含單硫(thiol，RSH)及雙硫(disulfide，RSSR)之胺基酸是生物體中相當重要的一類生化分子，測定它們在人體中含量之多寡，亦有許多臨床醫學上之意義。本實驗室劉榮忠學長所建立的毛細管電泳結合摻硼鑽石電極(CE-BDD)系統，已能成功地分離並偵測6種含硫胺基酸，但使用BDD電極必須施加較高的工作電位，使其偵測選擇性不佳，故在分析真實樣品時，將會碰到較嚴重的干擾問題。因此，為改善偵測選擇性，本研究將沿用劉榮忠學長的CE分離系統，但偵測系統則分別改用金/汞膜微電極脈波安培偵測法(pulse amperometric detection，PAD)及環-盤式金/汞膜雙微電極定電位安培偵測法，除能同時偵測6種單硫與雙硫胺基酸外，並能得到極佳的選擇性。

一、金/汞膜微電極脈波安培偵測法
在PAD的電位施加方式下，當施加初電位(Ei = -1.4 V)時，溶液中的RSSR會在金/汞膜微電極上發生還原反應而生成RSH，而RSH在施加電位轉變為末電位(Ef = - 0.1 V)時會在金/汞膜電極上發生氧化反應，故在此時同時記錄產生之氧化電流訊號即可測得RSSR；而溶液中原本的RSH亦可在施加末電位時直接測得其氧化電流訊號。故利用PAD配合CE，以金/汞膜單電極系統即可同時分析測定RSH及RSSR，而達到雙電極電化學偵測法的效果。
本系統測定6種含硫胺基酸，其電泳遷移時間(RSD＜0.3﹪)及偵測訊號(RSD＜3﹪)之再現性皆不錯，理論板數皆達10萬以上。線性範圍有兩個級數，偵測極限與文獻中能同時測定單硫與雙硫胺基酸之CE-EC系統相近或更佳。此外，對Cl－、維生素C、葡萄糖、果糖、尿酸、尿素、肌肝酸及3種胺基酸(methionine、histidine及serine)做干擾探討時，發現只有維生素C會對6種含硫胺基酸中的GSSG造成干擾。最後，我們應用此方法成功地分析尿液中CSSC及CSH的含量，分別為21.8 ppm及7.9 ppm。

二、環-盤式金/汞膜雙微電極定電位安培偵測法
以環-盤式金/汞膜雙微電極定電位安培偵測法並配合CE分離系統，可同時分析測定單硫與雙硫胺基酸。其原理是當溶液中的RSSR流經上游盤狀電極(Ed = -1.6 V)時，會發生還原反應而生成RSH；而生成之RSH在流經下游環狀電極(Er = 0.0 V)時會發生氧化反應，故記錄環狀電極上產生之氧化電流訊號，即可測得RSSR；溶液中原本的RSH亦可直接在環狀電極上，測得其氧化電流訊號。以本系統測定6種含硫胺基酸，其電泳遷移時間(RSD＜0.2﹪)及偵測訊號(RSD＜3﹪)之再現性皆不錯，理論板數皆達11萬以上。線性範圍有兩個級數，其偵測極限與文獻中能同時測定單硫與雙硫胺基酸之CE-EC系統相比是最低的。此外，對Cl－、維生素C、葡萄糖、果糖、尿酸、尿素、肌肝酸及3種胺基酸(methionine、histidine及serine)做干擾探討時，發現只有維生素C會對6種含硫胺基酸中的GSSG造成干擾。最後，我們應用此方法成功地分析尿液中CSSC及CSH的含量，分別為11.0 ppm及4.4 ppm。

None.

摘要………………………………………………………………………I
謝誌……………………………………………………………………III
目錄…………………………………………………………………….IV
圖目……………………………………………………………………VII
表目………………………………………………………………………X

壹、緒論…………………………………………………………………1
一、毛細管電泳…………………………………………………………1
二、含硫胺基酸…………………………………………………………8
三、含硫胺基酸的電化學偵測……………………………………….12
四、研究目的………………………………………………………….22

貳、實驗部分………………………………………………………….23
一、儀器設備………………………………………………………….23
二、藥品及溶液之配製……………………………………………….26
三、金/汞膜微電極之製作……………………………………………34
四、環-盤式金/汞膜雙微電極之製作……………………………….38
五、實驗過程………………………………………………………….39

