Synchronous fluorescence, UV–visible spectrophotometric, and voltammetric studies of the competitive interaction of bis(1,10-phenanthroline)copper(II) complex and neutral red with DNA

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.