Effect of pre-incubation time on riboflavin–candida albicans interactions prior to photodynamic inactivation assessed by spectrophotometry

Antimicrobial photodynamic inactivation (aPDI) has emerged as a promising approach for controlling Candida albicans, relying on the interaction between a photosensitizer, light, and molecular oxygen to generate cytotoxic reactive oxygen species. Photodynamic efficacy is strongly influenced by photosensitizer availability and its interaction with microbial cells before irradiation, making pre-incubation time a relevant parameter for treatment optimization. Riboflavin is a biocompatible, naturally occurring photosensitizer with favorable photochemical properties; however, the kinetics of its association with fungal cells and the influence of pre-incubation time remain poorly understood.

C. albicans ATCC 10231 suspensions (10⁵ CFU/mL) were incubated with 400 µmol L⁻¹ riboflavin in a 1:1 (v/v) ratio for different pre-incubation times (30–120 min) in 24-well plates under agitation (~400 rpm) and protected from light. Samples were then centrifuged at 4,000 rpm for 5 min, and riboflavin remaining in the supernatant was quantified spectrophotometrically at 450 nm.

Pre-incubation time markedly influenced riboflavin association with C. albicans. After 30 min, approximately 30% of the photosensitizer was associated with fungal cells, increasing to 75% after 60 min. A plateau was reached at 90 min, while at 120 min the available riboflavin was nearly depleted, suggesting that the initial concentration limited further time-dependent association.

Spectrophotometry provided a simple approach to optimize photosensitizer pre-incubation time, avoiding unnecessarily prolonged PS–microorganism contact. This strategy may be extended to other photosensitizers, particularly emerging molecules under investigation for photodynamic applications.