State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, PR China; University of Chinese Academy of Sciences, Beijing, 100049, PR China.
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, PR China; University of Chinese Academy of Sciences, Beijing, 100049, PR China.
Anal Chim Acta. 2019 Nov 4;1079:103-110. doi: 10.1016/j.aca.2019.06.036. Epub 2019 Jun 17.
Characterization of carbonaceous nanomaterials (CNMs) exposure is a key step and of great importance towards a better understanding of their toxicity and underlying mechanisms. However, it has been bottlenecked for lack of valid methods capable of quantifying cell-associated CNMs. Here, we developed a new economical and convenient method based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) that could accumulate graphene oxide (GO) at the interface between the loading well and the gel. The sharp black band formed there can be digitalized and the intensity quantified, which was proportional to the amount of GO loaded onto the gel. The method has a detection limit of 84.1 ng. We showed that the amount of GO in three different cell models, mouse macrophage cells (Raw264.7), human epithelial cells (A549) and mouse mesenchymal stem cells (MSC), could be accurately quantified by this assay, with the uptake rates decreasing in the order of MSC > Raw264.7 > A549. The results were consistent with the fluorescent imaging on cells exposed to fluorescence-labeled GO and TEM examination on ultrathin cell sections. The surprisingly highest uptake rate of MSC might be due to their abundant intracellular vesicles, which deserves further investigation. The novel method provides a complementary quantitative tool to the use of radioactive markers and fluorescent labeling of carbon nanomaterials and may facilitate the toxicological studies on carbon nanomaterials.
碳纳米材料（CNMs）暴露的特征描述是理解其毒性和潜在机制的关键步骤，具有重要意义。然而，由于缺乏能够定量检测细胞相关碳纳米材料的有效方法，这一过程一直受到限制。在这里，我们开发了一种基于十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）的新的经济便捷的方法，该方法能够在加载孔和凝胶之间的界面处积累氧化石墨烯（GO）。由此形成的锐利的黑色条带可以进行数字化，并对其强度进行定量，其与加载到凝胶上的 GO 量成正比。该方法的检测限为 84.1ng。我们表明，通过该方法可以准确地定量三种不同细胞模型（小鼠巨噬细胞（Raw264.7）、人上皮细胞（A549）和小鼠间充质干细胞（MSC））中的 GO 量，摄取率按 MSC > Raw264.7 > A549 的顺序降低。这些结果与细胞内荧光标记 GO 的荧光成像和超薄细胞切片的 TEM 检查结果一致。MSC 惊人的最高摄取率可能归因于其丰富的细胞内囊泡，这值得进一步研究。这种新方法为放射性标记和荧光标记碳纳米材料的使用提供了一种补充的定量工具，并可能有助于碳纳米材料的毒理学研究。
