Li L, McGown L B
Department of Chemistry, P.M. Gross Chemical Laboratory, Duke University, Durham, NC 27708-0346, USA.
J Chromatogr A. 1999 May 7;841(1):95-103. doi: 10.1016/s0021-9673(99)00292-7.
Investigations of fluorescence lifetimes of the dye 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoic acid (NBD-HA) and of DNA M13 primers labeled with NBD-HA, Cy3, rhodamine green and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene++ +-3-propionic acid (BODIPY-FL) dyes in polyacrylamide gels of various degrees of crosslinking and different crosslinkers, and in a cellulose sieving buffer with different organic modifiers, are described. The dependence of fluorescence lifetime on gel matrix and on experimental conditions was studied in order to identify which factors may be important for optimization of multiplex fluorescence lifetime detection. Lifetimes were determined in both batch solution and on-the-fly, on-column in CE. Results show that lifetimes of the primer-attached dyes remain constant in gels of different composition. Additionally, multiexponential fluorescence decays are observed for primer-attached dyes in batch solutions of the cellulose sieving buffers but are reduced to monoexponential decays when measured on-the-fly, on-column in CE. Lifetime detectability can be improved by addition of an organic modifier to the gel matrix.
本文描述了对染料6-[N-(7-硝基苯并-2-恶唑-1,3-二氮杂萘-4-基)氨基]己酸（NBD-HA）以及用NBD-HA、Cy3、罗丹明绿和4,4-二氟-5,7-二甲基-4-硼-3a,4a-二氮杂-s-茚满-3-丙酸（BODIPY-FL）染料标记的DNA M13引物，在不同交联度和不同交联剂的聚丙烯酰胺凝胶中，以及在含有不同有机改性剂的纤维素筛分缓冲液中的荧光寿命进行的研究。研究了荧光寿命对凝胶基质和实验条件的依赖性，以确定哪些因素对于优化多重荧光寿命检测可能是重要的。在批量溶液中以及在CE中柱上实时测定寿命。结果表明，附着引物的染料在不同组成的凝胶中的寿命保持恒定。此外，在纤维素筛分缓冲液的批量溶液中，附着引物的染料观察到多指数荧光衰减，但在CE中柱上实时测量时衰减为单指数衰减。通过向凝胶基质中添加有机改性剂可以提高寿命检测能力。
