臺灣博碩士論文加值系統

中文摘要
本研究之蛋白質晶片是以偵測引起細胞增殖、變異、發炎等 一系列免疫反應之微量細胞激素晶片。經環氧基改質之1”×3”玻片，在鋪設一級抗體後，以三明治免疫反應(immunoassay)捕捉抗 原-人類白血球間質素-6 (Interleukin 6)，再利用以生物素(biotin)修 飾之報告抗體-老鼠抗人類白血球間質素-6 (biotinylated mouse anti human IL6)，以鏈菌素(streptavidin)做為一架橋中間物來連結上另 ㄧ修飾有生物素之環狀DNA片段，以滾動循環DNA擴增技術 (Rolling-circle Amplification)複製出大片段之單股DNA產物，再雜 合(hybridization)帶螢光修飾之單股DNA，最後經掃描螢光訊號得 到免疫測試法之偵測結果。此訊號放大技術可用來降低晶片捕捉 目標蛋白質之濃度，尤其針對微量即可造成嚴重反應的低濃度細 胞激素-白血球間質素-6 (IL-6)，其蛋白質晶片(cytokine protein chip) 之製備即為此研究之主要目的。本研究詳細敘述滾動循環擴增技 術製備環狀模板之程序及測試方法，並指出細胞激素晶片製備時 所面對之挑戰。

Abstract
This research developed a protein biochip to detect trace human cytokines, which are important to small-secreted proteins mediating and regulating immunity, cellular differentiation and proliferation, and inflammation in response to an immune stimulus. A sandwich immunoassay was built on 1”×3” glass slides, of which surface was modified with epoxy and a primary antibody to capture the target, human cytokine IL-6. A circular DNA template was attached onto the crystal-consistent domain of the report antibody. Composed with the Rolling Circle Amplification (RCA) technique, this cytokine biochip was developed and read out by fluorescent labels. This study states the preparation process and the characterization of the circular-DNA template in detail and also points out the challenges during the development.

總目錄
中文摘要…………………………………………………………….Ⅰ
英文摘要………………………………………………………….....Ⅱ
謝誌......………….…………………………………………………..Ⅲ
總目錄……………………………………………………………….Ⅳ
圖目錄...….……………………………………………………….....Ⅶ
表目錄……………………………………………………………….Ⅸ
第一章、緒論
1-1 生物晶片之誕生…………………………………………………1
1-2 生物晶片種類……………………………………………………3
1-2.1微陣列晶片………………………………………………..4
1-2.2 微流道晶片( Microfluidic biochip )/Lab on a chip 整合型
晶片……………………………………………………….5
1-3 晶片訊號標記……………………………………………………6
1-4 抗體/抗原反應……………………………………………...........8
1-5 細胞激素(cytokines)之白血球間質素-6 (Interleukin-6)………..8
1-6 鏈菌素(streptavidin)與生物素(biotin)之作用…………………10
1-7 酵素連結免疫吸附分析法enzyme-linked immunosorbent assay
(ELISA)………………………………………………………...10
第二章、理論背景
2-1滾動循環擴增技術(rolling circle amplification，RCA)………..11
2-2白血球間質素6與抗體間之實驗設計……………………........13
2-3晶片塗佈表面化學……………………………………………...14
2-4 螢光顯色………………………………………………………..16
第三章、實驗方法與材料
3-1 材料……………………………………………………………..18
3-1.1 DNA引子、模板與探針………………………………18
3-1.2 晶片上所需之相關蛋白質…………………………….18
3-1.3 表面改質……………………………………………….19
3-1.4 ELISA ………………………………………………….19
3-1.5 電泳…………………………………………………….19
3-2 儀器及器材……………………………………………………..20
3-3 溶液製備………………………………………………………..21
3-4 實驗步驟………………………………………………………..23
3-4.1 抗原抗體間以ELISA做結合性測試…………………..23
3-4.2 生物晶片之製備………………………………………...25
3-4.3 抗原抗體晶片上結合性測試…………………………...30
3-4.4 液相RCA………………………………………………..33
3-4.4.1 環狀模板之製備 ………………………………33
3-4.4.2 環狀模板分子成形之驗證……………………..34
3-5 以晶片RCA偵測human IL6…………………………………..40
第四章、結果與討論
4-1 96微孔板抗原抗體間結合性之測試..……….…….…………..44
4-2 玻片表面清洗劑測試...….……………………………………..46
4-3 胺基與環氧基表面之改質……………………………………..46
4-4 晶片上抗原抗體間結合性測試………………………………..49
4-5 環狀模板構形之驗證………………………………………......53
4-6 固相RCA(Immuno-RCA)...........................................................57
第五章、結論……………………………………………………….61
第六章、參考文獻………………………………………………….64
第七章、附錄
7-1使用不同polymerase進行RCA……………………………….68
7-2 晶片掃描訊號值原始數據……………………………………..69

