Carbon Nanotubes Interference with Luminescence-Based Assays

Carbon nanotubes (CNTs) are one of the most promising nanomaterials synthesized to date. Thanks to their unique mechanical, electronic, and optical properties, they have found a wide application in electronics in the production of biosensors and nanocomposites. The functionalization of multiwalled carbon nanotubes (MWCNTs) is aimed at making them biocompatible by adding hydrophilic groups on their surface, increasing their solubility and thus rendering them applicable in the regenerative medicine. So far, there is conflicting information about carbon nanotubes in biological systems. This paper investigates the effect of functionalized, oxidized, multiwalled carbon nanotubes (MWCNT-Ox) on the cytotoxicity of normal human articular chondrocytes (NHAC-kn cell line). Since absorbance-based and fluorescence-based assays were shown to interfere with carbon nanotubes, luminescence-based tests were carried out, as they work on a different method of detection and provide advantages over the mentioned ones. Cell viability and reactive oxygen species (ROS) tests were carried out. The cell viability assay showed that with the increasing MWCNTs concentration, the number of viable chondrocytes was significantly decreasing. Exposure to MWCNT-Ox indicated oxidative stress in the lowest investigated concentration with a decreased amount of ROS with higher concentrations. However, control experiments with adenosine triphosphate (ATP) and H₂O₂-molecules that are detected by the assays-showed that carbon nanotubes interfere directly with measurement, thus rendering the results unreliable. To understand the exact interference mechanisms, further studies must be taken. In conclusion, this study shows that luminescence-based tests yield erroneous results, confirming that in vitro experiments in the literature concerning carbon nanotubes should be analyzed with caution.