Stability and sensitivity enhanced electrochemical in vivo superoxide microbiosensor based on covalently co-immobilized lipid and cytochrome c.

Enhanced stability and sensitivity of a superoxide anion radical (O(2)(•-)) microbiosensor were achieved through the sequential immobilization of lipid and cytochrome c (Cyt c) covalently bonded onto a conducting polymer layer that showed a clear quasi-reversible direct electron transfer (DET) process. The formal potential and the apparent standard rate constant were determined to be -0.24 V and 0.62 ± 0.05 s(-1), respectively. The detection of O(2)(•-) was attained through the catalytic activity of the haem group of Cyt c stabilized by coimmobilized lipid molecules (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-n-dodecanylamine (DGPD)). The linear dynamic range and the detection limit of the O(2)(•-) analysis were determined to be 0.2-6.0 nM and 30.0 ± 0.9 pM, respectively. The in vivo microbiosensor implanted into rat brain successfully determined the extracellular level of O(2)(•-) produced by acute and repeated injections of cocaine. The present O(2)(•-) microbiosensor could be an effective tool for monitoring the change in extracellular O(2)(•-) levels in response to stimulant drug exposure.