| 题名 | 光电及电化学发光传感器研究环境污染物导致的DNA特异性损伤 |
| 作者 | 吴一萍 |
| 学位类别 | 博士 |
| 答辩日期 | 2014-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 郭良宏 |
| 关键词 | 光电化学DNA传感器 电化学发光DNA传感器 DNA化学甲基化 8-羟基脱氧鸟苷 碱基缺失 DNA糖基化 photoelectrochemical DNA snesor electrochemiluminescence DNA sensor DNA chemical methylation 8-oxo-7 8-dihydro-2'-deoxyguanosine abasic sites DNA glycosylase |
| 其他题名 | Photoelectrochemical and Electrochemiluminescence DNA Sensor for the Detection of Specific DNA Damage Induced by Pollutants |
| 学位专业 | 分析化学 |
| 中文摘要 | DNA是生物体主要的遗传信息载体，它维持物种延续和调控生命过程。然而，无论是生命体正常代谢还是外源性化合物入侵，都有可能破坏DNA分子结构，造成损伤。未能得到及时修复的损伤会影响DNA稳定，进而引发肿瘤、癌症和一系列遗传性疾病。因此有必要建立可以定量检测DNA损伤的分析方法。目前可用于DNA损伤检测的方法有很多，电化学传感器(电化学、电化学发光及光电化学)因其设备简便、检测快速且易于实现高通量等优点吸引了越来越多科研工作者的关注。本文构建了两种可以特异性定量检测DNA损伤的电化学传感器，实现了对DNA化学甲基化损伤及DNA氧化损伤产物8-羟基脱氧鸟苷(8-oxodGuo)的测定。同时利用构建的电化学发光传感器评估了几种常见重金属离子对DNA修复酶E. coli formamidopyrimidine-DNA glycosylase (Fpg)活性的影响。此外，我们正在努力构建可以定量检测DNA碱基缺失的电化学传感器。 外源性烷基化试剂反应活性高，它们可以直接与DNA反应产生有遗传毒性的碱基。利用电化学传感器检测这些有害碱基存在两个难点：1) 甲基基团化学结构小，它们的加入不会破坏DNA双螺旋结构，很难直接检测； 2) 甲基基团化学惰性，不容易先标记后检测。本文的第一份工作发展了一种免标记特异性定量检测DNA化学甲基损伤的光电化学传感器。首先，通过高效液相色谱-质谱(HPLC-MS)检测结果发现DNA经过甲基化试剂甲基甲磺酸酯(MMS)暴露主要产生两种有害碱基，7-甲基鸟嘌呤(metGuo)和3-甲基腺嘌呤(metAde)，且两种碱基的含量随MMS浓度的增大而增加。利用传感器检测DNA化学甲基化损伤，先使用DNA糖基化酶Human Alkyladenine DNA Glycosylase (hAAG)将甲基化的嘌呤从DNA双链中剪切下来形成碱基缺失位点，接着利用脱嘌呤/脱嘧啶核酸内切酶Human Apurinic/Apyrimidinic Endonuclease (APE 1) 水解碱基缺失位点处DNA链中的磷酸二酯键，造成DNA单链断裂。通过两步酶反应，DNA点损伤被放大成链断裂，最后加入光电信号分子Ru(bpy)2(dppz)2+定量测定DNA链断裂。实验结果发现，随着MMS浓度增加，光电信号不断下降，表明DNA膜中有害碱基含量不断增加。对比传感器与HPLC-MS结果，两者相关系数为R2 = 0.943，表明构建的光电化学传感器定量检测DNA化学甲基化损伤非常可靠。该传感器甚至可以检测1 mM MMS导致的DNA损伤。假设表面反应效率和溶液一样，1 mM MMS暴露下，电极表面DNA膜中metAde含量约为42.5 fmol。此外，无论是对含有5-甲基胞嘧啶(5-mC)或8-羟基脱氧鸟苷(8-oxodGuo)的DNA膜，还是对经过其它烷基化试剂暴露的DNA膜，如乙基乙磺酸酯(EMS)和氧化苯乙烯(SO)，传感器都没有响应，表明特异性非常好。对于其它待测碱基，只要能找到对应的DNA糖苷酶，就可以利用该传感器实现检测。 我们实验室前期以精胺-生物素(spermine-biotin,SB)为捕获探针，钌标链霉亲和素(streptavidin-Ru)为信号分子，构建了可以特异性定量检测8-oxodGuo的光电化学传感器。该传感器在检测8-oxodGuo时涉及捕获和信号识别两步反应，步骤较多直接影响检测效率和灵敏度。因此，本文第二份工作就是设计合成一种新型的、双功能化学探针精胺-钌(spermine-Ru)，利用该探针构建了可以特异性定量测定8-oxodGuo的电化学发光传感器，并利用该传感器定量评估了重金属离子对DNA修复酶Fpg活性的影响。设计合成的化学探针由两部分组成，一部分为精胺，在氧化物溶液中精胺可以和8-oxodGuo发生共价反应；另一部分为三联吡啶钌螯合物，它具有优良的电化学发光特性。利用该探针通过一步反应即可实现对8-oxodGuo的捕获和标记。在构建电化学发光传感器时，首先通过巯基自组装的方式在金电极表面固定DNA单分子层。保证电极表面总碱基含量不变的前提下，不断稀释8-oxodGuo浓度，构建的传感器最终实现从500个正常碱基里检测1个8-oxodGuo。传感器检测Fenton反应引起的DNA氧化损伤时发现，100 μM Fe2+/400 μM H2O2浓度Fenton试剂大约可以使500个正常碱基中产生一个8-oxodGuo。Fpg可以有效剪切8-oxodGuo导致电化学发光信号下降。构建的传感器在定量评估重金属离子对Fpg蛋白活性影响时发现，Hg2+和甲基汞(MeHg)可以显著抑制Fpg蛋白的活性，而且两者之前存在良好的剂量-效应关系。利用Scatchard公式最终算得Hg2+和MeHg的半抑制浓度IC50，分别为14.04 μM 和4.34 nM。对于其它几种重金属离子Cd(II)，Pb(II)，Ni(II)和Co(II)，当它们的浓度低于1 mM时，基本不会影响Fpg蛋白的活性。 碱基缺失是一种常见的DNA损伤，它源于碱基与DNA磷酸骨架间N-糖苷键的断裂。碱基缺失主要有两种产生途径：1) DNA与外源性有毒化合物反应生成碱基加合物，加合物不稳定，直接脱落造成碱基缺失；2) DNA碱基切除修复途径中，糖苷酶识别并剪切DNA链中非正常碱基，形成碱基缺失。没有及时修复的碱基缺失会导致基因突变和细胞死亡，甚至引发机体癌变。因此我们尝试构建可以特异性定量检测DNA碱基缺失的电化学传感器。利用带有生物素的醛基反应探针Aldehyde Reative Probe (ARP) 共价捕获DNA中碱基缺失位点，然后以streptavidin-Ru作为信号分子，通过两步反应实现对DNA碱基缺失位点的捕获与标记。对于那些难以直接测定的非正常碱基加合物，可以先利用特异性的糖苷酶将它们转化成碱基缺失位点，然后进行定量测定。碱基缺失位点很不稳定，无法通过化学方法合成含有特定碱基缺失个数的DNA标准品，于是利用尿嘧啶糖苷酶剪切尿嘧啶制备含有特定个数碱基缺失位点的DNA标准品。初步实验表明利用尿嘧啶糖苷酶可以制得含有特定个数碱基缺失位点的DNA标准品。将ARP 探针与通过酶反应制得的含有碱基缺失位点的DNA标准品混合反应，一段时间后进行聚丙烯酰胺凝胶电泳(PAGE)，没有新条带出现。这可能是由于DNA和ARP反应条件没有最优化导致。接下来我们将优化两者的反应条件并进一步确认两者的反应活性。 通过实验室前期的工作加上本论文的研究，我们实现了利用光电及电化学发光传感器快速筛查环境污染物基因毒性和特异性定量检测DNA损伤的目的。这些工作可以为毒理学研究提供新的思路和手段。 |
