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题名: 电化学发光传感器特异性检测DNA损伤
作者: 冯 锐1
学位类别: 博士
答辩日期: 2017-12
授予单位: 中国科学院大学
授予地点: 北京
导师: 郭良宏
关键词: DNA碱基缺失,DNA氧化损伤,电化学发光传感器,传感器阵列,细胞毒性 ; Apurinic/apyrimidinic sites, DNA oxidative damage, Electrochemiluminescence sensor, Sensor array, Cytotoxicity
其他题名: Sensitive Detection of DNA damage by Electrochemiluminescence ensor
学位专业: 环境科学
中文摘要: 作为生物遗传信息的主要载体,DNA的稳定性对于维持生物的机体健康具有至关重要的意义;如果DNA损伤无法得到及时修复,将会引发各种疾病。在细胞的生命过程中,DNA损伤一般来自于内源性因素如碱基的氧化、脱胺基和外源性因素如紫外线、氧化应激、电离辐射或诱变剂等。同时,各种新型污染物层出不穷,研发一种不同污染物的基因毒性的高效筛查工具存在大量的社会需求。结合电化学发光传感器设备简便、检测快速且易于实现高通量等优点,本文首先采用利于微型化的电化学发光方法检测特定的DNA损伤;在此原理基础上,改进仪器设备,运用高通量的电化学发光传感器阵列,建立了一种检测特定DNA损伤的体外、乃至细胞内的特定DNA损伤类型的方法。 DNA常见的特定损伤有碱基缺失、碱基氧化、碱基加合和链的断裂等。其中,碱基缺失(AP sites)作为一种常见的DNA损伤,是由N-糖苷键断裂产生的;DNA链上的嘌呤和嘧啶会自发产生水解,由核糖的磷酸骨架上脱落。外源性损伤如UV、离子辐射、烷基化试剂、某些抗癌药物等能够不同程度地造成碱基脱落形成AP sites;各种N-糖苷键酶也可造成AP sites,在本文中,由于AP sites的DNA寡核苷酸链难于直接合成,研究人员设计了含U的DNA链,用UDG酶对其进行剪切形成数量已知的AP sites。此外,DNA构型中的碱基鸟嘌呤氧化电位及电离势都很低,因此很容易失去电子从而导致自身的氧化,而后发生质子转移、水合、与氧气反应等生成新的物种进而导致DNA氧化损伤,DNA氧化损伤产物之一8-羟基脱氧鸟苷(8-OHdG),常被作为DNA损伤标志物进行研究。 首先,本文构建了一种新型的定量DNA碱基缺失的电化学发光传感器,该传感器使用的化学探针能够特异性共价标记DNA脱碱基位点,同时连接具有电化学发光特性的信号分子,实现对DNA脱碱基位点的特异性定量检测;进而,采用化学物质暴露无损伤DNA链的方法,检测暴露后链上产生的AP sites数目,快速确定化学物质潜藏的基因毒性。传感器通过层层自组装的方式,首先,在金电极表面修饰了DNA单分子层,酶解处理DNA设计序列以形成含有AP sites的DNA标准品;然后,用化学探针对AP sites进行特异性共价标记。研究中,采用循环伏安法、交流阻抗法和计时电流法对金电极表面修饰的DNA膜进行表征,得到其膜密度为2.89×10-12 mol/cm2。采用非变性凝胶电泳法和基体辅助激光解吸离子-飞行时间质谱法分别验证了溶液中ARP探针对DNA中的AP sites的特异性捕获。通过电化学工作站收集电化学发光信号,构建传感器的电化学发光强度测定绘制工作曲线,得到的DNA中AP sites数目与电化学发光强度正相关,能够从512个碱基中检出1个AP site,依据膜密度计算出传感器的检测限达到2.8 fmol。利用该传感器考察了DNA烷基化试剂对DNA的损伤情况。 继而,运用实验室自主研发的电化学发光仪,其以塑料基板作为基底、通过丝网印刷法印刷电极对、具有96个的电极阵列;该仪器实现了96个样品的快速测定,一块阵列的检测时间不超过5 min,极大地提高了检测效率和节省了人力资源。利用该电化学发光传感阵列,本文构建了一种新型的通过定量8-OHdG而检测化学物质毒性的电化学发光方法。本文构建的方法能够快速定量基因组DNA中的8-OHdG,且利于微型化。本方法将DNA样品通过共价作用与包覆有特异性抗体的磁珠结合,然后信号分子标记进行信号检测。具体原理如下:采用抗体特异性结合细胞中的DNA中的8-OHdG,利用钌的配合物嵌入DNA链并产生电化学发光信号,将磁珠在外加磁场的作用下吸附至高通量微孔板,从而实现细胞中8-OHdG的特异性高通量检测。本文计算出抗体在磁珠表面的结合效率>70%;通过斑点实验验证了长链DNA在抗体表面的特异性结合;通过金属离子Fe2+暴露小鼠杂交骨髓瘤细胞SP2/0制备标准DNA样品,同时,采用HPLC/MS/MS对标准品中的8-OHdG含量进行测定;采用高通量电化学发光仪收集电化学发光信号,绘制工作曲线,得到的DNA中8-OHdG与电化学发光强度正相关,检测限达到0.05 nM。利用该方法直接考察了金属离子对细胞中基因组DNA的损伤情况。 再次,在上述方法的基础上,借鉴课题组前期构建的基于精胺钌发光探针特异性定量检测8-OHdG的类似的传感器原理:8-OHdG在弱氧化剂存在条件下形成醌类物质,精胺能够与该醌类物质发生特异性加成反应,连接到DNA链。由此,在阵列电极上构建传感器:磁珠直接捕获细胞中的DNA,精胺钌探针在弱氧化剂存在条件下与DNA中的氧化损伤8-OHdG结合,实现8-OHdG的高通量检测,该方法仍在探索过程中。 本文从细胞外和细胞内分别构建了两种可以特异性定量检测DNA损伤的电化学发光传感器,实现了对DNA损伤AP sites及DNA氧化损伤产物8-OHdG的定量测定,同时利用构建的电化学发光传感器评估了常见化学试剂对DNA的损伤情况。检测AP sites的方法能够实现电极表面fmol级的检测,检测8-OHdG的方法能够实现溶液中nM级的检测,力求满足现实毒性测定的需求,可用于化合物毒性的早期快速筛查。
英文摘要: As the main carrier of biogenetic information, the stability of DNA is of vital importance to the maintenance of organism's health. If DNA damage cannot be repaired in time, a variety of diseases would be occurred. During cell life, DNA damage is usually derived from endogenous factors such as alkaline oxidation, deamine and exogenous factors such as ultraviolet, oxidative stress, ionizing radiation or mutagenesis. Meanwhile, various new pollutants emerge, and there is a large amount of social demand for the development of a highly effective screening tool for the genetic toxicity of different pollutants. Based on the advantages of simple and fast detection of electrochemiluminescence (ECL) sensor, this paper uses the ECL method to detect specific DNA damage firstly. Based on this principle, equipment was improved and high flux ECL sensor array was established. New types of specific DNA damage detection sensor in and