人體尿蛋白在腎臟病前期診斷扮演重要角色，尿蛋白的自我檢測可以協助病患初步判斷疾病的嚴重程度。本研究擬製備電化學尿蛋白感測器，以牛血清白蛋白 (Bovine serum albumin, BSA) 為待測物，使用不同種硫醇在裸金電極上形成自組裝單分子層 (self-assembly monolayer, SAM)，以此修飾電極作為工作電極，利用伏安法及交流阻抗分析法分別探討不同白蛋白濃度在黃血鹽/赤血鹽系統的峰電流及阻抗變化，並利用傅立葉轉換紅外線光譜及接觸角量測儀分別分析修飾電極與BSA之間的鍵結及修飾電極親疏水性。
製備感測電極時，將1-(3-二甲氨基丙基)-3-乙基碳二亞胺 (EDC) 及N-羥基丁二醯亞胺 (NHS) 修飾在電極上改變硫醇單分子尾端官能基；感測時，此官能基與BSA合成胜肽鍵 (peptide)，經由循環伏安掃描將電極表面上未與硫醇分子形成胜肽鍵之白蛋白脫除。
結果顯示，電極在含浸白蛋白溶液前為不可逆反應，含浸白蛋白後隨著濃度提高，不可逆性上升、電子轉移阻抗 (Rct) 增大、電化學活性變小；在白蛋白溶液pH 5.5時，8-巰基辛酸 (8-mercaptooctanoic acid, 8-MOA) 修飾電極對白蛋白的靈敏度，從裸電極的0.666 ppm-1 (氧化峰) 及0.604 ppm-1 (還原峰) 提升至2.594 ppm-1 (氧化峰) 及2.614 ppm-1 (還原峰)，偵測上限為30 ppm。在相同pH，以12-巰基12烷酸 (12-mercaptododecanoic acid, 12-MDA) 修飾的電極靈敏度雖再提升至13.11 ppm-1 (氧化峰) 及11.25 ppm-1 (還原峰)，偵測上限卻降為6 ppm；考慮成人尿液中白蛋白含量約為20 ppm，因此本研究認為8-巰基辛酸最適合用以修飾電極作為電化學尿蛋白感測器。

Human urinary albumin played an important role in the earlier stage diagnosis of kidney disease. The self-examination of urine albumin could help patients in determining seriousness of kidney disease. In this study, an electrochemical urinary albumin sensor was prepared. Bovine serum albumin (BSA) was applied as the analytes, and various kinds of thiol were used to form self-assembly monolayers (SAMs) on gold electrode. Cyclic voltammetry (CV) and AC impedance analysis were used to determine peak potential, peak current and impedance in a Fe(CN)63-/Fe(CN)64- system. Fourier Transfer Infrared Spectroscopy (FT-IR) and contact angle goniometer were also used to analyze the bonding between modified electrode and BSA and hydrophobicity of modified electrode.
In preparing sensing electrode, N-(3-Dimethylaminopropyl)-N’- ethylcarbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide (NHS) were modified onto electrode to change the end group of thiols. Peptide form between BSA and thiol during sensing. After cyclic voltammetry, the BSA unbonding to the thiols on the electrode would desorb.
The result showed that redox reactions Fe(CN)63-/Fe(CN)64- on electrodes before immersing in BSA solution were irreversible reactions. After immersed in BSA solution and increasing the BSA concentration, the irreversibility of the redox reaction and the charge transfer resistance increased, and the electrode activity reduced. For 8-mercaptooctanoic acid modified electrode at pH 5.5, the BSA sensitivity was improved from 0.666 ppm-1 (oxidation) and 0.604 ppm-1 (reduction) with bare electrodes to 2.594 ppm-1 (oxidation) and 2.614 ppm-1 (reduction). The detecting upper limit of BSA was 30 ppm. For 12-mrecaptododecanoic acid modified electrode at pH 5.5, although the sensitivity was improved to 13.11 ppm-1 (oxidation) and 11.25 ppm-1 (reduction), the detecting upper limit of BSA decreased to 6 ppm. Since the normal concentration of albumin in urine is 20 ppm, the electrode modified with 8-MOA electrode is proposed the most suitable sensing electrode for the electrochemical urinary albumin sensor.

