Real-time monitoring of metabolically relevant biochemicals released in minuscule amounts is of utmost diagnostic importance. Superoxide anion as a primary member of reactive oxygen species, has physiological and pathological effects that depend on its concentration and release rate. Here we present fabrication and successfully testing of a highly sensitive electrochemical biosensor featuring a three-dimensional macroporous mesh of nanoporous gold tailored to measure the dynamics of extracellular superoxide concentration. Wide and accessible surface of the mesh combined with high porosity of the thin nanoporous gold coating enables capturing the analyte in pico- to nano-molar ranges. The mesh is functionalized with cytochrome-c (cyt-c) and incorporated as a working electrode to measure the release rate of drug-induced superoxides from C2C12 cells through a porous membrane. The device displays a considerably improved superoxide sensitivity of 7.29nAnM-1cm-2 and a low level of detection of 70pM. Such sensitivity is orders of magnitude higher than any similar enzyme-based electrochemical superoxide sensor and is attributed to the facile diffusion of the analyte through the well-spread nanofeatured gold skin. Superoxide generation rates captured from monolayer myoblast cultures containing about 4×104 cells, varied from 1.0 to 9.0nMmin-1 in a quasi-linear fashion as a function of drug concentration. This work provides a platform for the development of highly sensitive molecular electrochemical biosensors.
Keywords: Hierarchical nanostructure; Noninvasive electrochemical biosensor; Reactive oxygen species; Skeletal muscle cells; Superoxide anion monitoring.
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