博碩士論文 107324016 詳細資訊




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姓名 潘品憲(Pin-Hsien Pan)  查詢紙本館藏   畢業系所 化學工程與材料工程學系
論文名稱 利用抗原結合區段之抗體片段探針於矽奈米線場效電晶體來改善抗原檢測濃度極限之研究
(Improvements of detection limitation of antigen concentration by antibody fragment probe on silicon nanowire field effect transistor)
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摘要(中) 在場效電晶體進行生物分子檢測時,帶電目標物質在感測器表面的吸附所產生的電場會影響電晶體中導體的電流,藉由檢測電訊號的改變,可以對目標物質進行定量分析。目前場效電晶體生物感測器被廣泛應用於檢驗樣品內之核酸及蛋白質的研究上。然而,當樣品的鹽濃度過高(>100mM 離子強度)時,由於目標物質的表面電荷會被其周圍溶液的鹽離子屏蔽,場效電晶體檢測樣品內目標物質造成的電訊號將大幅降低,這種現象稱為德拜屏蔽效應(Debye screening effect)。而在德拜屏蔽效應下,帶電分子可以對導體產生電場效應作用的距離稱為德拜長度,因此若要檢測在場效電晶體表面的帶電物質,此帶電物質距離表面的位置需要部份或整體包含在德拜長度內。理論上,在生理鹽濃度(1xPBS)條件下的德拜長度僅不到1奈米,若要對生理溶液(如血液、血清)進行蛋白質檢測,作為其辨識分子探針的抗體已經具有相當大的尺寸(10~15奈米),當目標蛋白質與抗體結合時,其與表面距離將遠超德拜長度,因此,在生理溶液中,使用抗體作為場效電晶體的探針進行蛋白質檢測,會受德拜長度的限制而有檢測上的困難。
為了克服德拜長度的問題,我們嘗試在表面改質聚乙二醇(PEG)層,並且使用抗體片段作為探針。PEG可以顯著地改變水溶液中的介電性質,在許多生物感測器的研究中被用來延長德拜長度。具有PEG之表面亦有抗非專一性吸附的能力,可以降低非專一性吸附所造成的訊號干擾。再加上使用抗體片段作為探針而不使用完整抗體,期望本研究能利用PEG的特性以及抗體片段尺寸較小的性質,在接近生理鹽濃度(150mM BTP) 的情況,能克服德拜長度的限制而能進行檢測。
在本研究中,所使用的抗體片段需藉由酵素消化或是微生物蛋白質表達產生,因此實驗先嘗試以市售抗體片段作為探針進行檢測,並且同時發展抗體片段化技術。本研究的初步實驗中,在B淋巴球抗原CD19檢測的實驗結果,發現量測到的電訊號變化不一致,因而造成檢測定量上的困難。所幸的是,本實驗室Vu在2019的研究中[1],透過核適體(aptamer)對特定抗體的結合,除了讓檢測訊號放大外,也能讓檢測得到的電訊號有一致的變化方向。因此本研究後續的實驗將利用相同的核適體(R18)對兔源抗體有專一性結合的特性,對於使用PEG及抗體片段的方式進行抗原檢測,再做進一步的探討。
在進一步的實驗中,本研究將抗兔源抗體之完整抗體(Wab)及抗體片段(Fab)作為探針固定在檢測表面上對兔源抗體做檢測。從實驗結果中,也發現兔源抗體檢測的電訊號變化同先前檢測抗原CD19之結果,仍有電訊號變化不一致的現象,但在加入R18後的檢測結果發現,無論是使用完整抗體探針或抗體片段探針,產生的電訊號變化 (與baseline相比)都有產生一致的變化方向。再更進一步使用兩種探針對不同濃度(1pg/ml~10ng/ml)的兔源抗體做檢測,其實驗結果顯示,在各個濃度下使用Fab探針測得的電訊號變化量皆比使用Wab探針量測的變化量大,並且使用Fab探針能檢測之最低濃度可以下探到1pg/ml,比使用Wab探針之最低檢測濃度10pg/ml還低。因此從本研究的結果來看,若以多晶矽奈米線場效電晶體作為生物感測器檢測蛋白質,配合材料表面改質PEG及核適體的訊號放大,使用抗體片段作為生物探針將會使場效電晶體於蛋白質檢測上更具潛力。
摘要(英) Field effect transistor (FET)-based biosensors, which are widely used in the research of detecting nucleic acids and proteins, operate based on electrical signal generated by the adsorption of the charged substance on the sensing surface. The substance can be quantified by measuring the electrical variation. However, the charges of the detected molecules are shielded by the surrounding ions of high ionic strength conditions (>100mM), also known as screening effect, resulting in significantly weakened signal generated by the binding event between the targets and the probes. The length that electrostatic force of charged molecules exponentially declined is called the Debye length. In molecular detection by FET biosensors, the distance from the charged ligands and the sensing surface should be partially or fully within the Debye length. Consequently, protein detection in physiological environments (1xPBS) using antibodies as probes on field-effect transistors is extremely difficult and limited due to the bulky size of the antibodies (10–15 nm) and the small Debye length in this condition (< 1 nm). Fortunately, in the study of Vu in our laboratory in 2019, the specific binding of aptamers to antibodies not only stabilize the inconsistent trends but also amplify the variation of the electrical signal.
