【作者】 葛晨晨；

【导师】 曾令文；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 铜是生物体所必须的微量元素之一,其浓度高低直接影响人们的健康。在人体中铜离子主要以作为许多酶和蛋白(如超氧化物歧化酶、细胞色素氧化酶、多巴胺p-羟化酶、酪氨酸酶和铜蓝蛋白等)的催化辅助因子或结构组成部分来参与细胞中的新陈代谢过程,人体仅仅需要微量的铜就可以维持正常的生命活动。但是,铜缺乏或铜过量都会对健康产生不利的影响。铜缺乏一般伴随着其他营养元素的缺乏或者其生物拮抗物质的摄取过量,影响细胞内许多酶的正常功能,进而影响细胞的新陈代谢过程。铜过量通常是由于遗传性疾病或者由于环境重金属铜污染,误食了大量含铜的食物或吸入了含铜量高的气体所造成。人类的活动造成铜离子的大气污染和水体污染,最终都会回归到土壤中造成土壤污染,进而影响农作物的产量和质量。食物中的铜离子通过食物链的逐级放大过程,最终进入人体,对人类的健康产生危害。肝脏是储存铜离子的重要场所,也是铜排入胆汁的重要器官,铜过量会影响肝肾的正常代谢,造成胃肠道功能紊乱和溶血性贫血等；过多的铜离子若在脑、心脏等处沉淀,会造成神经退行性疾病和全身性的症状,包括威尔逊氏综合症和阿尔茨海默氏症。鉴于铜离子对人类健康,生态系统和食品安全方面会产生巨大的危害,在检测铜离子方面已投入了大量工作。美国环境保护局规定饮用水中铜离子的含量不能超过20μM。传统的用于检测重金属离子的方法主要有石墨烯原子吸收光谱法和电感耦合等离子原子发射光谱法。这两种方法对于重金属离子的检测准确可靠,但使用的仪器价格昂贵,需要经验丰富的技术人员来操作,因而限制了其在基层实验室的广泛应用。近年来,各种检测重金属离子的生物传感器不断出现,包括：荧光法,基于胶体金的比色法和动力学光散射法等。虽然与传统的检测方法相比,这些方法操作简便,检测灵敏度高,但是需要用到蛋白酶或者荧光标记的核酸,增加了检测的成本和反应体系的复杂性。因此,有必要构建一种简单、经济、快速的检测铜离子的生物传感器。真核生物基因组主要携带两种遗传学信息,一类是传统意义上的遗传信息,即DNA序列所携带的遗传信息,另一类指的是表观遗传学信息,不涉及DNA序列的改变,是可逆的并且可遗传的基因表达调控。组蛋白甲基化修饰是一种重要的表观遗传学机制,通常发生在组蛋白N末端赖氨酸和精氨酸残基上,具有广泛的生物学功能,与异染色质的形成、X染色体失活、转录调节、干细胞的维持和分化以及肿瘤的形成有关,这往往取决于组蛋白被甲基化的位置和甲基化的程度。近年来,伴随着表观遗传学的发展,一些检测组蛋白甲基化的技术随之产生,如染色质免疫共沉淀技术,或与PCR和生物芯片联合使用,以及western blot。这些检测方法准确可靠,但操作复杂,工作量大,因此,构建一种操作简便的生物传感器用于组蛋白甲基化的快速检测十分必要。本论文基于具有催化活性的脱氧核糖核酸酶构建了两种用于铜离子检测的生物传感器,包括以DNA自组装的非酶扩增荧光生物传感器以及基于点击化学和G四聚体的比色生物传感器；以单链DNA标记的胶体金为信号增强探针,构建了一种新型的信号增强的试纸条生物传感器,可用于组蛋白甲基化的快速检测。具体包括以下三方面的内容：(1)利用自组装的双链DNA串联体和SYBR Green Ⅰ染色法构建了非酶扩增的铜离子检测方法。反应机制基于铜离子能够特异性地识别并切断与铜离子依赖型DNA酶链杂交的底物链,释放出的靶核酸能够启动DNA自组装为长的双链DNA, SYBR Green Ⅰ染色后检测荧光强度。实验中,检测信号的放大不需要使用蛋白酶,实验操作简单方便。对铜离子的检测限为12.8pM,远低于美国环境保护局规定的饮用水中最大允许量20gM。(2)利用基于点击化学和具有辣根过氧化物酶活性的G四聚体的比色法构建了一种铜离子快速检测生物传感器。在反应体系中使用点击化学使修饰有叠氮基团和炔基基团的两条DNA序列形成G四聚体形成序列,之后加入高铁血红素和KCl,形成高铁血红素/G四聚体结构。由于高铁血红素/G四聚体具有HRP的活性,能催化其无色底物3,3’,5,5’-四甲基联苯胺(Tetramethylbenzidine, TMB)形成有颜色的底物,实验结果通过肉眼就能判别。定量分析时,将反应终溶液转移到酶标板中,用酶标仪读取溶液在450nm的光密度(Optical density, OD)值。对铜离子检测限为5.9nM,远低于美国环境保护局规定的饮用水中铜离子浓度的最大允许浓度20μM。检测体系简单迅速,特异性好,不受其他物质的干扰,结果肉眼可读,适合在基层实验室使用。(3)使用核酸标记的胶体金作为信号增强探针,构建了一种信号增强的试纸条生物传感器可用于组蛋白甲基化的快速、灵敏检测。传感器中用到了两种带有不同标记的胶体金颗粒：包括标记有单链DNA的金颗粒(信号增强探针)和同时标记有单链c-DNA和单克隆抗体的金颗粒(双标金颗粒),其中DNA的序列与c-DNA互补。实验时,首先通过双抗夹心法捕获蛋白,在检测线上形成三明治夹心复合物的结构,即抗体-蛋白-双标的金颗粒,则检测线因为胶体金颗粒的聚集而显红色。接着,再加入信号增强探针,这种金颗粒通过DNA和c-DNA的杂交而被固定在检测线区域,此时有更多的胶体金聚集,检测线的显色被加深。HeLa细胞中的组蛋白H3K9m3(tri-methylated lysine9of histone H3)被选来作为信号增强的试纸条检测的模型。实验结果表明,此方法在20ng的HeLa细胞组蛋白提取液中就能检测到H3K9me3,比传统的试纸条和western blot的灵敏度分别提高了10倍和15倍。并且这种信号增强的试纸条生物传感器具有普遍适用性,可以用来检测其他类型的组蛋白甲基化,如单甲基化、二甲基化和三甲基化的H3K4和H3K9,以及各种不同的物质,如核酸,蛋白,病毒,微生物和小分子等。更多还原

【Abstract】 Copper ion (Cu2+) is one of the essential micronutrient elements for human life, and the concentration level of Cu2+directly affects human’s health. As an essential micronutrient element, Cu2+is a necessary cofactor or structural component of numerous enzymes and proteins (such as superoxide dismutase, cytochrome oxidase, dopamine beta hydroxylase, tyrosinase, and ceruloplasmin) needed in metabolic processes, and only trace amount of Cu2+in the human body is able to maintain normal life activities. However, a lower or higher concentration of Cu2+can cause adverse health effects. For instance, in copper deficiency state, accompanied by the lack of other nutrients and excessive intake its biological antagonists, the activity of the enzymes and cell metabolism can be influenced. And a higher concentration of Cu2+retained in the body was usually caused by genetic diseases or the environment pollution, intaking too many foods containing