臺灣博碩士論文加值系統

酵素免疫分析法(Enzyme-Linked Immunosorbent Assay , ELISA)是現今相當重要的體外診斷技術，因為癌症的治癒率與接受治療的早晚有密切關係，為了提早發現，人們會定期至醫院做篩檢，因此體外診斷的需求逐年升高，在2016年的免疫檢測市場規模達到171億美元。在卵巢癌的檢測上，血紅素結合蛋白被作為癌症前置因子來判定患者罹癌之潛在風險，針對此蛋白質定量的酵素免疫分析法也已問世並採用。當此蛋白質在血液的濃度過高，表示有很高的風險罹癌，而在此蛋白質過少時，表示血液中溶血症狀嚴重。所以我們認為血紅素結合蛋白濃度的測量是重要的，目前的酵素免疫分析法主要是以UV-vis做為分析儀，並以比色法方式定量血紅素結合蛋白，而TMB2+這個化合物的生成，是顯色深淺的主因。不過，此法的偵測濃度範圍並不大。因此，我們嘗試以紅外線吸收光譜及表面增強拉曼光譜作為分析儀，改善現有酵素免疫分析法。在紅外線吸收光譜中，我們將各個標準檢液滴在二氟化鈣鹽片上，以真空法除去溶劑，並測得紅外線吸收光譜，我們可以看到TMB2+在1572 cm-1之吸收峰。而在表面增強拉曼光譜中，我們將標準檢液與金奈米粒溶液混合，而金奈米粒溶液可以增強TMB2+的訊號，因此測得1605cm-1的峰值，而這個峰值的強度會隨著TMB2+濃度上升而增加。根據以上，我們展示了FTIR與SERS作為ELISA分析儀的可行性，並增加此法可偵測的濃度範圍，我們非常期待這個新方法能夠使現今的ELISA技術更好，為醫療做出貢獻。

Enzyme-Linked Immunosorbent Assay (ELISA) is a very important in vitro diagnostic device (IVDs). Because the early treatment is able to improve the survival rate of cancer patients, where they could go to hospital for screening. Thus, the demand of in vitro diagnosis is increasing annually. In 2016, the global market of immunoassay reached USD 17.16 billion. In ovarian cancer, haptoglobin have been used as a prognosis factor to evaluate the risk of cancer, and we can find clinical tests based on haptoglobin ELISA kits. When the concentration of this protein in the blood is high, it means that increased risk of having ovarian cancer. In contrast, excessively low levels of haptoglobin indicates serious hemolysis. We believe that it is important to estimate the concentration of haptoglobin. However, The detection range of current ELISA reader is poor. Thus, we used FTIR spectroscopy and surface enhance Raman spectroscopy (SERS) as the readout of ELISA to improve the detection range of ELISA. In FTIR based ELISA, we dropped each standard sample on CaF2 window and the solvent was removed by vaccum. Subsequently, the samples was scanned by FTIR. It showed that a peak at 1572cm-1, which was the absorption of TMB2+, a product reacted by chromogen substrate(TMB) and HRP. In SERS, the sample was mixed with gold nanoparticles, as gold nanoparticle could enhance the signal of TMB2+. A peak at 1605 cm-1 was detected. It found that the intensity of the peak was increasing as the concentration of TMB2+ increased. In conclusion, we demonstrated the feasibility that FTIR and SERS could be used as ELISA readout, and the new method also expands the detection range of Hp. We look forward to improving existing ELISA methods by this research.

總目錄
誌謝 I
摘要 II
ABSTRACT III
總目錄 V
圖目錄 VIII
表目錄 XI
第一章 緒論 1
1.1 酵素免疫分析法(ENZYME-LINKED IMMUNOSORBENT ASSAY, ELISA) 2
1.1.1 酵素免疫分析法的歷史背景 2
1.1.2 呈色受質的原理與選用 3
1.1.3 酵素免疫分析法的種類 5
1.1.2.1 三明治法(Sandwich Method) 5
1.1.2.2 間接法(Indirect Method) 6
1.1.2.3 競爭法(Competitive Method) 7
1.1.2.4 本論文之酵素免疫分析方法 8
1.2表面增強拉曼散射(SURFACE-ENHANCED RAMAN SCATTERING, SERS) 9
1.2.1拉曼光譜之歷史背景與原理 9
1.2.2表面增強拉曼散射之歷史背景與原理 11
1.2.2.1 電磁增強效應(Electromagnetic Field Enhancement) 11
1.2.2.2 化學增強效應(Chemical Enhancement) 15
1.2.3表面增強拉曼散射之應用 16
1.3 傅立葉轉換紅外線光譜(FT-IR) 17
1.3.1 紅外線光譜之歷史背景 17
1.3.2 紅外線吸收光譜之基本原理 18
1.3.3紅外線吸收光譜之應用 20
1.4研究動機 21
第二章 實驗方法 22
2.1 化學藥品 22
2.2 酵素免疫分析法 23
2.2.1 酵素免疫分析法準備步驟 23
2.2.2 以UV-vis做為分析儀之操作步驟 26
2.2.3 以SERS做為分析儀之準備步驟 27
2.2.4 以FTIR做為分析儀之準備步驟 28
2.2.4.1 打片法 28
2.2.4.2 真空法 29
第三章 結果與討論 31
3.1 利用比色法定量血紅素結合蛋白(HAPTOGLOBIN, HP) 31
3.1.1 原理討論 31
3.1.2 在UV-Vis光譜中血紅素結合蛋白(Hp)濃度與吸收值之關係探討 33
3.2 以SERS為分析儀定量酵素免疫分析法 36
3.2.1 金奈米粒(GNPs)溶液對於拉曼峰值強度的影響 36
3.2.2 Hp濃度與其SERS強度的關係討論 38
3.3 以FTIR做為分析儀定量酵素免疫分析法 40
3.3.1 以溴化鉀粉末為基底觀察圖譜相關性 40
3.3.2 以真空法分析檢液的紅外線光譜 42
3.3.2.1 SP conjugate濃度與吸收峰強度的關係討論 42
3.3.2.2氮碳雙鍵吸收峰值與Hp濃度的關係討論 46
3.3.2.3 低濃度Hp與氮碳雙鍵吸收強度之關係討論 48
3.4 以FTIR與SERS做為分析儀之綜合分析 50
第四章 結論 52
第五章 參考文獻 53


















