.1 Microfabrication Techniques……………………………... 4
1.2.2 Polymer substrate Assembling………………………………. 6
1.3 The Fluid Control System on Polymer-based Microchip….7
1.4 Integration of Microfluidic Device with Mass Analyzer 8
1.4.1 The Aspects (mechanism) of Electrospray Ionization……8
1.4.2 The ESI Interface Designation………………………………12
1.4.3 The CE-ESI Interface……………………………………….. 12
1.4.4 The Coupling of Microchip with MS Detection……………14
1.5 Thesis Organization……………………………………………16
1.6 Reference…………………………………………………………18

Chapter 2. Plastic microchip electrophoresis for genetic screening: The analysis of polymerase chain reactions products of fragile X(CGG)n alleles

2.1 Introduction………………………………………………………24
2.2 Materials and Methods………………………………………… 26
2.2.1 Chemicals and Reagents……………………………………. 26
2.2.2 PCR Amplification………………………………….……… 26
2.2.3 Microchip System………………………………….……….. 27
2.3 Results and Discussion…………………………………….… 29
2.3.1 Nonradioative PCR and FMR 1 Genes………….…………. 29
2.3.2 Microchip Electrophoresis……………………….………… 30
2.4 Conclusions……………………………………………………… 33
2.5 Reference………………………………………………………… 34

Chapter 3. A disposable poly(methylmethacrylate)-based microfluidic module for protein identification by nanoelectrospray ionization-tandem mass spectrometry
3.1 Introduction……………………………………………………… 37
3.2 Material and Methods…………………………………………… 38
3.2.1 Chemicals and Reagents……………………………………… 38
3.2.2 Fabrication of Nano-ESI Microfluidic Module……………… 39
3.2.3 Sample Preparation, Injection, and Electroosmotic Pumping..40
3.2.4 Nano-ESI-MS/MS……………………………………………. 41
3.3 Results and Disscussion………………………………………………41
3.3.1 The Polymer-based Nano-ESI-MS Microfluidic Module……. 41
3.3.2 Signal Quality…………………………………………………. 42
3.3.3 Comparison with the Commercially Available Nanospray Tip.. 43
3.3.4 Protein Identification by Searching Sequence Database………44
3.4 Conclusion…………………………………………………………… 45
3.5 Reference…………………………………………………………….. 46

Chapter 4. Poly(dimethylsiloxane) (PDMS)-Based Microfluidic Device with ESI-MS Interface for Protein Identification
4.1 Introduction………….. ……………………………………………….50
4.2 Experiment Section…………………………………………………… 52
4.2.1 Chemicals…………………………………………………….. 52
4.2.2 Microchip Fabrication………………………………………… 52
4.2.3 EOF Measurement……………………………………………. 54
4.2.4 Sample Processing Scheme……………………………………. 54
4.2.5 Mass Spectrometry and Instrumentation…………………….. 55
4.3 Results and Discussion………………………………………………. 56
4.3.1 EOF…………………………………………………………… 56
4.3.2 Flow-Through Sampling………………………………………. 57
4.3.3 The Electrospray Interface……………………………………. 58
4.3.4 Sample Instruction, Separation and MS Detection of a Peptide Mixture………………………………………………………… 58
4.3.5 Protein Identification………………………………………….. 59
4.3.6 Promotion of Protein Identification Efficiency 60
4.4 Conclusion…………………………………………………………….. 62
4.5 Reference…………………………………………………………… 63