| 英文摘要 | DNA is the main genetic material for most living organics. It maintains the continuation of species and regulates the process of life. However, endogenous or exogenous sources may destroy the integrity of cellular DNA at any time. If unrepaired, the unstable DNA will result in a series of consequences, including tumor, cancer and inherited diseases. Therefore, it is necessary to develop methods for DNA damage analysis. With the distinctive advantages of simplicity, miniaturization and low instrument cost, electrochemical sensors have attracted the most attention for DNA damage detection recently. In this paper, we have constrcucted two novel bisosensors. They were used for quantification of DNA chemical methylation damage and 8-oxo-7,8-dihydro-2'-deoxyguanosine(8-oxodGuo) respectively. The latter was also applied to envaluate the effect of heavy metal ions on the activity of DNA repair enzymes quantitatively. Meanwhile, we are trying to develop another specific biosensor to quantify the abasic site. Exogenous chemicals may produce DNA methylation that is potentially toxic to living systems. Since the methyl group is small and chemically inert, methylated DNA bases are difficult to detect with biosensors. In the first section, a label-free photoelectrochemical sensor was developed for the selective detection of chemically methylated bases in DNA films. The sensor employed two DNA repair enzymes, human alkyladenine DNA glycosylase (hAAG) and human apurinic/apyrimidinic endonuclease (APE 1), to convert DNA methylation sites in DNA films on indium tin oxide electrodes into strand breaks. A DNA intercalator, Ru(bpy)2(dppz)2+ (bpy=2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine) was then used as the photoelectrochemical signal indicator to detect the DNA strand breaks. Its photocurrent signal was found to correlate inversely with the amount of 3-methyladenines (metAde) produced with a methylating agent, methylmethane sulfonate (MMS). The sensor detected the methylated bases produced with as low as 1 mM MMS, at which the amount of metAde on the sensor surface was estimated to be 42.5 fmol. Other DNA base modification products, such as 5-methylcytosine (5-mC), 8-oxodGuo and DNA adducts with ethyl and styrene groups, did not attenuate the photocurrent, demonstrating good selectivity of the sensor. This strategy can be utilized to develop sensors for the detection of other modified DNA bases with specific DNA repair enzymes. In the previous work, we have developed a photoelectrochemical DNA sensor for quantification of 8-oxodGuo by using spermine-biotin as a specific label in conjunction with a photoelectrochemical signal reporter. Though the sensor was available to quantify 8-oxodGuo, the labeling process before the detection was complicated and time-consuming. In my second work, a spermine conjugated ruthenium tris (bipyridine) derivative (spermine-Ru) was