臺灣博碩士論文加值系統

SPR是一新型光學感測器，近年來被廣泛應用於生物科技來研究生物分子間的相互作用。本研究利用德州儀器(TI)出品價格較便宜的SPR探討各類生物分子間的作用情形，由於TI之SPR感測金面上僅有一金膜，因此在固定化探針前必須將感測面加以修飾，增加表面上之官能基以利於探針之固定化，本論文首先研究在硫醇修飾層及在Con A修飾層上固定化之葡聚糖探針之非專一性吸附，發現在混合型硫醇修飾金面上固定化探針具有較高之專一性反應。在以SPR探討生物分子間的相互作用時分為四類：(1)蛋白質與醣類－以葡聚糖為探針不但可偵測到純的Con A，還可對Con A粗萃溶液進行活性測定，粉進一步推算出從刀豆中萃取Con A粗萃液其濃度為2750nM，而Con A與葡聚糖之間的親和常數(Ka)為1×106(1/M)。(2)蛋白質與細胞－將Con A共價固定化為探針後，可偵測不同的菌體細胞，並可對其間最佳作用條件做篩選，例如酸鹼值愈低愈有利於Con A與酵母菌之作用，且離子強度不會影響其間之作用。(3)蛋白質與蛋白質－以Protein A定向固定化抗組胺酸標籤之抗體，再對NusA及GM-CSF兩種帶有組胺酸標籤之蛋白質進行感測，可求得蛋白質與抗體間的親和常數(Ka)分別為1×107及1×108(1/M)。(4)蛋白質與核酸－分別將凝血蛋白酵素及核酸配體固定化，然後對其相對應之作用分子進行感測，感測之結果顯示核酸配體之構形不正確導致二種感測面皆不靈敏。
所使用過金面上固定化有特殊探針之SPR感測面，可在通臭氧的環境下並以紫外光照射得以再生，重新固定化新的探針後仍可使用，經硫醇修飾及蛋白質探針固定化之金面，可由AFM之表面粗糙度分析及ESCA之表面元素分析觀測到此再生方法效率。

Over the past decade, surface plasmon resonance (SPR) has emerged as a leading technology for the study and measurement of biomolecular binding events. In this work, we employed an integrally molded, SPR sensor invented at Texas Instruments (TI-SPR) to study the interaction between various biomolecules. The SPR transduction layer is comprised of a gold surface. Before immobilizing ligand to the SPR sensing surface, the sensor surface was modified with thiols to form a self-assembled monolayer (SAM). The modification can increase the functionality of the surface and help to immobilize the ligand onto the sensing surface.
Four different types of biomolecular interactions have been studied by using TI-SPR.
(1) Protein and Sugar- Dextran was used as sensing probe, it not only cloud detect the activity of pure Con A but also the activity of Con A in a crude extract solution. The concentration of Con A in the crude extract of Jack-beam meal was determined to be 2.75 mM.
(2) Protein and Cell- Immobilized Con A on the sensing surface can be used to detect the cells with different surface properties. The optimal interaction condition between Con A and yeast cell can be easily screened. The low pH is favorable for binding interaction and ionic strength does not affect the interaction.
(3) Protein and Protein- Protein A was used to direct the anti-his tag antibody and DMP was used to crosslink the Protein A and antibody. This SPR sensor could detect the proteins fused with his-tag, such as NusA and GM-CSF. The affinity constant between antibody and NusA is 1×107 (1/M) and 1×108 (1/M) for GM-CSF.
(4) Protein and DNA-Thrombin could interact with the specific DNA sequence. But the interacting response is not significant; probably the synthetic DNA does not exist in a favorable conformation.

