线粒体定位双光子荧光探针用于小鼠大脑炎症过程中H2O2的检测  

Mitochondria Located Two -Photon Fluorescent Probe for Detection of Hydrogen Peroxide in Mouse Brain Inflammation Process

作　　者：翟保评[1] 郝瑞林 何军 胡伟[2] 刘志洪[2] ZHAI Bao-ping;HAO Rui-lin;HE Jun;HU Wei;LIU Zhi-hong(Department of Chemistry,Xinzhou Teachers University,Xinzhou 034000;Key Laboratory of Analytical Chemistry for Biology and Medicine(Ministry of Education),College of Chemistry and Molecular Sciences,Wuhan University,Wuhan 430072)

机构地区：[1]忻州师范学院化学系,山西忻州034000 [2]武汉大学生物医学分析化学教育部重点实验室,化学与分子科学学院,湖北武汉430072

出　　处：《分析科学学报》2019年第5期529-537,共9页Journal of Analytical Science

基　　金：国家自然科学基金(No.21535005);忻州师范学院重点学科建设基础项目(No.XK201305)

摘　　要：本文利用具有线粒体定位功能的双光子荧光探针Mito-H2O2,实现对生物体中H2O2的高灵敏检测。在单光子和双光子模式下分别对探针和荧光团进行了检测,发现在弱碱性条件下,H2O2能够与探针硼酸酯发生亲核取代反应,得到荧光团Mito-green(neutral form),随即在弱碱性环境下去质子化形成Mito-green(anion form),通过增强分子内电荷转移(ICT)发出强的绿色荧光。三苯基膦基团能很好地通过细胞膜,定位于线粒体内膜上。实验结果表明,探针与H2O2反应后,Mito-H2O2最大发射峰的相对发射强度在60 min内增加了12倍(540 nm),最大双光子活性截面为20 GM(810 nm)。该探针检出限低至412 nmol/L,并且选择性高、细胞毒性低、光稳定性好。实验还考察了脂多糖(LPS)诱导神经免疫过程中小鼠大脑炎症过程中H2O2浓度的变化,发现LPS能够有效刺激小鼠大脑中H2O2的增加,加入抗炎药物NS-398能有效缓解小鼠大脑神经免疫。In this paper, a two-photon fluorescence probe Mito-H2O2 with mitochondrial localization is used to realize the high-sensitivity and real-time monitoring of hydrogen peroxide in organisms.Probe and fluorophore were detected in one photon and two-photon modes,respectively.It was found that under weak alkaline conditions,hydrogen peroxide reacted with borate of the probe to form a fluorophore Mito-green(neutral form),which was then deprotonated in a weakly alkaline environment to form Mito-green(anion form),releasing strong green fluorescence by enhancing intramolecular charge transfer(ICT).The triphenylphosphine group can pass well through the cell membrane and localize to the mitochondrial inner membrane.The experimental results show that the relative emission intensity of the maximum emission peak of Mito-H2O2 increases by 12-fold(540 nm) in 60 min after the probe reacts with hydrogen peroxide,and the maximum two-photon active cross section is 20 GM(810 nm).The probe has high sensitivity(detection limit as low as 412 nmol/L),high selectivity,low cytotoxicity and good light stability.We then examined the changes of the concentration of hydrogen peroxide in the brain of mice induced by LPS.It was found that LPS can effectively stimulate the increase of hydrogen peroxide in the brain of mice with a concentration-dependent manner. Our results also show that the addition of anti-inflammatory drug NS-398 can effective relieve neuroimmunity.

关 键 词：线粒体 双光子荧光 过氧化氢 神经免疫 抗炎药物NS-398 

分 类 号：O657.39[理学—分析化学]