Superoxide radical plays an important role in cell signaling. However, certain events can result in a large increase in superoxide concentration which has been linked to, among other conditions, inflammation, neurodegenerative diseases, and cancer. Consequently, in vivo detection of superoxide is of great interest. Previously, due to brittleness, instability, or size, superoxide sensors have been limited in their ability for in vivo work. We report the development of a flexible, micron-scale, superoxide sensor. Thin gold films are patterned on Kapton (TM) to form multiple electrodes that constitute the sensor. Cytochrome c was covalently anchored to the working electrode using a self-assembled monolayer of 3,3'-Dithiodipropionic acid di(N-hydroxysuccinimide ester). Calibration showed a linear response within the constraints imposed by using xanthine/xanthine oxidase as the superoxide source. Testing demonstrated that interference from physiological levels of NADH, citric acid, and uric acid to be insignificant. However, minor interference was seen in the presence of H(2)O(2) and glucose, and significant interference arose from ascorbic acid, a known radical scavenger. Qualitative observations provide insight into the preparation and cleaning of thin layer gold on Kapton (TM). (C) 2011 Elsevier B.V. All rights reserved.