Effects of gel material on fluorescence lifetime detection of dyes and dye-labeled DNA primers in capillary electrophoresis

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 labele d 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 ge ls of various degrees of crosslinking and different crosslinkers, and in a cellulose sieving buffer with different organic modifiers, are described. T he dependence of fluorescence lifetime on gel matrix and on experimental co nditions was studied in order to identify which factors may be important fo r 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 s ieving buffers but are reduced to monoexponential decays when measured on-t he-fly, on-column in CE. Lifetime detectability can be improved by addition of an organic modifier to the gel matrix. (C) 1999 Elsevier Science B.V. A ll rights reserved.