NTA-modified carbon electrode as a general relaying substrate to facilitate electron transfer of SOD: Application to in vivo monitoring of O2•- in a rat brain

Nitrilotriacetic acid (NTA)/histidine-tag (HT) technology has been first employed to facilitate the electron transfer of superoxide dismutase (SOD), and further been developed for in vivo monitoring of superoxide anion (O2(center dot-)) in a rat brain during cerebral ischemia/reperfusion processes. Direct electron transfer of SOD is greatly enhanced at NTA-modified electrode with a high rate constant (k(s)) of 24 +/- 1.1 s(-1). The formal potential (E-0') of SOD is estimated to be 5 +/- 2.5 mV vs. Ag vertical bar AgCl, because of which the E-0 value falls between the redox couples of O-2/O-2(center dot-) and O-2(center dot-)/H2O2, indicating that SOD is thermodynamically able to electrochemical catalyze the oxidation of O-2(center dot-) to O-2 and the reduction of O-2(center dot-) to H2O2. In addition, experimental results reveal that SOD is stably immobilized on NTA-modified electrode, and still maintains its biocatalytical activity toward O-2(center dot-). Thus, the electrochemical strategy for detection of O-2(center dot-) has been successfully established by the redox reaction of SOD at NTA-modified electrode followed by the chemical reaction of SOD toward O-2(center dot-) into O-2 and H2O2. The optimized O-2(center dot-) biosensor exhibits high selectivity, broad dynamic range from 10(-7) to 10(-4) M, low detection limit of 21 nM, and good stability and reproducibility. By taking the advantages of the developed strategy, as well as the characteristic of carbon material including biocompatible and easy to miniaturize, a reliable platform has been constructed for in vivo assaying of in the rat brain during ischemia and reperfusion processes. (c) 2012 Elsevier B.V. All rights reserved.