I. 利用毛細管電泳法分析透析病患血清中之親蛋白性尿毒素 II. 利用高分子奈米螢光顆粒分析人體血漿中之排鐵劑藥物

I. Development of capillary electrophoresis for determination of protein-bound uremic toxins in the serum of hemodialysis patients II. Synthesis of fluorescence polymer dots for analysis of deferiprone in human plasma

李芸維(Yun-Wei Lee)

指導教授 ： 吳秀梅

高雄醫學大學/藥學院/藥學系碩士班/碩士(2017年)

摘要

本論文建立一毛細管電泳法，應用於偵測透析 (hemodialysis) 病患血清中之親蛋白性尿毒素 (protein-bound uremic toxins)；另也建立一種化學感測器方法，利用高分子奈米螢光顆粒分析乙型地中海型貧血 (β-thalassemia) 病患血漿中之排鐵劑藥物含量。
本論文之第一部分為以微胞電動毛細管電泳法搭配電場放大樣品堆積及微胞掃集之線上濃縮技術 (Field-amplified sample stacking-sweeping-micellar electrokinetic chromatography, FASS-sweeping-MEKC) 分析4種常見於腎功能不全患者血清中之親蛋白性尿毒素，包含indole-3-acetic acid (IAA)、para-cresol sulfate (pCS)、3-indoxyl sulfate (3-INS) 及 hippuric acid (HA)。研究中利用化學計量實驗設計法 (chemometric experimental design) 輔助尋找最適化條件。其中磷酸鹽緩衝液濃度、磷酸鹽緩衝液pH值及SDS濃度皆對理想分離具有統計上之意義，經進一部探討，最適化條件為100.00 mM NaH2PO¬4 (pH 4.0) 緩衝液並含有150.00 mM SDS及7.50% methanol，於最佳條件下4個待測物可於15分鐘內完成分離。本法以標準添加法建立待測物檢量線，IAA之偵測極限為0.08 μg/mL，pCS為0.28 μg/mL，3-INS為0.34 μg/mL，HA為0.25 μg/mL。經方法確效，相對標準偏差 (RSD)皆小於14.15%，相對標準誤差 (RE)皆小於13.71%，並應用於分析100位透析病患血清中親蛋白性尿毒素之含量。
本論文之第二部分以高分子奈米螢光顆粒偵測乙型地中海型貧血病患血漿中之排鐵劑含量。將高分子螢光材料PFBT (poly(fluorene-alt-benzothiadiazole)) 及表面修飾材料PSMA (poly(styrene-co-maleic anhydride)) 以再沉澱法製備高分子奈米螢光顆粒之水分散溶液。本研究方法之最適化萃取條件為使用乙腈去蛋白搭配硫酸銨進行鹽析；最適化分析條件是以10.00 mM HEPES (pH 6.0) 緩衝溶液稀釋高分子奈米螢光顆粒，再利用50.00 μg/mL二價銅離子使高分子奈米螢光顆粒之螢光猝滅，加入經前處理並含有排鐵劑藥物deferiprone (DFP) 之血漿樣品，使螢光回復。本法之前處理時間僅需10分鐘，反應時間小於1分鐘，能夠快速且大量篩檢乙型地中海型貧血病患之血漿樣品。本法之偵測極限為2.50 μg/mL，定量分析範圍為5.00-30.00 μg/mL，經方法確效，相對標準偏差 (RSD)皆小於12.23%，相對標準誤差 (RE)皆小於14.82%，樣品經前處理後，具良好之專一性，並成功應用於分析2位乙型地中海型貧血病患血漿中之DFP濃度。

關鍵字

尿毒素 ； 毛細管電泳 ； 實驗設計法 ； 排鐵劑 ； 高分子奈米螢光顆粒

並列摘要

One capillary electrophoresis (CE) method had been developed for simultaneous determination of protein-bound uremic toxins in the serum of hemodialysis patients. The other method, chemical sensor, had also been developed for determination of iron chelating medicine in the plasma of β-thalassemia patients.
The first part of the thesis is determination of four uremic toxins in the serum of chronic kidney disease (CKD) patients, including indole-3-acetic acid (IAA), para-cresol sulfate (pCS), 3-indoxyl sulfate (3-INS), and hippuric acid (HA), by field-amplified sample stacking-sweeping-micellar electrokinetic chromatography (FASS-sweeping-MEKC). Chemometric experimental design was used to determine the optimal condition for analysis. The p value of the concentration of phosphate buffer and SDS and pH value of phosphate buffer were less than 0.05, indicating that the factors affected the separation in statistical significance. In a further discussion, the optimal condition was set at 100.00 mM NaH2PO¬4 (pH 4.0) containing 150.00 mM SDS and 7.50% MeOH. The separation could be finished within 15 minutes. The calibration curves were established by standard addition method. The limit of detection (LOD) of IAA, pCS, 3-INS and HA were 0.08, 0.28, 0.34, and 0.25 μg/mL, respectively. The relative standard deviation (RSD) was less than 14.15% and the relative error (RE) was less than 13.71%. The proposed method could be successfully applied in quantitative determination of the four uremic toxins in the serum of 100 hemodialysis patients.
The second part of the thesis is determination of iron chelating medicine in the plasma of β-thalassemia patients by fluorescent polymer dots (Pdots). The PFBT (poly(fluorene-alt-benzothiadiazole)) polymer dots functionalized with PSMA (poly(styrene-co-maleic anhydride)) polymer was synthesized by reprecipitation. The optimal condition for pretreatment of plasma samples was deproteinized with acetonitrile and salting-out with ammonium sulfate. The optimal condition for analysis is using 50.00 μg/mL copper ions to quench the fluorescence of the Pdots which diluted with 10.00 mM HEPES buffer (pH 6.0) and the fluorescence was recovered by adding the pretreated plasma samples containing deferiprone (DFP). The pretreatment time of plasma samples was only 10 minutes and the reaction time was less than 1 minutes, indicating that the proposed method could be applied in the rapidly screening plasma samples from β-thalassemia patients. The LOD was 2.50 μg/mL and the linear range was 5.00-30.00 μg/mL. The RSD and RE were lower than 12.23% and 14.82%, respectively. The specificity of the method was good. The proposed method could be successfully applied in quantitative determination of the DFP in the plasma of 2 β-thalassemia patients. The specificity test was ongoing.

並列關鍵字

uremic toxins ； capillary electrophoresis ； experimental design ； iron chelation ； polymer dots

參考文獻

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