A Comparative Analysis of Absorbance- and Fluorescence-Based 1,3-Diphenylisobenzofuran Assay and Its Application for Evaluating Type II Photosensitization of Flavin Derivatives

Singlet oxygen is a type of reactive oxygen species that is typically generated via type II photosensitization reactions. Since 1,3-diphenylisobenzofuran (DPBF), a commonly used chromogenic probe, exhibits peak absorbance at 410 nm for singlet oxygen detection, it severely interferes with blue light irradiation and compounds that absorb in this wavelength region. This study investigated developing and validating a fluorescence-based method using DPBF to quantitatively analyze the type II photosensitizing property of riboflavin (RF) and its heterocyclic flavin derivatives. DPBF fluorescence-based analysis provided more sensitive and practical results than traditional colorimetric methods. It effectively overcomes spectral interference from colored photosensitizers, such as RF and its derivatives, under blue light irradiation (λ peak 447 nm). This method permitted more effective measurement of their activity without interference from their intrinsic color and maintained high linearity and low variation across different sample concentrations, even with short irradiation times. The type II photosensitizing potency of the tested compounds under blue light was consistently ranked as follows: RF > flavin mononucleotide > flavin adenine dinucleotide > lumiflavin > lumichrome. The results suggest that the DPBF fluorescence-based assay is a more effective approach than colorimetric analysis, making it a practical and reproducible tool for assessing the type II photosensitizing properties of diverse compounds. This study also provides a refinement of an existing probe-based assay for relative comparisons under visible light conditions.