參考文獻
[1] 行政院經建會人力規劃處, "2010年至2060年人口推計及分析," 2010.
[2] 行政院衛生署, "99年主要死因分析," 2011.
[3] D.R. Smith, 一般泌尿科學: 合記出版社, 1987.
[4] 蘇裕謀, "腎臟疾病處方判讀及處置," 台北醫學大學, 2010.
[5] D.C. Scheid, L.H. McCarthy, F.H. Lawler, R.M. Hamm, and K. Reilly, "Screening for microalbuminuria to prevent nephropathy in patients with diabetes: a systematic review of the evidence," J Fam Pract, vol. 50, pp. 661-668, 2001.
[6] T. Seiyama, A. Kato, K. Fujiishi, and M. Nagatani, "A new detector for gaseous components using semiconductive thin films," Analytical Chemistry, vol. 34, pp. 1502-1503, 1962.
[7] B.J. Hwang, "Solid-state electrolyte based electrochemical-gas sensors," Chinese Chemical Society, vol. 59, pp. 207-217, 2001.
[8] X. Wang, W. Liu, J. Liu, F. Wang, J. Kong, S. Qiu, C. He, and L. Luan, "Synthesis of Nestlike ZnO Hierarchically Porous Structures and Analysis of Their Gas Sensing Properties," Acs Applied Materials & Interfaces, vol. 4, pp. 817-825, 2012.
[9] L. K. Randeniya, P.J. Martin, and A. Bendavid, "Detection of Hydrogen Using Multi-walled Carbon-nanotube Yarns Coated with Nanocrystalline Pd and Pd/Pt Layered Structures," Carbon, vol. 50, pp. 1786-1792, 2012.
[10] J. Lee, S. Mubeen, C.M. Hangarter, A. Mulchandani, W. Chen, and N.V. Myung, "Selective and Rapid Room Temperature Detection of H(2)S Using Gold Nanoparticle Chain Arrays," Electroanalysis, vol. 23, pp. 2623-2628, 2011.
[11] N. Yamazoe, "Toward innovations of gas sensor technology," Sensors and Actuators B: Chemical, vol. 108, pp. 2-14, 2005.
[12] T. Takeuchi, "Oxygen sensors," Sensors and Actuators, vol. 14, pp. 109-124, 1988.
[13] L. C. Clark Jr. and C. Lyons, "Electrode Systems for Continuous Monitoring in Cardiovascular Surgery," Annals of the New York Academy of Sciences, vol. 102, pp. 29-45, 1962.
[14] 李佳錚, "自組裝單層膜修飾電極在尿蛋白生醫感測之應用," 碩士, 化學工程學系碩博士班, 國立成功大學, 台南市, 2010.
[15] A.J. Bard and L.R. Faulkner, Electrochemical methods: fundamentals and applications vol. 2: Wiley New York, 1980.
[16] 楊婉鈴, 李博仁, and 陳逸聰, "無標記奈米級生物感測器," 化學, vol. 69, pp. 199-209, 2011.
[17] 陳旻政, 林家毅, and 林昌賢, "奈米線場效電晶體平台製作及生物分子感測," 奈米通訊, vol. 18, pp. 37-43, 2011.
[18] 杜振豪, "以電鍍法製作氧化鋅奈米柱陣列做為EGFET葡萄糖感測膜," 碩士, 材料科學工程學系, 國立清華大學, 新竹市, 2011.
[19] 謝振傑, "光纖生物感測器," 物理雙月刊, vol. 28, p. 704, 2006
[20] S. Yin and P. Ruffin, "Fiber Optic Sensors," in Wiley Encyclopedia of Biomedical Engineering, ed: John Wiley & Sons, Inc., 2006.
