面向第三代纳米孔基因测序技术的DNA摩擦学行为研究

发布时间：2018-06-01 03:58

本文选题：纳米摩擦学 + 粘着　；参考：《西南交通大学》2015年博士论文

【摘要】：作为遗传信息的载体,DNA是生命在进化过程中最为重要的一种物质。快速准确地探测DNA的序列不仅可以帮助人们有效地预防和治疗如糖尿病、血友症和癌症等遗传学疾病,还能有助于人类解开自身的生命起源之谜。自上世纪70年代起,人们已先后发展了三代DNA测序技术。第一代DNA测序技术基于Sanger法,耗时15年完成了人类基因组计划,直接花费约30亿美元。第二代DNA测序技术以高通量为特点,仅需一周、花费不到100万美元就能完成人类基因组的测序。近年来备受关注的基于纳米孔的第三代DNA测序技术,通过检测DNA碱基通过纳米孔时所产生的特征阻塞电流进行测序。凭借超快速、高精度、低成本的优势,纳米孔测序技术有望完成美国国立卫生研究院(NIH)设定的只花费1000美元、在24小时内完成个人基因组测序的目标。尽管如此,纳米孔测序技术目前仍存在一些关键问题亟需解决,如DNA分子过孔速度过快以至于难以对阻塞电流信号进行精确地检测。研究表明,增大DNA分子与纳米孔孔壁间的摩擦力可以在一定程度上降低其过孔速度。然而,以往的研究主要集中于DNA表面自组装、分子形貌、DNA分子链的力学稳定性以及基于DNA分子特定功能器件的研发,有关DNA分子摩擦学行为的研究却鲜见报道。因此,为了全面、深入地理解DNA分子与纳米孔材料之间的摩擦作用机制,亟需系统地开展DNA摩擦学行为的研究。相关研究成果不仅可以促进基于纳米孔的第三代DNA测序技术的发展和应用,同时也能够丰富和完善生物纳米摩擦学的理论基础。本论文借助原子力显微镜(AFM),针对DNA分子/纳米孔材料摩擦副,重点研究了不同纳米孔材料、溶液NaCl浓度、溶液pH值、分子链长对DNA分子摩擦学行为的影响规律及作用机制。首先,成功制备出表面吸附有DNA分子的二氧化硅针尖,利用其研究了溶液环境中DNA与几种典型纳米孔材料之间的摩擦学行为,进而提出了纳米孔材料选择的优化建议。在此基础上,以原子级平整的云母表面为基底成功制备出DNA自组装薄膜,并使用AFM的氮化硅针尖系统研究了溶液NaCl浓度、溶液pH值以及分子链长对DNA摩擦学行为的影响规律及作用机制。同时,在东南大学陈云飞教授课题组的帮助下,对不同pH值溶液中DNA分子通过氮化硅纳米孔的过孔情况进行了测试,以验证本论文有关溶液pH值优化的研究结果。论文的主要结论及创新点如下：(1) 成功制备出表面吸附有DNA分子的二氧化硅针尖,揭示出不同纳米孔材料与DNA分子之间摩擦学行为的变化规律。使用3-氨丙基三乙氧基硅烷偶联剂修饰二氧化硅针尖,将DNA分子自组装于其表面,成功地制备出表面吸附有DNA分子的二氧化硅针尖。在此基础上,使用该针尖系统研究了DNA分子与5种典型的纳米孔材料(氮化硅、石英、APS-石英、石墨烯、石墨)之间的摩擦学行为。结果表明,在相同载荷下5种纳米孔材料与DNA分子之间的摩擦力从大到小依次为：APS-石英氮化硅石英石墨烯石墨。其中石墨烯和石墨由于拥有特殊的微观层状结构,其与DNA之间的摩擦力明显小于其余3种纳米孔材料；通过不同纳米孔材料表面的接触角测试,发现液下环境中疏水表面有可能在一定程度上增大DNA分子与材料间的摩擦力；通过APS-石英、氮化硅和石英表面力曲线的测试,发现带正电的APS-石英表面与DNA分子之间会产生静电引力,有助于增大DNA-SiO2针尖与基底间的相互作用力,进一步导致摩擦力的增大。该部分研究结果针对纳米孔材料的选择提出相关建议,也为后续DNA摩擦行为研究中针尖材料的选择提供了参考。(2) 阐明了NaCl溶液浓度对DNA分子构象及摩擦行为的影响规律。在云母表面成功制备出DNA自组装薄膜,使用氮化硅针尖测试了溶液的NaCl浓度对DNA分子构象及摩擦行为的影响规律。结果显示,随着溶液中带正电的Na离子浓度的增大,越来越多的DNA分子骨架外侧磷酸基团所带的负电荷被屏蔽,DNA分子从较为伸展的构象逐渐转变为“线圈盘绕”的构象。此外,在相同载荷下,当溶液NaCl浓度较低时,由于DNA分子的刚度较高,针尖与DNA分子间的摩擦力较大。可见,较低的NaCl浓度能使DNA分子更为伸展,有利于DNA分子在良好的姿态下进入并通过纳米孔,同时,较大的摩擦力利于降低DNA分子的过孔速度。因此,较低的NaCl浓度更利于DNA分子的纳米孔测序。(3) 揭示了溶液pH值对DNA分子构象及摩擦行为的作用机制。在不同pH值的溶液中,使用氮化硅针尖在DNA薄膜表面进行了粘着与摩擦行为的测试,揭示了溶液pH值对DNA分子构象及摩擦力的影响机制。研究表明,溶液pH值的变化将对DNA分子碱基之间,以及DNA分子与氮化硅针尖之间的相互作用力产生影响,从而会进一步影响DNA分子的构象及其与氮化硅针尖的摩擦行为。当pH较低时,DNA分子与氮化硅针尖之间存在静电引力,在一定程度上增大了两者之间的相互作用力,导致摩擦力增大；当pH值较高时,DNA分子与针尖之间存在静电斥力,在一定程度上平衡了部分外加载荷,导致摩擦力降低。此外,不同pH值下DNA构象的变化也能影响摩擦力的大小。低pH值下DNA分子的吸附构象主要为“平躺型”,针尖在剪切时会受到较大的阻力；高pH下DNA分子的吸附构象主要为“链圈型”,并且部分分子链伸展至溶液中,针尖在剪切时受到的阻力较小。在低pH值溶液中,平躺的构象有利于DNA分子的一端进入并保持良好的姿态通过纳米孔；同时,低pH值溶液中DNA分子与氮化硅纳米孔材料的摩擦力较大,利于降低DNA分子的过孔速度。最后,不同pH值溶液中DNA穿越纳米孔的实验也同样验证了较低的溶液pH值能降低DNA分子的过孔速度,因此,较低的溶液pH值更利于DNA分子的纳米孔测序。(4) 研究表明链长对DNA分子摩擦学行为的影响不大,纳米孔测序对DNA分子的读取长度不受摩擦限制。使用不同链长的DNA分子在云母表面制备出自组装薄膜,采用氮化硅针尖研究了链长对DNA分子构象及摩擦行为的影响规律。结果表明,DNA分子以链圈和平躺共存的构象吸附于APS修饰的云母基底表面；随着链长的增大,DNA分子更为伸展,利于DNA分子以良好的姿态进入并通过纳米孔。在较低的载荷范围内,链长对DNA分子与氮化硅针尖之间的摩擦力影响不大,DNA分子的过孔速度受链长的影响较小。因此,从摩擦学的角度来看,纳米孔测序的读取长度不受限制。综上所述,本论文系统研究了纳米孔材料、溶液NaCl浓度、溶液pH值以及分子链长对DNA分子摩擦学行为的影响规律和作用机制,研究结果不仅丰富了生物纳米摩擦学的理论基础,而且为基于纳米孔的第三代DNA测序技术的优化提供了理论依据。
[Abstract]:As a carrier of genetic information, DNA is the most important substance in the evolution of life. Rapid and accurate detection of DNA sequences can not only help people effectively prevent and treat genetic diseases such as diabetes, hemophilia and cancer, but also help to unravel the mystery of the origin of life. Since the 70s of last century, people The three generation of DNA sequencing technology has been developed successively. The first generation of DNA sequencing technology