以毛細管電泳結合線上濃縮法監測去氧胞苷二磷酸與去氧胞苷三磷酸

本研究以毛細管區帶電泳結合線上濃縮法，測定胞核苷二磷酸(CDP, cytidine 5’ -diphosphate) 經核醣核苷酸還原酶反應後生成去氧胞苷二磷酸(dCDP, 2’-deoxycytidine 5’-diphosphate) 和去氧胞苷三磷酸(dCTP, 2’-deoxycytidine 5’ -triphosphate) 濃度，藉此分析核醣核苷酸還原酶酵素活性。為了增加胞核苷二磷酸與去氧胞苷二磷酸兩者間遷移速率的差異，本研究在背景電解質溶液添加苯硼酸及環糊精；並利用環糊精的疏水性腔室包覆苯硼酸形成錯合物，而且苯硼酸會與核醣核苷酸上的雙醇基形成硼酯錯合物，使得樣品裡的胞核苷二磷酸和腺核苷三磷酸(ATP, adenosine 5’ -triphosphate) 質量增加和電泳遷移速率變慢。如此一來在鹼性背景電解質環境裡，不需要樣品前處理就可以分離腺核苷三磷酸、胞核苷二磷酸、去氧胞苷二磷酸和去氧胞苷三磷酸。為了提高偵測靈敏度，本實驗利用陽離子型聚合物作為毛細管管壁改性劑，使電滲流流動方向跟負電荷分析物泳動方向相同，相較於壓力進樣，電進樣可以在短時間內選擇性導入更大量分析物，而且利用樣品區帶及電泳緩衝液間不同的黏度及離子強度，在分析物進入毛細管時，因遷移速率的不同而堆積濃縮，進而提高偵測靈敏度。在本實驗研究中分別探討苯硼酸與羥丙基-α-環糊精濃度、乙烯二胺四醋酸二鈉(EDTA) 濃度、聚乙烯亞胺(PEI-2k) 濃度、電泳緩衝液的pH值、電泳緩衝液濃度與電動進樣電壓與時間及分離電壓等變因。以最佳化條件進行實驗可於7分鐘內即可完成分析。dCTP 與dCDP 線性範圍在0.5-100 μM 之間，相關係數為0.9996和0.9994，偵測極限可達0.19μM和1.04 μM。

關鍵字

毛細管電泳、去氧胞苷二磷酸、去氧胞苷三磷酸、苯硼酸、環糊精

並列摘要

In this study, we developed a capillary zone electrophoretic method combined with online concentration for determination of 2’-deoxycytidine 5’-diphosphate (dCDP) and 2’-deoxycytidine 5’ -triphosphate (dCTP), which are the products of cytidine 5’ -diphosphate (CDP) reduction catalyzed by ribonucleotide reductase. In order to increase the mobility difference of dCDP and CDP, phenylboronic acid and hydroxypropyl-α-cyclodextrin (HP-α-CD) were added into the background electrolyte (BGE). The hydrophobic interaction between HP-α-CD and the phenyl group of phenylboronic acid as well as the condensation of borate with ribonucleotide makes the mobility of CDP-phenylboronic acid-HP-α-CD complex decreased, so we can directly separate dCDP and dCTP from CDP and other ribonucleotides. We also used a cationic polymer, polyethylenimine, to dynamically coat the inner surface of the uncoated capillary, which made the electroosmotic flow reversed. Therefore electrokinetic injection could introduce much more sample into capillary than pressure injection. Because the viscosity and conductivity difference between the sample zone and the BGE zone, when alanlytes move into BGE, their velocities decrease, which results in online preconcentration and the improvement of the detection limit. Herein, we investigated the effects of phenylboronic acid, HP-α-CD, ethylenediaminetetraacetic acid, polyethylenimine and BGE. Under optimal conditions, it takes 7 min to finish the analysis. The linear range of this method was 0.5-100 μM for dCDP and dCTP, and the correlation coefficients were 0.9996 and 0.9994 for dCTP and dCDP, respectively. The detection limits were 0.19μM and 1.04 μM for dCTP and dCDP, respectively.