Regional Centre of Advanced Technologies and Materials, Czech Advanced Technology and Research Institute (CATRIN), Palacký University Olomouc, Šlechtitelů 27, 779 00 Olomouc, Czech Republic.
Department of Physical Chemistry, Faculty of Science, Palacký University Olomouc, 17. Listopadu 12/1192, 771 00 Olomouc, Czech Republic.
Int J Mol Sci. 2021 Jul 20;22(14):7750. doi: 10.3390/ijms22147750.
The scope of application of carbon nanomaterials in biomedical, environmental and industrial fields is recently substantially increasing. Since in vitro toxicity testing is the first essential step for any commercial usage, it is crucial to have a reliable method to analyze the potentially harmful effects of carbon nanomaterials. Even though researchers already reported the interference of carbon nanomaterials with common toxicity assays, there is still, unfortunately, a large number of studies that neglect this fact. In this study, we investigated interference of four bio-promising carbon nanomaterials (graphene acid (GA), cyanographene (GCN), graphitic carbon nitride (g-CN) and carbon dots (QCDs)) in commonly used LIVE/DEAD assay. When a standard procedure was applied, materials caused various types of interference. While positively charged g-CN and QCDs induced false results through the creation of free agglomerates and intrinsic fluorescence properties, negatively charged GA and GCN led to false signals due to the complex quenching effect of the fluorescent dye of a LIVE/DEAD kit. Thus, we developed a new approach using a specific gating strategy based on additional controls that successfully overcame all types of interference and lead to reliable results in LIVE/DEAD assay. We suggest that the newly developed procedure should be a mandatory tool for all in vitro flow cytometry assays of any class of carbon nanomaterials.
碳纳米材料在生物医学、环境和工业领域的应用范围最近大幅增加。由于体外毒性测试是任何商业用途的第一步，因此拥有一种可靠的方法来分析碳纳米材料的潜在有害影响至关重要。尽管研究人员已经报道了碳纳米材料对常见毒性测定的干扰，但不幸的是，仍有大量研究忽视了这一事实。在这项研究中，我们研究了四种有生物应用前景的碳纳米材料（石墨烯酸（GA）、氰基石墨烯（GCN）、石墨相氮化碳（g-CN）和碳点（QCDs））在常用的 LIVE/DEAD 测定中的干扰。当应用标准程序时，这些材料会引起各种类型的干扰。带正电荷的 g-CN 和 QCDs 通过形成游离聚集体和固有荧光特性产生虚假结果，而带负电荷的 GA 和 GCN 则由于 LIVE/DEAD 试剂盒中荧光染料的复杂猝灭效应导致错误信号。因此，我们开发了一种新方法，使用基于附加对照的特定门控策略，成功克服了所有类型的干扰，并在 LIVE/DEAD 测定中得到了可靠的结果。我们建议，新开发的程序应成为所有类别的碳纳米材料的体外流式细胞术测定的强制性工具。
