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

Abstract

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−) 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 (ks) of 24±1.1s−1. The formal potential (E0′) of SOD is estimated to be 5±2.5mV vs. Ag|AgCl, because of which the E0 value falls between the redox couples of O2/O2− and O2−/H2O2, indicating that SOD is thermodynamically able to electrochemical catalyze the oxidation of O2− to O2 and the reduction of O2− to H2O2. In addition, experimental results reveal that SOD is stably immobilized on NTA-modified electrode, and still maintains its biocatalytical activity toward O2−. Thus, the electrochemical strategy for detection of O2− has been successfully established by the redox reaction of SOD at NTA-modified electrode followed by the chemical reaction of SOD toward O2− into O2 and H2O2. The optimized O2− biosensor exhibits high selectivity, broad dynamic range from 10−7 to 10−4M, low detection limit of 21nM, 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 O2− in the rat brain during ischemia and reperfusion processes.