based on the Sanger method has completed the human genome project for 15 years and costs about $3 billion. The second generation DNA sequencing technology is characterized by high throughput, requiring only one week and less than 1 million dollars to complete the sequencing of the human genome. The third generation DNA sequencing technology in nanoscale has been sequenced by detecting the characteristic blocking current produced by the DNA base through the nanopore. With the advantage of super fast, high precision and low cost, the nanoscale sequencing technology is expected to complete the US National Institutes of Health (NIH) for only $1000 and complete the individual genome within 24 hours. In spite of this, there are still some key problems to be solved in nanoscale sequencing technology. For example, the speed of the DNA molecules is too fast to accurately detect the blocking current signal. The study shows that the increase of the friction force between the DNA and the pore wall of the nanopore can be reduced to a certain extent. The research focused on the surface self-assembly of DNA, the molecular morphology, the mechanical stability of the DNA molecular chain and the development of the specific functional devices based on the DNA molecules. The research on the tribological behavior of the DNA molecules is rarely reported. Therefore, in order to understand the mechanism of the friction between the DNA molecules and the nanomorp materials, it is urgent to systematically understand the mechanism of the friction between the DNA molecules and the nanomorp materials. The research on the tribological behavior of DNA can not only promote the development and application of the third generation DNA sequencing technology based on nanoscale, but also enrich and improve the theoretical basis of the biological nano tribology. This paper focuses on the study of the friction pairs of DNA molecules / nanoscale materials with the help of atomic force microscopy (AFM). The influence law and mechanism of nano pore material, solution NaCl concentration, solution pH value and molecular chain length on the tribological behavior of DNA molecules. First, the silicon dioxide tip with DNA molecules adsorbed on the surface was successfully prepared, and the tribological behavior between DNA and several typical nanoscale materials in the solution environment was studied, and then the nanopore was proposed. On this basis, DNA self assembled film was successfully prepared on the basis of atomic level smooth mica surface, and the effect of NaCl concentration of solution, pH value of solution and molecular chain length on the tribological behavior of DNA and the mechanism of action were studied by AFM silicon nitride needle point system. At the same time, Chen Yunfei teaching at Southeast University was used. In order to verify the results of the study on the optimization of the pH value of the solution, the main conclusions and innovation points of this paper are as follows: (1) the successful preparation of the silicon dioxide tip with DNA molecules on the surface was successfully prepared, and the different nanometers were revealed. The change law of tribological behavior between the pore material and the DNA molecule. Using the 3- ammonia propyl triethoxy silane coupling agent to