臺灣博碩士論文加值系統

摘要

D-Penicillamine(D-PA)與 DL-Tiopronin(DL-Tio)藥物已經廣泛用於治療多種疾病，如：威爾森氏症(Wilson’s disease)、重金屬疾病(鉛、砷、汞和鋅等)、胱氨酸尿症、抗纖維化的硬皮病和類風溼關節炎等。在本研究中，我們使用Tween 20金奈米粒子(Tween 20-AuNPs)萃取技術可在複雜的基質下選擇性濃縮胺基硫醇類的特性，再結合聚合物堆積法搭配毛細管電泳成功偵測藥錠與尿液中這四種分析物。最佳化條件為，10 mL 的樣品水溶液，使用 200 µL 200倍 Tween 20-AuNPs 為萃取吸附材料、20 µL Dithiothreitol(DTT)的取代劑，靜置反應後離心取出上清液，經過衍生化試劑 fluorenylmethyloxycarbonyl 衍生反應 2分鐘後即可上機偵測，其分離最佳方法是緩衝溶液中添加 25 mM β-cyclodextrin 和 0.5 %v/v polyethylene oxide(PEO)，使用此方法可以得到最低偵測極限為 1.8~4.0 nM 與好的線性範圍 7.5~3000 nM (R2 > 0.9961)。此結果顯示，本研究方法是便宜、快速、方便用於分析物的偵測。

Abstract

D-Penicillamine(D-PA) and DL-Tiopronin(DL-Tio) are thiol drugs widely used for treating many diseases. In this paper, a simple method for the detecting of DL-Tio and D-PA is described. Tween 20-capped gold nanoparticles (Tween 20-AuNPs) were used as sCctive probes for chiral thiol drugs from an aqueous solution. Tween 20-AuNPs selectively extracted and enriched the thiol drugs from a complicated matrix, because the Tween 20 capped layer effectively inhibited nonspecific adsorption. A 10.0 mL chiral thiol drug solution was extracted using 200× AuNPs (200 µL), and the extracted aminothiols derivatized with fluorenylmethyloxycarbonyl at pH 10.0 were detected using capillary electrophoresis (CE) with UV detection. After extracting and centrifuging, dithiothreitol was used for detaching the chiral thiol drugs from the AuNPs surface. On-line concentration and separation of the detached chiral thiol drugs were performed using 25 mM β-cyclodextrin and 0.5%(v/v) polyethylene oxide as an additive in CE. Under optimal extraction conditions, the limits of detection at a signal-to-noise ratio of 3 for four chiral thiol drugs ranged 1.8–4.0 nM, and satisfactory linearity was obtained at 7.5–3000 nM (R2 > 0.996). These results suggest that the present method is inexpensive, rapid, easy, accurate.

