Simultaneous Electrochemical Detection of Primary Reactive Oxygen and Nitrogen Species Released by Cell Populations in Integrated Microdevices
Résumé
Innovative microdevices were designed to monitor electrochemically primary reactive oxygen (ROS) and reactive nitrogen species (RNS) released by populations of aerobic cells. Taking advantage of the space confinement and microelectrodes properties, only few experiments were sufficient to provide significant statistical data relative to the average behavior of cells during oxidative stress bursts. In this study, platinum-black coated platinum (Pt/Pt-black) electrodes were first microfabricated and optimized to reach optimal performances during the electrochemical detection of four primary species H2O2, NO•, ONOO-and NO2 −. The results demonstrated that relative ROS/RNS contents in synthetic mixtures could be easily assessed at selected detection potentials. Under given experimental conditions, the Pt/Pt-black electrodes allowed detection limits down to 10 nM with high sensitivities and long-term stability of the electrode responses. The electrochemical detection of ROS/RNS released by cell populations was then implemented with Pt/Pt-black microelectrodes integrated into a multi-wells microdevice and a microfluidic device. As an important cell type, macrophages secretion triggered by calcium ionophore was chosen for assessing the performances, sensitivity and specificity of the detections. In comparison to some previous evaluations obtained from single-cell measurements, reproducible and relevant determinations were achieved. However, separating emitting cells from the detection area in the microfluidic device seems a better approach to avoid any perturbations of cell behaviors by electrode operations. Furthermore, the investigation of any biological effects during oxidative stress of living cells is facilitated. As a proof of concept, we reported the analysis of the influence of a NO synthase inhibitor during the perfusion culture.