微陣列蛋白質晶片利用傳統免疫分析法(Immunoassay)，應用在快速且平行的疾病篩檢上，此法已被公認為檢測能力強大的技術。但是其所遇到的挑戰，為檢測流程中的參數不易掌控，導致檢測的結果不夠穩定。在傳統生物晶片檢測模式中，主要利用生物血清來作為鍵結阻絕試劑，以達到有效的檢測目的。因為其具有非常良好的阻絕效果，所以此方法已經廣泛地被使用在流行病學與感染性疾病的偵測，成為一公認的標準檢測分析法。不過由於鍵結阻絕試劑不良的沾黏問題、鍵結阻絕時間耗時太久及螢光影像暈開拖尾等缺點，導致辨識率及檢測靈敏度降低。

有鑑於此，本文提出的新型生醫分子鍵結阻絕模式，主要利用氣相式生長奈米自組裝單分子膜(FOTS)方法，取代傳統廣為使用的生物血清分子鍵結阻絕模式，進一步的使蛋白質晶片檢測靈敏度提高。所以在本論文裡針對免疫分析流程的步驟，分別作了不同實驗項目參數研究，來進一步改善蛋白質晶片檢測性能。主要可分為: 蛋白質晶片檢測流程參數調變、蒸鍍自組裝單分子膜方法、FOTS與APTS鍵結品質、生物血清與FOTS分子對蛋白質鍵結阻絕效果與非特異性吸附特性、鍵結阻絕時間與效果探討、FOTS鍵結阻絕與生物晶片相容性等參數。

利用上述的參數調變有效的提升了下列的性能: (1)將點上蛋白質後的試片，放置在37℃及相對濕度55%腔體內，保持蛋白質點濕潤度。並以預加熱37℃的PBST來進行清洗，可以將使物理吸附殘留在空白試片上的螢光強度，由原先約20%降低到2~3%。(2)利用真空蒸鍍系統，得到的自組裝單分子膜品質較佳，所需要的成膜時間也較短。依據不同的自組裝單分子種類，只需要數分鐘到數十分鐘，就能得到品質良好的自組裝單分子膜。(3)由於鍵結阻絕試劑與蛋白質分子間的沾黏現象越嚴重，則越降低第一級抗體與第二級抗體間辨識能力。本論文比較傳統生物血清(MILK、BSA)與FOTS自組裝單分子，這三種鍵結阻絕試劑與蛋白質分間的沾黏狀況，得到MILK最高，其次為BSA，而FOTS的沾黏現象最低。在傳統的生物血清鍵結阻絕法中，以HS、MILK、BSA得到的噪訊比，分別為31、25.2及21.6。但本文所提出之FOTS新穎型鍵結阻絕方法，可達到更高的噪訊比35.4，並且能夠得到不拖尾、不暈開的清晰螢光結果，避免檢體間的干擾與交互污染發生。(4)本論文開發不同壓克力盒設計來蒸鍍FOTS以達到鍵結阻絕效果，在第二代壓克力盒設計中，僅需要5分鐘的鍵結阻絕時間，就能夠達到高於傳統生物血清鍵結阻絕的噪訊比，及良好的螢光訊號值，各螢光訊號點的標準差約為65。且FOTS在5分鐘時，鍵結阻絕效果達到最佳接合效率2.3，高於利用傳統生物血清的接合效率0.5~1.0。(5)利用蒸鍍FOTS進行蛋白質檢測流程後，觀測試片上蛋白質訊號點呈現親水，而其餘的地方因為蒸鍍FOTS而呈現疏水。故表示FOTS蒸鍍進行鍵結阻絕，對蛋白質晶片檢測機制沒有受到破壞，所以此法可成功的相容於檢測流程中。

因此本研究成功地利用了氣相式生長奈米自組裝單分子膜方法，改善生物血清使用在傳統生物晶片的缺點，並且達到良好的生醫分子鍵結阻絕效果。因此已進一步提升了生物晶片的性能，如增加其檢測靈敏度、提高生醫分子間辨識率、縮短鍵結阻絕時間、及得到清晰不受干擾的螢光呈像。綜合上述之優勢，氣相式生長奈米自組裝單分子膜方法，對於生物晶片之免疫檢測，提供一重要且關鍵性的鍵結阻絕模式。故本研究證實了此新穎技術於生物晶片上，足以取代三十年來所仰賴的傳統生物血清的鍵結阻絕方法。

[ ] http://www.sinphar.com/medical/no30/product_01.html
[2] Yiwen Li, Nidhi Nath, and W. Monty Reichert, “Parallel Comparison of Sandwich and Direct Label Assay Protocols on Cytokine Detection Protein Arrays”, Anal. Chem. 2003, 75, 5274-5281.
