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类型 基础研究 预答辩日期 2018-03-08
开始(开题)日期 2014-03-06 论文结束日期 2017-12-05
地点 机械学院424室 论文选题来源 973、863项目     论文字数 8.5 (万字)
题目 纳米孔在DNA及蛋白质分子检测应用中的关键技术研究
主题词 纳米孔,DNA,蛋白质,折叠,降速
摘要 纳米孔单分子传感器相比其他传统单分子检测技术而言具有低成本高通量的优势。当一个带电生物分子在外加电场力的作用下电泳穿过纳米孔时,由于其物理占位作用及其与纳米孔壁间的强相互作用使得过孔电流被调制,通过对调制电流的分析可以获取待测分子的带电特性,形状以及取向性等特征。因此,纳米孔被广泛应用于一些单分子的检测中,如核酸分子,蛋白质,核酸与蛋白的结合物等,甚至用于单个DNA碱基以及蛋白质氨基酸的识别。然而,因为核酸分子及蛋白质分子的带电状态,取向性以及二级结构等都会产生不同的调制电流,使得实现高精度的核酸分子以及蛋白质分子检测具有极大的挑战性。为了丰富纳米孔单分子检测领域的基础理论研究,为将来实现高精度的纳米孔单分子检测传感器的设计与制造提供数据支撑,本学位论文主要聚焦于研究纳米孔对核酸分子及蛋白质分子的检测和离子电流阻塞机理的研究。本文主要研究了DNA电泳穿过固态纳米孔及生物纳米孔的动力学行为过程;采用原子力显微镜对纳米孔接入电阻进行了系统的研究,并成功的实现了对DNA过孔的降速以及高精度操控;最后用纳米孔对蛋白质的折叠状态,折叠-解折叠转变甚至蛋白质折叠中间态进行了实时检测。本文的主要研究内容和结论如下: (1)采用了分子动力学模拟的方法研究了双链DNA分子以及单链DNA均聚物电泳穿过石墨烯纳米孔的动力学过程。研究发现双链DNA穿过石墨烯纳米孔的过孔速度可以通过调节外加电压和纳米孔的尺寸来进行有效调节。较低的外加电压使得作用在DNA上的电场力也随之降低,因此可以降低DNA的过孔速度。当DNA穿过较大纳米孔时,其过孔时间也会相应增加,这是由于较大纳米孔两侧的电势差相比小孔而言相对较小,当DNA在孔内时,作用在DNA上的电场力也相对较小。此外,通过分子动力学模拟进一步研究了2纳米石墨烯纳米孔对由10个碱基组成的单链DNA均聚物的检测。由于四种碱基的电荷量及尺寸差异较小,在噪声的影响下从单纯的离子电流堵塞幅值上并不能区分出不同的单链DNA均聚物。然而,通过检测分析poly(dA)10, poly(dC)10, poly(dG)10和poly(dT)10的过孔时间却能对四种单链DNA均聚物进行区分。 (2)基于上述理论研究,纳米孔的尺寸可以影响DNA的过孔速度,为了验证这一理论结果,本文用实验的方法研究了氮化硅纳米孔尺寸对poly(dT)30过孔速度的影响。研究表明,当将纳米孔的直径从4.8纳米增加到10.8纳米的时候,poly(dT)30穿过氮化硅纳米孔的速度可以降低至少一半。这一结果和上述的分子动力学仿真结果相吻合。此外,poly(dT)30过孔产生的阻塞电流幅值随着纳米孔直径的增加而逐渐降低,这与采用经典的阻塞离子电流模型得到的结果也相一致。 (3)本文研究了由胸腺嘧啶组成的柔性单链DNA均聚物在高浓度条件下电泳穿过α-溶血素纳米孔的输运机理。通过对阻塞电流信号分析发现,poly(dT)20会产生两种幅值的阻塞电流信号,分别由poly(dT)20的过孔以及其与α-溶血素纳米孔的前庭碰撞产生。两种阻塞电流的幅值分别随着外加电压的增加而线性增加。虽然外加电压不同,但是相对阻塞电流幅值却不随着外加电压的改变而发生变化。poly(dT)20与α-溶血素纳米孔前庭的碰撞时间也并不随着外加电压的变化而改变。然而poly(dT)20的过孔速度却随着外加电压的增加而逐渐降低;这主要是由于胸腺嘧啶的碱基堆叠效应较差,当外加电压升高后,作用在poly(dT)20的作用力提高,使得其非常容易发生团簇,使过孔时间延长,分子动力学模拟结果进一步证实了这一实验现象。 (4)离子电流阻塞机理是设计和指导纳米孔单分子传感器的重要基础,本文通过将纳米孔单分子传感器和原子力显微镜进行集成,研究了当一个物体放置在纳米孔附近区域时对纳米孔电阻产生的影响。研究结果表明在纳米孔周围存在一个半球区域,在区域内原子力显微镜探针会阻塞部分离子电流,而当探针处于半球区域外时,离子电流不受探针的影响。探针在越靠近纳米孔的位置其阻塞电流幅值也就越大。研究还发现纳米孔周围半球区域的半径稍低于离子的捕获半径,而且其半径值随着外加电压的增加而线性增加,随着孔径的增加而呈现出抛物线增长。值得注意的是这一半径值与探针的工作模式以及扫描速度无关。此外,基于实验数据的分析本文提出了一套完善的理论模型对阻塞物体距离纳米孔的位置以及其几何形状对离子电流的影响进行了精确描述,有限元分析的方法进一步验证了本文所提模型的正确性。 (5)DNA过孔速度过快一直是实现DNA测序传感器设计与制造的一大障碍,为了解决这一难题,本文设计了一套集成了纳米孔和原子力显微镜的且具有纳米精度的操控系统来降低DNA穿过固态纳米孔的速度甚至实现对DNA运动方向的控制。通过巯基-金或者生物素-链霉亲和素的强相互作用将DNA绑定在AFM探针针尖上,结合AFM高精度的进给系统操控探针精确移动,可以将DNA的过孔速度降低至2埃/毫秒;因为目前现有的离子电流信号检测频率可达5兆赫兹,这一速度可以保证有足够多的采样点来提取待测分子的结构信息。本套测试系统的另外一个优势是可以控制DNA的运动方向,只要DNA不从探针尖端剥落便可以一直反复实验,对DNA阻塞电流进行反复测量降低测量误差。 (6)蛋白质折叠与解折叠直接关系到蛋白质的折叠路径,而对蛋白质折叠路径的探索一直是制药领域难以攻克的挑战。尽管传统单分子检测方法可以检测蛋白质的折叠过程,但是其目前缺乏高通量而且需要复杂的化学修饰。本文采用分子动力学模拟的方法从理论上说明纳米孔检测技术可以用于检测区分蛋白质的折叠程度。分子动力学模拟结果表明,蛋白质的解折叠态相比其折叠态会阻塞更多的离子电流,这主要是由于离子迁移率在近蛋白质表面发生骤降引起的。本文还基于分子动力学模拟的结果提出了一套具有原子精度的理论模型用于计算蛋白质不同折叠态的阻塞电流,理论计算结果表明,蛋白质折叠态和解折叠态产生的阻塞电流在考虑蛋白质取向性以及构象性变化的情况下依然能被区分开来,通过对一系列蛋白质折叠动力学轨迹的分析进一步证实了纳米孔离子电流的测量可以实时检测蛋白质折叠态与解折叠态的转变过程,甚至可以识别出蛋白质折叠中间态。
英文题目 Nanopore Sensing of DNA and Protein Molecules by Theoretical Simulations and Experimental Studies
英文主题词 nanopore,DNA,protein,folding,slow down
英文摘要 Nanopore sensor has the advantage of both high-throughput and low-cost compared to other traditional methods for characterization of single molecules. In a typical nanopore meas-urement, biomolecules are electrophoretically driven through a nanopore one at a time by an external electric field, due to the physical occupation as well as the strong interaction between the molecules and the nanopore wall, the modulated ionic current can reflect the information, including charge state, shape, orientation and so on, carried by the molecules. Thus, nanopore sensor has been widely used to detect and characterize DNA molecules, single proteins, pro-tein-DNA complexes and even single nucleotide in a single DNA strand