以電化學技術探討青光眼患者與白內障患者前房液中抗壞血酸含量

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研究生：	王鴻儒 Wang, Hung-Ru.
論文名稱：	以電化學技術探討青光眼患者與白內障患者前房液中抗壞血酸含量 Bio-electrochemical analysis L-ascorbate concentration in aqueous humor between glaucoma/cataract patients aqueous humor
指導教授：	柯美蘭 Ko, Mei-Lan
口試委員:	陳榮治 Chen, Jung-Chih 董瑞安 Doong, Ruey-an
學位類別：	碩士 Master
系所名稱：	原子科學院 - 生醫工程與環境科學系 Department of Biomedical Engineering and Environmental Sciences
論文出版年：	2020
畢業學年度：	108
語文別：	中文
論文頁數：	61
中文關鍵詞：	抗壞血酸、白內障、青光眼、生物電化學、前房液
外文關鍵詞：	ascorbate, cataract, glaucoma, bio-electrochemistry, aqueous humor
相關次數：	點閱：3 下載：0
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抗壞血酸 (ascorbate) 在生物體內可藉由代謝作用產出。抗壞血酸分為 (L) 左旋與 (D) 右旋，本實驗針對具有生物活性的左旋抗壞血酸作進一步研討，而左旋的抗壞血酸又稱為維生素C。青光眼 (glaucoma) 為全球導致失明第二高眼疾-但其病因與其生化路徑尚未明確，近年各醫學期刊紛紛提出青光眼與氧化壓力有關。本研究欲將具有抗氧化壓力的抗壞血酸在不同病因(白內障、青光眼)在前房液含量的差別，並進一步研究前房液中抗壞血酸含量變化與青光眼的關係。
目前檢測青光眼的方法是散瞳後經由眼科醫師目測視神經杯盤比或是做視野圖檢查或做OCT(optical coherence tomography)光學相干斷層掃描來判定是否青光眼，但通常疾病已形成，藉由電化學的方式研究證明抗壞血酸於前房液中的濃度與青光眼之間的關係，以便對青光眼的病因或視神經做進一步研究因果關係。藉由施加一循環電位在不同的抗壞血酸與等單位活性抗壞血氧化酶的結合，呈現出青光眼與白內障病患前房液中抗壞血酸的氧化峰（anodic peak），並與不同濃度之抗壞血酸回歸曲線回推出病患前房液中抗壞血酸濃度。
與預測相同的是白內障併青光眼病患之前房液抗壞血濃度相較於只有白內障前房液抗壞血酸濃度來得更低，更進一步確認青光眼與氧化壓力的相關性。

Vitamins are essential nutrients required for various biological processes in the body. Because they cannot be synthesized in the body, vitamins must be ingested in foods. Ascorbate has been classified to two versions, Dextro (D) and Levo (L) form (Vitamin C) , and there’s only vitamin C has bioactivity. Vitamin C is also an antioxidant, protecting the body from the deleterious effects of free radicals. Glaucoma is the second-leading cause of blindness after cataract and its pathogenesis was still largely unknown, this year medical journal surmise glaucoma might relate with oxidation pressure .due to anti-oxidative capacity of ascorbate we will investigate the ascorbate concentration difference between cataract, glaucoma patients and their relationship. The common glaucoma diagnosis are Ophthalmoscopy after mydriasis or visual field test and optical coherence tomography , but optical nerve already has been lost due to glaucoma. We got various anodic peak from glaucoma/cataract patients aqueous samples by Cyclic voltammetry. Contrast with concentration linear regression we found lower ascorbate concentration in glaucoma patients compare with cataract patients .confirm relationship between oxidative stress and glaucoma.

目錄
中文摘要……………………………………………………………………………I
Abstract……………………………………………………………………………III
謝誌………………………………………………………………………………V
目錄………………………………………………………………………………VI
圖目錄……………………………………………………………………………VIII
表目錄………………………………………………………………………………X
第一章 前言…………………………………………………………………………1
1-1 青光眼…………………………………………………………………1
1-2 氧化壓力………………………………………………………………4
1-3 研究動機………………………………………………………………6
第二章 基礎理論……………………………………………………………………8
2-1 電化學系統……………………………………………………………8
2-2 電化學原理……………………………………………………………9
2-3 電化學聚合法…………………………………………………………13
2-4 指數增益配體進化法…………………………………………………15
2-5 核酸序列合成…………………………………………………………18
VII
第三章 材料與方法………………………………………………………………20
3-1 適體篩選……………………………………………………………20
3-2 化學核酸合成…………………………………………………………22
3-3 抗壞血酸電極製作……………………………………………………24
3-4 繪已知濃度抗壞血酸檢量線…………………………………………26
3-5 前房液檢體的採集及分析……………………………………………27
第四章 結果與討論………………………………………………………………29
4-1 抗壞血酸氧化酶電極…………………………………………………29
4-2 以適體取代抗壞血酸氧化酶…………………………………………33
4-3 以酸性溶液修飾碳電極表面…………………………………………36
4-4 以電化學聚合法形成多巴胺膜取代羧酸基修飾……………………39
4-5 抗壞血酸溶液 PH 值與量測穩定性…………………………………43
4-6 人體樣本的分析與高效液相色譜儀的測量結果……………………44
第五章 結論與未來展望……………………………………………………………55
5-1 結論……………………………………………………………………55
5-2 未來展望………………………………………………………………57
參考文獻……………………………………………………………………58
VIII
圖目錄
圖 1-1-1 視神經節細胞受損機制…………………………………………………1
圖 1-1-2 前房液正常、不正常排出比較圖………………………………………2

