利用毛細管電泳檢測胺基酸、生物鹼及分枝桿菌

Determinations of Amino Acids, Alkaloids, and Mycobacterium Species by Capillary Electrophoresis

張柏齡(Po-Ling Chang)

指導教授 ： 張煥宗

國立臺灣大學/理學院/化學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.10207

摘要

本論文主要著重於以毛細管電泳（capillary electrophoresis, CE）為分析工具針對胺基酸、生物鹼做檢測，並結合分子生物技術鑑定分枝桿菌及奴卡氏菌。本文之第一章主要介紹毛細管電泳基本原理和應用；以及分枝桿菌之重要性與臨床檢驗方法。第二章則敘述以發光二極體誘發螢光（light-emitting-diode induced fluorescence, LEDIF）、naphthalene-2,3-dicarboxaldehyde（NDA）作為衍生化試劑，針對胺基酸進行管柱內之衍生化、樣品堆積及分離。和傳統管柱外方法相較，此法呈現出較少的衍生化副產物效應。若以gamma-aminobutyric acid（GABA）為分析物，其可偵測濃度之線性範圍包含10–5到10–8 M，且偵測極限可達4 nM。第三章吾人則組裝發光二極體誘發螢光及電化學發光（electrochemiluminescence, ECL）之裝置並以此同時檢測胺基酸和生物鹼。在此雙重偵測器之毛細管電泳裝置中，ECL偵測器置於毛細管之出口端並與LEDIF差距12公分。一級胺基酸同樣以NDA作為衍生化試劑，二、三級胺則以Ru(bpy)32+作為ECL之電化學試劑。以此經濟裝置對標準樣品作分析時，胺基酸和生物鹼的偵測極限分別為49 nM–0.2 μM和0.66–4.7 μM。第四章的研究內容則著重於以LEDIF檢測肝病病人腹水樣品之分支鏈胺基酸（branched-chain amino acids, BCAAs）。經由聚環氧乙烯（poly(ethylene oxide), PEO）改善白胺酸及異白胺酸的分離效率，吾人間接證明聚環氧乙烯與胺基酸間厭水性作用力的存在。若對腹水之BCAAs作定量分析，其回收率為83.7％–134％。此外，一天之內的精準度為1.7％–5.8％、天與天之間的精準度則為2.2％–7.4％。第五章則是以聚合酶鏈鎖反應（polymerase chain reaction, PCR）及限制片段長度多型性（restriction fragment length polymorphism, RFLP）結合CE對分枝桿菌進行菌種的鑑定。在本章中，總共12個菌種（含52個菌株）皆可在只用一種限制酵素-Hae III的情況下達到鑑定的目的，而最小可測得之DNA片段為12 bp。另外，只要菌種之熱休克蛋白（65-kDa heat shock protein, hsp65）基因有額外的Hae III限制部位或一個Hae III限制部位的差異都可清楚呈現於電泳圖中。第六章則是以同樣方法鑑定少見之分枝桿菌菌種及奴卡氏菌。在此章中，一樣只用一種限制酵素的情況下，12種少見之分枝桿菌及7種奴卡氏菌都能清楚無誤地呈現出完整的RFLP圖譜於電泳圖上。且預估之DNA片段大小與實際之DNA定序結果完全吻合。在第七章中，吾人以蜂巢式聚合酶鏈鎖反應（nested PCR）進行分枝桿菌之鑑定，且證實nested PCR可達到單分子的偵測靈敏度。然而，DNA取樣時受限於波氏分佈（Poisson distribution）的影響，因此會有陽性率不如預期的情況發生。除了靈敏度的改善，吾人亦證實了nested PCR有助於減少非特異性引子-雙體的產生及樣品中PCR抑制物的負面影響。此外，本方法亦證實可以在不經過細菌培養的情況下直接對痰液中的分枝桿菌菌種進行檢測及鑑定。