[3] Yong Huang, Naveep S. Sekhon, James Borninski, Ning Chen and Boris Rubinsky, “Instantaneous, quantitative single-cell viability assessment by electrical evaluation of cell membrane integrity with microfabricated devices ”, Sensors and Actuators A, 105, 31-39, (2003)
[4] 成大生物科技中心網頁http://www.ncku.edu.tw/~cbst/biochip.htm
[5] 陽明大學網頁http://binfo.ym.edu.tw/styang/seminar/proarray/array_farbication.htm
[6] http://brc.se.fju.edu.tw/plans/homework/49.doc
[7] Wlad Kusnezow, Anette Jacob, Alexandra Walijew, Frank Diehl, Jorg D. Hoheisel, “Antibody microarrays: An evaluation of production parameters”, Proteomics 2003, 3, 254–264.
[8] 孫偉銘, "利用微波電漿活化聚丙烯不織布纖維表面以固定蛋白質", 1999.
[9] Ulman, A., "An Introduction to Ultrathin Organic Film", Academic Press, San Diego, 1991.
[10] David L. Allara., "Critical issues in applications of self-assembled monolayers", Biosensors & Bioelectronics 1995, 10, 771-783.
[11] Sung-Il Chang, Yun-Seok Choi and Jun-Bo Yoon, “Self-assembled monolayer-assisted thin metal polishing for fabricating uniform 3D microstructures”, J. Micromech. Microeng. 15 (2005) 1027–1032
[12] Ulman, A., "Formation and Structure of Self-assembled Monolayers", Chem. Rev., 1996, 96, 1533-1554.
[13] Bierbaum, K.; Grunze, M.; Baski, A. A.; Chi, L. F.; Schrepp, W.; Fuchs, H., "Growth of self-assembled n-alkyltrichlorosilane films on Si(100) investigated by atomic force microspcopy", Langmuir, 1995, 11, 2143-2150.
[14] Resch, S. R.; Grasserbauer, M.; Friedbacher, G.; Vallant, TH.; Brunner, H.; Mayer, U.; Hoffmann, H.; "In situ and ex situ AFM investigation of the formation of octadecylsiloxane monolayer", Appl. Surface Sci., 1999, 168-175.
[15] Silberzan, P.;Leger, L; Ausserre, D.; Bematter, J. J., "Silanation of silica surfaces: A new method of constructing pure or mixed monolayers", Langmuir, 1991, 7, 1647-1651.
[16] Grange, J.; D.; Markham, J. L.; Kurkjian, C. R., "Effects of surface hydration on the deposition of ilane monolayers on silica", Langmuir, 1993, 9, 1749-1753.
[17] Brzoska, J.B., Shahidzadeh, N., Rondelez, F., "Evidence of transition temperature for the optimum deposition of drafted monolayer coatings", Nature, 1992, 360, 712-713.
[18] WASSERMAN, S. R.;Tao, Y. T.;Whiteside, J. M. "Structure and reactivity of alkylsiloxane monolayers formed by reaction of alkyltrichorosilanes on silicon substrates", Langmuir, 1989, 5, 1074-1087.
[ 9] Hoffmann, H.; Mayer, U.; Kischanitz, A., "Structure of alkysiloxane monolayers on silicon surfaces investigated by exernal reflection infrared spectroscopy", Langmuir, 1995, 11, 1304-1312.
