A porous polyaniline nanotube sorbent for solid-phase extraction of the fluorescent reaction product of reactive oxygen species in cells, and its determination by HPLC
A porous polyaniline nanotube sorbent for solid-phase extraction of the fluorescent reaction product of reactive oxygen species in cells, and its determination by HPLC
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多孔聚苯胺纳米管吸附剂固相萃取细胞内活性氧荧光反应产物及其HPLC测定
DOI：
10.1007/s00604-018-3000-6
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发表时间：
2018-10-01
影响因子：
5.7
通讯作者：
Guo, Yong
中科院分区：
文献类型：
--
作者：
Niu, Panhong;Li, Feifei;Guo, Yong
本文介绍了一种提取和检测活性氧物种(ROS)与二氯二氢荧光素二乙酸酯(DCFH-DA)反应生成的荧光反应产物(DCF)的方法。采用多孔聚苯胺纳米管(PPN)提取DCF，其比表面积和孔体积较大，有利于吸附。其他吸引人的特征包括合适的孔径分布、疏水表面和有助于吸附DCF的电子吸引基团。采用多种方法对PPN的形态进行表征。在最佳条件下，在0.08~1.0M浓度范围内进行DCF法，校正曲线的相关系数为0.999。标准DCF溶液的检出限为20 nm。与用于固相萃取(SPE)的商用吸附剂(如市售的Carbon或Welchrom(R)C18)相比，使用这种新型吸附剂可获得更好的回收效果(92%)和更长的重复使用时间(30倍)。用阿霉素和X射线照射体外刺激HepG2和Hela细胞产生ROS。用DCFH-DA稳定ROS，用DCF定量。固相萃取后，用高效液相色谱法测定DCF，计算ROS浓度。
A method is described for extracting and detecting the fluorescent reaction product (2,7-dichlorofluorescein, DCF) that is formed by reaction of reactive oxygen species (ROS) with dichlorodihydrofluorescein diacetate (DCFH-DA). DCF is extracted by using porous polyaniline nanotubes (PPN) which have a large specific surface and pore volume which favor the adsorption capacity. Additional attractive features include an appropriate pore size distribution, hydrophobic surface, and electron-attracting groups which contribute to DCF adsorption. A variety of methods was applied to characterize the morphology of PPN. Under optimal conditions and by performing DCF in 0.08-1.0M concentrations, the correlation coefficient of the calibration plot is 0.999. The limits of detection for standard DCF solutions is 20nM. Compared with commercial sorbents for solid-phase extraction (SPE) such as commercially available carbon or Welchrom (R) C18, the use of the new sorbent results in better retraction recovery (92%) and longer reuse times (30 times). Doxorubicin and X-ray radiation were used to externally stimulate the ROS production in HepG2 and Hela cells. ROS was stabled by DCFH-DA and quantified by DCF. Following SPE, DCF was detected by HPLC and the concentration ROS was calculated.