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系統識別號 U0026-1806201909451000
論文名稱(中文) 應用電性鑑別偵測法定量檢測人類絨毛膜促性腺激素之研究
論文名稱(英文) Quantitative Analysis Based on Impedance Measurement for Human Chorionic Gonadotropin
校院名稱 成功大學
系所名稱(中) 工程科學系
系所名稱(英) Department of Engineering Science
學年度 107
學期 2
出版年 108
研究生(中文) 陳心瑜
研究生(英文) Hsin-Yu Chen
學號 N96064484
學位類別 碩士
語文別 中文
論文頁數 111頁
口試委員 指導教授-林裕城
口試委員-林弘萍
口試委員-李哲明
中文關鍵字 電性鑑別  三明治免疫分析技術  金奈米粒子  側流免疫層析法  高分子相轉換 
英文關鍵字 Electro-microchip  Sandwich-immunoassay  Gold nanoparticles  Lateral flow assay  Polymer phase inversion 
學科別分類
中文摘要 本研究利用電性鑑別法應用於人類絨毛膜促性腺激素(Human Chorionic Gonadotropin, hCG)三明治免疫反應之檢測,結合阻抗式檢測電極晶片並利用金奈米粒子抗體標定技術,以助於偵測待測檢體之免疫反應並定量阻抗量測分析結果鑑別。實驗中利用電感、電容、電阻量測儀(LCR Meter)偵測阻抗晶片之電性訊號,藉由阻抗值改變分析判斷免疫反應變化,避免免疫色層分析法使用人眼辨別造成之誤差,並建立電性量測三明治免疫分析系統。為避免硝化纖維膜與檢測電極間間隙造成電阻抗訊號量測不穩定的狀態發生,直接將硝化纖維膜透過高分子溶液相轉換方式固定於檢測電極表面,成功利用阻抗變化、金奈米粒子標定技術和三明治免疫分析,達成進行人類絨毛膜促性腺激素(hCG)定量分析。其免疫檢測阻抗分析檢測試劑晶片之極限達6.25 mIU/mL,成功地突破檢測閾值25 mIU/mL限制,且電性阻抗與hCG濃度之檢量線決定係數(Coefficient of Determination)達0.9899,即表示本研究可利用此線性分析轉換曲線進行定量量測分析,且於免疫檢測分析上提供了一個全新的方向與思維。
英文摘要 This study, a novel electrode biochip for immunoassay was applied to detect sandwich-immunoassay of human chorionic gonadotropin, a gold nanoparticles (AuNPs) as a label of antibody to enhance immunoassay impedance quantitative analysis detection. Compared with traditional enzyme-linked immunosorbent assay (ELISA), we utilized the LCR meter instrument to detect the electric signal on electrode biochip. By detecting the impedance changes because of sandwich-immunoassay reaction, a sandwich-immunoassay detection system was established. In order to avoid a gap between a nitrocellulose membrane and electrode bringing about the unstable electric signal, a nitrocellulose membrane was fixed on a surface of electrode chip. In the study, combined impedance changing detection and gold nanoparticles labelled in sandwich-immunoassay achieves quantitative the concentration of human chorionic gonadotropin(hCG) antigen. The results show that the cut-off value decreased from 25 mIU/mL to 6.25 mIU/mL. The coefficient of determination between electric signal and concentration of hCG has come to 0.9899.