[20] Mark Metzke, Jane Z. Bai, and Zhibin Guan, “A Novel Carbohydrate-Derived Side-Chain Polyether with Excellent Protein Resistance”, J. AM. CHEM. SOC. 2003, 125, 7760-7761
[21] Filip Frederixa, Kristien Bonroya, Gunter Reekmansa,Wim Laureyna, Andrew Campitellia, Mikhael A. Abramovb, Wim Dehaenb, Guido Maes, “Reduced nonspecific adsorption on covalently immobilized protein surfaces using poly(ethylene oxide) containing blocking agents”, J. Biochem. Biophys. Methods 58 (2004) 67–74
[22] David J. Vanderah, Hongly La, Jessica Naff, Vitalii Silin, and Kenneth A. Rubinson, “Control of Protein Adsorption: Molecular Level Structural and Spatial Variables”, J. AM. CHEM. SOC. 2004, 126, 13639-13641
[23] Subramanian Balamurugan, Linnea K. Ista, Juchao Yan, Gabriel P. Lo´pez, Jo¨ rg Fick, Michael Himmelhaus, and Michael Grunze , “Reversible Protein Adsorption and Bioadhesion on Monolayers Terminated with Mixtures of Oligo(ethylene glycol) and Methyl Groups”, J. AM. CHEM. SOC., June, 2005
[24] L. Spencer Roach, Helen Song, and Rustem F. Ismagilov, “Controlling Nonspecific Protein Adsorption in a Plug-Based Microfluidic System by Controlling Interfacial Chemistry Using Fluorous-Phase Surfactants”, Anal. Chem. 2005, 77, 785-796
[25] Kathryn L. Brogan, Jae Ho Shin, and Mark H. Schoenfisch, “Influence of Surfactants and Antibody Immobilization Strategy on Reducing Nonspecific Protein Interactions for Molecular Recognition Force Microscopy”, Langmuir, 2004, 20, 9729-9735.
[26] Yiwen Li, Nidhi Nath, and W. Monty Reichert, “Parallel Comparison of Sandwich and Direct Label Assay Protocols on Cytokine Detection Protein Arrays”, Anal. Chem. 2003, 75, 5274-5281.
[27] Jung-Mu Kim, Chang-Wook Baek, Jae-Hyoung Park, Dong-Sik Shin, Yoon-Sik Lee and Yong-Kweon Kim , “Continuous anti-stiction coatings using self-assembled monolayers for gold microstructures”, J. Micromech. Microeng. 12 (2002) 688–695
[28] Alexander Y. Fadeev and Thomas J. McCarthy, “Self-Assembly Is Not the Only Reaction Possible between Alkyltrichlorosilanes and Surfaces: Monomolecular and Oligomeric Covalently Attached Layers of Dichloro- and Trichloroalkylsilanes on Silicon”, Langmuir 2000, 16, 7268-7274
[29] Gun-Young Jung, Zhiyong Li, Wei Wu, Yong Chen, Deirdre L. Olynick, Shih-Yuan Wang, William M. Tong, and R. Stanley Williams, “Vapor-Phase Self-Assembled Monolayer for Improved Mold Release in Nanoimprint Lithography”, Langmuir, 2004
[30] Y. Masuda, T. Koumura, T. Okawa, and K. Koumoto, “Micropatterning of Ni particles on a BaTiO3 green sheet using a self-assembled monolayer”, Journal of Colloid and Interface Science 263 (2003) 190–195
[31] Yoshitake Masuda, Won-Seon Seo, Kunihito Koumoto, “Deposition mechanism of anatase TiO2 from an aqueous solution and its site-selective deposition”, Solid State Ionics 172 (2004) 283–288
[32]古燕華,”蛋白質微陣列反應晶片材質與自我組裝單層分子之表面處理研究”, 國立清華大學碩士論文, 2001.
