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 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.