and single amino acid in a single peptide. However, there remain some challenges in the area of detecting DNA and protein molecules due to no consensus on the ionic current signal that is related to the corre-sponding molecule, as the charge state, the orientation and secondary structure of the molecule will all induce various blockade currents. To enrich the fundamental research in the area of sin-gle molecule detection by nanopores and facilitate the development of nanopore sensor, in this thesis, we mainly focused on the nucleic acid molecules and protein molecules detection by us-ing nanopores and studied the mechanism of current modulation theory. We firstly investigated the DNA transport dynamics through both solid-state nanopores and biological nanopores. Then the mechanism of nanopore access resistance was studied by using atomic force microscopy and we also realized slowing down DNA transport through solid-state nanopores and manipulation of DNA translocation direction by using atomic force microscopy. Finally, we used nanopore to characterize protein folding states, protein folding-unfolding transitions and protein folding in-termediates. Our main results and conclusions obtained by using both theoretical and experi-mental methods are summarized as below: 1. Using molecular dynamics simulations, graphene nanopores were used to investigate dsDNA transport dynamics through nanopores and to differentiate the ssDNA homopolymers composed of different nucleotides. The translocation speed of a dsDNA through the graphene nanopore could be controlled by adjusting the appropriate applied bias and the diameter of the nanopore. A smaller bias could slow down the dsDNA translocation through the nanopore due to the small electric force acting on the dsDNA. It was also found that the dsDNA translocation speed decreased with the nanopore diameter, it is because the potential drop across a larger na-nopore is a bit smaller compared to a smaller nanopore. Molecular dynamics simulations were also performed to study the translocation process of four ssDNAs with ten identical bases through graphene nanopore with diameter of 2 nm. Due to the similar dimension of the four nu-cleotides, the blockage current was unlikely to provide a distinguishable signal for the homo-polymers. However, by simply monitoring and analyzing the translocation time of poly(dA)10, poly(dC)10, poly(dG)10 and poly(dT)10 though the nanopore, each ssDNA could be identified and characterized. 2. As found above that the nanopore dimension could influence the translocation speed of DNA through the nanopore, to validate this interesting finding, we investigated the nanopore size effect on translocation of poly(dT)30 through Si3N4 membrane using experimental method. It was found that the speed of the poly(dT)30 transport through Si3N4 nanopores can be slowed down by half through increasing the nanopore diameter from 4.8 nm to 10.8 nm. The results were consistent with our simulation results. Besides, the current blockage induced by DNA passing through the nanopore was less obvious as pore diameter is larger, which is in good agreement with the theoretical prediction. 