Superoxide microsensor integrated into a Sensing Cell Culture Flask microsystem using direct oxidation for cell culture application

• Fully integrated superoxide sensor for the measurement of free radical release from stimulated T-47D breast cancer cells.
• Enzymeless amperometric sensing of superoxide radical in complex media by direct oxidation on gold electrodes.
• Polymer membrane inhibits unspecific adsorption by molecular weight cut-off of interfering substances.
• Integration into conventional tissue culture flask enables direct cell growth on-chip and measurement of pericellular superoxide in situ.
• Thin-film microsensor on transparent substrate for optical inspection of the cells.

A new electrochemical sensor system for reliable and continuous detection of superoxide radical release from cell culture was developed utilizing direct oxidation of superoxide on polymer covered gold microelectrodes. Direct superoxide oxidation was demonstrated to provide robust measurement principle for sensitive and selective reactive oxygen species (ROS) quantification without the need for biocomponent supported conversion. Sensor performance was investigated by using artificial enzymatic superoxide production revealing a sensitivity of 2235 A M−1 m−2. An electrode protection layer with molecular weight cut-off property from adsorbed linear branched polyethylenimine was successfully introduced for long term and selectivity improvement. Thin-film based sensor chip fabrication with implemented three-electrode setup and full integration into the technological platform Sensing Cell Culture Flask was described. Cell culturing directly on-chip and free radical release by phorbol-12-myristate-13-acetate (PMA) stimulation was demonstrated using T-47D human breast cancer carcinoma cell model. Transient extracellular superoxide production upon stimulation was successfully observed from amperometric monitoring. Signal inhibition from scavenging of extracellular superoxide by specific superoxide dismutase (SOD) showed the applicability for selective in vitro ROS determination. The results confirm the possibility of direct superoxide oxidation, with exclusion of the main interfering substances uric acid and hydrogen peroxide. This offers new insights into the development of reliable and robust ROS sensors.