[21] 余聲宏, "光纖漸逝波感測器應用於微量生物分子檢測," 碩士, 醫學工程研究所, 國立陽明大學, 台北市, 2004.
[22] J. Melendez, R. Carr, D. Bartholomew, H. Taneja, S. Yee, C. Jung, and C. Furlong, "Development of a surface plasmon resonance sensor for commercial applications," Sensors and Actuators B: Chemical, vol. 39, pp. 375-379, 1997.
[23] D.A. Schultz, "Plasmon resonant particles for biological detection," Current Opinion in Biotechnology, vol. 14, pp. 13-22, 2003.
[24] 劉盈村, "光纖式表面電漿子共振生醫微感測器之研發," 碩士, 醫學工程學研究所, 國立臺灣大學, 台北市, 2002.
[25] T. Nomura and M. Iijima, "Electrolytic determination of nanomolar concentrations of silver in solution with a piezoelectric quartz crystal," Analytica Chimica Acta, vol. 131, pp. 97-102, 1981.
[26] T. Nomura, "Single-drop method for determination of cyanide in solution with a piezoelectric quartz crystal," Analytica Chimica Acta, vol. 124, pp. 81-84, 1981.
[27] 廖昌昱, "以微製程技術製備鋯鈦酸鉛壓電式微重量感測器," 碩士, 電機工程研究所, 國立中央大學, 桃園縣, 2007.
[28] U.E. Spichiger-Keller, "Chemical Sensors and Biosensors for Medical and BiologicalApplications," Data Processing, vol. 1, 1998.
[29] E.M. Damsgaard, A. Froland, O.D. Jorgensen, and C.E. Mogensen, "Microalbuminuria as predictor of increased mortality in elderly people," British Medical Journal, vol. 300, pp. 297-300, 1990.
[30] P.T. Sawicki, L. Heinemann, and M. Berger, "Comparison of methods for determination of microalbuminuria in diabetic patients," Diabetic Medicine, vol. 6, pp. 412-415, 1989.
[31] D. Caballero, E. Martinez, J. Bausells, A. Errachid, and J. Samitier, "Impedimetric immunosensor for human serum albumin detection on a direct aldehyde-functionalized silicon nitride surface," Analytica Chimica Acta, vol. 720, pp. 43-48, 2012.
[32] H.J. Chen, Z.H. Zhang, L.J. Luo, and S.Z. Yao, "Surface-imprinted chitosan-coated magnetic nanoparticles modified multi-walled carbon nanotubes biosensor for detection of bovine serum albumin," Sensors and Actuators B: Chemical, vol. 163, pp. 76-83, 2012.
[33] C.J. VanDelden, J.M. Bezemer, G.H.M. Engbers, and J. Feijen, "Poly(ethylene oxide)-modified carboxylated polystyrene latices - Immobilization chemistry and protein adsorption," Journal of Biomaterials Science-Polymer Edition, vol. 8, pp. 251-268, 1996.
[34] R.G. Nuzzo, L.H. Dubois, and D.L. Allara, "Fundamental studies of microscopic wetting on organic surfaces. 1. Formation and structural characterization of a self-consistent series of polyfunctional organic monolayers," Journal of the American Chemical Society, vol. 112, pp. 558-569, 1990.
[35] C.E.D. Chidsey and D.N. Loiacono, "Chemical functionality in self-assembled monolayers: structural and electrochemical properties," Langmuir, vol. 6, pp. 682-691, 1990.
[36] W.C. Bigelow, D.L. Pickett, and W.A. Zisman, "Oleophobic monolayers: I. Films adsorbed from solution in non-polar liquids," Journal of Colloid Science, vol. 1, pp. 513-538, 1946.
