臺灣博碩士論文加值系統

本研究利用銀染奈米金粒子，擴大電流訊號，以作DNA檢測。首先利用微機電製程在玻璃表面製作間距為140μm之鋁質微電極。玻璃經表面改質處理，增加與探針DNA之結合性。加入修飾有Biotin之目標DNA進行雜交，再與Streptavidin-金粒子結合。最後利用銀染沉積加大金屬顆粒，以增大電流訊號。
本研究針對電極對長度、銀染劑之比例與濃度、銀染溫度、銀染時間、銀染次數及銀染顯影時間等因素進行實驗探討，求出最佳參數，有效區分雜交DNA之電流訊號及控制組之偽訊號。目前技術可測出0.1μM之DNA。

In this study, the silver enhancement technique coupled with the
electric current measurement is employed for DNA detection. First,
aluminum electrodes with a gap of 140μm are facilitated on the surfaces
of glass substrates using MEMS technology. The surface of glass is
modified to improve the immobilization of probe DNA. The target DNA
labeled with biotin is hybridized with probe DNA and conjoined with
Streptavidin-gold . The silver is deposited on the nano-gold particles, which
increases the size of metal particles and forms a circuit between electrodes.
The effects of silver solution (ratio, volume and concentration),
reaction conditions (temperature, duration and number of repetition) and
the reaction duration of fixer on the silver enhancement are examined.
Optimal operation conditions, which increase the signal-to-noise ratio, are
obtained. Target DNA with a concentration of 0.1μM can be identified.

目錄
中文摘要……………………………………………………………………i
英文摘要……………………………………………………………………ii
目錄…………………………………………………………………………iii
圖目錄………………………………………………………………………vi
表目錄………………………………………………………………………x

第一章 序論………………………………………………………………...1
1.1 前言………………………………….…………………………….1
1.2 研究動機及目的…………………….…………………………….6
1.3 文獻回顧…………………………………………………………..7
1.3.1 DNA 檢測方式……………………………………………….7
1.3.2 銀染反應…………………………………………………….11
1.4 內容大綱及簡介…………………………………………………15

第二章 研究方法與原理………………………………………………….18
2.1 DNA 訊號檢測及方式…………………………………………..18
2.1.1 DNA 基本性質特性………………………………………...18
2.1.2 DNA 檢測法………………………………………………...18
2.2 玻璃表面改質…………………………………………………....23
2.2.1 改質簡介…………………………………………………….23
2.2.2 玻璃表面改質與DNA固定(Immobilization)之關係……….26
2.3 銀染檢測原理及運用……………………………………………28
第三章實驗方法…………………………………………………………...30
3.1 電極製程技術………..…………………………………………..30
3.1.1 光罩設計與製作…………………………………………….30
3.1.2 玻璃清洗製程……………………………………………….33
3.1.3 微影(Lithography)製程……………………………………..35
3.1.4 金屬薄膜沈積……………………………………………….40
3.1.5 掀舉法(Lift off)……………………………………….……..41
3.2 玻璃表面處理………………………………………………...….43
3.2.1 玻璃表面處理……………………………………………….43
3.2.2 矽烷化(Silanization)反應…………………………………...44
3.2.3 醛化(Glutaraldehyde)反應………………………………….45
3.3 固定DNA、雜交反應、Streptavidin-Gold 接合及銀染反應……46
3.3.1 固定(Immobilization) DNA及還原反應（Reduction Reaction）…………………………………………………...46
3.3.2 雜交反應…………………………………………………….48
3.3.3 Biotin與Streptavidin-Gold 接合…………………………..49
3.3.4 銀染反應…………………………………………………….50
3.4 製程鑑定…………………………………………………………51
3.4.1 玻璃表面改質效果 - 親疏水性質…………………………51
3.4.2 Biotin與Streptavidin-Gold 接合鑑定……………………….52
3.4.3 銀染反應效果……………………………………………….54
第四章結果與討論………………………………………………………...56
4.1 微電極製作之結構觀察與討論…………………………………56
4.1.1 濺鍍功率對於電極製作之影響………………………….…56
4.1.2 吸附層對於剝離製程之影響…………………………….....57
4.2 微電極長度對於訊號之影響……………………………………59
4.3 銀染反應參數探討-以單股DNA為例..…………………………60
4.3.1 銀染試劑量、比例探討……………………………………...60
4.3.2 銀染試劑反應時間、反應溫度之探討……………………...63
4.3.3 銀染次數之影響…………………………………………….66
4.3.4 控制組之電流訊號………………………………………….67
4.3.5 銀染顯影時間對於訊號的影響 …………………………...68
4.4 雜交DNA經銀染反應之電流訊號………………………….…..70
4.4.1 實驗組之訊號…………..…………………………………...70
4.4.2 雜交DNA之電流訊號……..…………………...…………...72
4.4.3 不同濃度目標DNA之電流…………………………………75
4.4.4 未知樣品測試……………………………………………….77
第五章 結論與未來方之研究方向……………………………………….79
5.1 結論……………………………………………………………..79
5.2 未來之研究方向.…………………………………………….…81
參考文獻 ………………………………………………………………….83
附錄A. 實驗流程…………………………………………………………87
附錄B. 實驗試劑…………………………………………………………89
附錄C. 實驗藥劑、DNA及螢光分子……………………………………90
附錄D. 儀器設備…………………………………………………………92