論文目次 摘要 I
EXTENDED ABSTRACT II
誌謝 IX
縮寫表 X
目錄 XII
表目錄 XVII
圖目錄 XVIII
第一章 緒論 1
1-1 研究背景 1
1-2 免疫分析法 4
1-2-1 免疫分析基本理論 5
1-2-2 抗體與抗原定義 6
1-2-3 抗原與抗體結合力 8
1-2-4 免疫分析檢測種類 9
1-2-5 免疫分析偵測方法 10
1-2-6 傳統酵素聯結免疫吸附分析法-ELISA 13
1-3 生物電阻抗分析技術 15
1-4 側流層析免疫反應檢測試劑 16
1-5 文獻回顧 19
1-5-1 人類絨毛膜促性腺激素 19
1-5-2 hCG於人體之來源與作用 20
1-5-3 hCG於妊娠檢測之應用 22
1-5-4 hCG於腫瘤標記之應用 23
1-5-5 人類絨毛膜促性腺激素驗孕免疫反應檢測試劑 23
1-5-6 相轉換法原理 24
1-5-7 乾式相轉換法 25
1-5-8 相轉換法成膜機制 26
1-6 研究動機與目的 28
1-7 研究架構 29
第二章 阻抗式檢測電極晶片設計與整合製程 31
2-1 電極晶片之等效電路模型 31
2-2 電極晶片之檢測電極材料與設計 32
2-3 檢測試劑之反應區薄膜 36
2-4 檢測試劑結構與製程 38
2-5 金奈米粒子-二抗膜製備與樣品墊處理 40
2-6 阻抗式檢測電極晶片製作 42

第三章 實驗與研究方法 44
3-1 實驗儀器與設備 44
3-1-1 渦旋混合器 44
3-1-2 電子天秤 45
3-1-3 加熱型攪拌器 46
3-1-4 真空烘箱系統 47
3-1-5 微量注射幫浦 48
3-1-6 硝化纖維膜塗佈平台建立 49
3-1-7 二氧化碳雷射雕刻機 50
3-1-8 真空冷凍乾燥機 51
3-1-9 桌上型掃描式電子顯微鏡 53
3-1-10 LCR高精度量測儀 54
3-2 實驗藥品 56
3-3 實驗方法 58
3-3-1 檢測試劑之反應區薄膜製程開發與製作 59
3-3-1-1 硝化纖維溶液製備與塗佈 59
3-3-1-2 硝化纖維膜相轉換濕度研究 62
3-3-1-3 檢測試劑之反應區薄膜製程 63
3-3-1-4 硝化纖維膜結構研究 64
3-3-2 檢測試劑整合製作 66
3-3-3 檢測電極晶片阻抗量測平台 67
3-3-4 三明治免疫阻抗分析實驗-hCG-GC濃度測試 69
3-3-5 三明治免疫阻抗分析實驗-hCG-Ab濃度測試 70
3-3-6 三明治免疫阻抗分析實驗-反應時間 71
3-3-7 三明治免疫阻抗分析實驗-頻率研究 72
3-3-8 三明治免疫阻抗分析實驗-hCG檢測 73
第四章 結果與討論 74
4-1 相轉換之相對濕度與孔隙率 74
4-1-1 硝化纖維膜相轉換濕度 74
4-1-2 硝化纖維膜結構研究 77
4-1-3 硝化纖維膜厚度研究 79
4-2 三明治免疫阻抗分析實驗 81
4-2-1 hCG-GC濃度測試 81
4-2-2 hCG-Ab濃度測試 84
4-2-3 反應時間測試 87
4-2-4 頻率測試 88
4-2-5 hCG阻抗分析檢測 94
第五章 結論與未來展望 99
5-1 結論 99
5-2 未來展望 101
參考文獻 102
參考文獻 [1] T. Porstmann and S. T. Kiessig, “Enzyme immunoassay techniques and overview,” Journal of Immunological Methods, 150, pp. 5-21, 1992.
[2] A. Chaubey and B. D. Malhotra, “Mediated biosensors,” Biosensors and Bioelectronics, 17, pp. 441-456, 2002.
[3] 何敏夫,臨床生化學,合記圖書出版社, 1992。
[4] R. H. Garrett and C. M. Grisham, Biochemistry, Saunders College Publishing, 1995.
[5] 廖國棠,金奈米粒子標記物在免疫分析、DNA 序列分析及微管道晶片系統分析上的應用,國立中山大學化學研究所博士論文,民國九十四年。
[6] E. Engvall and P. Perlmann, “Enzyme-linked immunosorbent assay, Elisa. 3. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes,” Journal of Immunology, 109, pp. 129-135, 1972.
