用于核酸损伤和化合物基因毒性检测的新型DNA电化学传感器

利用电化学方法检测DNA损伤和污染物基因毒性具有重要意义．根据分子膜层层自组装的原理,将受化学损伤的小牛胸腺DNA固定于氧化铟锡电极表面,制备出核酸传感器界面．使用DNA嵌入剂二联吡啶二吡啶并3,2- a;2’,3’-c]吩嗪钌Ru(bpy)2dppz2+]作为电信号指示剂,与核酸膜结合后,进行电化学检测．由于嵌入剂具有很高的DNA结合能力和双链特异性,与DNA的结合数量在损伤前后发生变化．在电化学检测中,采用已研究成功的高倍数信号放大机制,用电子给予体草酸还原Ru(bpy)2dppz3+,使之循环产生电流信号．这样,嵌入剂与DNA结合数目的微量变化得到灵敏检测．在工作中,分别采用石英晶体微天平和电化学方法对DNA的表面固定进行了表征,计算出未损伤DNA的固定量为3．2 ng·mm-2,损伤DNA的固定量为4．2 ng·mm-2．对两种核酸膜进行电化学检测,发现在信号放大剂草酸溶液中,损伤DNA的电流信号显著高于未损伤DNA,前者是后者的2．2倍．通过荧光分析,得到Ru(bpy)2 dppz2+与损伤DNA的结合常数(K=1．07×107 M-1)和结合比(0．68)．嵌入剂与损伤DNA结合物的荧光强度是未损伤DNA 的1．5倍,说明损伤后嵌入剂结合量增加了1．5倍．损伤DNA电化学检测信号增高的主要原因是由于信号指示剂嵌入电极表面核酸数量的增加．利用高结合能力、高选择性的电化学嵌入剂,结合高倍数信号放大机制,可以对核酸损伤进行灵敏检测．

Electrochemical DNA Sensor for the Detection of DNA Damage Chemical Toxicity

DNA damage can be formed through reactions with environmental pollutants directly or indirectly, leading to carcinogenesis and mutagenesis in organism. So the detection of DNA damage and chemical loxicity of pollutants are of great significance. Among various methods, electrochemical method has become the preferential one for sensor detection due to its advantages of simple, rapid and sensitive detection. In this report, calf thymus DNA was immobilized onto indium-doped tin oxide electrode through layer-by-layer selfassembly, which functioned as the interface of an electrochemical DNA sensor. A DNA intercalator, Ru (bpy)2dppz2+ (bpy =2, 2'-bipyridine, dppz = dipyrido 3, 2-a; 2',3' -c] phenazine) was selected as electrochemical indicator to react with the DNA layer. Electrochemical measurement was performed with the help of a signal amplified system in which oxalate serving as sacrifice electron donor that reduces Ru(bpy)2dppz3+ to Ru(bpy)2dppz2+and amplified the signal. The combination of high-affinity, high-selectivity indicator and signal amplification mechanism permitted the detection of small changes in DNA binding after it was damaged by styrene oxide. In the work, quartz crystal microbalance (QCM) and electrochemical method were used to verify the immobilization of DNA layer, and determine the amount of DNA immobilized. The amount of immobilized DNA was estimated by QCM to be 3.2 ng·mm-2 for intact DNA and 4.2 ng·mm-2 for damaged DNA respectively. Electrochemical signal of the intercalator bound to damaged DNA film was found to be about 2.2 times higher than the intact DNA. The binding constant and binding ratio of Ru(bpy)2dppz2+ to damaged DNA were obtained for the first time by fluorescence, which are K=1.07×107 M-1and 0.68 base pair per intacalator molecule respectively. The fluorescent intensity of Ru-dppz bound to damaged DNA is about 1.5-fold higher than intact DNA, suggesting that the amount of bound Ru-dppz increased 1.5 times after DNA damage. Therefore, the difference of electrochemical signals is mainly assigned to the change of the amount of Ru-dppz bound to DNA before and after damage.