苯乙烯致DNA间接损伤效应的光电化学生物传感器快速检测

Enzyme-Incorporated Photoelectrochemical Sensors for the Rapid Detection of DNA Damage Induced by Styrene

许多有机化合物自身没有致癌毒性,但进入生物体内,经过体内代谢酶的催化后转化成活性中间体,与DNA形成共价化合物.这些间接的损伤会最终导致DNA分子结构和功能的变化,对人类的健康造成威胁.因此,建立一种快速有效的方法检测间接致癌化合物对DNA的损伤,成为当前研究的热点.论文建立了一种新型的光电化学生物传感器来检测有机化合物苯乙烯对DNA的间接损伤效应,该传感器以层层自组装的方式将光电信号分子、双链DNA和血红蛋白组装在半导体电极上.在H2O2存在的条件下,传感膜中的血红蛋白可将苯乙烯转化为氧化苯乙烯,氧化苯乙烯扩散到膜内,与DNA形成加合物,引起DNA结构变化,导致光电分子光电流信号的增加.实验中,将修饰好的电极置于终浓度为2mM H2O2和2%苯乙烯(体积比)的混合液(pH7.3的磷酸缓冲液配制)中反应一段时间后,在电解质溶液中进行光电流检测.实验结果表明,光电流信号随着反应时间逐渐升高,在30min后趋于稳定,表明苯乙烯在传感膜上的氧化和DNA的损伤反应基本完成.与反应前相比,反应后光电流增加了40%,并且酶催化苯乙烯生成的氧化苯乙烯经紫外可见光谱得到验证.论文建立的光电化学生物传感器模拟了体内DNA损伤反应过程,能快速有效地检测苯乙烯对DNA的间接损伤效应,有望为有机化合物潜在基因毒性的风险评估提供一个快速筛查工具。

Although many chemicals have been found not to be direct carcinogens, in vivo some of them are transformed by metabolizing enzymes into active intermediates and form covalent adducts with DNA which are deleterious to human health. It is therefore of great significance to establish a fast and effective method to detect DNA damage induced by indirect carcinogens. In this paper a novel enzyme-incorporated photoelectrochemical sensor was developed for the detection of DNA damage indirectly induced by styrene. The multi-layer sensing film was constructed on tin oxide electrodes using layer-by-layer electrostatic self-assembly, and consisted of photoelectrochemical signal indicator, double-stranded DNA and hemoglobin. In the presence of hydrogen peroxide, hemoglobin in the sensor film catalyzes styrene into styrene oxide, which reacts with the bases of ds-DNA in the same film to form covalent adducts. This results in conformation change of DNA and consequently increased photocurrent. In the experiment, the sensor was incubated in mixture of H202 （terminal concentration： 2mM）and styrene （terminal concentration： 2%, volume to volume）（the mixtrue was prepared with PBS of pH 7.3）for DNA damage, and then detected in electrolyte. The results showed that the photocurrent increased with the incubation time and reached its maximum after 30rain, when the signal was 40% higher than before the damage. Conversion of styrene into styrene oxide by hemoglobin in the presence of hydrogen peroxide was confirmed by UV-Vis absorbance measurement. It can be concluded that this photoelectrochemical sensor can effectively detect DNA damage induced indirectly by styrene and has the potential of becoming a screening tool for the rapid assessment of the genotoxicity of organic compounds.