圖目錄
(圖1)圖示分子信標(Molecular beacon)之作用……………………..7
(圖2) RCA示意圖…………………………………………………..12
(圖3)晶片上免疫分析示意圖………………………………………13
(圖4)表面改質劑(3-Glycidyloxypropyl)-trimethoxysilane (GLYMO) 與玻片之反應……………………………………………………….15
(圖5)生物晶片製作…………………………………………………26
(圖6)晶片點陣位置…………………………………………………28
(圖7)製備RCA所需之環狀模板…………………………………..34
(圖8)RCA-primer(實線)與template(虛線)經雜合後，可能產生三種形式之產物...................………………………………………….….36
(圖9)產物I經RCA後由urea-PAGE分析應有之結果……………37
(圖10)產物II經RCA後由urea-PAGE分析應有之結果....……...38
(圖11)產物III經RCA後由urea-PAGE分析應有之結果……..…39
(圖12) RCA晶片點陣位置…………...…………………………….42
(圖13)蛋白質與96孔板間吸附關係………………....……………..45
(圖14)(左)poly-L-lysine與(右)GLYMO改質之晶片表面，以不同濃度Cy5 labeled goat anti rabbit IgG點陣於二表面，比較二者之訊號與背景值…………………………………………………………….48
(圖15)一級抗體(rabbit anti human-IL6)與抗原(human-IL6)間結合性測試……………………………………………………………….50
(圖16)一級抗體(mouse anti human-IL6)與抗原(human-IL6)間結合性測試……………………………………………………………….51
(圖17)抗原一系列濃度稀釋(human-IL6)與二級抗體(goat anti rabbit IgG conjugated Cy5)之非專一性結合驗證………………………...52
(圖18)將各階段實驗產物進行10 % Urea – PAGE………...………54
(圖19)RCA產物之驗證……………………………….……………56
(圖20)固相RCA對human IL6之測試……………..……………..58
(圖21)固相RCA對human IL6之測試..……………………………59
(圖22)合併圖22與23之固相RCA對human IL6測試………….60
(圖23)於RCA使用不同DNA polymerase…………………………68

表目錄
(表1)晶片上免疫反應鍵結之操作流程………………………..…..32
(表2)晶片上各點之欲反應樣品……………………………………43
(表3)為圖14之ELISA原始數據…………………………..…….…69
(表4)為圖16之晶片訊號原始數據……………………….……..…69
(表5)為圖17之晶片訊號原始數據………………………….…..…69
(表6)為圖22之晶片訊號原始數據……………………………..….69
(表7)為圖23之晶片訊號原始數據……………………………..….70

[1] Hunger, H., Schmidt G. Flachmeier, C., Behrendt, G., Coutelle, C., “High- sensitivity protein detection by a new "contact-copy" method using a protein A-neomycin phosphotransferase II fusion protein.” Analytical biochemistry 186 (1) (1990) 159-64.

[2] King, V. Rolling circle amplification. Handbook of Immunohistochemistry and In Situ Hybridization of Human Carcinomas 2 (2005) 73-81.

