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Analysis of trinitrophenylated adenosine and inosine using capillary electrophoresis-laser induced fluorescence detection and gamma-cyclodextrin.

机译:使用毛细管电泳-激光诱导的荧光检测和γ-环糊精分析三硝基苯基化腺苷和肌苷。

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摘要

Adenosine (Ado) and adenine ribonucleotides are essential in cell metabolism and energy production, cellular signaling, and DNA and RNA synthesis. The biosynthesis of these molecules takes place in both the intracellular and extracellular space via transphosphorylation reactions catalyzed by several distinct kinase enzymes like adenylate kinase. Several analytical detection methodologies have been developed to monitor these molecules in biological tissue, including both liquid chromatography (LC) and capillary electrophoresis (CE) techniques. However, many of these methodologies are limited by separation resolution and sample injection volume requirements. This thesis presents a novel capillary electrophoresis-laser induced fluorescence detection (CE-LIF) method with high separation power to analyze Ado and Inosine (Ino), a metabolite of Ado, by derivatization with 2,4,6-trinitrobenzenesulfonic acid to form fluorescent trinitrophenylated complexes of Ado (TNP-Ado) and Ino (TNP-Ino). The development and validation of the CE-LIF method, optimization of the trinitrophenylation reaction, and fluorescence enhancement of TNP-Ado and TNP-Ino with gamma-cyclodextrin will be discussed. Detection limits were 1.6 muM for Ado and 4 muM for Ino in rat brain tissue. Large-volume sample stacking (LVSS) was employed to further enhance the sensitivity of the CE-LIF method, with detections limits of 310 nM and 159 nm for Ado and Ino, respectively. The CE-LIF method offers promise for the analysis of Ado, Ino and potentially other adenine ribonucleotides in small volume generating biological experiments like in vivo microdialysis and single cell metabolomics.
机译:腺苷(Ado)和腺嘌呤核糖核苷酸在细胞代谢和能量产生,细胞信号传导以及DNA和RNA合成中至关重要。这些分子的生物合成在细胞内和细胞外空间中通过几种不同的激酶(如腺苷酸激酶)催化的转磷酸化反应发生。已经开发了几种分析检测方法来监测生物组织中的这些分子,包括液相色谱(LC)和毛细管电泳(CE)技术。但是,这些方法中的许多方法都受到分离分辨率和样品进样量要求的限制。本文提出了一种新的毛细管电泳-激光诱导荧光检测(CE-LIF)方法,该方法具有高分离能力,可通过2,4,6-三硝基苯磺酸衍生化而形成Ado和Ado的代谢产物肌苷(Ino)。 Ado(TNP-Ado)和Ino(TNP-Ino)的三硝基苯配合物。将讨论CE-LIF方法的开发和验证,三硝基苯基化反应的优化以及用γ-环糊精增强TNP-Ado和TNP-Ino的荧光。大鼠脑组织中Ado的检出限为1.6μM,Ino的检出限为4μM。采用大体积样品堆叠(LVSS)进一步增强了CE-LIF方法的灵敏度,Ado和Ino的检出限分别为310 nM和159 nm。 CE-LIF方法为小剂量分析Ado,Ino和潜在的其他腺嘌呤核糖核苷酸提供了希望,可进行诸如体内微透析和单细胞代谢组学的生物学实验。

著录项

  • 作者单位

    University of Alaska Fairbanks.;

  • 授予单位 University of Alaska Fairbanks.;
  • 学科 Biochemistry.;Neurosciences.;Analytical chemistry.
  • 学位 M.S.
  • 年度 2016
  • 页码 112 p.
  • 总页数 112
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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