Chapter 5. Conclusions…………………………………….…………………… 66
5.1 Overview of the dissertation…………………………………………66
5.2 Future Works………………….……………………………………… 67
1.Manz, A., Miyahara, Y., Miura, J., Watanable, Y., Miyagi, H., Sata, K.,
Sens. Actuators, 1990, B1, 249-255.
2.Figey, D., Pinto, D., Anal. Chem., 2000, 72, 330A-335A.
3.Bruin, G. J., Electrophoresis, 2000, 21, 3931-3951.
4.Dolnik, V., Liu, S., Jovanovich, S., Electrophoresis, 2000, 21, 41-54.
5.Terry, S. C., Jerman, J. H., Angell, J. B., IEEE Trans, Electron Device,
1979, ED-26, 1880
6.Jacobson, S. C., Hergenroder, R., Moore, A. W., Ramsey, J. M., Anal.
Chem., 1994, 66, 4127-4132.
7.Jacobson, S. C., Koutny, L. B., Hergenroder, R., Moore, A. W., Ramsey, J.
M., Anal. Chem., 1994, 66, 3472-3476.
8.Jacobson, S. C., Koutny, L. B., Hergenroder, R., Moore, A. W., Ramsey, J.
M., Anal. Chem., 1994, 66, 1114-1118.
9.Khandurina, J., Mcknight, T. E., Jacobson, S. C., Waters, L. C., Foote,
R. S., Ramsey, J. M., Anal. Chem., 2000, 72, 2995.
10.Wang, C., Oleschuk, R., Ouchen, F., Li, J., Thibault, P., Harrison, D.
J., Rapid Commun. Mass Spectrom., 2000, 14, 1377-1383.
11.Fan, Z. H., Harrison, D. J., Anal. Chem., 1994, 66, 177-184.
12.Kameoka, J., Craighead, H. G., Zhang, H., Henion, J., Anal. Chem., 2001,
73, 1935-1941.
13.Yao, S., Anex, D. S., Caldwell, W. B., Arnold, D. W., Smith, K. B.,
Schultz, P. G., Proc. Natl. Acad. Sci. USA, 1999,96, 5372.
14.Effenhauser, C. S., Paulus, A., Manz, A., Widmer, H. M., Anal. Chem,
1994, 66, 2949-2953.
15.Becker, H., Gartner, C., Electrophoresis, 2000, 21,12-16.
16.Martin, R. S., Gawron, A. J., Lunte, S. M., Anal. Chem., 2000, 72,
3196-3202.
17.Ocvirk, G., Munroe, M., Tang, T., Oleschuk, R., Westra, K., Harrison, D.
J., Electrophoresis, 2000, 21, 107-115.
18.Effenhauser, C. S., Bruin, G. J. M., Paulus, A., Ehart, M., Anal. Chem.,
1997, 69, 3451-3457.
19.Duffy, D. C., McDonald, J. C., Schueller, O. J. A., Whitesides, G. M.,
Anal. Chem., 1998, 70, 4974-4984.
20.Martynova, L., Locascio, L. E., Gaitan, M., Kramer, G. W., Christensen,
R. G., MacCrehen, W. A., Anal. Chem., 1997, 69, 4783-4789.
21.Ford, S. M., McWhorter, S., Davies, J., Soper, S. A., Klopf, M.,
Calderon, G., Saile, V. J., Microcolumn Sep., 1998, 10, 413-422.
22.Roberts, M. A., Rossier, J. S., Becier, P., Girault, H., Anal. Chem.,
1997, 69, 2035-2042.
23.Fan, Z. H., Harrison, J. D., Anal. Chem., 1994, 66, 177-184.
24.Effenhauser, C. S., Bruin, G. J. M., Paulus, A., Electrophoresis, 1997,
18, 2203-2213.
25.He, B., Tait, N., Regnier, F., Anal. Chem., 1998, 70, 3790-3797.
26.Campana, A. M. G., baeyens, W. R. G., Aboul-Enein, H. Y., Zhang, X., J.
Microcolumn Sep., 1998, 10, 339-355.
27.Martynova, L., Locascio, L. E., Galtan, M., Kramer, G. W., Christensen,
R. G., MacCrehan, W. A., Anal. Chem., 1997, 69, 4783-4789.
28.Lee, G. B., Chen, S. H., Huang, G. R., Sung, W. C., Lin, Y. H., Sensors
and Actuators B, 2001, 75, 142-148.
29.Effenhauser, C. S., Bruin, G. J. M., Paulus, A., Ehrat, M., Anal. Chem.,
1997, 69, 3451-3457.
30.Duffy, D. C., McDonald, J. C., Schueller, O. J. A., Whitesides, G. M.,
Anal. Chem., 1998, 70, 4974-4984.
31.Roberts, M. A., Rossler, J. S., Bercier, P., Girault, H., Anal. Chem.,
1997, 69, 2035-2042.
32.Soper, S. A., J. Chromatogr. 1999, 853, 107-120.
33.Dolnik, V., Liu, S., Jovanovich, S., Electrophoresis, 2000, 21, 41-54.
34.Harrison, D. J., Fluri, K., Seiler, K., Fan, Z., Effenhauser, C. S.,