圖目錄
圖1-1 山葵過氧化酶的循環反應[7] 4
圖1-2 酵素免疫分析 – 三明治法之流程示意圖 5
圖1-3 酵素免疫分析 – 間接法之流程示意圖 6
圖1-4 酵素免疫分析 – 競爭法之流程示意圖 7
圖1-5 本論文酵素免疫分析法之流程示意圖 8
圖1-6 拉曼訊號相對應不同的能階示意圖，線的粗細程度大約表示其發生機率 10
圖1-7 表面增強拉曼效應與波長半徑比&表面距離之關係示意圖 11
圖1-8 銀半圓柱半徑大小與銀片厚度關係圖[18] 13
圖1-9 在圖1-8中D大小與入射能量對應增強訊號之關係圖(D = 2R~4R) [18] 13
圖1-10 在最大增強效應的條件下(E = 2.7EV, D = 2R)電場之分岐狀況[18] 14
圖1-11 在圖1-8中D大小與入射能量對應增強訊號之關係圖(D = 1.2R~2R)[18] 14
圖1-12 分子吸附於金屬表面之能階圖及其變化路徑[19] 15
圖2-1 S1至S5 HAPTOGLOBIN 標準檢液之配製示意圖 24
圖2-2 標示S1至S6的微量盤示意圖 25
圖2-3 加入25 ΜL HP 標準液及25 ΜL 1X BIOTINYLATED HP 後之示意圖 25
圖2-4 6個加入23毫克溴化鉀粉末的玻璃管 28
圖2-5 6個加入1ΜL檢液的微離心管 30
圖2-6 真空法後在二氟化鈣上留下乾燥的檢液物質 30
圖3-1 加入STOP SOLUTION後溶液內的TMB變為TMB2+ 32
圖3-2 加入顯色液後產生藍色錯合物及顏色差別 32
圖3-3 四甲基聯苯胺在酵素免疫分析法所進行的反應 32
圖3-4 本實驗進行競爭ELISA後之標準檢液 34
圖3-5 HP 濃度與450NM可見光吸收值之關係圖 35
圖3-6 HP 濃度對數值與450NM可見光吸收度對數值之關係圖 35
圖3-7 TMB2+在2X金奈米粒溶液所呈現之SERS圖譜 37
圖3-8 檢液加入金奈米粒溶液所造成之顏色變化 37
圖3-9 HP濃度與1605CM-1處拉曼強度之關係圖 39
圖3-10 HP 濃度對數值與1605CM-1拉曼強度對數值之相關圖 39
圖3-11 S1-S4在1095CM-1及1636CM-1處的吸收強度比較 41
圖3-12 以HP濃度對應吸收強度/吸收峰面積之相關圖: (A)不同濃度HP對應之吸收峰面積(1523 CM-1-1689 CM-1), 1636 CM-1(B)不同濃度HP對應之吸收峰面積(971 CM-1-1186 CM-1), 1095 CM-1(C)不同濃度HP對應之吸收強度, 1636 CM-1(D)不同濃度HP對應之吸收強度, 1095 CM-1。 41
圖3-13 A1取1ΜL於二氟化鈣鹽片並以真空除去溶劑後的紅外線光譜 43
圖3-14 A1(CAF2)與S4(KBR)紅外線光譜比較 44
圖3-15 A1至A5以直接真空法測得之紅外線光譜 45
圖3-16 A1至A3 SP CONJUGATE 濃度對應吸收峰積分值之相關圖 45
圖3-17 S1至S6 的紅外線光譜比較 47
圖3-18 S4至S6 HP濃度與吸收峰積分值之對應圖 47
圖3-19 S5至S9 的紅外線光譜比較 49
圖3-20 S5至S9 的HP濃度對應吸收峰積分值之相關圖 49
圖3-21 以SERS測得S1至S11的HP濃度對應拉曼強度之相關圖 50
圖3-22 以FTIR測得S1至S11的HP濃度吸收峰積分值之相關圖 51
圖3-23 S1至S11不同HP濃度與SERS & FTIR訊號之相關圖 51

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