Simultaneous intra- and extracellular superoxide monitoring using an integrated optical and electrochemical sensor system

A new integrated optical and electrochemical sensor system for simultaneous monitoring of intra- and extracellular superoxide (O₂^-) was developed using an array-based cell chip. For in vitro assays, A172 human glioblastoma cells were transferred into the cell chip and stimulated by phorbol 12-myristate 13-acetate (PMA). Intracellular O₂^- generation was detected via fluorescence image analysis with a dye probe, dihydrorhodamine 123 (DHR 123). Extracellular O₂^- was detected using an amperometric sensor constructed by immobilisation of cytochrome c using a binder, 3,3′-dithiobis(sulphosuccinimidylpropionate), to attach the redox protein onto the surface of electrodeposited Au electrodes incorporated into the optically transparent cell chip. The simultaneous intra- and extracellular production of O₂^- was successfully observed from PMA-stimulated A172 cells and inhibited by superoxide dismutase (SOD). The quantification of O₂^- concentration based on a mathematical model study and possible applications using the sensor system developed were discussed. The results confirm that there was no detectable interference or crosstalk between the optical and electrochemical assays. Feasibility of the integration of the two methods, optical and electrochemical, and the neutralisation of the intra- and extracellular O₂^- levels by SOD have been demonstrated.