Silver Nanoparticles and Metallic Silver Interfere with the Griess Reaction: Reduction of Azo Dye Formation via a Competing Sandmeyer-Like Reaction

Abstract

Silver (Ag) is the most common nanomaterial (NM) in consumer products. Much research has been focused on elucidating the potential impact of Ag-containing NMs on human health, e.g., cytotoxicity, genotoxicity, or proinflammatory responses. In the case of proinflammatory responses, a frequently used end point is the induction of nitric oxide (NO), which is indirectly quantified as nitrite (NO₂^-) with the Griess reaction. After preliminary studies in a macrophage-like cell culture system showed anomalous false negative results in the presence of silver nanoparticles (Ag NPs), we studied the influence of Ag on the detection of NO₂^- in a cell-free environment. Solutions containing a known concentration of NaNO₂ were prepared in H₂O, PBS, or complete cell culture medium (CCM) and analyzed using the Griess reaction in the presence of Ag in its metallic or ionic state. In Milli-Q H₂O, the impact of salts on the detection was investigated using NaCl and KBr. After completion of the Griess reaction, the samples were analyzed spectrophotometrically or chromatographically. It was found that the presence of metallic but not ionic Ag interfered with the quantification of NO₂^-. The effect was more pronounced in PBS and H₂O containing NaCl or KBr. The chromatographical analysis provided evidence of a competing reaction consuming the intermediate diazonium salt, which is critical to the Griess reaction. These findings demonstrate yet another substantial interference of NMs with a frequently used in vitro assay. If gone unnoticed, this interference might cause false negative results and an impaired hazard assessment of Ag NMs.