This paper describes the first use of frequency-domain fluorescence lifetime for multiplex detection of DNA restriction fragments in capillary electrophoresis (CE). The fragments were labeled with monomeric intercalating dyes that can be excited by either the 488- or 514-nm line of an argon ion laser and have lifetimes in the range of 0.5-2.5 ns. We were able to achieve multiplex lifetime detection in the CE separation of a restriction fragment digest and a DNA size ladder in the same run, for fragments shorter than 700 bp. Different gel buffer systems, including a modified polyacrylamide gel and several tris-borate-EDTA/hydroxyethylcellulose (TBE/HEC) gels, were investigated for separation and detection of the dye-labeled DNA fragments. Best results for both electrophoretic resolution and lifetime detection were obtained using a gel containing 1% high molecular weight (90,000-105,000) HEC and 0.3% low molecular weight (24,000-27,000) HEC in TBE buffer.

β-Cyclodextrin (β-CD) is a seven-unit cyclic sugar molecule with a torroidal shape, a hydrophilic exterior, and a hydrophobic cavity. It has been hypothesized that pyrene (P) forms a P(β-CD)2 complex in which the pyrene lies in between the two wide, or secondary, rims of the two β-CDs. This dimer structure appears to be stabilized in the crystal state by hydrogen bonds between –OH groups on the rims of the adjacent β-CDs. We have chosen to test this hypothesis by comparing the fluorescence properties of pyrene in the presence of substituted β-CDs with these properties in the presence of β-CD. These properties include the shape (I/III vibronic band ratio) and lifetime of pyrene fluorescence and the effect on the pyrene I/III ratio of additives such as an iodide ion and a fluorinated alcohol. The CD derivatives in this investigation include those with no wide rim substitution and those with substitution on both the wide and narrow rims of β-CD. Our data indicate that the a P(β-CD)2 complex forms only when there is no substitution on the wide rim, which confirms the proposed structure for this complex.

The use of novel intercalating dyes as labels in DNA restriction fragment analysis by capillary electrophoresis with frequency-domain fluorescence lifetime detection is described. The dyes, including one mono-intercalating dye with three positive charges and three bis-intercalating, homodimeric dyes with four positive charges, were excited by the 488 nm line of an argon ion laser and exhibited lifetimes in the range of 1-3 ns. The separations were performed using a gel containing 1% high-molecular-weight (HMW) hydroxyethylcellulose (HEC) (90,000-105,000) and 0.3% low-molecular-weight (LMW) HEC (24,000-27,000) in Tris-borate-EDTA buffer (TBE). Multiplex lifetime detection of mixtures of dye-labeled DNA restriction fragment digests and size standard fragments was achieved. Compared to previous results obtained with several mono-intercalating dyes of lesser charge (McIntosh, S. L., Nunnally, B. K., Nesbit, A. R., Deligeorgiev, T. G., Gadjev, N. I., McGown, L. B., Anal. Chem. 2000, 72, 5444-5449), the present dyes provided a wider range of lifetimes and better lifetime discrimination in multiplex detection. There was no evidence of dye exchange during the capillary electrophoresis experiment.