Development of a nanostructured sensor for monitoring oxidative stress in living cells

Abstract

Oxidative burden is elevated in the lung of COPD patients and is associated with aging and chronic inflammation. When overcoming physiological levels, reactive oxygen species (ROS) cause cell damage and sustain inflammation. Both lung epithelium and alveolar macrophages contribute to ROS generation. Currently, ROS generation is measured using fluorescent probes and colorimetric/fluorimetric assays. We present an amperometric nanostructured sensor for real-time detection of hydrogen peroxide (H2O2) released by living cells. The H2O2 sensing performance was evaluated through the current vs time response of platinum rod at a working potential of −0.45 V vs saturated calomel electrode acting as a reference. The detection current was related to the bulk concentration of H2O2 in solution. Using this sensor, H2O2 release was measured in conditioned medium from THP-1 macrophages and 16-HBE bronchial epithelial cells exposed to different stimuli (lipopolysaccharide (LPS), cigarette smoke extract (CSE), nigericin). Results were compared with those obtained by flow cytometry using the same cells stained with Carboxy-H2DCFDA and MitoSOXTM Red. The addition of LPS, CSE and nigericin resulted in a significant increase of the cathodic current due to the reduction of H2O2 indicating an increase in H2O2 release. The results paralleled those obtained by flow cytometry. The proposed nanostructured sensor offers several advantages over current methods: (i) real time release of H2O2 is monitored without disturbing cell growth; (ii) quantitative data can be generated using a calibration curve; (iii) the method is highly sensitive, fast and cost effective; (iv) potential use for in vivo monitoring of oxidative stress.