Screening for Oxidative Stress Elicited by Engineered Nanomaterials: Evaluation of Acellular DCFH Assay

The DCFH assay is commonly used for measuring free radicals generated by engineered nanomaterials (ENM), a well-established mechanism of ENM toxicity. Concerns exist over susceptibility of the DCFH assay to: assay conditions, adsorption of DCFH onto ENM, fluorescence quenching and light scattering. These effects vary in magnitude depending on ENM physiochemical properties and concentration. A rigorous evaluation of this method is still lacking. The objective was to evaluate performance of the DCFH assay for measuring ENM-induced free radicals. A series of diverse and well-characterized ENM were tested in the acellular DCFH assay. We investigated the effect of sonication conditions, dispersion media, ENM concentration, and the use of horseradish peroxidase (HRP) on the DCFH results. The acellular DCFH assay suffers from high background signals resulting from dye auto-oxidation and lacks sensitivity and robustness. DCFH oxidation is further enhanced by HRP. The number of positive ENM in the assay and their relative ranking changed as a function of experimental conditions. An inverse dose relationship was observed for several Carbon-based ENM. Overall, these findings indicate the importance of having standardized assays for evaluating ENM toxicity and highlights limitations of the DCFH assay for measuring ENM-induced free radicals.