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Fang, Y., Cai, H., Wang, Y., and He, P.: Electrochemical Detection of DNA Hybridization based on Silver-Enhanced Gold Nanoparticle Label. Analytica Chimica Acta 2002, 469:165-172.

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Kangatharalingam, N. and Essenberg, M.: Modified Pyrogallol-Initiated Immunogold Silver Enhancement Technique Applicable to Prokaryotes. Journal of Microbiological Methods 2000, 41:211-217.

Kohler, A., Lauritzen, B. and Van Noorden, C.J.: Signal Amplification in Immunohistochemistry at the Light Microscopic Level Using Biotinylated Tyramide and Nanogold Silver Staining. The Journal of Histrochemistry and Cytochemistry 2000, 48(7):933-941.

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Scopsi, L. and Larsson, L.: Increased Sensitivity in Immunocytochemistry Effects of Double Application of Antibodies and Silver Intensification on Immunogold and Peroxidase Antiperoxidase Staining Techniques. Histochemistry 1985, 82:321-329.

Southern, E.M.: Detection of Specific Sequences among DNA Fragments Separated by Gel Electrophoresis. Journal of molecular biology 1975, 98:503-517.

Springall, D.R., Grimelius, L. and Polak, J.M.: The Potential of the Immunogold Silver Staining Method for Paraffin Sections. Histochemistry 1984, 81:603-608.

Su X., Li, S.F.Y. and O'Shea, S.J.: Au Nanoparticle and Silver Enhancement Reaction Amplified Microgravimetric Biosensor. Chemical Communications 2001:755-756.

Teasdale, J.P., Holgate, C.S. and Cowen, P.N.: Identification of Oestrogen Receptors in Cells of Paraffin-Processed Breast Cancers by IGSS. Histochemistry 1987, 87:185-187.

Wang, J., Xu, D. and Polsky, R.: Magnetically-Induced Solid-State Electrochemical Detection of DNA Hybridization. Journal of the American Chemical Society 2002, 124:4208-4209.

Al-hazzaa, A.A., Bowen, I.D. and Birchall, M.A.: A Comparison of the Antihistion and APOP-TAG Technique for Demonstrating APOPTOSIS with Option for Silver Enhancement. Cell Biology International 1998, 22(4):271-276.

Caswell, K.K., Wilson, J.N., Bunz, U.H. and Murphy, C.J.: Preferential End-to-End Assembly of Gold Nanorods by Biotin-Streptavidin Connectors. Journal of American Chemical Society 2003, 125:13914-13915.

Cobbe, S., Connolly, S., Ryan, D.,Nagle, L., Eritja, R., and Fitzmaurice, D.: DNA-Controlled Assembly of Protein-Modified Gold Nanocrystals. Journal of Physical Chemistry B 2003, 107:470-477.

Connolly, S., Rao, S. N. and Fitzmaurice, D.: Characterization of Protein Aggregated Gold Nanocrystals. Journal of Physical Chemistry B 2000, 104:4765-4776.

Danscher, G.: Localization of Gold in Biological Tissue a Photochemical Method for Light and Electronmicroscopy. Histochemistry 1981, 71:81-88.

