本論文研究分為兩個部分，第一個部分為利用修飾有黃體素（progesterone）的微脂球應用於研發偵檢黃體素的免疫生物感測分析系統。研究中所使研發的偵檢平台有二：(一)樣品中之黃體素與修飾有黃體素之微脂球，同步競爭固定在硝化纖維材質之免疫分析試片上抗原捕捉區的黃體素抗體結合位，為競爭型的免疫分析法；(二)則是在可拋棄式網版印刷電極上進行抗體的定錨，再利用競爭型免疫分析法的原理進行測量。經過多種變因最佳化實驗，免疫分析試片所達到的偵測極限為 108 pg/mL，線性範圍為 10-6~10-11g/mL；而拋棄式網版印刷電極系統所達到的偵測極限則為0.83 pg/mL，線性範圍為10-4~10-13g/mL。由於男性血液中的黃體素量大約為0.3~0.97 ng/mL；而女性的黃體素量在濾泡期約為0.15~0.7ng/mL，黃體期則為2.0~25 ng/mL，這些指標皆在本研究所發展的兩種偵檢平台之有效偵測範圍內。
第二個部分為利用修飾有黃體成長素(Luteinizing hormone)抗體的微脂球用於偵測黃體成長素的免疫生物感測分析系統。研究中利使用可拋棄式網版印刷電極進行抗體的定錨，再加入黃體成長素使之成為抗體-抗原複合物。最後加入修飾有黃體成長素的微脂球形成三明治型的免疫分析進而測量黃體成長素的含量。經過多種變因最佳化實驗，我們得到偵測極限為0.23 mIU/mL，線性範圍為0.1~1000 mIU/mL，由於男性血液中的黃體成長素大約為1.24~7.8 mIU/mL；而女性的黃體素量在濾泡期約為1.68~15 mIU/mL，在黃體期約為0.61~16.3 mIU/mL，在排卵時則為21.9~56.6 mIU/mL，這些指標皆在本研究所發展的偵測器之有效偵測範圍內。此外，經過黃體素與黃體成長素的混和測試，黃體素和黃體成長素皆能維持單一感測器時的靈敏度而不受另一分析物干擾。因此本研究預期可對人體血液中之黃體成長素以及黃體素進行簡單、快速、靈敏的定量分析。

There are two parts of work included in this study. The first part of work deals with the fabrication of immunosensors for the detection of progesterone, based on a
liposomal competitive assay. Two different sensing platforms were presented herein for the development of the progesterone sensors, one is nitrocellulose
membrane-based immunochromatographic approach with optical density measurement; and the other is screen-printed electrode strip with electrochemical detection. Immobilized anti-progesterone antibody on the detection surface, the
progesterone-modified liposome, and the sample progesterone altogether in a competitive manner form a perfect immunosensing platform for the detection of progesterone. With nitrocellulose membrane as the detection surface, the calibration curve for progesterone shows a linear range from 10-6 to 10-11 g/mL with a limit of detection (LOD) of 108 pg/mL. As in the case of electrochemical immunoassay, the calibration curve for progesterone depicts a linear range from 10-4 to 10-13 g/mL with LOD of 0.83 pg/mL. It was reported that the concentration of progesterone is about
0.3~0.97 ng/mL in male blood; and in case of female, 0.15~0.7 ng/mL was found in follicular phase and 2.0~25 ng/mL in luteal phase. Both immunochromatographic and
electrochemical method clearly shows high selectivity and sensitivity towards progesterone detection in human blood samples.