目錄
中文摘要.......................................................I
英文摘要.....................................................III
致謝.......................................................... V
目錄..........................................................VI
圖目錄........................................................IX
表目錄.......................................................XII
第一章前言
1.1研究背景....................................................1
1.2研究內容及目的..............................................3
第二章表面電漿子共振(Surface Plasmon Resonance；SPR)
2.1共振原理....................................................8
2.2 SPR感測分子間親和作用及動力參數估算.......................15
第三章 文獻回顧
3.1 SPR感測面上之探針固定化...................................21
3.1.1直接法...................................................21
3.1.2間接法...................................................22
3.2 SPR偵測生物分子間交互作用.................................33
3.2.1外源凝集素(Lectin)-醣蛋白質..............................33
3.2.2抗體與抗原...............................................40
3.2.3 蛋白質與DNA.............................................45
3.3感測金面之再生.............................................48
第四章 實驗材料設備與方法
4.1藥品.......................................................49
4.2實驗裝置...................................................52
4.2.1 SPR感測器...............................................52
4.2.1.1 SPR感測器之配件.......................................52
4.2.1.2 SPR感測器之內部元件...................................52
4.2.2 其他實驗裝置............................................56
4.3實驗方法...................................................57
4.3.1探針之固定化.............................................57
4.3.1.1直接固定化蛋白質探針...................................57
4.3.1.2間接固定化探針.........................................58
4.3.2不同類型之生物分子間的親和作用...........................61
4.3.2.1半刀豆球蛋白 A(Con A)-葡聚糖...........................61
4.3.2.2半刀豆球蛋白 A(Con A)-酵母菌...........................62
4.3.2.3抗組胺酸標籤抗體-組胺酸標籤蛋白........................63
4.3.2.4凝血蛋白酵素- 抗凝血蛋白酵素之核酸配體.................67
4.3.3金面之再生...............................................69
第五章 結果與討論
5.1 SPR感測面上之探針固定化...................................70
5.1.1羥基硫醇修飾面上之葡聚糖探針固定化.......................70
5.1.2Con A修飾面上之葡聚糖探針固定化..........................70
5.2 SPR偵測不同類型生物分子間的親和作用.......................75
5.2.1蛋白質與醣類：半刀豆球蛋白 A-葡聚糖......................75
5.2.2蛋白質與細胞：半刀豆球蛋白 A-酵母菌......................81
5.2.3蛋白質-蛋白質：抗組胺酸標籤抗體-組胺酸標籤蛋白...........83
5.2.4蛋白質-核酸：凝血蛋白酵素- 抗凝血蛋白酵素之核酸配體......91
5.3感測金面之再生.............................................97
第六章 結論與建議............................................103
參考文獻.....................................................106
圖目錄
圖 1.1 (A)葡聚醣修飾法 (B)混合式SAM修飾法......................7
圖 2.1 Kretschmann 稜鏡組.....................................11
圖 2.2反射係數與入射角之關係圖................................12
圖 2.3 利用SPR觀測不同濃度糖類之折射率........................13
圖 2.4 不同種類金屬面之SPR曲線................................14
圖 2.5 SPR感測流程圖.. .......................................19
圖 2.6 親和作用之動力分析.. ..................................20
圖3.1 (A)SAM (B)葡聚糖之固定化及羧基化........................24
圖 3.2葡聚糖硫醇化之步驟......................................27
圖3.3 混合型SAM 之可能排列方式................................30
圖3.4 Con A結構示意圖 .. .....................................39
圖3.5 免疫球蛋白(IgG)結構圖...................................43
圖3.6 Protein A與抗體(IgG)的共價鍵結..........................44
圖4.1 SPR 裝置圖..............................................53
圖4.2 感測單元.. .............................................54
圖4.3 感測面單元之設計........................................54
圖4.4 感測器之設計............................................55
圖4.5 以波長595nm之紫外光測量並製作蛋白質檢量線...............66
圖5.1 Con A修飾層上固定化葡聚糖探針後之專一性測試.............74
圖5.2 直接將Con A固定於金面利用SPR觀測濃度的葡聚糖與Con A之作 用 .................................... ......................77
圖5.3利用共價固定化之Con A將葡聚糖探針固定化再通入Con A溶液偵測........................................................... 78
圖5.4先利用Con A與葡聚糖探針作用再通入Con A溶液偵測後計算親和常數(Ka) .......................................................79
圖5.5 葡聚糖探針對不同濃度之Con A粗萃液偵測...................80
圖5.6 酸鹼值對酵母菌吸附至Con A感測面上產生應答之影響.........82
圖5.7 以DMP將Protein A與抗體交聯製備免疫型感測器之探針........86
圖5.8 A . 培養生產NusA之E.coli的生長曲線 B.NusA之純化 C.蛋白質電泳............................................................87
圖5.9 以免疫型感測器偵測NusA之SPR偵測圖.......................88
圖5.10 A . 培養生產GM-CSF之Yeast的生長曲線 B.GM-CSF之純化 C.蛋白質電泳. ......................................................89
圖5.11 以免疫型感測器偵測GM-CSF之SPR偵測圖....................90
圖5.12 Streptavidine修飾金面以利帶有生物素標籤之核酸配體探針固定化. ..........................................................93
圖5.13核酸配體探針與凝血蛋白酵素之SPR偵測圖...................94
圖5.14凝血蛋白酵素探針之固定化................................95
圖5.15凝血蛋白酵素探針與核酸配體之SPR偵測圖...................96
圖5.16 再生前後感測面之共振角測量.............................99
圖5.17 AFM分析圖(A)金面，(B)混合型硫醇修飾面，(C) 混合型硫醇修飾面再生後.....................................................100
圖5.18 再生前之ESCA分析......................................101
圖5.19 再生後之ESCA分析......................................102
表目錄
表 3.1 羧基化葡聚糖上蛋白質之固定化數量.......................25
表 3.2 各種蛋白質在混合形式SAM上之固定化......................31
表 3.3 各種混合型式SAM之表面性質..............................32
表 3.4 SPR量測各生物分子間作用系統............................36
表 3.5 各種固定化抗體探針之方法...............................42
表 3.6 已篩選出之配體.........................................47
表5.1 不同修飾面之感測器與蛋白質間之SPR測試結果...............72
表5.2 混合型SAM上Con A探針與不同蛋白質及菌體之作用............73

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