[33] Yue Wang, Shubo Han, Alejandro L. Briseno, Raymond J. G. Sanedrin, and Feimeng Zhou, “A modified nanosphere lithography for the fabrication of aminosilane/polystyrene nanoring arrays and the subsequent attachment of gold or DNA-capped gold nanoparticles”, J. Mater. Chem., 2004, 14, 1-8
[34] Ganesan Prabusankar, Ramaswamy Murugavel, and Ray J. Butcher, “Synthesis, Spectral Studies, and Structural Characterization of a New Organosilanetriol, Its Amine Complexes, and a Surface Lewis Basic Cubic Aluminosilicate”, Organometallics 2005, 24, 2124-2128
[35] Alex C. C. Chang, Steven S. C. Chuang, McMahan Gray, and Yee Soong, “In-Situ Infrared Study of CO2 Adsorption on SBA-15 Grafted with γ-(Aminopropyl) triethoxysilane”, Energy & Fuels 2003, 17, 468-473
[36] Mieko Matsuzawa, Shuzo Tokumitsu, Wolfgang Knoll, and Hiroyuki Sasabe, “Effects of Organosilane Monolayer Films on the Geometrical Guidance of CNS Neurons”, Langmuir 1998, 14, 5133-5138
[37] Y. Masuda, M. Itoh, T. Yonezawa, and K. Koumoto, “Low-Dimensional Arrangement of SiO2 Particles”, Langmuir 2002, 18, 4155-4159
[38] Z. F. Li and E. Ruckenstein, “Patterned Conductive Polyaniline on Si(100) Surface via Self-Assembly and Graft Polymerization”, Macromolecules 2002, 35, 9506-9512
[39] Michael L. Chabinyc, J. Christopher Love, Joseph H. Thywissen, Federico Cervelli, Mara G. Prentiss, and George M. Whitesides, “Self-Assembled Monolayers Exposed to Metastable Argon Beams Undergo Thiol Exchange Reactions”, Langmuir 2003, 19, 2201-2205
[40] Florin Andrei Nae, Nagahiro Saito, Atsushi Hozumi, and Osamu Takai, “High-Resolution Submicron Patterning of Self-Assembled Monolayers Using a Molecular Fluorine Laser at 157 nm”, Langmuir 2005, 21, 1398-1402
[41] Hiroyuki Sugimura, Kazuya Ushiyama, Atsushi Hozumi, and Osamu Takai, “Micropatterning of Alkyl- and Fluoroalkylsilane Self-Assembled Monolayers Using Vacuum Ultraviolet Light”, Langmuir 2000, 16, 885-888
[42] Nagahiro Saito, Sun-Hyung Lee, Ishizaki Takahiro, Junko Hieda, Hiroyuki Sugimura,| and Osamu Takai, “Surface-Potential Reversibility of an Amino-Terminated Self-Assembled Monolayer Based on Nanoprobe Chemistry”, J. Phys. Chem. B 2005, 109, 11602-11605
[43] S. L. Brandow, M.-S. Chen, R. Aggarwal, C. S. Dulcey, J. M. Calvert, and W. J. Dressick, “Fabrication of Patterned Amine Reactivity Templates Using 4-Chloromethylphenylsiloxane Self-Assembled Monolayer Films”, Langmuir 1999, 15, 5429-5432
[44] http://www.escoproducts.com/html/s1-uv_fused_silica.html
[45] K.-M. Byun et al, Thin Solid Films, 376, 26-31 (2000).