圖 2-1-1 電化學系統與訊號………………………………………………………8
圖 2-2-1 氧化還原與電子動向……………………………………………………9
圖 2-2-2 電極表面與電解質溶液………………………………………………10
圖 2-2-3 3 電極與 2 電極系統比較……………………………………………11
圖 2-2-4 電位隨時間改變關係……………………………………………………12
圖 2-3-1 電極表面多巴胺電化學聚合……………………………………………13
圖 2-3-2 多巴胺聚合物導電膜……………………………………………………14
圖 2-4-1 指數增益配體進化法流程圖……………………………………………15
圖 2-4-2 適體與配體的結合………………………………………………………16
圖 2-4-3 適體與多巴胺形成希夫鹼並固定於電極表面…………………………17
圖 2-5-1 核酸序列合成步驟………………………………………………………18

圖 4-1-1 糊精不同濃度冷卻情形…………………………………………………30
圖 4-1-2 具方向性抓取抗體與不具方向性抓取抗體比較圖……………………32
圖 4-1-3 以糊精固定抗壞血酸氧化酶製成電極循環伏安圖………………32
IX
圖 4-2-1 以適體取代抗壞血酸氧化酶製成電極循環伏安圖……………………34
圖 4-2-2 以 Plamsense4 測量不同濃度市售抗壞血酸 PBS 溶液循環伏安圖…34
圖 4-2-3 適體電極偵測不同濃度市售抗壞血酸 PBS 溶液所繪製檢量線………35
圖 4-4-1 多巴胺電聚合循環伏安圖………………………………………………39
圖 4-4-2 多巴胺模修飾過載情形…………………………………………………40
圖 4-4-3 使用較小電位窗電聚合多巴胺膜後測量穩定度………………………41
圖 4-4-4 電極表面以不同物質修飾後阻抗值………………………………42
圖 4-5-1 適體電極偵測不同 PH 值氧化還原溶液量穩定度…………………….43
圖 4-6-1 以高效液相色譜儀偵測市售抗壞血酸的 PBS 溶液的訊號……………44
圖 4-6-2 高效液相色譜儀偵測各濃度市售抗壞血酸的 PBS 溶液後所繪製檢量線
………………………………………………………………………………………45
圖 4-6-3 以高效液相色譜儀分析不同眼疾前房液中抗壞血酸濃度的平均值…51
圖 4-6-4 以適體電極分析不同眼疾前房液中抗壞血酸濃度的平均值…………51
圖 4-6-5 同組別不同測試方法結果比較……………………………………52
圖 4-6-6 以適體電極測量不同族群及年齡條件的前房液中抗壞血酸濃度…52
圖 4-6-7 以適體電極測量不同族群及年齡條件的前房液中抗壞血酸濃度…53
X
表目錄
表 4-1-1 不同抗壞血酸所混合抗壞血酸氧化酶單位數…………………………30
表 4-1-2 不同濃度抗壞血酸與抗壞血酸氧化酶反應時間………………………31
表 4-3-1 各種酸類羧酸基修飾條件………………………………………………36
表 4-3-2 以不同條件硫酸修飾碳電極表面的測量穩定度………………………37
表 4-3-3 以不同條件硝酸修飾碳電極表面的測量穩定度………………………37
表 4-3-4 以不同條件鹽酸修飾碳電極表面的測量穩定度………………………37
表 4-6-1 受試者人數及男女比……………………………………………………46
表 4-6-2 以適體電極測量前房液中抗壞血酸濃度的受試者列表及訊號………47
表 4-6-3 以高效液相色譜儀測量前房液中抗壞血酸濃度的受試者列表及訊號.48
表 4-6-4 以適體電極測量前房液中抗壞血酸濃度的受試者列表及訊號 49
表 4-6-5 以適體電極測量前房液中抗壞血酸濃度的受試者列表及訊號 50
表 5-1-1 適體電極與高效液相色譜儀比較表…………………………………56
                                

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