參、結果與討論……………………………………………………….44
一、金/汞膜微電極－脈波安培偵測法………………………………44
1. CE分離系統實驗條件最佳化………………………………………44
(1)標準樣品配製溶液對分離系統之影響……………………………44
(2)堆疊(Stacking)效果………………………………………………46
2. 偵測系統實驗條件最佳化…………………………………………49
(1)工作電極表面至毛細管出口的距離之選擇………………………49
(2)還原電位( Ei )之選擇……………………………………………49
(3)還原時間( ti )之選擇……………………………………………53
(4)氧化電位( Ef )之選擇……………………………………………53
(5)氧化時間( tf )之選擇……………………………………………56
3. 實驗最佳化條件……………………………………………………56
4. 系統之再現性、校正曲線及偵測極限……………………………62
5. 測定6種含硫胺基酸之干擾探討………………………………….72
6. 真實尿液樣品之分析………………………………………………75
7. 結論…………………………………………………………………80

二、環-盤式金/汞膜雙微電極－定電位安培偵測法……………….81
1. CE分離系統實驗條件最佳化………………………………………81
2. 偵測系統實驗條件最佳化…………………………………………81
(1)工作電極表面至毛細管出口的距離之選擇………………………81
(2)上游盤狀電極還原電位( Ed )之選擇……………………………83
(3)下游環狀電極氧化電位( Er )之選擇……………………………86
3. 實驗最佳化條件……………………………………………………86
4. 系統之再現性、校正曲線及偵測極限……………………………90
5. 測定6種含硫胺基酸之干擾探討………………………………….97
6. 真實尿液樣品之分析…………………………………………….103
7. 環-盤式雙微電極的收集效率(collection efficiency)…….107
8. 結論……………………………………………………………….111

肆、總結………………………………………………………………112
伍、參考文獻…………………………………………………………114

陸、附錄………………………………………………………………118
一、金/汞膜微電極循環伏安法對6種含硫胺基酸之電化學探討…118
二、金/汞膜微電極脈波伏安法對6種含硫胺基酸之電化學探討…122
三、金/汞膜微電極循環伏安法對AA及UA之電化學探討………….126
四、毛細管電泳金/汞膜微電極定電位偵測胺基酸之探討……….126
五、毛細管電泳電化學偵測法不同天之間的再現性………………129

1. F. Foret, L. Krivankova and P. Bocek, "Capillary Zone Electrophoresis", VCH, Weinheim, Germany, 1993.

2. 卓振源“電化學偵測法在毛細管電泳上的應用 ”碩士論文，中山大學化學所，1993。

3. 洪千惠“毛細管電泳電化學偵測法使用銅微電極分析尿液中胱胺酸及半胱胺酸之研究 ”碩士論文，中山大學化學所，1996。

4. 莊美英“毛細管電泳脈波安培偵測法使用金汞膜微電極同時測定半胱胺酸及胱胺酸之研究 ”碩士論文，中山大學化學所，1998。

5. 柯均耀“毛細管電泳電化學偵測法使用金汞雙微電極同時測定半胱胺酸及胱胺酸之研究 ”碩士論文，中山大學化學所，1999。

6. 劉榮忠“摻硼鑽石電極結合安培偵測法在毛細管電泳系統中分析含硫胺基酸之研究 ”碩士論文，中山大學化學所，2004。

7. C. Bayle, E. Causse and F. Couderc, Electrophoresis, 25(2004)1457. “Determination of aminothiols in body fluids, cells, and tissues by capillary electrophoresis”

8. J. Lock and J. Davis, Trends. Anal. Chem., 21(2002)807. “The determination of disulphide species within physiological fluids”

9. P. C. White, N. S. Lawrence, J. Davis and R. G. Compton, Electroanalysis, 14(2002)89. “Electrochemical determination of thiols : a perspective”

10. O. Chailapakul, W. Siangproh, B. V. Sarada, C. Terashima, T. N. Rao, D. A. Tryk and A. Fujishima, Analyst, 127(2002)1164. “The electrochemical oxidation of homocysteine at boron-doped diamond electrodes with application to HPLC amperometric detection”

11. C. Terashima, T. N. Rao, B. V. Sarada, Y. Kubota and A. Fujishima, Anal. Chem., 75(2003)1564. “Direct electrochemical oxidation of disulfides at anodically pretreated boron-doped diamond electrodes”

12. P.J. Vandeberg and D.C. Johnson, Anal. Chem., 65(1993)2713. “Pulsed electrochemical detection of cysteine, cystine, methionine, and glutathione at gold electrodes following their separation by liquid chromatography”

13. W. R. LaCourse and G. S. Owens, Anal. Chim. Acta, 307(1995)301. “Pulsed electrochemical detection of thiocompounds following microchromatographic separations”

14. T. R. I. Cataldi and D. Nardiello, J. Chromatogr. A, 1066(2005)133. “A pulsed potential waveform displaying enhanced detection capabilities towards sulfur-containing compounds at a gold working electrode”

