3.3.3. Fluorescence Spectroscopy

A survey of the emission fluorescence spectra of pyrene was performed using a Cary Eclipse G9800A fluorescence spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) at 25 °C. The excitation wavelength was 335 nm. Emission spectra were registered in the range of 350–500 nm with a constant scanning speed (120 nm/min). A cuvette with a 1 cm width was used in all measurements. Fluorescence intensities of the first (I_I) and third (I_III) vibrational peaks of pyrene at 373 nm and 384 nm, respectively, were extracted from the collected spectra.

Fluorescence spectra of the oligonucleotide–ethidium bromide complexes were recorded in the range from 500 to 700 nm at an excitation wavelength of 480 nm. A 0.8 mL sample containing 0.5 µM of EB and a 10 µM concentration of oligonucleotide (counting on 1 nucleotide unit) in 4 mM Tris–HCl buffer (pH = 8.0) was equilibrated for 10 min at 25 °C. After that, a specific aliquot of the amphiphile solution was added to the mixture, and the fluorescence emission spectrum was registered [52,53]. Each concentration dependence was performed 2 times, and the standard deviation of the results was less than 2%. The following equation was used for the quantitative evaluation of the binding degree of oNu to amphiphile:

where I_free and I_bound are the intensities of fluorescence of free EB and probe bound to oNu, respectively; and I_obs is the fluorescence intensity observed during titration measurements at a specific amount of amphiphile added.