modify the silica pinpoint, the DNA molecules are self assembled on the surface of the silicon dioxide, and the silica pinpoints with DNA molecules adsorbed on the surface have been successfully prepared. On this basis, the DNA molecules and 5 typical kinds of nanofilms are studied using this needle point system. The tribological behavior between the mesoporous materials (silicon nitride, quartz, APS- quartz, graphene, graphite). The results show that the friction between the 5 nanoscale materials and the DNA molecules under the same load is in turn: APS- quartz silicon nitride quartz graphene graphite. Among them, graphene and graphite have special microstructure, and they are with D The friction between NA is significantly less than the other 3 kinds of nanoscale materials. Through the contact angle test of the surface of different nanoscale materials, it is found that the hydrophobic surface in the underwater environment may increase the friction between the DNA and the material to a certain extent. Through the test of the surface force curve of APS- quartz, silicon nitride and quartz, the positive APS- stone is found. The electrostatic attraction between the DNA and the surface of the UK will help to increase the interaction between the tip and the substrate of the DNA-SiO2, and further lead to the increase of the friction force. The results of this study provide some suggestions for the selection of nanoscale materials, and provide a reference for the selection of the tip materials in the subsequent DNA friction study. (2) clarified The influence of the concentration of NaCl solution on the conformation and friction behavior of DNA molecules. The DNA self assembled film was successfully prepared on the mica surface. The influence of NaCl concentration on the conformation and friction of DNA was measured with the NaCl concentration of the silicon nitride. The results showed that as the concentration of positive Na ions in the solution increased, more and more DNA fractions were found. The negative charge of the phosphoric group outside the subframework is shielded, and the DNA molecules gradually change from the more extensional conformation to the "coil coiled" conformation. In addition, when the concentration of the solution NaCl is low, the friction force between the needle tip and the DNA molecule is larger when the concentration of the DNA molecule is high. The lower NaCl concentration can make the DNA molecule More extensional, it is beneficial for DNA molecules to enter and pass through nanoscale in good posture, and the larger friction force is beneficial to reducing the pore velocity of DNA molecules. Therefore, the lower NaCl concentration is more beneficial to the nanoscale sequencing of DNA molecules. (3) the mechanism of the pH value of the solution on the conformation and friction of DNA molecules is revealed. In the solution of different pH values. On the surface of the DNA film, the adhesion and friction behavior of the silicon nitride needle was tested. The influence mechanism of the solution pH value on the conformation and friction of the DNA molecule was revealed. The study shows that the change of the pH value of the solution will affect the interaction between the DNA molecular base and the interaction between the DNA molecule and the