應用毛細管電泳分析APTS衍生之幾丁寡醣

Abstract

近年來，幾丁聚醣之應用極廣，遍及人類生活的各個細節，因此建立一快速而準確度高的分析方法，將是十分重要的課題。 本研究利用毛細管電泳結合雷射誘發螢光偵測器(capillary electrophoresis-laser induced fluorescence,CE-LIF)分析聚合度1~6的幾丁寡醣標準品(chitosan-oligosaccharides)，為克服分析物沒有發光團，且本身不帶電的問題，選用負三價的螢光衍生試劑9-aminopyrene-1,4,6-trisufonate(APTS)經過還原胺化(reductive amination)反應而衍生，條件為37℃下反應16小時。研究中對電泳分離時緩衝溶液的種類、緩衝溶液酸鹼度和濃度都加以討論。成功的利用pH 4.6濃度為60 mM醋酸鹽緩衝溶液和pH 10.2濃度為40 mM硼酸鹽緩衝溶液，分別在20分鐘以及33分鐘內完成分析，偵測極限可達20 amol。更進一步，將幾丁聚醣真實樣品利用酵素水解以及微波酸水解過程消化成寡醣，其中微波酸水解僅花費50秒，利用功率500 W的家用微波爐即可完成，相較傳統耗時的加熱板酸水解，節省許多時間。水解後的產物不需任何處理同樣和APTS衍生，在最佳電泳條件下得到和標準品相呼應的結果。最後，使用鹼性緩衝溶液在晶片上分析APTS-幾丁單醣衍生產物，單醣之移動時間僅需40秒，並與衍生試劑波峰間有好的分離效率，成功證明晶片電泳分析醣分子之可行性，文末並對晶片實驗提出心得和檢討。

A capillary electrophoresis method with laser-induced fluorescence(LIF) detection for analyzing chitosan oligosaccharides are described. Chitosan oligosaccharides were derivatized with 9-aminopyrene-1,4,6-trisulfonate(APTS) via reductive amination. The effects of buffer types, buffer pH values, and buffer concentrations on the separation are discussed. The APTS-derivatized chitosan oligosaccharides were successfully separated by using a pH 4.6-60 mM acetic/sodium acetate buffer or a pH 10.2-40 mM borate buffer. Moreover, the oligosaccharides of real samples were produced by two ways. First,chitosan polysaccharides were digested by chitosanase. Second, chitosan polysaccharides were treated by 500 w microwave for 50 seconds. Using the optimized APTS labeling procedure and the CE-LIF method applied to digested products was demonstrated. Finally, four kinds of medicinal tablets are analyzed . Moreover, analysis glucosamine by microchip electrophoresis is achieved within 40 secs. There is great potential for the use of Chip-CE to analyse oligosaccharides in the future.