out of cells had been made. Nucleic acids experience a variety of perturbations, including depurination/depyrimidines, oxidation, modification, and strand cleavage. Considered to be one of the most abundant lesions in genomic DNA, apurinic/apyrimidinic sites (AP sites), which are lesions in DNA formed by loss of a nucleobase from oxidative stress or carcinogen adducts, result from the cleavage of the Nglycosylic bond that connects the purine or pyrimidine base to the deoxyribose sugar. Generally, AP sites are derived from spontaneous depurination and the action of various physical and chemical agents, including UV and irradiation, ionizing radiation, oxidative stress, mutagens, heterocyclic N-oxides of the tirapazamine family, organometallic oxidants and the anticancer antibiotics. Furthermore, excision of altered nucleotides by glycosylases might be a significant mechanism for generation of AP sites. Among these glycosylases, uracil-DNA glycosylase (UDG) is the most efficient DNA glycosylase and is responsible for the removal of most U residues from DNA. In this paper, because the DNA oligonucleotide chain is difficult to synthesize directly, the researchers have designed the DNA strand containing U, and the enzyme has been used to shear the base of the known base. In addition, the oxidation potential and ionization potential of guanine in DNA are all very low. Guanine is easy to lose electronic leading to oxidation, and then proton transfer, hydration, react with oxygen to generate new species resulting in DNA oxidative damage. As one of DNA oxidative damage product, 8-hydroxy-2’-deoxyguanosine (8-OHdG) is a typical biomarker of DNA oxidative damage. Firstly, a new chemical toxicity sensor was developed based on the ECL quantification of AP sites in a DNA monolayer with a covalent aldehyde reactive probe (ARP). The standard AP sites were generated by enzymatic treatment. Then the AP sites detection was achieved by labeling them with a specific probe. The photoelectric signal was collected by the electrochemical workstation. The final detection limit reached fM grade. The technique described here provides a sensitive and selective method for AP sites quantification and can be applied to screen DNA damages by exogenous chemicals. In the sensor, a uracil-containing DNA duplex was first immobilized on a gold electrode by self-assembly. The duplex was then reacted with uracil-DNA glycosylase (UDG) to convert uracils into AP sites. ARP was employed to tag the AP site with a biotin. After reacting with a ruthenium complex labeled streptavidin, ECL was measured for quantitative analysis. The DNA monolayer was characterized by cyclic voltammetry, electrochemical impedance spectroscopy and chronocoulometry, and its density was measured to be 2.89×10-12 mol/cm2. Characterization of the reaction product between ARP and DNA AP sites in solution by nondenaturing polyacrylamide gel electrophoresis and mass spectrometry confirmed successful biotinylation. ECL intensity of the labeled DNA monolayer on the electrode was found to correlate with the number of AP sites, and the detection limit was estimated to be about 1 lesion in 512 DNA bases, which meant that 2.8 fmol AP bases on the electrode were detected. The damage of DNA alkylation agent to DNA was investigated by using the sensor. The sensor successfully detected the AP sites in normal DNA induced by methylmethane sulfonate (MMS), a carcinogenic chemical. The novel combination of