Real-time in vitro detection of cellular H2O2 under camptothecin stress using horseradish peroxidase, ionic liquid, and carbon nanotube-modified carbon fiber ultramicroelectrode

Abstract

Single-walled carbon nanotubes (SWCNTs), horseradish peroxidase (HRP), and 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM·BF4) were employed to construct a cellular H2O2 sensor based on direct electron transfer. At a working potential of −0.35V, HRP-BMIM·BF4/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/Mcm2. The apparent Michaelis-Menten constant (Km,app) was estimated to be as low as 15.4μM, which suggested that HRP molecules entrapped in the BMIM·BF4 and SWCNTs immobilized at the carbon fiber ultramicroelectrode maintained a very high affinity. Because of the extremely small dimension and low working potential, HRP-BMIM·BF4/SWCNTs/CFUME enabled direct amperometric real-time monitoring of H2O2 in HeLa cells treated with the anticancer drug, camptothecin, without requiring complex data processing and extra surface coatings to prevent interference. HRP-BMIM·BF4/SWCNTs/CFUME testing clearly showed that the H2O2 level significantly increased in HeLa cells under camptothecin stress. When HeLa cells were cultured in medium without camptothecin, the H2O2 level remained stable during the whole measurement process. These results indicate that HRP-BMIM·BF4/SWCNTs/CFUME could be a powerful tool for real-time investigation of cellular H2O2 level, especially under anticancer drug stress. This method could provide in-depth insights regarding the occurrence, development, and apoptosis of tumors.