Real-time in vitro detection of cellular H_2O_2 under camptothecin stress using horseradish peroxidase, ionic liquid, and carbon nanotube-modified carbon fiber ultramicroelectrode

摘要

Single-walled carbon nanotubes (SWCNTs), horseradish peroxidase (HRP), and l-butyl-3-methylimidazolium tetrafluoroborate (BMIMBF_4) were employed to construct a cellular H_2O_2 sensor based on direct electron transfer. At a working potential of -0.35V, HRP-BMIMBF_4/SWCNTs/CFUME showed a dynamic range of up to 10.2μM with a low detection limit of 0.13μM (S/N=3) and a high sensitivity of 4.25A/Mcm~2. The apparent Michaelis-Menten constant (K_(m.app)) was estimated to be as low as 15.4μM, which suggested that HRP molecules entrapped in the BMIMBF_4 and SWCNTs immobilized at the carbon fiber ultramicroelectrode maintained a very high affinity. Because of the extremely small dimension and low working potential, HRP-BMIMBF_4/SWCNTS/CFUME enabled direct amperometric real-time monitoring of H_2O_2 in HeLa cells treated with the anticancer drug, camp tothecin, without requiring complex data processing and extra surface coatings to prevent interference. HRP-BMIMBF_4/SWCNTS/CFUME testing clearly showed that the H_2O_2 level significantly increased in HeLa cells under camptothecin stress. When HeLa cells were cultured in medium without camptothecin, the H_2O_2 level remained stable during the whole measurement process. These results indicate that HRP-BMIMBF_4/SWCNTs/CFUME could be a powerful tool for real-time investigation of cellular H_2O_2 level, especially under anticancer drug stress. This method could provide in-depth insights regarding the occurrence, development, and apoptosis of tumors.