copper or inhaled the gas containing high concentration of copper ion. Both of atmospheric and water pollution by Cu2+can eventually cause soil pollution, which further affects the yields and quality of crops. Copper ion accumulated in the crops and animals was taken into the body through the food chain expansion process step by step, resulting severely harm to human health. The liver is an important place to store copper ions and help Cu2+releasing into the bile, as a consequence, excessive Cu2+will affect the normal metabolism of liver and kidney, causing gastrointestinal disturbance and hemolytic anemia as well as loss of cognition in the elderly and individuals with Wilson’s disease. Hence, considerable efforts have been devoted to the detection of copper ion due to the high concentration of copper ion has high toxicity on human health, ecosystem and food safe. The maximum level of Cu2+in drinking water permitted by the U.S. Environmental Protection Agency (EPA) is20μM.Classical approaches for the detection of Cu2+, such as graphite furnace atomic absorption spectrometry and inductive coupled plasma atomic emission spectroscopy, have been used as standard procedures. However, the requirement of costly instruments and highly trained personnel prevent their use in many laboratories. Some new methods and technologies, including fluorescence methods, colorimetric method based gold nanoparticles, and dynamic light scattering technique, et al., have been developed in recent years. Although these methods are effective, the use of DNA ligase and fluorophore labeled oligonucleotides is not only expensive but also increases the complexity of the assay. Hence, the development of an inexpensive and sensitive method for Cu2+detection is urgently needed.Eukaryotic genome carrys two kinds of genetic information, one is the nucleic acid sequence carrying genetic information in the conventional sense, the other is epigenetic information, which mainly refers to the reversible and heritable variation of gene function without DNA sequence changes. Histone methylation is an important epigenetic modification, and the commonly modified residues are lysine (K) and arginine (R) at the N-terminal tails of core histones. Previous studies have shown that histone methylation plays important roles in the formation of heterochromation, X-chromosome inactivation, transcriptional regulation, maintenance and differentiation of stem cells, and tumorigenesis, all of which depend on the degree and position of histone methylation. Some new techniques for histone methylation detection emerged in recent years with the development of epigenetics, such as western blot, ChIp (chromatin immunoprecipitation), or ChIp combines with PCR and microarray. These strategies are accurate and reliable except for the tedious operation and heavy workload. Hence, it is necessary to develop a much more simple method for the rapid detection of histione methylation.This paper describes two biosensors for Cu2+detection making full use of the catalytic activity of DNAzyme, including a fluorescence biosensor based on self-assembled DNA concatamers for signal amplification, and a colorimetric biosensor using Cu+-catalyzed click chemistry and hemin/G-quadruplex DNAzyme. An enhanced strip biosensor using DNA functionalized gold nanoparticles as an enhancer probe for rapid and sensitive detection of histone methylation was also constructed. Three