Squalene-based oxygen-scavenging system for enhancing fluorescent dye stability for biosensing applications

Photobleaching impedes the application of fluorescent dyes in long-term imaging. While enzymatic oxygen scavenging enhances photostability, it induces progressive acidification of the solution. Here, we report a new oxygen-scavenging system comprising squalene (SQ) and Trolox that improves dye stability without perturbing pH. DPPH and ABTS radical cation scavenging assays verified the antioxidant activity of SQ, with 0.7–1% concentrations achieving 35% radical scavenging capacity. The system maintains pH stability for more than 18 min, in contrast to the rapid acidification observed with glucose oxidase/catalase. Total internal reflection fluorescence (TIRF) imaging of cyanine 3 (Cy3) and cyanine 5 (Cy5) revealed that SQ with Trolox performs comparably to enzymatic systems while permitting extended illumination, establishing a pH-neutral strategy for enhanced photostability.