[37] D.L. Allara and R.G. Nuzzo, "The application of reflection infrared and surface enhanced raman spectroscopy to the characterization of chemisorbed organic disulfides on gold," Journal of Electron Spectroscopy and Related Phenomena, vol. 30, pp. 11-11, 1983.
[38] Y.N. Xia and G.M. Whitesides, "Soft lithography," Angew. Chem., Int. Ed., vol. 37, pp. 551-575, 1998.
[39] A.N. Parikh, D.L. Allara, I.B. Azouz, and F. Rondelez, "An intrinsic relationship between molecular structure in self-assembled n-alkylsiloxane monolayers and deposition temperature," Journal of Physical Chemistry, vol. 98, pp. 7577-7590, 1994.
[40] J.P. Folkers, C.B. Gorman, P.E. Laibinis, S. Buchholz, G.M. Whitesides, and R.G. Nuzzo, "Self-assembled monolayers of long-chain hydroxamic acids on the native oxides of metals," Langmuir, vol. 11, pp. 813-824, 1995.
[41] J.J. Hickman, P.E. Laibinis, D.I. Auerbach, C. Zou, T.J. Gardner, G.M. Whitesides, and M.S. Wrighton, "Toward orthogonal self-assembly of redox active molecules on platinum and gold: selective reaction of disulfide with gold and isocyanide with platinum," Langmuir, vol. 8, pp. 357-359, 1992.
[42] Q. Wang, D. Dong, and N.Q. Li, "Electrochemical response of dopamine at a penicillamine self-assembled gold electrode," Bioelectrochemistry, vol. 54, pp. 169-175, 2001.
[43] Y. Sato and F. Mizutani, "Determination of real composition of 3-mercaptopropionic acid and 1-octadecanethiol mixed self-assembled monolayers by using electrochemical and electrochemical quartz crystal microbalance measurements," Electroanalysis, vol. 10, pp. 633-637, 1998.
[44] A. Ulman, "An introduction to ultrathin organic films: from langmuir blodgett to self assembly," Chem. Rev, vol. 96, p. 1533, 1996.
[45] L.H. Dubois and R.G. Nuzzo, "Synthesis, structure, and properties of model organic surfaces," Annual Review of Physical Chemistry, vol. 43, pp. 437-463, 1992.
[46] L.H. Dubois, B.R. Zegarski, and R.G. Nuzzo, "Fundamental studies of microscopic wetting on organic surfaces. 2. Interaction of secondary adsorbates with chemically textured organic monolayers," Journal of the American Chemical Society, vol. 112, pp. 570-579, 1990.
[47] L.R. de Astudillo, L. Rivera, R. Brito-Gómez, and R.J. Tremont, "Electrochemical study of 1,4-benzoquinone on gold surface modified," Journal of Electroanalytical Chemistry, vol. 640, pp. 56-60, 2010.
[48] H. Sellers, A. Ulman, Y. Shnidman, and J.E. Eilers, "Structure and binding of alkanethiolates on gold and silver surfaces: implications for self-assembled monolayers," Journal of the American Chemical Society, vol. 115, pp. 9389-9401, 1993.
[49] A. Ulman, "Formation and structure of self-assembled monolayers," Chemical reviews, vol. 96, p. 1533, 1996.
[50] 劉仁材, "自組裝單層膜技術於光學式及電梳式生物感測器之應用研究," 博士, 化學工程與材料工程研究所, 國立中央大學, 桃園縣, 2009.
[51] P.E. Laibinis and G.M. Whitesides, "Omega-terminated alkanethiolate monolayers on surfaces of copper, silver, and gold have similar wettabilities," Journal of the American Chemical Society, vol. 114, pp. 1990-1995, 1992.
[52] K.W. Kittredge, M.A. Fox, and J.K. Whitesell, "Effect of alkyl chain length on the fluorescence of 9-alkylfluorenyl thiols as self-assembled monolayers on gold," The Journal of Physical Chemistry B, vol. 105, pp. 10594-10599, 2001.
