4.5.3. Competitive Binding Assay with Ethidium Bromide (EB) through Fluorescence Spectroscopy

The fluorescence spectra were recorded using 1.6 mL samples containing 10 µM CT-DNA, 2 µM EB, and increasing amounts (0–40 µM with an increment of 5 µM) of tested compounds; the samples were prepared by keeping the components in contact for 10 min at room temperature, under continuous stirring and in the dark. The excitation wavelength was 500 nm, and the spectra were recorded between the wavelengths of 520 and 800 nm. A Jasco FP 6500 fluorometer and quartz cells with a 1 cm route length were employed. The classical Stern–Volmer equation (Equation (3)) was used to interpret the obtained data [104]:

F₀ and F are the fluorescence intensities of the EB–DNA samples in the absence and presence of the tested compounds, respectively; K_SV is the Stern–Volmer constant, which was computed as the slope from the plot of F₀/F vs. [Q], where [Q] is the chemical concentration.

The binding ratio was plotted against the logarithm of the concentration in comparison with a control sample exposed solely to DMSO.

For fitting data points, the modified Hill function with offset (Origin^®) was used: where y represents the binding ratio (F/F₀), x represents the concentration, A₁ represents the minimum of y values, A₂ represents the maximum of y values, K₅₀ represents the concentration corresponding to 50% binding or the half maximum binding constant, and n represents the Hill coefficient [97].