covalent probe and ECL measurement in a sensor configuration therefore provides unique advantages in selectivity and sensitivity, and can be potentially employed in the screening of chemicals for their genetic toxicity. Secondly, the ECL instrument developed independently by the laboratory is in the base of plastic substrate. The electrode array including 96 electrodes is printed by the screen printing method. The instrument realized the rapid determination of 96 samples, and the detection time of an array was less than 5 min, which greatly improved the detection efficiency and saved human resources. By using this ECL sensing array, a novel ECL method to detect the toxicity of chemical substances by quantitative 8-OHdG has established. Currently, technologies for 8-OHdG detection have high performance liquid chromatography - mass spectrometry technology (HPLC/MS/MS), high performance liquid chromatography with electrochemical detection (HPLC/ECD) and enzyme-linked immunoassay. Among them, enzyme-linked immunoassay major determine 8-OHdG in DNA fragments released during DNA repair. Although detect 8-OHdG in genomic DNA, HPLC/MS/MS and HPLC/ECD methods need complicated sample pretreatment and expensive instruments. In this paper, 8-OHdG in genomic DNA can be rapidly quantitative and are easy to be miniaturization. In this method, DNA samples are combined with magnetic beads coated with specific antibody by covalent action, and then signal molecule is linked. The principle is as follows: the antibody specificity combined with 8-OHdG in DNA of the cells. Ruthenium complexes insert double chains to produce ECL signals. Under magnetic field, the magnetic beads are adsorbed to high flux micro pore board, so as to realize the specificity, high-throughput 8-OHdG detection in cells. In this paper, the combined efficiency of antibody and magnetic beads is more than 70%. The specific binding of long strand DNA to antibody is verified by dot experiment. The standard DNA samples are prepared by Fe2+ exposed SP2/0 cells. The content of 8-OHdG in standard products is determined by HPLC/MS/MS. Using high-flux ECL to collect ECL signals, working curve is made. It was found that 8-OHdG is positively correlated with ECL intensity and the detection limit is 0.05 nM. This method is used to investigate the damage of metal ions to the genomic DNA in cells. Thirdly, 8-OHdG would transformed to quinones under the condition of weak oxidant. Spermine can specificity reacted with the quinones connecting to the DNA chain. Thus, in our sensor array, magnetic beads directly coat with the DNA in cells. spermine-Ru probe under the condition of weak oxidant capture DNA oxidative damage (8-OHdG). The high-throughput detection of 8-OHdG has been realized. This method is still under exploration. From extra-cellular and intra-cellular respectively, this paper builds two ECL sensors for specific quantitative detection of DNA damage. AP sites of DNA damage and DNA oxidative damage product 8-OHdG are measured. Meanwhile, toxicity of common chemical reagents is evaluated. AP sites detection can realize fmol level on the electrode surface, and 8-OHdG detection can realize nM level in the solution, all can meet the needs of toxicity measurement in reality. Rapid screening of chemical toxicity can be realized.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/38618
Appears in Collections:环境化学与生态毒理学国家重点实验室_学位论文

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