Danscher, G. and Norgaard, J.O.: Light Microscopic Visualization of Colloidal Gold on Resin-Embedded Tissue. The Journal of Histrochemistry and Cytochemistry 1983, 31(12):1394-1398.

Fang, Y., Cai, H., Wang, Y., and He, P.: Electrochemical Detection of DNA Hybridization based on Silver-Enhanced Gold Nanoparticle Label. Analytica Chimica Acta 2002, 469:165-172.

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Hayat, M.A.: Colloidal Gold: Principles, Methods, and Applications 1989, San Diego:Academic Press

Holgate, C.S., Jackson, P., Cowen, P.N. and Bird, C.C.: Immunogold Silver Staining: New Method of Immunostaining with Enhanced Sensitivity. The Journal of Histrochemistry and Cytochemistry 1983, 31(7):938-944.

Kangatharalingam, N. and Essenberg, M.: Modified Pyrogallol-Initiated Immunogold Silver Enhancement Technique Applicable to Prokaryotes. Journal of Microbiological Methods 2000, 41:211-217.

Kohler, A., Lauritzen, B. and Van Noorden, C.J.: Signal Amplification in Immunohistochemistry at the Light Microscopic Level Using Biotinylated Tyramide and Nanogold Silver Staining. The Journal of Histrochemistry and Cytochemistry 2000, 48(7):933-941.

Liesi, P., Pierre, J., Vilja, P. and Grosveld, F.: Specific Detection of Neuronal Cell Bodies: In Situ Hybridization with a Biotin Labeled Neurofilamnet cDNA Probe. The Journal of Histrochemistry and Cytochemistry 1986, 34(7):923-926.

Mirkin, C.A, Taton, T.A. and Letsinger, R.L.: Scanometric DNA Array
Detection with Nanoparticle Probes. Science 2000, 289:1757-1760.

Mirkin, C.A., Park, S.J. and Taton, T.A.: Array-Based Electrical Detection of DNA with Nanoparticle. Science 2002, 295(22):1503-1506.

Mirkin,C.A., Mucic, R.C., Storhoff, J.J. and Letsinger, R.L.: DNA-Directed Synthesis of Binary Nanoparticle Network Materials. American Chemical Society 1988, 120(48):12674-12675.

Sastry, M., Lala, N.P. and Chavan, S.P.: Optical Absorption Study of the Biotin-Avidin Interaction on Colloidal Silver and Gold Particles. Langmuir 1998, 14:4138-4142.

Schena, M.: Microarray Analysis 2003, New Jersey: Johin Wiley & Sons Inc.

Schena, M., Davis, R.W. and Brown, P.O.: Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray. Science 1995, 270:467-470.

Scopsi, L. and Larsson, L.: Increased Sensitivity in Immunocytochemistry Effects of Double Application of Antibodies and Silver Intensification on Immunogold and Peroxidase Antiperoxidase Staining Techniques. Histochemistry 1985, 82:321-329.

Southern, E.M.: Detection of Specific Sequences among DNA Fragments Separated by Gel Electrophoresis. Journal of molecular biology 1975, 98:503-517.

Springall, D.R., Grimelius, L. and Polak, J.M.: The Potential of the Immunogold Silver Staining Method for Paraffin Sections. Histochemistry 1984, 81:603-608.

Su X., Li, S.F.Y. and O'Shea, S.J.: Au Nanoparticle and Silver Enhancement Reaction Amplified Microgravimetric Biosensor. Chemical Communications 2001:755-756.

Teasdale, J.P., Holgate, C.S. and Cowen, P.N.: Identification of Oestrogen Receptors in Cells of Paraffin-Processed Breast Cancers by IGSS. Histochemistry 1987, 87:185-187.

Wang, J., Xu, D. and Polsky, R.: Magnetically-Induced Solid-State Electrochemical Detection of DNA Hybridization. Journal of the American Chemical Society 2002, 124:4208-4209.

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林易賞:表面聲波生物感測器開發與製作2003, 基隆:國立台灣海洋大學機械與輪機工程學系碩士論文

莊達人: VLSI 製造技術2000,台北:高立圖書有限公司

翁稟霖:生物科技概論2000, 新加坡:亞洲湯姆生國際出版

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