Nonenzymatic Electrochemical Sensor with Ratiometric Signal Output for Selective Determination of Superoxide Anion in Rat Brain

There is still an urgent need to develop reliable analytical methods of O₂^•– in vivo for deeply elucidating the roles of O₂^•– playing in the brain. Herein, a nonenzymatic electrochemical sensor with ratiometric signal output was developed for an in vivo analysis of O₂^•– in the rat brain. Diphenylphosphonate-2-naphthol ester (ND) was designed and synthesized as a specific recognition molecule for the selective determination of O₂^•–. An anodic peak ascribed to the oxidation of 2-naphthol was generated via the nucleophilic substitution between ND and O₂^•– and was increased with the increasing concentration of O₂^•–. Meanwhile, the inner reference of methylene blue (MB) was co-assembled at the electrode surface to enhance the determination accuracy of O₂^•–. The anodic peak current ratio between 2-naphthol and MB exhibited a good linear relationship with the concentration of O₂^•– from 2 to 200 μM. Because of the stable molecule character of ND and its specific reaction with O₂^•–, the developed electrochemical sensor demonstrated excellent selectivity toward various potential interferences in the brain and good stability even after storage for 7 days. Accordingly, the present electrochemical sensor with high selectivity, high stability, and high accuracy was successfully exploited in monitoring the levels of O₂^•– in the rat brain and that of the diabetic model followed by cerebral ischemia.