摘要..............................................................................................................................Ⅰ
Abstract........................................................................................................................Ⅱ
目錄..............................................................................................................................Ⅲ
圖目..............................................................................................................................Ⅴ
表目..............................................................................................................................Ⅵ
壹、緒論........................................................................................................................1
一、威爾森氏症(Wilson’s Disease) .........................................................................1
二、分析物簡介........................................................................................................2
(1) DL- Penicillamine (DL-PA) ............................................................................3
(2) DL-Tiopronin (DL-Tio) ...................................................................................3
三、藥物分析方法....................................................................................................5
四、毛細管電泳簡介................................................................................................9
五、毛細管電泳分離模式........................................................................................12
5.1毛細管區帶電泳 (CZE)..............................................................................12
5.2微胞電動層析法(MEKC) ...........................................................................13
六、樣品線上濃縮堆積法 (sample stacking) .......................................................14
6.1 等速電泳法 (Isotachophoresis, ITP) ........................................................15
6.2 場放大堆積法 (Field-Amplified Sample Stacking, FASS) ......................16
6.3 大體積樣品堆積法 (Large Volume Sample Stacking, LVSS) .................18
6.4 掃集堆積法 (Sweeping stacking) .............................................................19
6.5 聚合物堆積法 (polymer stacking) ............................................................20
七、環糊精( cyclodextrin ) 簡介............................................................................22
八、萃取方法...........................................................................................................24
8.1 液-液萃取 ( Liquid-liquid extraction, LLE ) ..............................................25
8.2 固相萃取( Solid-phase extraction, SPE ) ....................................................25
8.3 固相微萃取( Solid-phase microextraction, SPME ) ....................................26
8.4 液相微萃取( Liquid-phase microextraction, LPME ) .................................27
8.5 磁固相微萃取法 ( Magnetic solid-phase extraction, MSPE)......................28
九、金奈米粒子 ( gold nanoparticle ) 介紹與萃取…………………………….28
9.1 奈米介紹…………………………………………………………………...28
9.2 金奈米粒子置備與表面修飾……………………………………………...29
十、研究動機與目的………………………………………………………………31
貳、實驗……………………………………………………………………………..32
一、儀器設備……………………………………………………………………..32
二、實驗藥品……………………………………………………………………..33
三、實驗方法……………………………………………………………………..35
3.1 CE-UV 偵測系統………………………………………………………….35
3.2 儲存溶液配置……………………………………………………………...36
3.3 緩衝溶液配置……………………………………………………………...37
3.4 合成金奈米粒子 ( AuNPs)………………………………………………..38
3.5 金奈米粒子萃取過程 …………………………………………………….38
參、結果與討論………………………………………………………………………41
一、目的…..………………………………………………………………………41
二、毛細管電泳分離條件…………………………………………………..……41
2.1 緩衝溶液 pH 值的探討…………………………………………..………42
2.2 環糊精的選擇與探討…………………………………………..………….45
2.3 高分子聚合物環氧乙烷(ethylene oxide, PEO)對分析物分離探討……...50
2.4 含 PEO 的緩衝溶液(Tris Borate, TB)濃度探討…………..……..……....52
三、毛細管電泳樣品線上濃縮條件…………..……..…………………………..55
3.1注入時間探討………………………………………………………………55
四、Tween 20-AuNPs 萃取條件探討……………………………………………58
4.1Tween 20-AuNPs濃度……………………………………………………...58
4.2取代劑DTT濃度探討……………………………………………………..60
4.3樣品體積探討………………………………………………………………62
五、目標分析物的定量、靈敏度、精密度……………………………………..64
5.1標準品直接分析……………………………………………………………64
5.2線上濃縮技術………………………………………………………………65
5.3樣品經過奈米粒子萃取後分析…………………………………………....65
5.4結合奈米粒子萃取與線上濃縮…………………………………………....66
肆、結論……………………………………………………………………………..75
參考文獻……………………………………………………………………………..76






圖目錄
圖1.電滲流驅動與壓力區動支層流差異 11
圖2.毛細管區帶電泳圖 12
圖3.毛細管微胞電動層析法 ( MEKC ) 示意圖 14
圖4.等速電泳法示意圖 16
圖5.場放大樣品堆積法示意圖 17
圖6.大體積樣品堆積法示意圖 18
圖7.大體積樣品堆積法示意圖 19
圖8.聚合物堆積法示意圖 21
圖9. 環糊精示意圖 23
圖10.毛細管電泳示意圖 36
圖11.金奈米粒子萃取過程示意圖 40
圖 12. 緩衝溶液 pH 值探討 44
圖 13. 緩衝溶液添加不同濃度 β-CD 對分析物解析度結果示意圖 47
圖 14. 緩衝溶液添加不同環糊精對分析物分離結果示意圖 48
圖 15. 緩衝溶液添加不同環糊精對分析物分離結果示意圖 49
圖 16. 緩衝溶液添加PEO對分析物分離結果示意圖 51
圖 17. 緩衝溶液濃度對分析物分離結果示意圖 53
圖 18. 線上濃縮堆積法-秒數對訊號面積 57
圖 19. 金奈米粒子萃取濃度對分析物結果示意圖 59
圖 20. DTT濃度對分析物結果示意圖 61
圖 21. 樣品體積對分析物結果示意圖 63
圖22.金奈米粒子萃取結合聚合物堆積法 71
圖23樣品經過Tween 20-AuNPs萃取結合聚合物堆積法後最低濃度 73


表目錄
表 1.分析物結構 ………………………………………………………………..…...4
表2. DL-PenA和DL-Tio分析物應用於不同前處裡、衍生化和偵測儀器之分析…..8
表3.緩衝溶液 Tris Borate 最佳化條件 ……………………..……………………53
表4.標準品直接分析 ………………………………………………………………67
表5.標準品使用聚合物堆積法進行濃縮 …………………………………………68
表6.標準品使用Tween 20-AuNPs 萃取 ………………………………………….69
表7.標準品使用Tween 20-AuNPs 萃取結合聚合物堆積法……………………. 70
表8.次與次的標準品回收率與相對標準偏差(n=3)………………………………73
表9.天與天標準品回收率與相對標準偏差(n=3) ………………………………...74

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