15. G. S. Owens and W. R. LaCourse, J. Chromatogr B, 695(1997)15. “Pulsed electrochemical detection of thiols and disulfides following capillary electrophoresis”

16. T. S. Hsi and M. Y. Chuang, J. Chin. Chem. Soc., 45(1998)827. “Preliminary study on reverse pulse amperometric detection in capillary electrophoresis for simultaneous determination of cysteine and cystine”

17. W. A. Kleinman and J. P. Richie Jr., J. Chromatogr B, 672(1995)73. “Determination of thiols and disulfides using high-performance liquid chromatography with electrochemical detection”

18. B. L. Lin, L. A. Colon, R.N. Zare, J. Chromatogr A, 680(1994)263. “Dual electrochemical detection of cysteine and cystine in capillary zone electrophoresis”

19. M. Zhong and S. M. Lunte, Anal. Chem., 71(1999)251. “Tubular-wire dual electrode for detection of thiols and disulfides by capillary electrophoresis/electrochemistry”

20. I. G. Casella, M. Contursi and E. Desimoni, Analyst, 127(2002)647. “Amperometric detection of sulfur-containing compounds in alkaline media”

21. W. Zhang, F. Wan, W. Zhu, H. Xu, X. Ye, R. Cheng and L. T. Jin, J. Chromatogr B, 818(2005)227. “Determination of glutathione and glutathione disulfide in hepatocytes by liquid chromatography with an electrode modified with functionalized carbon nanotubes”

22. J. Zhou, T. J. O’Shea and S. M. Lunte, J. Chromatogr. A, 680(1994)271. “Simultaneous detection of thiols and disulfides by capillary electrophoresis-electrochemical detection using a mixed-valence ruthenium cyanide-modified microelectrode”

23. T. J. O’Shea and S. M. Lunte, Anal. Chem., 66(1994)307. “Chemically modified microelectrodes for capillary electrophoresis/electrochemistry”

24. M. Windholz, S. Budavari, R. F. Blumetti and E. S. Otterbein, "The Merck Index", 南山堂出版社, 1983.

25. W. Jin and Y. Wang, J. Chromatogr. A, 769(1997)307. “Determination of cysteine by capillary zone electrophoresis with end-column amperometric detection at a gold/mercury amalgam microelectrode without deoxygenation”

26. A. Pastore, R. Massoud, C. Motti, A. L. Russo, G. Fucci, C. Cortese and G. Federici, Clin. Chem., 44(1998)825. “Fully automated assay for total homocysteine, cysteine, cysteinylglycine, glutathione, cysteamine, and 2-mercaptopropionylglycine in plasma and urine”

27. S. Zhang, W. L. Sun, W. Zhang, W. Y. Qi, L. T. Jin, K. Yamamoto, S. Tao and J. Jin, Anal. Chim. Acta, 386(1999)21. “Determination of thiocompounds by liquid chromatography with amperometric detection at a Nafion/indium hexacyanoferrate film modified electrode”

28. P. Ventura, R. Panini, M. C. Pasini, G. Scarpetta and G. Salvioli, Pharmacol. Res., 40(1999)345. “N-acetyl-cysteine reduces homocysteine plasma levels after single intravenous adminidtration by increasing thiols urinary excretion”

29. T. Inoue and J. R. Kirchhoff, Anal. Chem., 74(2002)1349. “Determination of thiols by capillary electrophoresis with amperometric detection at a coenzyme pyrroloquinoline quinone modified electrode”

30. P. Lochman, T. Adam, D. Friedecky, E. Hlidkova and Z. Skopkova, Electrophoresis, 24(2003)1200. “High-throughput capillary electrophoretic method for determination of total aminothiols in plasma and urine”

31. K. Amarnath, V. Amarnath, K. Amarnath, H. L. Valentine and W. M. Valentine, Talanta, 60(2003)1229. “A specific HPLC-UV method for the determination of cysteine and related aminothiols in biological samples”

32. C. F. Yeh, S. J. Jiang and T. S. Hsi, Anal. Chim. Acta, 502(2004)57. “Determination of sulfur-containing amino acids by capillary electrophoresis dynamic reaction cell inductively coupled plasma mass spectrometry”

33. M. Zhong, J. Zhou, S. M. Lunte, G. Zhao, D. M. Giolando and J. R. Kirchhoff, Anal. Chem., 68(1996)203. “Dual-electrode detection for capillary electrophoresis/electrochemistry”

34. D. C. Chen, S. S. Chang and C. H. Chen, Anal. Chem., 71(1999)3200. “Parallel-opposed dual-electrode detector with recycling amperometric enhancement for capillary electrophoresis”