Chen Jian-Bo, Zhang Hui-Xian, Guo Xiao-Feng, Wang Hong, Zhang Hua-Shan
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan, P. R. China.
Institute of Chemical Materials, CAEP, Mianyang, P. R. China.
Electrophoresis. 2016 Feb;37(4):609-15. doi: 10.1002/elps.201500341. Epub 2015 Dec 9.
Precise measurement of nitric oxide (NO) is of great importance to understand the function of NO in liver and the mechanism of liver injury. 8-(3',4'-Diamino phenyl)-3,5-(2-hydroxyphenyl)-dimethylene pyrrole (BOPB), a fluorescent probe in the red region (>600 nm) newly developed in our group, has good photostability and excitation/emission wavelength of 622/643 nm matching well with commercial 635 nm semiconductor laser of CE-LIF detection. Therefore, BOPB was used in CE-LIF for the determination of NO in mice liver. Both derivatization and separation conditions were optimized. Derivatization reaction of BOPB and NO was carried out in pH 7.4 PBS buffer at 35°C for 12 min and the separation of NO derivative of BOPB (BOPB-T) was achieved within 7.0 min in pH 9.0 running buffer containing 15 mM H3 BO3 -NaOH and 15 mM SDS. Good linearity was found in the range of 1.0 × 10(-9) -5.0 × 10(-7) M with the LOD of 0.02 nM. The proposed method was applied to the analysis of NO in real samples, and NO concentration was obviously increased in acute liver injury of mice. Compared to existing derivatization-based CE-LIF methods for NO, this method has lower LOD and less background interference owing to detection wavelength of BOPB in the red region.
精确测量一氧化氮（NO）对于理解NO在肝脏中的功能以及肝损伤机制具有重要意义。8-(3',4'-二氨基苯基)-3,5-(2-羟基苯基)-二亚甲基吡咯（BOPB）是我们小组新开发的一种红色区域（>600 nm）的荧光探针，具有良好的光稳定性，其激发/发射波长为622/643 nm，与CE-LIF检测的商用635 nm半导体激光器匹配良好。因此，BOPB被用于CE-LIF法测定小鼠肝脏中的NO。对衍生化和分离条件进行了优化。BOPB与NO的衍生化反应在pH 7.4的PBS缓冲液中于35°C进行12分钟，在含有15 mM H3BO3-NaOH和15 mM SDS的pH 9.0运行缓冲液中，7.0分钟内实现了BOPB的NO衍生物（BOPB-T）的分离。在1.0×10(-9)-5.0×10(-7) M范围内发现良好的线性关系，检测限为0.02 nM。该方法应用于实际样品中NO的分析，小鼠急性肝损伤时NO浓度明显升高。与现有的基于衍生化的CE-LIF法测定NO相比，该方法由于BOPB在红色区域的检测波长而具有更低的检测限和更少的背景干扰。