[53] X. Lu, H. Yuan, G. Zuo, and J. Yang, "Study of the size and separation of pinholes in the self-assembled thiol-porphyrin monolayers on gold electrodes," Thin Solid Films, vol. 516, pp. 6476-6482, 2008.
[54] E. Klein, P. Kerth, and L. Lebeau, "Enhanced selective immobilization of biomolecules onto solid supports coated with semifluorinated self-assembled monolayers," Biomaterials, vol. 29, pp. 204-214, 2008.
[55] Y.S. Chen and J.H. Huang, "Electrochemical sensing of bovine serum albumin at self-assembled SWCNTs on gold," Diamond and Related Materials, vol. 18, pp. 516-519, 2009.
[56] M. Baker, A.J. Palmer, W.R. MacGillivray, and R.T. Sang, "Lithographic pattern formation via metastable state rare gas atomic beams," Nanotechnology, vol. 15, p. 1356, 2004.
[57] J. Xin, K. Mitsunori, S. Taku, and Y. Yasushi, "Positive and negative patterning of ethanethiol, decanethiol, and hexadecanethiol self-assembled monolayers by using a metastable helium beam," Thin Solid Films, vol. 464–465, pp. 420-424, 2004.
[58] 胡啟章, 電化學原理與方法 vol. 2. 台北: 五南圖書出版股份有限公司, 2007.
[59] 陸瑞東, "以聯氨還原製備質子交換膜燃料電池鉑 鎳/碳陰極之研究," 碩士, 化學工程學系碩博士班, 國立成功大學, 台南市, 2005.
[60] D.M. Ruthven, Principles of adsorption and adsorption processes: Wiley-Interscience, 1984.
[61] H.O. Finklea, D.A. Snider, J. Fedyk, E. Sabatani, Y. Gafni, and I. Rubinstein, "Characterization of octadecanethiol-coated gold electrodes as microarray electrodes by cyclic voltammetry and ac impedance spectroscopy," Langmuir, vol. 9, pp. 3660-3667, 1993.
[62] V. Ganesh, S.K. Pal, S. Kumar, and V. Lakshminarayanan, "Self-assembled monolayers (SAMs) of alkoxycyanobiphenyl thiols on gold—A study of electron transfer reaction using cyclic voltammetry and electrochemical impedance spectroscopy," Journal of Colloid and Interface Science, vol. 296, pp. 195-203, 2006.
[63] S. Campuzano, M. Pedrero, C. Montemayor, E. Fatás, and J.M. Pingarrón, "Characterization of alkanethiol-self-assembled monolayers-modified gold electrodes by electrochemical impedance spectroscopy," Journal of Electroanalytical Chemistry, vol. 586, pp. 112-121, 2006.
[64] C.A. Widrig, C. Chung, and M.D. Porter, "The electrochemical desorption of n-alkanethiol monolayers from polycrystalline Au and Ag electrodes," Journal of Electroanalytical Chemistry, vol. 310, pp. 335-359, 1991.
[65] S.I. Imabayashi, D. Hobara, T. Kakiuchi, and W. Knoll, "Selective replacement of adsorbed alkanethiols in phase-separated binary self-assembled monolayers by electrochemical partial desorption " Langmuir, vol. 13, pp. 4502-4504, 1997.
[66] M.M. Walczak, C.A. Alves, B.D. Lamp, and M.D. Porter, "Electrochemical and X-ray photoelectron spectroscopic evidence for differences in the binding sites of alkanethiolate monolayers chemisorbed at gold," Journal of Electroanalytical Chemistry, vol. 396, pp. 103-114, 1995.
[67] A. Badia, S. Arnold, V. Scheumann, M. Zizlsperger, J. Mack, G. Jung, and W. Knoll, "Probing the electrochemical deposition and/or desorption of self-assembled and electropolymerizable organic thin films by surface plasmon spectroscopy and atomic force microscopy," Sensors and Actuators B: Chemical, vol. 54, pp. 145-165, 1999.