research themes are included in this paper and listed as follows:(1) An enzyme-free and label-free fluorescence turn on biosensor for amplified Cu2+detection has been constructed based on self-assembled DNA concatamers and SYBR Green Ⅰ. The reaction mechanism is based on the cleavage of Cu2+-specific DNAzyme, the released target DNA triggering the formation of dsDNA concatamers and using SYBR Green I as a signal reporter. The detection limit for Cu2+(12.8pM) is six orders of magnitude lower than the United States EPA limit of Cu2+in drinkable water (20μM). The whole reaction process does not need any protein enzyme and fluorescent-labeled DNA, making the system more simple and cost-effective.(2) The accurate and rapid detection of Cu2+using Cu+-catalyzed click chemistry and hemin/G-quadruplex horseradish peroxidise (HRP)-mimicking DNAzyme by the naked eye without using typically bulky instruments was developed. The reaction mechanism is based on the formation of a G-quadruplex-forming sequence using Cu+-promoted click chemistry between azide-and alkyne-modified short G-rich sequences, followed by the self-assembly of hemin/G-quadruplex DNAzyme in the presence of hemin and K+in aqueous solution. This G-quadruplex DNAzyme can catalyze its colorless substrate TMB into a colored product. Hence, the whole process of color change of the solution can be monitored by the naked eye. For quantitative analysis, the resulting yellow solution was transferred into a96-well microtiter plate. The optical density (OD) value of the yellow solution at450nm in each well was recorded using a microplate reader. The LOD of our strategy (5.9nM) is much lower than the EPA limit of Cu2+in drinkable water (20μM). In comparison with previously reported Cu2+-specific DNAzyme/substrate based methods for Cu2+detection, the presence of Cu2+can be evaluated by the naked eye, and the whole reaction process does not need long manipulation time and complicated procedure, making the system more simple and convenient.(3) An enhanced strip biosensor using DNA functionalized gold nanoparticles (AuNP-DNA) as an enhancer probe for rapid and sensitive detection of histone methylation has been successfully constructed. Another dual labeled AuNPs used in this assay was functional ized with an antibody and another oligonucleotide (c-DNA) simultaneously. The sequence of the c-DNA is complementary to the DNA on the enhancer probe. The mechanism of the enhanced biosensor is based on the formation of an antibody/target/dual labelled AuNP sandwich structure on the test zone, and a red band can be observed firstly, then AuNPs-DNA solution was added onto the strip to hybridize with the c-DNA on the surface of dual labelled AuNPs. In this case, the red band on the test zone and control zone deepened significantly due to accumulation of more AuNPs. Tri-methylated lysine9of histone H3(H3K9me3) in HeLa cells was chosen as our target. With this biosensor, we can visually detect H3K9me3in20ng of histone extract from HeLa cells in15min without instrumentation, which is10-fold and15-fold lower detection limit than the conventional strip biosensor and western blot, respectively. In addition, the principle of the enhanced strip biosensor can be applied to detect other types of histone methylation, such as mono-, di-or tri-methylated H3K4and H3K9, as well as other analytes, such as nucleic acids, proteins, virus, microorganisms, and small molecules.更多还原