[7] T. Watanabe, Y. Ohkuno, H. Matsuoka, H. Kimura, Y. Sakai, Y. Ohkaru, T. Tanaka and Y. Kitaura, “Development of a simple whole blood panel test for detection of human heart-type fatty acid-binding protein,” Clinical Biochemistry, 34, pp. 257-263, 2001.
[8] M. Hedenfalk, P. Adlercreutz, and B. Mattiasson, “Modulation of the measuring range of a radioimmunoassay using an organic water two phase system,” Analytica Chimica Acta, 341, pp. 269-274, 1997.
[9] F. Hardy, L. Djavadi-Ohaniance and M. E. Goldberg, “Measurement of antibody/antigen association rate constants in solution by a method based on the enzyme-linked immunosorbent assay,” Journal of immunological methods, 200, pp. 155-159, 1997.
[10] P. Onnerfjord, S. Eremin, J. Emneus and G. Marko-Varga, “Fluorescence polarisation for immunoreagent characterization,” Journal of immunological methods, 213, pp. 31-39, 1998.
[11] J. A. Schmid and A. Billich, “Simple method for high sensitivity chemiluminescence ELISA using conventional laboratory equipment,” BioTechniques, 22, pp. 278, 1997.
[12] C. A. Janeway, P. Travers, M. Walport, and M. J. Shlomchik, Immunobiology, 5th edition, Garland Science, 2001.
[13] I. Bronstein, J. C. Voyta, G. H. G. Thorpe, L. J. Kricka and G. Armstrong, “Chemiluminescent assay of alkaline phosphatase applied in an ultrasensitive enzyme immunoassay of thyrotropin,” Clinical Chemistry, 35, pp. 1441-1446, 1989.
[14] E. Ishikawa, S. Hashida, T. Kohno and K. Hirota, “Ultrasensitive enzyme immunoassay,” Clinica Chimica Acta, 194, pp. 43-55, 1990.
[15] R. F. Kushner and D. A. Schoeller, “Estimation of total body water by bioelectrical impedance analysis,” American Journal of Clinical Nutrition, 44, pp. 417-424, 1986.
[16] R. W. McNaught and J. T. France, “Studies of the biochemical basis of steroid sulphatase deficiency: preliminary evidence suggesting a defect in membrane-enzyme structure,” Journal of steroid biochemistry, 13, pp. 363-373, 1980.
[17] A. J. Lapthorn, D. C. Harris, A. Littlejohn, J. W. Lustbader, R. E. Canfield, K. J. Machin, F. J. Morgan and N. W. Isaacs, “Crystal structure of human chorionic gonadotropin,” Nature, 369, pp. 455-461, 1994.
[18] J. G. Pierce and T. F. Parsons, “Glycoprotein Hormones: Structure and Function,” Annual Reviews Biochemistry, 50, pp. 465-495, 1981.
[19] S. Birken, Y. Maydelman, M. A. Gawinowicz, A. Pound, Y. Liu and A. S. Hartree, “Isolation and characterization of human pituitary chorionic gonadotropin,” Endocrinology, 137, pp. 1402-1411, 1996.
[20] L. A. Cole and A. Kardana, “Discordant Results in Human Chorionic Gonadotropin Assays,” Clinical Chemistry, 38, pp. 263-270, 1992.
[21] R. Bellisario, R. B. Carlsen and O. P. Bahl, “Human Chorionic Gonadotropin Linear Amino Acid Sequence of The α Subunit,” Journal of Biological Chemistry, 248, pp. 6796-6809, 1973.
[22] R. B. Carlsen, O. P. Bahl and N. Swawinathan, “Human Chorionic Gonadotropin Linear Amino Acid Sequence of The β Subunit,” Journal of Biological Chemistry, 248, pp. 6810-6827, 1973.
[23] https://zh.wikipedia.org/wiki/%E4%BA%BA%E7%BB%92%E6%AF%9B%E8%86%9C%E4%BF%83%E6%80%A7%E8%85%BA%E6%BF%80%E7%B4%A0
[24] L. A. Cole, “New discoveries on the biology and detection of human chorionic gonadotropin,” Reproductive Biology and Endocrinology, 7, pp. 8, 2009.