synthesized which could effectively trap 8-oxodGuo through a one-step reaction and acted as a signal reporter as well. On basis of the probe, a sensitive and selective electrochemiluminescence (ECL) sensor was developed for 8-oxodGuo selective quantification. In the sensor, a lesion-containing DNA film was deposited on a gold electrode surface through thiol self-assembly. The luminescence signal was found to be depended on the amount of 8-oxodGuo on the surface and the final estimated detection limit was about 1 lesion in 500 bases or 5 fmol of 8-oxodGuo. The sensor was used to assess oxidatively damaged DNA, finding there was one 8-oxodGuo lesion in 500 bases after the DNA film exposure to 100 μM Fe2+/400 μM H2O2. Moreover, the impact of metal compounds on the activity of E. coli formamidopyrimidine-DNA glycosylase (Fpg) was investigated with the sensor. The luminescence signal was founded to recover gradually when Fpg was pre-incubated with different concentrations of the metal compounds. This suggested that inactivation of Fpg might be the mechanism accounting for the inhibition of oxidatively damaged DNA repair in the presence of toxic metal compounds. The obtained result indicated that Hg (II) and methylmercury (MeHg) inhibited the activity of Fpg significantly with IC50 values of 14.04 μM and 4.34 nM, respectively. But the activity of Fpg was not affected by Cd (II), Pb (II), Ni (II) and Co (II) when their concentrations were less than 1 mM. Abasic sites (apurinic/apyrimidinic or AP sites) are common lesions in DNA which are resulted from the cleavage of N-glycosylic bond between the bases and the sugar-phosphate backbone. Abasic sites can be formed by spontaneous depurination of the unstable adducts,but also occur as intermediates in base excision repair. If unrepaired, the abasic sites may be harmful to the organics. So development of methods for Abasic sites detection is urgent demanded. We are trying to construct a biosensor for Abasic site quantification. The aldehyde reative probe was employed to trap Abasic site and ruthenium tris (bipyridine) labeled streptavidin was introduced as a signal reporter. The assay might be generally applicable to the quantification of other tough lesions as long as the lesions can be converted into Abasic site with the help of the DNA glycosylase. Using uracil DNA glycosylase, we have obtained the standard DNA which contaning different number of Abasic sites. But after the incubation of the standaed DNA with the probe, no new lag band appeared in the polyacrylamide gelelectrophoresis (PAGE) experiment. The work is still in progress. Under our continuous effort, we have constructed a variety of biosensors for assessing the genotoxicity of chemicals and qutifiying the specific DNA damage. All these may provide new strategies and methods for the research of toxicology. |
| 公开日期 | 2015-07-07 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/15629]  |
| 专题 | 生态环境研究中心_环境化学与生态毒理学国家重点实验室 |
| 推荐引用方式 GB/T 7714 | 吴一萍. 光电及电化学发光传感器研究环境污染物导致的DNA特异性损伤[D]. 北京. 中国科学院研究生院. 2014. |
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