The experiments of this study are designed using the same aptamer as signal amplifier for SiNWFET immunosensors modified with PEG and antibody fragments (Fab). PEG not only can extend the Debye length of FET biosensors by modulating the dielectric property of solutions but also is able to reduce the interference caused by non-specific bio-species by preventing their adsorption onto the sensing surface. It is expected that SiNWFET immunosensors utilizing compact structure of Fab as probes, the advantage from the characteristics of PEG, and signal enhancement by aptamer and can overcome the Debye length of the operating environment equivalent with the physiological concentration (150mM BTP) and remarkably generate electrical variation after capturing the antigen.
The whole antibody (Wab) and Fab are immobilized as probes on the surface to detect the rabbit IgG in this study. The commercially available Fab are employed as sensing elements for antigen detection by FET while the procedure to synthesize the Fab from the Wab is simultaneously developed. The empirical results reveal that the electrical signal changes of the detection of rabbit antibody are inconsistent, which is similar to previous detection of CD19 antigen. The results after adding R18 showed that the electrical signal changes (compared to baseline) have a consistent direction no matter which kind of probes are used. Furthermore, the Wab and Fab are used as probes to detect rabbit antibodies of different concentrations (1pg/ml~10ng/ml). The experimental results showed that the electrical signal changes using the Fab probe are more significant than those using Wab at each concentration. And the lowest concentration that can be detected using the Fab probe can reach to 1pg/ml, which is lower than the detection limitation using the Wab probe. The results of this study indicate that in combination with signal amplification of aptamer and surface modification of PEG, using Fab as probes is more promising, compared to Wab, for protein detection by SiNWFET biosensors.
關鍵字(中) ★ 矽奈米線場效電晶體
★ 抗體片段探針
★ 混和自組裝單層膜
關鍵字(英) ★ silicon nanowire field effect transistor
★ antibody fragment probe
★ mixed self-assemble monolayers
論文目次 摘要 i
Abstract iv
誌謝 vi
目錄 viii
圖目錄 xii
表目錄 xvi
第一章 緒論 1
第二章 文獻回顧 3
2.1疾病檢測-Immunoassay 3
2.1.1酵素結合免疫吸附分析法(ELISA) 5
2.1.2免標定免疫檢測(label-free immunoassay) 9
2.2 矽奈米線場效電晶體生物感測器 11
2.3抗體分子 17
2.3.1 抗體分子概論 17
2.3.2 抗體結構 19
2.3.3 抗原結合區段(Fab region) 20
2.3.4 可結晶區段(Fc region) 20
2.3.5 抗體探針種類 21
2.4 抗體片段(antibody fragment)的製程 25
2.4.1 酵素消化反應之抗體 25
2.4.2 重組(蛋白)抗體 26
2.5 晶片表面改質 28
2.5.1 自組裝單層膜表面改質技術 28
2.5.2 表面分子固定化 31
2.5.3 矽氧烷-聚乙二醇(Silane-PEG)於自組裝單層膜之應用 32
2.6 電訊號的增幅及抗體位向的導正 36
第三章 實驗藥品、儀器設備與方法 39
3.1 實驗藥品 39
3.1.1 FET晶片表面改質與檢測 39
3.1.2 ELISA 抗體親和力檢測 40
3.1.3 抗體片段化實驗 40
3.1.4 電泳 41
3.2 儀器設備 43
3.3 晶片表面改質 43
3.3.1 晶片表面清洗及氧電漿處理 43
3.3.2 修飾silane-PEG(-NH2 & -OH (1:10) mixing) 44
3.3.3 修飾GA(glutaraldehyde) 44
3.3.4 探針固定化 44
3.4 FET電性測量 46
3.5 ELISA 抗體親和力(affinity test)實驗 47
3.6 抗體片段化實驗 49
3.7 SDS-PAGE 蛋白質電泳 51
第四章 結果與討論 54
4.1 表面改質之分析 54
4.2 抗體片段探針與完整抗體探針的比較 60
4.2.1抗體親和力對檢測的影響 60
4.2.2對特定抗原進行檢測及比較 63
4.2.3 添加R18進行電性檢測及比較 66
4.3 酵素切割抗體的最佳化 70
4.3.1 SDS-PAGE結果探討 71
4.3.2 酵素切割抗體親和力檢測之結果探討 72
第五章 結論與未來展望 75
5.1 結論 75
5.2 未來展望 76
第六章 參考文獻 78
第七章 附錄 84
7.1 SPR測定抗體親和力實驗 84
7.1.1 SPR實驗器材、藥品、儀器以及操作程序 84
7.1.2 SPR實驗結果與現象探討 86
7.2 COB實驗 90
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指導教授 陳文逸(Wen-Yih Chen) 審核日期 2020-7-30
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