[25] R. Hoermann, G. Spoettl, R. Moncayo and K. Mann, “Evidence for the presence of human chorionic gonadotropin (hCG) and free beta-subunit of hCG in the human pituitary,” Journal of Clinical Endocrinology & Metabolism, 71, pp. 179-186, 1990.
[26] J. J. Gregory and J. L. Finlay, “Alpha-fetoprotein and beta-human chorionic gonadotropin: their clinical significance as tumour markers,” Drugs, 57, pp. 463-467, 1999.
[27] U. A. Kayisli, B. Selam, O. G. Kayisli, R. Demir and A. Arici, “Human chorionic gonadotropin contributes to maternal immunotolerance and endometrial apoptosis by regulating Fas-Fas ligand system,” Journal of Immunology, 171, pp. 2305-2313, 2003.
[28] J. Askling, G. Erlandsson, M. Kaijser, O. Akre and A. Ekbom, “Sickness in pregnancy and sex of child,” Lancet, 354, pp. 2053, 1999.
[29] X. G. Fan and Z. Q. Zheng, “A Study of Early Pregnancy Factor ctivity in Preimplantation,” American Journal of Reproductive Immunology, 37, pp. 359-364, 1997.
[30] http://www.fertilityplus.com/faq/hpt.html
[31] https://www.fda.gov.tw/tc/includes/GetFile.ashx?id=f636694174340898429
[32] L. A. Cole, Y. X. Wang, M. Elliott, M. Latif, J. T. Chambers, S. K. Chambers and P. E. Schwartz, “Urinary Human Chorionic Gonadotropin Free β-Subunit and β-Core Fragment: A New Marker of Gynecological Cancers,” Cancer Research, 48, pp. 1356-1360, 1988.
[33] Y. Kato and G. B. Braunstein, “β-core fragment is a major form of immunoreactive urinary chorionic gonadotropin in uman pregnancy,” Journal of Clinical Endocrinology & Metabolism, 66, pp. 1197-1201, 1988.
[34] M. Mulder, Basic Principles of Membrane Technology, Kluwer Academic Publishers, 1996.
[35] T. Matsuura, Synthetic membranes and membrane separation processes, CRC Press, 1993.
[36] Z. Sun, Z. Yang, Z. Wang and C. Li, “The role of pre-evaporation in the preparation process of EVOH ultrafiltration membranes via TIPS,” Journal of Membrane Science, 563, pp. 238-246, 2018.
[37] X. Wang and Z. Wang, “Wide liquid-liquid phase separation region enhancing tensile strength of poly (vinylidene fluoride) membranes via TIPS method with a new diluent,” Polymer, 141, pp. 46-53, 2018.
[38] J. T. Jung, J. F. Kim, H. H. Wang, E. D. Nicolo, E. Drioli and Y. M. Lee, “Understanding the non-solvent induced phase separation (NIPS) effect during the fabrication of microporous PVDF membranes via thermally induced phase separation (TIPS),” Journal of Membrane Science, 514, pp. 250-263, 2016.
[39] H. A. Tsai, C. Y. Kuo, S. L. Su, D. M. Wang and J. Y. Lai, “The morphological evolution of solvent-containing PMMA membranes in various solvent removal processes,” Journal of Membrane Science, 345, pp. 288-297, 2009.
[40] D. M. Wang and J. Y. Lai, “Recent advances in preparation and morphology control of polymeric membranes formed by nonsolvent induced phase separation,” Current Opinion in Chemical Engineering, 2, pp. 229-237, 2013.
[41] S. H. Chen, R. M. Liou, Y. Y. Lin, C. L. Lai and J. Y. Lai, “Preparation and characterizations of asymmetric sulfonated polysulfone membranes by wet phase inversion method,” European Polymer Journal, 45, pp. 1293-1301, 2009.
[42] A. Venault, M. B. Ballad, Y. T. Huang, Y. H. Liu, C. H. Kao and Y. Chang, “Antifouling PVDF membrane prepared by VIPS for microalgae harvesting,” Chemical Engineering Science, 142, pp. 97-111, 2016.