silicon nitride needle. The conformation of DNA molecules and the friction behavior of the silicon nitride needle are affected. When the pH is low, there is an electrostatic force between the DNA and the tip of the silicon nitride needle, which, to a certain extent, increases the interaction between the two and causes the friction force to increase. When the pH value is high, there is an electrostatic repulsion between the DNA molecule and the needle tip, and the equilibrium is balanced to a certain extent. The friction force is reduced by partial loading. In addition, the change of DNA conformation under different pH values can also affect the size of the friction force. The Adsorption Conformation of DNA molecules at low pH values is mainly "flat", and the tip of the needle will be subjected to greater resistance in shear; the adsorption structure of DNA molecules under high pH is mainly "chain ring", and some molecular chains are used. In the solution, the tip of the needle is less resistant to shear. In a low pH value solution, the flat conformation is beneficial to the entry of the DNA molecule and maintain a good posture through the nanopore; at the same time, the friction force of the DNA molecule in the low pH solution is larger than that of the silicon nitride nanomaterials, which is beneficial to the reduction of the pore velocity of the DNA molecule. Finally, the difference in the pore velocity of the DNA molecule is different. The experiment of DNA through the nanoport in pH value solution also verified that the lower pH value of the lower solution can reduce the pore velocity of the DNA molecule. Therefore, the lower pH value is beneficial to the nanoscale sequencing of the DNA molecules. (4) the study shows that the chain length has little effect on the tribological behavior of the DNA molecules, and the reading length of DNA molecules by nanomorp sequencing is not limited by the friction limit. The self assembled film was prepared on the mica surface using DNA molecules with different length of chain. The effect of chain length on the conformation and friction of DNA was studied by the tip of silicon nitride. The results showed that the conformation of DNA molecules coexisted peacefully on the surface of the APS modified mica surface. With the increase of the chain length, the DNA molecules were more extended. In a relatively low load range, the chain length has little effect on the friction between the DNA molecules and the silicon nitride pinpoint, and the pore velocity of the DNA molecule is less affected by the chain length. Therefore, from the tribological point of view, the reading length of the nanoscale sequencing is not limited. In summary, this is the case. The influence rule and mechanism of the nano pore material, the concentration of solution NaCl, the pH value of the solution and the length of the molecular chain on the tribological behavior of DNA molecules are systematically studied in this paper. The results not only enrich the theoretical basis of the biological nano tribology, but also provide a theoretical basis for the optimization of the third generation DNA sequencing technology based on the nanopore.
【学位授予单位】：西南交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：Q523

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