Shanghai State Key Laboratory of Green Chemistry and Chemical Processes, Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, Dongchuan Road 500, Shanghai 200241, China.
Analyst. 2019 Mar 21;144(6):2150-2157. doi: 10.1039/c9an00079h. Epub 2019 Feb 12.
As a reactive oxygen species (ROS), peroxynitrite (ONOO) generated by nitric oxide (NO) and superoxide anion (O˙) 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.
作为一种活性氧（ROS），由一氧化氮（NO）和超氧阴离子（O˙）生成的过氧亚硝酸盐（ONOO）在大脑的生理和病理过程中发挥重要作用。然而，体内缺乏可靠和持久的分析方法仍然是理解大脑中 ONOO 信号通路的一个瓶颈。在这项工作中，开发了一种比率型电化学生物传感器，用于实时监测和准确量化大鼠脑缺血后 ONOO。首先，设计并合成了一种新型有机分子 4-(S-(6-巯基己基)苯并噻唑-6-基)-7-(二乙氨基)-2-(4-(哌嗪基二 ferrocformamide-1-基)苯基)色烯翁（HEMF），其具有对 ONOO 的特异性识别基团和作为电活性基团的 ferrocene，用于高选择性测定 ONOO。随着 ONOO 浓度的增加，ferrocene 的氧化峰减小，响应时间在 15 s 内迅速响应，因为 HEMF 分子中的吡喃鎓基团特异性地与 ONOO 反应，导致 ferrocene 基团从 HEMF 分子中通过开环反应丢失。同时，优化了 5'-MB-GGCGCGATTTT-SH-3'（SH-DNA-MB）作为内参分子，在避免环境影响的同时，能够准确地定量 ONOO。ferrocene 和 MB 的氧化峰电流比与 ONOO 的浓度在 20.0 nM 至 2.0 μM 之间表现出良好的线性关系。所实现的检测限低至 12.1 ± 0.8 nM。由于吡喃鎓基团与 ONOO 之间的特异性反应，开发的生物传感器对大脑中的其他 ROS、金属离子、氨基酸和生物活性物质等潜在干扰具有显著的选择性。因此，本研究中的比率型电化学生物传感器具有出色的分析性能，包括高时间分辨率、高选择性和准确性，结合碳纤维微电极（CFME）的独特特性，如高空间分辨率和良好的生物相容性，成功地应用于实时测定大鼠脑缺血后 ONOO 的浓度。