[43] Y. S. Su, C. Y. Kuo, D. M. Wang, J. Y. Lai, A. Deratani, C. Pochat and D. Bouyer, “Interplay of mass transfer, phase separation, and membrane morphology in vapor-induced phase separation,” Journal of Membrane Science, 338, pp. 17-28, 2009.
[44] G. E. Gaides and A. J. Mchugh, “Gelation in an amorphous polymer: a discussion of its relation to membrane formation,” Polymer, 30, pp. 2118-2123, 1989.
[45] H. Sun, S. Liu, B. Ge, L. Xing and H. Chen, “Cellulose nitrate membrane formation via phase separation induced by penetration of nonsolvent from vapor phase,” Journal of Membrane Science, 295, pp. 2-10, 2007.
[46] M. Mulder, Basic Principles of Membrane Technology, Kluwer Academic Publishers, 1996.
[47] R. M. Boom, T. V. Boomgaard and C. A. Smolders, “Mass transfer and thermodynamics during immersion precipitation for a two-polymer system. Evaluation with the system PES-PVP-NMP-water,” Journal of Membrane Science, 90, pp. 231-249, 1994.
[48] J. J. van Aartsen and C. A. Smolders, “Light scattering of polymer solutions during liquid-liquid phase separation,” European Polymer Journal, 6, pp. 1105-1112, 1970.
[49] H. Caquineau, P. Menut, A. Deratani and C. Dupuy, “Influence of the Relative Humidity on Film Formation by Vapor Induced Phase Separation,” Polymer Engineering and Science, 43, pp. 798-808, 2003.
[50] J. Y. Kim, Y. D. Kim, T. Kanamori, H. K. Lee, K. J. Balk and S. C. Kim, “Vitrification phenomena in polysulfone/NMP/water system,” Journal of Applied Polymer Science, 71, pp. 431-438, 1999.
[51] 郭純因,非溶劑誘導相分離製備具雙連續結構微孔膜及其成膜機制之研究,中原大學化學工程學系博士學位論文,民國九十七年。
[52] M. A. Hayat, Colloidal Gold: Principles, Methods, and Applications, Academic Press, 1989.
[53] O. Niwa, M. Morita and H. Tabei, “Electrochemical Behavior of Reversible Redox Species at Interdigitated Array Electrodes with Different Geometries: Consideration of Redox Cycling and Collection Efficiency,” Analytical Chemistry, 62, pp. 447-452, 1990.
[54] O. Niwa, M. Morita and H. Tabei, “Highly sensitive and selective voltammetric detection of dopamine with vertically separated interdigitated array electrodes,” Electroanalysis, 3, pp. 163-168, 1991.
[55] A. L. Ahmad, S. C. Low, S. R. Abd Shukor, A. Ismail and A. R. Sunarti, “Development of lateral flow membranes for immunoassay separation,” Desalination and Water Treatment, 5, pp. 99-105, 2009.
[56] A. L. Ahmad, S. C. Low, S. R. Abd Shukor and A. Ismail, “INVESTIGATING MEMBRANE MORPHOLOGY AND QUANTITY OF IMMOBILIZED PROTEIN FOR THE DEVELOPMENT OF LATERAL FLOW IMMUNOASSAY,” Journal of Immunoassay and Immunochemistry, 33, pp. 48-58, 2012.
[57] 賴君義,以非溶劑誘導相分離來製備具雙連續結構之微孔膜-機制探討、製程設計及應用評估,行政院國家科學委員會專題研究計畫成果報告,民國九十八年。
[58] A. L. Ahmad, S. C. Low and S. R. Abd. Shukor, “Effects of membrane cast thickness on controlling the macrovoid structure in lateral flow nitrocellulose membrane and determination of its characteristics,” Scripta Materialia, 57, pp. 743-746, 2007.
[59] A. L. Ahmad, S. C. Low, S. R. Abd Shukor and A. Ismail, “Optimization of Membrane Formulation and Process Variables via Crossed-Design Concept in Design of Experimental (DOE),” Separation Science and Technology, 44, pp. 2870-2893, 2009.
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