Highly selective determination of bioactive compounds based on intra molecular fluorescence resonance energy transfer detection.

基于分子内荧光共振能量转移检测的生物活性化合物的高选择性测定。

• 批准号: 12672100
• 负责人: NOHTA Hitoshi
• 金额: $ 2.05万
• 依托单位: Fukuoka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12672100/
• 关键词: Fluorometry; Fluorescence resonance energy transfer; Fluorescence derivatization; Indoleamine; Catecholamine

I have developed highly selective HPLC method for the determination of tyrosine, tryptophan and their related compounds (L-DOPA, catechdlamines, 5-hydroxytryptamine, etc.). The compounds were precolumn-derivatized with a commercially available fluorogenic reagent for amines by usual manner. Each derivative afforded intramolecular fluorescence resonance energy transfer (FRET) from the tyrosyl or tryptophoryl moiety (donor) to the labeled fluorophore (acceptor) ; the acceptor fluorescence was observed with the excitation of the donor at 280 run. The derivatives were separated on a reversed-phase column and then effectively detected by monitoring their FRET. Through the screening study of 15 fluorogenic reagents, ο-phthalaldehyde (with 2-mercaptoethanol), Dansyl chloride, 4, 5-dimetholy-ο-phthalaldehyde and DAMTC afforded efficient FRET ; ο-phthalaldehyde was selected as a highy selective, sensitive and practical reagent. The FRET detection method was highly selective and sensitive (limits of detection ; 20-70 fmol on clumn) by comparison with the previous methods detecting native fluorescence of the compounds or typical fluorescence of the acceptor. This method could be successfully applied to human urine sample without any tedious pretre atment.

我已经开发了高选择性的HPLC法测定酪氨酸，色氨酸及其相关化合物（L-多巴，儿茶胺，5-羟色胺等）。化合物通过常规方式用市售的胺荧光试剂进行柱前衍生化。每个衍生物提供分子内荧光共振能量转移（FRET）从酪氨酰基或胆固醇酰基部分（供体）的标记的荧光团（受体）;受体荧光观察与供体的激发在280 nm。在反相柱上分离衍生物，然后通过监测它们的FRET有效地检测。通过对15种荧光试剂的筛选研究，邻苯二甲醛（含2-巯基乙醇）、丹磺酰氯、4，5-二甲氧基邻苯二甲醛和DAMTC均具有良好的荧光共振能量转移性能，选择邻苯二甲醛作为一种选择性高、灵敏度高、实用性强的荧光试剂。与检测化合物的天然荧光或受体的典型荧光的先前方法相比，FRET检测方法具有高度选择性和灵敏度（检测限; 20-70 fmol/柱）。该方法可成功地应用于人体尿液样品，无需任何繁琐的前处理。