The second part of work describes the development of an electrochemical immunosensor for Luteinizing hormone based on liposomal sandwich assay. In this study, the anti-Luteinizing hormone antibody was immobilized on the screen printed carbon electrode firstly; follow by the sequential addition of sample Luteinizing hormone and anti-Luteinizing hormone-tagged liposome to form a sandwich immunocomplex. The calibration curve for Luteinizing hormone obtained using this newly 􀂱developed assay has a linear range 0.1 to 1000 mIU/mL with LOD of 0.23 mIU/mL. It has been reported that the level of Luteinizing hormone is about 1.24~7.8 mIU/mL in males and in case of females, 1.68~15 mIU/mL was observed in follicular phase and 0.61~16.3 mIU/mL in luteal phase; 21.9~56.6 mIU/mL before ovulation. It is clear that the detection limit of our immunosensor falls well under this range. Our ultimate goal is to develop an immunosensor for co-detection of Luteinizing hormone and progesterone; therefore, the possible interference from each other must be investigated. We found that the present design is able to detect Luteinizing hormone in the presence progesterone without occurrence of significant interference, and vice
versa. We thus conclude that sensors developed herein offer rapid, simple, and sensitive detection approaches and hold promise for the future of healthcare. Such
sensors can be further extended to be point-of-care, in-vitro diagnostic kits for the detection of progesterone and Luteinizing hormone, and become an useful ovulation calculator, helping to pinpoint when one ovulates.

中文摘要·················································································································· Ⅰ
英文摘要(Abstract) ································································································· Ⅱ
目錄·························································································································· Ⅳ
圖目錄······················································································································ Ⅵ
表目錄······················································································································ Ⅷ
第一章 緒論 ·············································································································· 1
1-1 前言 ···················································································································· 1
1-2 黃體素、黃體成長素與不孕症 ········································································· 2
1-2-1 不孕症(infertility) ····················································································· 2
1-2-2 黃體素（progesterone） ·········································································· 4
1-2-3 黃體成長素 （luteinizing hormone） ···················································· 5
1-3 免疫分析法 (Immunoassay) ·············································································· 7
1-3-1 抗體與抗原 ······························································································· 7
1-3-2 免疫分析法之介紹 ················································································· 10
1-3-3 免疫分析法的分類 ················································································· 11
1-4 微脂球 (Liposome)··························································································· 15
1-4-1 微脂球之演進 ························································································· 15
1-4-2 微脂球之組成 ························································································· 16
1-4-3 微脂球之種類 ························································································· 17
1-4-4 微脂球之製備 ························································································· 19
1-4-5 微脂球之表面修飾 ················································································· 21
1-5- 電化學生物感測器 (Electrochemical biosensor) ·········································· 22
1-5-1 生物感測器 ···························································································· 22
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1-5-2 電化學方法 ···························································································· 23
1-5-3 伏安法 ···································································································· 29
第二章 利用免疫分析試片和網版印刷電極偵測黃體素 ···································· 32
2-1 前言 ··················································································································· 32
2-2 實驗器材與方法 ······························································································· 33
2-2-1 藥品試劑 ································································································· 33
2-2-2 實驗儀器 ································································································· 34
2-2-3 實驗方法與步驟 ····················································································· 35
2-3 實驗結果與討論 ······························································································· 43
2-3-1 免疫分析試片 ························································································· 43
2-3-2 網版印刷電極 ························································································· 52
2-3-3 免疫分析試片與網版印刷電極之比較 ················································· 60
第三章 利用網版印刷電極偵測黃體成長素 ························································ 62
