Simultaneous and sensitive analytical methods of guanine-containing nucleic acid-related compounds by means of new selective fluoresence reaction

利用新型选择性荧光反应同时灵敏地分析含鸟嘌呤核酸相关化合物的方法

• 批准号: 04807157
• 负责人: KAI Masaaki
• 金额: $ 1.22万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04807157/
• 关键词: Guanine; Nucleic acid-related compound; High-performance liquid chromatography; Fluorescence detection; Chemiluminescence detection; Phenylglyoxal; Selective reagent; Sensitive analysis

Pre-column and post-column fluorescence derivatization methods for the high-performance liquid chromatographic (HPLC) determination of guanine-containing compounds have been studied on the basis of the fluorescence reaction with phenylglyoxal (PGO). The detection limits for the compounds tested were 140-720 fmol and 3-10 pmol per 100-ul injection volume in the pre- and post-column derivatization methods, respectively. The PGO reagent was also applied to the measurement of the nuceobase-pair of guanine-cytosine in double-stranded DNAs.Alkoxyphenylglyoxals as the analogues of PGO were synthesized and found to be more sensitive reagents for the guanine-containing compounds. A novel chemiluminogenic method was developed on the basis of the fluorescence reaction of the guanine-containing compounds with PGO in a phosphate buffer (pH 6.0) at 37 ﾟC for 20 min, followed by a chemiluminescence reaction with N, N-dimethylformamide in a weakly alkaline medium. The chemiluminescence method was approximately 20-fold more sensitive than the fluorimetric one.

基于与苯乙二醛(PGO)的荧光反应，研究了鸟嘌呤类化合物的柱前和柱后荧光衍生化方法。在柱前衍生化方法和柱后衍生化方法中，被测化合物的检出限分别为每100微升进样量140-720fmoL和3-10pmo1。将该试剂应用于双链DNA中鸟嘌呤-胞嘧啶核碱基对的测定，合成了与PGO类似的烷氧基苯乙醛，对含鸟嘌呤类化合物具有更高的灵敏度。在37ﾟC的磷酸盐缓冲溶液中，鸟嘌呤类化合物与丙二醛发生荧光反应20min，然后在弱碱性介质中与N，N-二甲基甲酰胺发生化学发光反应，建立了一种新的化学发光分析方法。化学发光法的灵敏度大约是荧光法的20倍。

项目成果

M.Kai: "Chemiluminescence determination of guanine and its nucleosides and nucleotides by reaction with phenylglyoxal" Anal.Chim.Acta. 287. 75-81 (1994)

M.Kai：“通过与苯基乙二醛反应化学发光测定鸟嘌呤及其核苷和核苷酸”Anal.Chim.Acta。

大庭義史: "Alkoxyphenylglyoxals as fluorogenic reagents selective for guanine and its nucleosides and nucleotides in liquid chromatography" Analytical Chimica Acta. 287. 215-221 (1994)

Yoshifumi Ohba：“烷氧基苯基乙二醛作为液相色谱中鸟嘌呤及其核苷和核苷酸的荧光试剂”，《分析化学学报》287. 215-221 (1994)。

甲斐雅亮: "Fluorometric assay of double-stranded DNA by phenylglyoxal and chloroacetoaldehyde reactions" Analytical Sciences. 10(印刷中). (1994)

Masaaki Kai：“通过测定苯乙二醛和氯乙醛反应进行双链 DNA 荧光测定”，《分析科学》10（出版中）。

米倉さゆり: "Determination of guanine and its nucleosides and nucleotides in human erythrocytes by high-performance liquid chromatography with postcolumn fluorescence derivatization using phenylglyoxal reagent" Journal of Chromatography. 654. 19-24 (1994)

Sayuri Yonekura：“使用苯基乙二醛试剂通过柱后荧光衍生化的高效液相色谱法测定人红细胞中的鸟嘌呤及其核苷和核苷酸”《色谱杂志》654. 19-24 (1994)。

甲斐雅亮: "Chemiluminescence determination of guanine and its nucleosides and nucleotides using phenylglyoxal" Analytical Chimica Acta. 287. 75-81 (1994)

Masaaki Kai：“使用苯基乙二醛进行鸟嘌呤及其核苷和核苷酸的化学发光测定”《分析化学学报》287. 75-81 (1994)。