Fluorescence imaging and spectroscopy for real-time, in-situ characterization of interactions of free radicals with oil-in-water emulsions

The overall goal of this study was to develop a rapid method based on optical imaging and spectroscopy for real time characterization of interactions of free radicals with oil in water emulsions. The method was based on encapsulation of a lipid soluble, free radical sensitive dye that transforms from a non-fluorescent to a fluorescent state upon reaction with hydroxyl radicals. Using this approach, interaction of hydroxyl radicals generated using the Fenton's reagents in aqueous phase with free radical sensitive dye encapsulated in the oil phase of whey protein isolate (WPI) stabilized emulsion was characterized. The results based on fluorescence imaging and spectroscopy showed a linear increase in fluorescence of free radical sensitive dye during the initial time period (~ 30 min) of incubation with the Fenton's reagents. The results showed a high degree of correlation between spectroscopy and quantitative fluorescence imaging measurements. Increase in fluorescence of free radical sensitive dye was a function of concentration of ferrous ions and hydrogen peroxide. Incorporation of EDTA reduced the rate of increase in fluorescence of the dye due to the chelation of metal ions (p < 0.05). The addition of α-tocopherol in the lipid phase also significantly reduced the rate of increase in fluorescence (p < 0.05). Lipid oxidation was measured to validate propagation of radicals in the oil phase of the emulsion. The results showed a positive correlation between an increase in fluorescence of the free radical sensitive dye and the level of lipid peroxidation. In summary, the method developed in this study is sensitive for real time quantitative measurement of interactions of free radicals with emulsions.