結合pH值界面堆積與形成離子對錯合物於毛細管電泳線上濃縮並偵測胸苷單磷酸

胸苷三磷酸(Thymidine triphosphate, TTP)為合成DNA的四種前驅物之一，係由胸苷單磷酸(Thymidine monophosphate, TMP)磷酸化而來。胸苷單磷酸形成的路徑可分為生合成(de novo pathway)與回收(salvage pathway)兩類；其中胸苷激酶(Thymidine Kinase, TK)是回收路徑中的重要酵素，因此胸苷激酶活性可作為DNA合成與細胞增生之指標。本實驗以毛細管電泳結合線上預濃縮方法偵測樣品溶液中之胸苷單磷酸並藉以推估胸苷激酶之活性。在pH 8.0的樣品溶液中，分析物胸苷單磷酸與腺苷三磷酸(adenosine triphosphate, ATP)皆為帶負電的離子。我們首先利用電泳緩衝溶液與樣品緩衝溶液之間pH值的差異(pH junction)，使TMP與ATP之磷酸根所帶的負電荷減少，降低其電泳動速率，進而造成分析物堆積的效果。另外，我們在電泳緩衝溶液中加入陽離子聚合物Hexadimethrinebromide (HDB)與β-環糊精(β-cyclodextrin)做為修飾劑使用。β-環糊精的加入可以解決TMP與腺苷單磷酸(adenosine monophosphate, AMP)因質荷比過於相近而無法被分離開之問題。而陽離子聚合物HDB不但可以使電滲流反向，縮短分析時間，還會與帶負電荷的分析物形成離子對錯合物(ion complex)，導致質荷比增加。因分析物本身的帶電量不同，所以與HDB產生不同程度的結合後，可以在樣品緩衝溶液與電泳緩衝溶液的界面上產生堆積；如此可提高樣品注射量，並降低偵測極限至0.85μM。本實驗分別對於陽離子修飾劑的添加量、電泳緩衝溶液的濃度與pH值還有β-環糊精的添加量進行探討，並進一步尋找出最佳分離環境。利用最佳分離環境，可於7分鐘之內完成分析，TMP定量的線性範圍是0.5μM至50μM；顯示本方法可迅速且靈敏的偵測TMP的生成，以利將來應用於細胞中TK之活性分析。

關鍵字

毛細管電泳；胸苷單磷酸； pH值界面堆積；離子錯合物； hexadimethrine bromide

並列摘要

The code of life – DNA is composed by deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), thymidine triphosphate (TTP), and deoxyguanosine triphosphate (dGTP). There are two ways to form thymidine monophosphate (TMP) which is further phosphorylated to TTP. One is de novo pathway, and the other is salvage pathway. In the salvage pathway, thymidine is phosphorylated to TMP catalyzed by thymidine kinase (TK). Therefore, the activity of TK is a marker of DNA synthesis and cell proliferation. This study combined capillary electrophoresis (CE) with online preconcentration techniques to monitor TMP. TMP and adenosine triphosphate (ATP) are negatively charged in the sample buffer at pH 8.0. The dynamic pH junction between the background electrolyte and the sample buffer results in the decrease of the charges of analytes. Addition of hexadimethrine bromide (HDB) into the background electrolyte reverses the electroosmotic flow and speeds up the migration of negatively charged analytes. Furthermore, HDB could form ion complex with negatively charged analytes, which increases mass/charge ratios of the analytes. Besides, β-cyclodextrin (β-CD) was added to resolve TMP from adenosine monophosphate (AMP). Several parameters affecting the separation were optimized including the concentration of HDB, the concentration and the pH value of the background electrolytes and the concentration of β-CD. This method is linear in the range of 0.5μM to 50μM and the limit of detection is 0.85μM for TMP. This method can be applied to determine the cellular TK activity in future.