Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
1176051 | Analytical Biochemistry | 2006 | 12 Pages |
Constant wavelength synchronous fluorescence spectroscopy (CW–SFS), UV–visible absorption spectroscopy, and cyclic and differential pulse voltammetry were applied to investigate the competitive interaction of DNA with the bis(1,10-phenanthroline)copper(II) complex cation ([Cu(phen)2]2+) and a fluorescence probe, neutral red dye (NR), in a tris–hydrogen chloride buffer (pH 7.4). The results show that both the [Cu(phen)2]2+and the NR molecules can intercalate competitively into the DNA double-helix structure. The cyclic voltammetry method showed that both anodic and cathodic currents of [Cu(phen)2]2+ decreased on addition of the DNA and the intercalated [Cu(phen)2]2+–DNA complex formed (β = (4.14 ± 0.24) × 103). CW–SFS measurements were facilitated by the use of the three-way resolution of the CW–SFS for NR, [Cu(phen)2]2+, and NR–DNA. The important constant wavelength (CW) interval, Δλ, was shown to vary considerably when optimized (135, 58, and 98 nm for NR, NR–DNA, and [Cu(phen)2]2+, respectively). This approach clearly avoided the errors that otherwise would have arisen from the common assumption that Δλ is constant. Furthermore, a chemometrics approach, parallel factor analysis (PARAFAC), was applied to resolve the measured three-way CW–SFS data, and the results provided simultaneously the concentration information for the three reaction components, NR, [Cu(phen)2]2+, and NR–DNA, for the system at each equilibrium point. The PARAFAC analysis indicated that the intercalation of the [Cu(phen)2]2+ molecule into the DNA proceeds by exchanging with the NR probe and can be attributed to two parallel reactions. Comprehensive information was readily obtained; the replacement of the intercalated NR commenced immediately on introduction of [Cu(phen)2]2+, approximately 50% of NR was replaced by [Cu(phen)2]2+ at a concentration of 0.45 × 10−5 mol L−1, and nearly all of the NR was replaced at a [Cu(phen)2]2+ concentration of 2.50 × 10−5 mol L−1. This work has the potential to improve extraction of information from the fluorescence intercalator displacement (FID) assay.