We evaluated the intracellular NAD(P)H:quinone oxidoreductase (NQO) activity of single HeLa cells by using the menadione-ferrocyanide double-mediator system combined with scanning electrochemical microscopy (SECM). The double-mediator system was used to amplify the current response from the intracellular NQO activity and to reduce menadione-induced cell damage. The electron shuttle between the electrode and menadione was mediated by the ferrocyanide/ferricyanide redox couple. Generation of ferrocyanide was observed immediately after the addition of a lower concentration (10. μM) of menadione. The ferrocyanide generation rate was constant for 120. min. At a higher menadione concentration (100. μM), the ferrocyanide generation rate decreased within 30. min because of the cytotoxic effect of menadione. We also investigated the relationship between intracellular reactive oxygen species or glutathione levels and exposure to different menadione concentrations to determine the optimal condition for SECM with minimal invasiveness. The present study clearly demonstrates that SECM is useful for the analysis of intracellular enzymatic activities in single cells with a double-mediator system.
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