Riboflavin photosensitized inactivation of lambda phage in PBS: An action spectrum and mechanistic investigation

Current industrial work includes investigating methods for reducing the risks of blood borne pathogens within the donated blood supply. The Ohio State University has been working in collaboration with Navigant Biotechnology to investigate the wavelength dependence of the viral kill of one of these processes. The combination of riboflavin and monochromatic light was used in conjunction with a microbiological plaque assay to determine the amount of viral kill at each wavelength.; Only 0.1 J/mL is needed to obtain up to 2.5 logs of lambda phage inactivation with wavelengths of 304 nm or lower. This is due to the direct absorption of radiation by the phage. In this region, the addition of riboflavin retards the amount of viral inactivation by screening the phage from receiving the light. Although the amount of inactivation is reduced, the mechanism of inactivation may actually be quite different than direct irradiation of phage.; When light used to irradiate the system has a wavelength of at least 308 nm, the energy required achieving the same range of viral inactivation increases to 5 J/mL. The reason for this increase in the required amount of energy is because the mechanism of viral inactivation changes from direct phage absorption to sensitization. The action spectra obtained with various amounts of riboflavin indicate that there is no sensitizer concentration dependence on the action spectra. The concentration insensitivity in this region is due to the energy at which the action spectra were obtained. At energy doses higher than 5 J/mL, a concentration effect is observed. The profile of the action spectrum does not exactly follow the absorption spectra of aqueous riboflavin or the major photoproduct, lumichrome.; The amount of photobleaching of riboflavin did not follow its aqueous absorption spectrum. Instead, the amount of wavelength dependent RB photobleaching agreed very well with the perturbed fluorescence-excitation scan of riboflavin. The excitation scan perturbation of riboflavin was due to a strong inner filter effect of the flavin. The inner filter effect of the solution being irradiated in combination with absorption shift due to phage-bound flavin is the source of the shape of the action spectrum.