Real-time monitoring of peroxynitrite (ONOO^-) in the rat brain by developing a ratiometric electrochemical biosensor

As a reactive oxygen species (ROS), peroxynitrite (ONOO^-) generated by nitric oxide (NO) and superoxide anion (O₂^-) plays important roles in physiological and pathological processes in the brain. However, the lack of reliable and durable analytical methods in vivo is still a bottleneck to understanding the signal pathway of ONOO^- in the brain. In this work, a ratiometric electrochemical biosensor was developed for real-time monitoring and accurate quantification of ONOO^- in the rat brain followed by cerebral ischemia. Firstly, a novel organic molecule, 4-(S-(6-mercaptohexyl)benzothioate-6-yl)-7-(diethylamino)-2-(4-(piperazinyl diferroformamide-1-yl)phenyl)chromenylium (HEMF), with a specific recognition group toward ONOO^- and ferrocene as an electroactive group, was designed and synthesized for determination of ONOO^- with high selectivity. The oxidation peak of ferrocene decreased with increasing concentration of ONOO^-, with a rapid response within 15 s, because the pyrylium group in HEMF molecule specifically reacted with ONOO^-, resulting in the loss of the ferrocene group from the HEMF molecule through a ring-opening reaction. Meanwhile, 5′-MB-GGCGCGATTTT-SH-3′ (SH-DNA-MB) was optimized as an inner reference molecule, enabling accurate quantification of ONOO^- while avoiding environmental effects. The oxidation peak current ratio between ferrocene and MB demonstrated good linearity with concentrations of ONOO^- from 20.0 nM to 2.0 μM. The achieved detection limit was as low as 12.1 ± 0.8 nM. The developed biosensor showed remarkable selectivity against potential interferences in the brain, such as other ROS, metal ions, amino acids and bioactive species, due to the specific reaction between the pyrylium group and ONOO^-. As a result, the present ratiometric electrochemical biosensor with significant analytical performance, including high temporal resolution, high selectivity and accuracy, combined with the unique characteristics of carbon fiber microelectrode (CFME), such as high spatial resolution and good biocompatibility, was successfully applied in real-time determination of ONOO^- in rat brains followed by global cerebral ischemia.