[46] C. P. Tripp, P. Kazmaier, and M. L. Hair, “A Low Frequency Infrared and ab Initio Study of the Reaction of Trichlorosilane with Silica”, Langmuir 1996, 12, 6404-6406
[47] J. R. Combes, L. D. White, and C. P. Tripp, “Chemical Modification of Metal Oxide Surfaces in Supercritical CO2: In Situ Infrared Studies of the Adsorption and Reaction of Organosilanes on Silica”, Langmuir 1999, 15, 7870-7875
[48] M. Jason Kelly, Joseph H. Han, Charles B. Musgrave, and Gregory N. Parsons, “In-Situ Infrared Spectroscopy and Density Functional Theory Modeling of Hafnium Alkylamine Adsorption on Si-OH and Si-H Surfaces”, Chem. Mater. 2005, 17, 5305-5314
[49] Myung M. Sung, G. Jonathan Kluth, and Roya Maboudian, “Formation of alkylsiloxane self-assembled monolayers on Si3N4”, J. Vac. Sci. Technol. A 17.2., Mar/Apr 1999
[50] Max Muhlhauser, Marcus Gastreich, Christel M. Marian,“Structural Properties of (Trichlorosilyl)-Amino-Dichloroborane”
[51] A. S. Maria Chong and X. S. Zhao, “Functionalization of SBA-15 with APTES and Characterization of Functionalized Materials”, J. Phys. Chem. B 2003, 107, 12650-12657
[52] J. L. Blin,, C. Ge´rardin, L. Rodehu¨ ser, C. Selve, and M. J. Ste´be´, “Influence of Alkyl Peptidoamines on the Structure of Functionalized Mesoporous Silica”, Chem. Mater. 2004, 16, 5071-5080
[53] José L. Foschiera, Tania M. Pizzolato and Edilson V. Benvenutti, “FTIR Thermal Analysis on Organofunctionalized Silica Gel”, J. Braz. Chem. Soc., Vol. 12, No. 2, 159-164, 2001.
[54] H. Ma¨ ckel and R. Lu¨demann, “Detailed study of the composition of hydrogenated SiNx layers for high-quality silicon surface passivation”, JOURNAL OF APPLIED PHYSICS VOLUME 92, NUMBER 5, 1 SEPTEMBER 2002
[55] Marco A. Schiavon, Gian Domenico Sorar_u, I. V al_eria P. Yoshida, “Synthesis of a polycyclic silazane network and its evolution to silicon carbonitride glass”,Journal of Non-Crystalline Solids 304 (2002) 76–83
[56] Y. Q. Wang, Y. G. Wang, L. Cao, and Z. X. Cao, “High-efficiency visible photoluminescence from amorphous silicon nanoparticles embedded in silicon nitride”, APPLIED PHYSICS LETTERS VOLUME 83, NUMBER 17 27 OCTOBER 2003.
[57] J. Chen, A. J. Steckl, and M. J. Loboda, “In Situ N2-Doping of SiC Films Grown on Si(111) by Chemical Vapor Deposition from Organosilanes”, Journal of The Electrochemical Society, 147 (6) 2324-2327 (2000).
[58] Young-Bae Park, Shi-Woo Rhee, “Optoelectronic properties of fluorine-doped silicon nitride thin films”, Journal of Non-Crystalline Solids 343 (2004) 33–38.
[59] Kimihisa MATSUMOTO, Kenji IMAKITA, Minoru FUJII and Shinji HAYASHI, “Photoluminescence from Si Nanocrystals Embedded in SiOxNy Thin Films”, Japanese Journal of Applied Physics Vol. 44, No. 50, 2005, pp. L 1547–L 154
[60] J. F. Moulder, W. F. Stickle, P. E. Sobol and K. D. Bomben “Handbook of X-Ray Photoelectron Spectroscopy”, Physical Electronics, 1995
[61] W. Robert Ashurst, C. Carraro and R. Maboudian, “Vapor Phase Anti-Stiction Coatings for MEMS”, IEEE Transactions on Device and Materials Reliability, Vol. 3, No. 4, Dec. 2003
[62] C. Louro, A. Cavaleiro, F. Montemor, “How is the chemical bonding of W_Si_N sputtered coatings?”, Surface and Coatings Technology 142-144 (2001) 964-970
[63] http://science.scu.edu.tw/micro/1024/learn/02micro_bio/chao000/chao019.htm
[64] http://polymer.che.ncku.edu.tw/papers/E-Nano/E050.pdf#search='PLLA%26PS'
[65]徐怡琳, “利用矽烷類混合式自組裝單分子膜提高蛋白質晶片上抗體與檢體的接合效率”, 國立清華大學碩士論文, 2003.