Online Monitoring of Superoxide Anions Released from Skeletal Muscle Cells Using an Electrochemical Biosensor Based on Thick-Film Nanoporous Gold

Online detection and accurate quantification of superoxide anions released from skeletal muscle tissue is important in both physiological and pathological contexts. Above certain physiologically redundant levels, superoxides may exert toxic effects. Here we present design, fabrication, and successful testing of a highly sensitive electrochemical superoxide biosensor based on nanoporous gold (NPG) immobilized with cytochrome-c (cyt-c). A significant 14-fold enhancement in the biosensor sensitivity was achieved using NPG instead of nonporous gold, enabling the device to quantify minuscule levels of superoxides. Such improvement was attributed to the very large surface-to-volume ratio of the NPG network. The average values of superoxide sensitivity and analytical limit of detection (LOD) were 1.90 ± 0.492 nA nM–1 cm–2 and 3.7 nM, respectively. The sensor was employed to measure the rates of superoxide release from C2C12 myoblasts and differentiated myotubes upon stimulation with an endogenous superoxide-prod...