關鍵字

毛細管電泳 ； 雷射誘發螢光 ； 發光二極體誘發螢光 ； 電化學發光 ； 胺基酸 ； 生物鹼 ； 結核分枝桿菌

並列摘要

In the dissertation, two major topics focus on amino acids and/or alkaloids determination and identification of Mycobacterium and Nocardia species by capillary electrophoresis (CE). Chapter 1 introduces the principles and applications of capillary electrophoresis along with the importance of Mycobacterium tuberculosis and relative clinical examinations. Chapter 2 states the in-column derivatization, stacking, and separation of amino acids (AA) by capillary electrophoresis in conjunction with light-emitting-diode induced fluorescence (LEDIF) using naphthalene-2,3-dicarboxaldehyde (NDA). In comparison with the off-column approach to the analysis of amino acids, our proposed method provides a lower degree of interference from polymeric NDA compounds and other side products. As a result, the plot of the peak height as a function of γ-aminobutyric acid (GABA) concentration is linear over the range from 10–5 to 10–8 M, with the limit of detection
being 4 nM. Chepter 3 describes the determination of alkaloids and amino acids using capillary electrophoresis in conjunction with sequential light-emitting-diode
induced fluorescence and electrochemiluminescence (ECL) detections. In the CE-LEDIF-ECL system, the ECL detector was located in the outlet of the capillary, while the LEDIF detector was positioned 12 cm from the outlet. NDA was used to form fluorescent AA–NDA derivatives from amino acids possessing primary amino groups, while Ru(bpy)32+ was used to obtain ECL signals for analytes having secondary and tertiary amino groups. This low-cost CE-LEDIF-ECL system allows the analysis of these AA–NDA derivatives and alkaloids at concentrations in the ranges 49 nM–0.2 μM and 0.66–4.7 μM, respectively. In chapter 4, we have
developed a convenient separation method of branched-chain amino acids (BCAAs) from ascites of patients suffer liver diseases. Amino acids was labeled by NDA with CN- as nucleophil, the derivatives were then introduced to capillary by hydrodynamic injection and separated by linear polymer under electroosmotic flow. The recovery data range from 83.7% to 134% over three amino acids and five different
concentrations. The within-day precisions of BCAAs were range from 1.7% to 5.8%, while between-day precisions were 2.2% to 7.4%. In part of identification of M. tuberculosis, we have demonstrated the separation of DNA or restriction fragments digested from the mycobacterial gene encoding for the 65-kDa heat shock protein (hsp65) by capillary electrophoresis as described in chapter 5. Using a pair of
unlabeled primers, Tb11 and Tb12, and only one restriction enzyme, HaeIII, a total of 52 reference and clinical strains encompassing 12 Mycobacterium species were investigated. The electrophoretic separation of high-resolution CE required less than 20 minutes and was capable of identifying fragments as small as 12 bp. A good agreement of measurement was observed between the sizes of restriction fragments resolved by CE and the real sizes deduced from the sequence analysis. Distinct differentiations were also well demonstrated between some species and subspecies by an extra HaeIII digestion site. Furthermore, in chapter 6, additional patterns of 12 less common Mycobacterium and 7 Nocardia species were analyzed and collected for the database of identification purpose. A good agreement of measurement was observed between the sizes of restriction fragments resolved by CE and the real sizes deduced from the sequence analysis. Some closely related species exhibiting similar biochemical characteristics could also be well discriminated by a different or extra HaeIII digestion site. Finally, chapter 7 focus on improve the sensitivity and differentiation in rapidly identifying a small amount of mycobacteria directly from sputum that has significant implications for reducing tuberculosis transmission. In the present study, PCR is replaced with nested PCR (nPCR) in which the primers and
other optimizations are redesigned. As the results shown, the implementation of nPCR in PCR-RFLP assay (PRA) with CE (PRACE) is not only able to detect the presence of mycobacterial DNA less than ten copies, but differentiate the species as well. Both Mycobacterium tuberculosis and mycobacteria other than tuberculosis could be identified even without DNA extraction or in the presence of inhibitors. The least interference of primer-dimers improved by nPCR also contributes to the excellent specificity of RFLP patterns.

並列關鍵字

Capillary electrophoresis ； Laser-induced fluorescence ； Light-emitting-diode induced fluorescence ； electrochemiluminescence ； amino acid ； alkaloid ； Mycobacterium tuberculosis

參考文獻

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