Multi-Chambers Microsystem for Simultaneous and Direct Electrochemical Detection of Reactive Oxygen and Nitrogen Species Released by Cell Populations

Electrochemical quantification of four reactive oxygen and nitrogen species (e.g., H2O2, ONOO−, NO and NO2−) emitted from macrophages populations was completed using a four-chambers microchip. This microchip was fabricated from a glass substrate equipped with four sets of microband electrodes. Each set consisted of a platinum-black coated working electrode combined to an Ag/AgCl reference and a Pt counter electrodes. A cover made of polydimethylsiloxane was designed with four wells whose open bottoms fitted over each set. Loading, culture and stimulation of cells were conducted within each well. After stimulation of macrophages by a calcium ionophore, amperometric responses were then monitored using a multipotentiostat to detect one species per well simultaneously. The results showed good reproducibility and were quite consistent with averaged amperometric detections obtained from single cell measurements. This methodology allowed drastically diminishing the number of experiments necessary to reach a statistical significance from n=100–160 experiments required for single cell detection compared to n=5 experiments to obtain the mean behavior of the cell population. This work demonstrated that this simple and versatile microchip could be used for quantification of species in oxidative stress investigations on cell populations as well as a complementary tool to compare behaviors between single cell and cell population for investigations involving many experimental variables.