Manz, A., Science, 1993, 261, 895-897.
35.Ford, S. M., J. Chromatogr. 1998, 10, 413-422.
36.Chen, Y. H., Wang, W. C., Yang, K. C., Chang T. T., Chen, S. H., Clinical
Chemistry, 1999, 45(11), 1938-1943.
37.Yamashita, M., Fenn, J. B., J. Phys. Chem., 1984, 88, 4451.
38.Yamashita, M., Fenn, J. B., J. Phys. Chem., 1984, 88, 4671.
39.Smith, R. D., Loo, J. A., Edmonds, C. G., Barinaga, C. J., Udseth, H. R.,
Anal. Chem., 1990, 62, 882.
40.Guevremont, R., Siu, K. W. M., LeBlanc, J. C. Y., Berman, S. S., J. Am.
Soc. Mass. Spectrm., 1992, 3, 216.
41.Loo, J. A., Ogozalek, R. R., Light, J. K., Edmonds, C. G., Smith, R. D.,
Anal. Chem., 1992, 64, 81.
42.Loeb, L. B., Kip, A. F., Hudson, G. G., Bennet, W. H., Phys. Rev., 1941,
60, 714.
43.Taylor, G. I., Proc. R Soc. London A., 1964, A280, 383.
44.Kebarle, P., Tang, L., Anal. Chem., 1993, 22, 972A.
45.Bruins, A. P., Covery, T. R., Henion, J. D., Anal. Chem., 1987, 59, 2642.
46.Banks, J. F., Shen, S., Whitehouse, C. M., Fenn, J. B., Anal. Chem.,
1994, 66, 406.
47.Banks, J. F., Quinn, J. P., Whitehouse, C. M., Anal. Chem., 1994, 66,
3688.
48.Olivares, J. A., Nguyen, N. T., Yonker, C. R., Smith, R. D., Anal. Chem.,
1987, 59, 1230.
49.Smith, R. D., Olivares, J. A., Nguyen, N. T., Udseth, H. R., Anal. Chem.,
1988,60, 436.
50.Lee, E. D., Muck, W., Henion, J. D., Covey, T. R., Biomed. Environ.
MassSpectrom. 1989, 18, 844.
51.Cai, J., Henion, J. D., J. Chromatogr. A, 1995, 703, 667.
52.Wilm, M., Mann, M., Anal. Chem., 1996, 68: 1-8.
53.Xue, Q., Foret, F., Dunayevskiy, Y. M., Zavracky, P. M., McGruer, N. E.
and Karger, B. L., Anal. Chem., 1997, 69, 426-430.
54.Ramsey, R. S. and Ramsey, J. M., Anal. Chem., 1997, 69, 1174-1178.
55.Chen, S. H., Sung, W. C., Lee, G. B., Lin, Z. Y., Chen, P. W., and Liao,
P. C., Electrophoresis, 2001, 22, 3972-3977.
56.Valaskovic, G. A., Kelleher, N. L., Little, D. P., Aaserud, D. J. and
Mclafferty, F. W., Anal. Chem., 1995, 67, 3802-3805.
57.Xu, N., Lin, Y., Hofstadler, S. A., Matson, D. W., Charles, J. C. and
Smith, R. D., Anal. Chem., 1998, 70, 3553-3556.
58.Xiang, F., Lin, Y., Jenny, W., Matson, D. W. and Smith, R. D., Anal.
Chem., 1999 71, 1485-1490.
59.Wen, J., Lin. Y., Xiang, F., Matson, D. W., Udseth, H. R. and Smith, R.
D., Electrophoresis, 2000, 21, 191-197.
60.Meng, Z., Qi, S., Soper, S. A. and Limbach, P. A., Anal. Chem., 2001, 73,
1286-1291.
61.Svedberg, M., Pettersson, A., Nilsson, S., Bergquist, J., Nyholm, L.,
Nikolajeff, F. and Markides, K., Anal. Chem., 2003, 75, 3934-3940.
62.Rohner, T. C., Rossier, J. S., Girault, H. H., Anal. Chem., 2001, 73,
5353-5357.
63.Kameoka, J., Orth, R., Llic, B., Czaplewski, D., Wachs, T. and Graighead,
H. G., Anal. Chem., 2002, 74, 5897-5901.
64.Gobry, V., van Oostrum, J., Martinelli, M., Rohner, T. C., Reymond, F.,
Rossier, J. S. and Girault, H. H., Proteomics, 2002, 2, 405-412.
65.Liu, H., Felten, C., Xue, Q., Zhang, B., Jedrzejewski, P., Karger, B. L.
and Foret, F., Anal. Chem., 2000, 72, 3303-3310.
66.Kim, J. S., Knapp, D. R., Electrophoresis, 2001, 22, 3993-3999.
67.Yuan, C. H., Shiea, J., Anal. Chem., 2001, 73, 1080-1083.
68.Kameoka, J., Graighead, H. G., Zhang, H. and Henion, J., Anal. Chem.,
2001, 73: 1935-1941.
69.Chan, J. H., Timperman, A. T., Qin, D. and Aebersold, R., Anal. Chem.,
1999, 71, 4437-4444.
連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top