胺、馬兜鈴酸及DNA之毛細管電泳分析

Capillary electrophoresis for the analyses of amines, aristolochic acids and DNA

黃銘峰(Ming-Feng Huang)

指導教授 ： 張煥宗

國立臺灣大學/理學院/化學研究所/博士(2006年)

https://doi.org/10.6342/NTU.2006.02421

摘要

中文摘要
本論文主要是利用毛細管電泳的分離技術，結合雷射誘導螢光的偵測方式，分別針對胺類分子、馬兜鈴酸以及DNA 片段，開發出快速、高解析度且高靈敏度的分析技術。論文內容可分為四個部分：(1)利用毛細管電泳結合間接螢光偵測的方式，成功分離並偵測六種胺類分子。我們使用pH 3.5 的溶液，其中包括5.0% 甲醇, 0.1 mM 硫酸, 0.1mM cresyl violet, and 0.3 mM 鋰離子作為毛細管電泳的電解質溶液，來分離六種胺類分子，因為胺類分子並不具有螢光性質，因此利用與其具有相同官能機且淌度相近的螢光染料crysyl violet，在電泳過程中進行取代，因此可以得到胺間接螢光偵測的訊號。利用此方法其偵測極限可以達到µM 的程度，如果搭配pH junction 的濃縮方式，可降低其偵測極限達19 倍。(2) 馬兜鈴酸類化合物是常見於中藥中的一類成分，其具有腎毒性及致癌的危害，因為馬兜鈴酸為具有硝基苯的化合物，本身不具有螢光，因此在0.1 M 的鹽酸溶液中，利用鐵粉將硝基還原成胺基，被還原的馬兜鈴酸在390 nm 的光源激發下，會釋放出473 nm 的螢光。利用毛細管電泳暨雷射誘導螢光的技術，可在50.0 mM sodium tetraborate、10.0 mM SDS，pH 9.0 的條件下，在12 分鐘內分析出兩種自然界最常見的馬兜鈴酸類化合物－AA-I 與AA-II，其偵測極限可達到8.2 Nm (AA-I) and 5.4 nM (AA-II)，我們並II利用此技術分析61 種實際中藥材樣品中，並分析出其中24 種含有馬兜鈴酸成分。(3) 在第三部分中，我們利用加入金奈米粒子(GNPs)的低黏度聚環氧乙烷溶液來分離DNA 片段，我們發現當加入56 nm 的金奈米粒子後，分析片段大小為51 個鹼基對到2176 個鹼基對的marker DNA，可在5 分鐘內解析出其中28 個片段，另外針對較大片段的DNA，如片段大小為5 千到4 萬個鹼基對的5 kb ladder DNA，也可在30 分鐘內成功分離出其8 個片段，證明這是個高分離解析度與快速的DNA 分析技術。(4) 針對大小在數千個鹼基對以上的大DNA 片段，我們開發出新的快速且高解析度的毛細管電泳分離技術。我們在金奈米粒子的表面修飾聚合物分子，稱之為GNPPs。將GNPPs 填入到毛細管中作為DNA 的分離介質，DNA 藉由與金奈米
粒子間的作用力，以及與金奈米粒子上所修飾的聚合物分子所產生的摩擦力而分離，利用此技術我們可在5 分鐘內解析出大小為0.12 到23 kbp 的lambda DNA 的所有片段，另8.27 到48.5 kbp 的HMD DNA也可在6 分鐘之內分離出其所有片段。與傳統分析大片段DNA 的平板凝膠電泳或變換電壓式的毛細管電泳相較，其分析時間可從至少數小時縮短到數分鐘。

關鍵字

毛細管電泳 ； 胺 ； 馬兜鈴酸 ； DNA

並列摘要

Abstract
This thesis focuses on developing efficient and sensitive capillary electrophoresis-laser induced fluorescence (CE-LIF) techniques for amines, aristolochic acid, and DNA. We developed a novel method for the analysis of amines under acidic conditions by CE in conjunction with indirect laser-induced fluorescence (CE-ILIF). The analysis of six amines by CE-ILIF using a solution, pH 3.5, containing 5.0% methanol, 0.1 mM sulfuric acid, 0.1 mM cresyl violet, and 0.3 mM lithium was complete in 5 min, with the limits of detection (LOD) on the level of µM.To further improve the sensitivity, on-line concentration based on pH junction has
been demonstrated. When injecting the sample prepared in a solution of 0.2 mM sulfuric acid, pH 3.3, at 15 kV for 60 s to the above-mentioned solution, the LODs for the amines down to sub µM and the sensitivity improvements up to 19-fold when compared to that injecting at 15 kV for 5 s.

Aristolochic acid (AA), a naturally occurring nephrotoxin and carcinogen, has been associated with the development of urothelial cancer in humans. Two predominant forms of AA are 8-methoxy-6-nitrophenanthro-(3,4-d)-1,3-dioxolo-5-
carboxylic acid (AA-I) and 6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AA-II). Owing to lack of intrinsic fluorescence characteristics of oxidized AAs
(OAAs), the RAAs reduced from OAAs in 10.0 mM HCl containing iron powder is required prior to CE analysis. The RAAs exhibit fluorescence at 477 nm when excited
at 390 nm. By using 50.0 mM sodium tetraborate (pH 9.0) containing 10.0 mM SDS,the determination of aristolochic acid I and aristolochic acid II by CE-LIF has been
achieved within 12 min. The CE-LIF provides the LODs of 8.2 and 5.4 nM for AA-I and AA-II, respectively. The successful analysis of 61 medicinal samples and dietary supplements shows that the present CE-LIF is practical for the determination of AA-I and AA-II in real samples.

Reproducible, rapid, and high-resolution DNA separations have been achieved using low-viscosity PEO solutions containing GNPs ranging in diameter from 3.5 to 56 nm. The separation of DNA ranging in size from 8 to 2176 base pairs (bp) was accomplished in 5 min using 0.2% PEO(8 MDa) containing 56-nm GNPs. We have also demonstrated the separations of the DNA fragments ranging from 5 to 40 kbp
using 0.05% PEO(2 MDa) containing 13-nm GNPs or 0.05% PEO(4 MDa) containing 32-nm GNPs.

To separate long double strands of DNA by CE, with high efficiency, high speed, simplicity, and reproducibility, we describe a CE technique, which we call nanoparticle-filled CE (NFCE), using polymer-modified gold nanoparticles (GNPPs)
in this topic. The gold nanoparticles were modified with poly(ethylene oxide) via noncovalent bonding to form GNPPs. The separations of high molecular weight (HMW) DNA with sizes ranging from 8.27 to 48.5 kbp and λ DNA (0.12–23.1 kbp) were accomplished within 6 and 5 min, respectively. The separation speed and resolution are greater than those by pulsed CE and slab gel electrophoresis. This is the first example for separating such high DNA molecules by CE-LIF using GNPPs.

The results present in this thesis clearly demonstrate that CE-LIF based techniques are practical for analysis of amines, aristolochic acid, and DNA, with the advantages of rapidity, sensitivity, and reproducibility. The examples of separating DNA using PEO containing GNPs and GNPPs open the avenue of using nanoparticles for improved resolution and reproducibility for analysis of DNA. It is our strongly
belief that the technique should be further applied to analysis of small solutes such as amines and acids and macromolecules like proteins.

並列關鍵字

Capillary ； elecrophoresis ； amines ； aristolochic acids ； DNA

參考文獻

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