Determinations of Amino Acids, Alkaloids, and Mycobacterium Species by Capillary Electrophoresis
Date Issued
2007
Date
2007
Author(s)
Chang, Po-Ling
DOI
en-US
Abstract
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.
Subjects
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
Capillary electrophoresis
Laser-induced fluorescence
Light-emitting-diode induced fluorescence
electrochemiluminescence
amino acid
alkaloid
Mycobacterium tuberculosis
SDGs
Type
thesis
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