[68] E. Boubour and R.B. Lennox, "Potential-induced defects in n-alkanethiol self-assembled monolayers monitored by impedance spectroscopy," The Journal of Physical Chemistry B, vol. 104, pp. 9004-9010, 2000.
[69] Y.C. Yang, Y.P. Yen, L.Y. Ou Yang, S.L. Yau, and K. Itaya, "Elucidation of the deposition processes and spatial structures of alkanethiol and arylthiol molecules adsorbed on pt(111) electrodes with in situ scanning tunneling microscopy," Langmuir, vol. 20, pp. 10030-10037, 2004.
[70] J.A. Williams and C.B. Gorman, "Alkanethiol reductive desorption from self-assembled monolayers on gold, platinum, and palladium substrates," The Journal of Physical Chemistry C, vol. 111, pp. 12804-12810, 2007.
[71] J.R. Brown, "Structure of bovine serum-albumin," Federation Proceedings, vol. 34, pp. 591-591, 1975.
[72] T. Peters Jr, R.C. Feldhoff, and R.G. Reed, "Immunochemical studies of fragments of bovine serum albumin," Journal of Biological Chemistry, vol. 252, pp. 8464-8468, 1977.
[73] S. Era, H. Ashida, S. Nagaoka, H. Inouye, and M. Sogami, "CD-resolved secondary structure of bovine plasma albumin in acid-induced isomerization*," International Journal of Peptide and Protein Research, vol. 22, pp. 333-340, 1983.
[74] M.Y. Khan, "Direct evidence for the involvement of domain III in the NF transition of bovine serum albumin," Biochemical Journal, vol. 236, p. 307, 1986.
[75] S. Era, K.B. Itoh, M. Sogami, K. Kuwata, T. Iwama, H. Yamada, and H.W. Atari, "Structural transition of bovine plasma albumin in the alkaline region the N-B transition," International Journal of Peptide and Protein Research, vol. 35, pp. 1-11, 1990.
[76] M. Dockal, D.C. Carter, and F. Rüker, "Conformational transitions of the three recombinant domains of human serum albumin depending on pH," Journal of Biological Chemistry, vol. 275, pp. 3042-3050, 2000.
[77] J.M. Lee, H.H.L. Edwards, C.A. Pereira, and S.I. Samii, "Crosslinking of tissue-derived biomaterials in 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)," Journal of Materials Science: Materials in Medicine, vol. 7, pp. 531-541, 1996.
[78] L.H.H.O. Damink, P.J. Dijkstra, M.J.A.V. Luyn, P.B. Wachem, P.V. Nieuwenhuis, and J. Feijen, "Cross-linking of dermal sheep collagen using a water-soluble carbodiimide," Biomaterials, vol. 17, pp. 765-773, 1996.
[79] S. Balasubramanian, A. Revzin, and A. Simonian, "Electrochemical desorption of proteins from gold electrode surface," Electroanalysis, vol. 18, pp. 1885-1892, 2006.
[80] T. Ignat, M. Miu, I. Kleps, A. Bragaru, M. Simion, and M. Danila, "Electrochemical characterization of BSA/11-mercaptoundecanoic acid on Au electrode," Materials Science and Engineering B-Advanced Functional Solid-State Materials, vol. 169, pp. 55-61, 2010.
[81] H.D. Wang, Q. Yang, C.H. Niu, and I. Badea, "Protein-modified nanodiamond particles for Layer-by-Layer assembly," Diamond and Related Materials, vol. 20, pp. 1193-1198, 2011.
[82] T. Miyazawa, T. Shimanouchi, and S. Mizushima, "Characteristic infrared bands of monosubstituted amides," The Journal of Chemical Physics, vol. 24, p. 408, 1956.
[83] G. Herzberg and J.W.T. Spinks, Molecular spectra and molecular structure: Prentice-Hall, 1966.