應用毛細管電泳與晶片電泳分析三種寡醣

Abstract

碳水化合物醣類分子因其具生理活性的特質，及於自然界中扮演多重及重要的角色，使得研究碳水化合物相關分子日益重要。本論文研究以毛細管電泳與晶片電泳技術分析木聚醣(xylan)、幾丁聚醣(chitosan)與幾丁質(chitin)經酸水解或酵素水解後所得寡醣分子。所選取的衍生試劑分別為3-aminonaphthalene-2,7-disulfonic acid (3-ANDA)、p-nitrobenzylhydroxylamine (PNB)與9-aminopyrene-1,4,6-trisulfonic acid (APTS)。 以毛細管電泳分析3-ANDA/木醣寡醣衍生產物實驗中，首先針對四項衍生反應條件：衍生試劑、醋酸濃度，反應溫度與反應時間進行研究。結果顯示使用0.2 M，3-ANDA 及30 % (vol/vol) 醋酸於90℃下與木聚寡醣反應2小時之反應效率最佳。同時探討了磷酸、三乙基胺濃度與毛細管溫度對電泳分離的影響。所得到最適化電泳分離條件為pH 2.1，250 mM磷酸溶液中加入2.0 % (vol/vol) 三乙基胺，毛細管溫度於40℃下，可解析聚合度1-17之木聚寡醣。並以此聚合度1-17之3-ANDA-木醣寡醣電泳圖作為鑑定木聚醣與酵素反應之特異性。 幾丁聚醣經酵素水解後所得寡醣分子與PNB試劑進行衍生反應後所得產物以毛細管電泳分析研究中，除探討最佳反應條件以簡化PNB試劑與幾丁寡醣之衍生反應外，同時也研究緩衝溶液pH值及硼酸鹽濃度對電泳分離的影響。在最適化電泳分離條件下可解析聚合度1-16之幾丁寡醣。 在本研究中，同時以毛細管電泳及晶片電泳技術分析聚合度1-6之幾丁質寡醣。實驗結果中發現，APTS與幾丁質寡醣之衍生於37℃下反應16小時所得衍生產物量最佳。在毛細管電泳方法中，分別使用兩種緩衝溶液系統，boric acid/borax與citric acid/Na2HPO4，雙重鑑定幾丁聚醣經兩種不同酵素水解後所得寡醣產物。聚合度1-6之APTS-幾丁質寡醣於晶片電泳技術中，使用citric acid/Na2HPO4/Brij35緩衝溶液系統，在施加-600 V/cm電場強度下，可於45秒內完成分離，APTS-幾丁質單糖之理論板數可高達960,000。除了以同時壓製兩條注射微流道增加進樣量以提高偵測靈敏度外，加入中性界面活性劑Brij35於電泳緩衝溶液中修飾低親水性之聚甲基丙烯酸甲酯(俗稱壓克力，PMMA)膠片微流道內壁，可驅動較多親水性高之寡醣分子進入分離微流道中，及使用低離子強度之樣品基質溶液以達到線上堆積、濃縮而進一步降低偵測極限。估計使用2.0 mm 進樣長度之PMMA膠片結合晶片電泳技術於最適化分離條件下分析APTS-幾丁質單糖之偵測極限值可達5.4 amole。

In this study, analysis of xylan-, chitosan- and chitin-oligosaccharides after acid hydrolysis or enzymatic digestion by capillary electrophoresis and microchip electrophoresis. The selected derivatizing reagents were 3-aminonaphthalene-2,7-disulfonic acid (3-ANDA), p-nitrobenzylhydroxyl- amine (PNB) and 9-aminopyrene-1,4,6-trisulfonic acid (APTS)。 The derivation conditions including concentration of 3-ANDA or acetic acid, reaction temperature, and time are discussed. The effects of pH value of buffer solution, concentration of TEA, and running temperature on the electrophoretic mobility of xylan oligomers are discussed. In addition, determination of xylan-oligosaccharides both by acid hydrolysis and enzymatic of xylan polysaccharides is presented. An improved procedure has been developed for the rapid derivatization of oligosaccharides produced by chitosanase digestion of chitosan polysaccharides with PNB. The derivation conditions including concentration of reagent, reaction temperature, and time are discussed. The effects of pH value and concentration of buffer solution on the electrophoretic mobility of chitosan oligomers are described. The chitosan-oligosaccharides with degree of polymerization (DP) 1-16 were analyzed under the optimized separation conditions. Analyses of DP 1-6 chitin-oligomers both by capillary electrophoresis (CE) and microchip electrophoresis with laser-induced fluorescence (LIF) were investigated. Chitin oligomers were derivatized with APTS at 37℃for sixteen hours (optimized conditions). The APTS-chitin oligosaccharides were analyzed using either an acidic citric acid/phosphate buffer or an alkaline borate buffer with CE method. The electrophoretic separation can be successfully separated in 45 seconds by using citric acid/phosphate/Brij35 buffer with PMMA microchip electrophoresis applied -600 V/cm electric field strength. Improved detection limit was achieved by using sample stacking method, modify microchannel surface by adding Brij35 surfactant and extending sample loading volume by creating simultaneously two injection channels on PMMA substrate. The detection limit of APTS-chitin monomer is 5.4 attomole can be obtained by using 2.0 mm sample loading length PMMA chip with microchip electrophoresis under the optimized separation conditions. Moreover, the specificity of enzyme digestion on chitin polysaccharides using the optimized microchip electrophoresis was demonstrated.