[3] Paul M. Lizardi, Xiaohua Huang et al”Mutation detection and single-molecμle counting using isothermal rolling-circle amplification.” Nature genetics , 19 , (1998) 225-232

[4] Xiao-bo Zhong , Paul M. Lizardi , Xiao-hua Huang , Patricia L. Bray-Ward , and David C. Ward .”Visualization of oligonucleotide probes and point mutations in interphase nuclei and DNA fibers using rolling circle DNA amplification.” PNAS Vol.98 No.7 3940-3945 (2001)

[5] Hsin-Yun Hsu, Yi-You Huang.”RCA combined nanoparticle-based optical detection technique for protein microarray: a novel approach.” Biosensors and Bioelectronics 20 (2004) 123–126

[6] Barry Schweitzer , Steven Wiltshire , Jeremy Lambert , Shawn O’Malley , Kari Kukanskis , Zhengrong Zhu , Stephen F. Kingsmore , Paul M. Lizardi , and David C. Ward “Immunoassays with rolling circle DNA amplification: A versatile platform for ultrasensitive antigen detection.” PNAS Vol.97 No.18 10113-10119 (2000)

[7] Weiping Shao, Zhimin Zhou, Isabelle Laroche, Hong Lu, Qiuling Zong , Dhavalkumar D. Patel, Stephen Kingsmore, and Steven P. Piccoli. “Optimization of Rolling-circle Amplified Protein Microarray for Multiplexed Protein Profiling.” Journal of Biomedicine Biotechnology (2003)

[8] Dobrovolskaia, E.; Gam, A.; Slater, J. E., “Competition enzyme-linked immunosorbant assay (ELISA) can be a sensitive method for the specific detection of small quantities of allergen in a complex mixture.” Clinical and Experimental Allergy 36(4) 525-530 (2006)

[9] Jean A. Nemzek , Javed Siddiqui , Daniel G. Remick “Development and optimization of cytokine ELISAs using commercial antibody pairs.” Journal of Immunological Methods 255 149-157 (2001)

[10] Ekins, R., Chu, F., Biggart, E.,“Fluorescence spectroscopy and its application to a new generation of high sensitivity, multimicrospot ,multianalyte, immunoassay.” Clin. Chim. Acta, 194 (1990)

[11] Barbara I. Fall , Bernadette Eberlein-Konig , Heidrun Behrendt, Reinhard Niessner, Johannes Ring, and Michael G. Weller, “Microarrays for the Screening of Allergen-Specific IgE in Human Serum.” Anal. Chem., 75, 556-562(2003)
[12] Ji-Yen Cheng, Chien-Ju Hsieh, Yung-Chuan Chuang and Jing-Ru Hsieh “Performing microchannel temperature cycling reactions using reciprocating reagent shuttling along a radial temperature gradient.” Analyst 130, 931-940(2005)

[13] Mats Nilsson, Mats Gμllberg, Fredrik Dahl, Karoly Szuhai, Anton K.Raap , “Real-time monitoring of rolling circle amplification using a modified molecμlar beacon design.” Nucleic Acids Research,Vol.30, No.14 e66. (2002)

[14] 免疫學，王政光編著，新文京開發所出版, 2004

[15] Daisuke Furuya, Atsuhito Yagihashi, Tomomi Yajima, Daisuke Kobayashi, Kunzo Orita, Masashi Kurimoto, Naoki Watanabe.“An immuno-polymerase chain reaction assay for human interleukin-18.” Journal of Immunological Methods 238 (2000) 173-180 .

[16] Stefan Beyer, Patrick Nickels, Friedrich C. Simmel“Periodic DNA Nanotemplates Synthesized by Rolling Circle Amplification.” Nano Lett, (2005) Vol.5, No.4 , 719-722

[17] Jean A. Nemzek, Javed Siddiqui, Daniel G. Remick.”Development and optimization of cytokine ELISAs using commercial antibody pairs.” Journal of Immunological Methods 255 149-157 (2001)
[18] Joerger and Hendrickson;”Analyte detection with DNA-labeled antibodies and polymerase chain reaction.” Clinical Chemistry, 1371-1377 (1995)

[19] Grit Festag, Andrea Steinbr¨uck, AndreasWolff,1 Andrea Csaki,
Robert M¨oller, and Wolfgang Fritzsche1.“Optimization of Gold Nanoparticle Based DNA Detection for Microarrays.” Journal of Fluorescence,Vol.15, No.2, (2005)

[20] Heiko Kuhn, Maxim D. Frank-Kamenetskii “Template-independent ligation of single-stranded DNA by T4 DNA ligase.” FEBS Journal 272 (2005) 5991-6000