3-1 前言 ··················································································································· 62
3-2 實驗器材與方法 ······························································································· 63
3-2-1 藥品試劑 ································································································· 63
3-2-2 實驗儀器 ································································································· 64
3-2-3 實驗方法與步驟 ····················································································· 65
3-3 實驗結果與討論 ······························································································· 69
3-3-1 網版印刷電極偵測黃體成長素 ···························································· 69
3-3-2 黃體素與黃體成長素混合液的定量分析 ············································· 78
第四章 結論與未來展望 ························································································ 81
第五章 參考資料 ···································································································· 82
附錄一 修飾有黃體素抗體的微脂球之計算························································ 91
附錄二 修飾有黃體成長素抗體的微脂球之計算················································ 92
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圖目錄
圖1-1 衛生署接受人工生殖治療統計週期數 ····················································· 2
圖1-2 黃體素之結構 ····························································································· 4
圖1-3 月經週期 ····································································································· 6
圖1-4 抗體之結構 ································································································· 8
圖1-5 抗體的分類 ································································································· 9
圖1-6 競爭型免疫分析法 ··················································································· 13
圖1-7 三明治型免疫分析法示意圖 ··································································· 14
圖1-8 微脂球之結構 ··························································································· 15
圖1-9 常見脂質結構圖 ······················································································· 16
圖1-10 微脂球的種類 ··························································································· 18
圖1-11 電位法偵測之基本裝置圖 ······································································· 24
圖1-12 電流法偵測之基本裝置圖 ······································································· 26
圖1-13 在頻率􀈦 下之電極電流與電位隨時間變化之關係圖 ···························· 28
圖1-14 最簡單之等效電路圖 ··············································································· 28
圖2-1 修飾有黃體素的微脂球製備 ··································································· 36
圖2-2 免疫分析試片偵測黃體素實驗流程圖 ··················································· 38
圖2-3 聚乙稀亞胺與戊二醛反應圖 ··································································· 40
圖2-4 戊二醛與黃體素抗體反應圖 ··································································· 41
圖2-5 網版印刷電極偵測黃體素實驗流程圖 ··················································· 42
圖2-6 修飾有黃體素的微脂球之粒徑分布圖 ··················································· 44
圖2-7 甲基藍(MB)之標準濃度曲線圖 ······························································ 45
圖2-8 固定於試片之黃體素抗體的濃度與訊號強度百分比 ··························· 48
圖2-9 微脂球數量與訊號強度百分比 ······························································· 49
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圖2-10 溶液總體積與訊號強度百分比 ······························································· 48
圖2-11 免疫分析試片用於黃體素檢量線 ··························································· 51
圖2-12 修飾有黃體素的微脂球之粒徑分布圖 ··················································· 53
圖2-13 固定於電極之黃體素抗體的濃度與電流訊號強度百分比 ··················· 56
圖2-14 微脂球稀釋倍數與電流訊號強度百分比 ··············································· 56
圖2-15 反應時間與電流訊號強度百分比 ··························································· 57
圖2-16 網版印刷電極用於黃體素檢量線 ··························································· 59
圖3-1 修飾有黃體成長素抗體的微脂球製備 ··················································· 66
圖3-2 網版印刷電極偵測黃體成長素實驗流程圖 ··········································· 68
圖3-3 修飾有黃體成長素抗體的微脂球之粒徑分布圖 ··································· 70
圖3-4 K4Fe(CN)6 之標準濃度曲線圖 ································································· 71
圖3-5 固定於電極之黃體成長素抗體的濃度與電流訊號關係圖 ··················· 74
圖3-6 微脂球稀釋倍數與電流訊號強度百分比 ··············································· 74
圖3-7 反應時間與電流訊號強度百分比 ··························································· 75
圖3-8 網版印刷電極用於黃體成長素檢量線 ··················································· 77
圖3-9 添加黃體成長素對於黃體素偵測比較圖 ··············································· 79
圖3-10 黃體成長素吸附於網版印刷電極測試 ··················································· 79
圖3-11 添加黃體素對於黃體成長素偵測比較圖 ··············································· 80
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表目錄：
表1-1 不孕症之原因 ······························································································· 3
表1-2 各種伏安法電位波形與電流應答關係 ····················································· 31
表2-1 修飾有黃體素的微脂球之特性分析(免疫分析試片) ······························· 46
表2-2 修飾有黃體素的微脂球之特性分析(網版印刷電極) ······························· 54
表2-3 免疫分析試片與網版印刷電極之比較 ····················································· 60
表2-4 偵測黃體素方法之文獻比較 ····································································· 61
表3-1 修飾有黃體成長素的微脂球之特性分析 ················································· 72

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