3. The electrophoretic transport mechanism of flexible DNA homopolymer composed of thymines through the α-hemolysin nanopores in high concentration potassium chloride solution was studied. Two obvious current blockades were found to be induced by poly(dT)20 transloca-tion and collision events. Both blockade currents increase linearly with the applied bias. How-ever, the relative blockade currents are almost kept the same though variable voltages were ap-plied. The collision times of poly(dT)20 in the luminal site of the pore remain constant for dif-ferent voltages. The translocation speed of poly(dT)20 through the nanopore decreases as the applied bias increases. It is because as the potential increases, the drag force acting on the ho-mopolymer is much easier to help it crumple into a cluster due to the poor stacking of thymine residues compared to homopolymers consisting of other nucleotides. Molecular dynamics simu-lations further confirmed the experimental results. 4. The current modulation mechanism is significant for designing the single molecule sen-sor based on nanopores. In this thesis, we studied how an occluding object placed near a na-nopore affects its access resistance by integrating a nanopore sensor with an atomic force mi-croscopy. It was found that there exists a critical hemisphere around the nanopore, inside which the tip of an atomic force microscopy would affect the ionic current. The radius of this hemi-sphere, which is a bit smaller than the theoretical capture radius of ions, increases linearly with the applied bias voltage and quadratically with the nanopore diameter, but is independent of the operation modes and scanning speeds of the atomic force microscopy. A theoretical model was also proposed to describe how the tip position and geometrical parameters affect the access re-sistance of a nanopore. 5. A measuring system that integrates nanopore sensor with atomic force microscopy was designed to control DNA translocation through solid-state nanopores. By attaching the dsDNA strand to the AFM probe tip using thiol-Au or biotin-streptavidin interaction, we can slow down DNA translocation speed to 2 ?/ms, this is slow enough to gain sufficient data for extracting structure information as the bandwidth for the ionic current measurement has reached to 5 MHz by now. Another advantage of the designed measuring system is that, we can realize even reversing the DNA translocation direction and repeatedly dragging out the DNA strand and threading it through the nanopore for many times as long as the DNA is not exfoliated from the AFM probe tip. 6. Single-molecule studies of protein folding hold keys to unveiling protein folding pathways and elusive intermediate folding states - attractive pharmaceutical targets. Although conventional single-molecule approaches can detect folding intermediates, they presently lack throughput and require elaborate labeling. Here, we theoretically show that measurements of ionic current through a nanopore containing a protein can report on the protein’s folding state. Our all-atom molecular dynamics simulations show that the unfolding of a protein lowers the nanopore ionic current, an effect that originates from the reduction of ion mobility in proxim-ity to a protein. Using a theoretical model, we show that the average change in ionic current produced by a folding?unfolding transition is detectable despite the orientational and con-formational heterogeneity of the folded and unfolded states. By analyzing millisecond-long all-atom MD simulations of multiple protein transitions, we show that a nanopore ionic current recording can detect folding?unfolding transitions in real time and report on the structure of folding intermediates.
学术讨论
主办单位时间地点报告人报告主题
东南大学 2013年10月11日 机械楼424室 陈伟宇 Van der Waals 作用对双层薄膜切向热导率的影响I
东南大学 2013年12月19日 机械楼302室 袁志山 DNA测序电极设计与制作传感技术联合国家重点实验室设备与工艺介绍
东南大学 2014年4月11日 机械楼424室 陈伟宇 Van der Waals 作用对双层薄膜切向热导率的影响II
东南大学 2014年9月19日 机械楼410室 司伟 Manipulation of ssDNA transport through Si3N4 nanopores by Atomic Force Microscope
东南大学 2014年9月25日 机械楼302室 章寅 基于固态纳米通道的单分子传感器设计与制造
东南大学 2015年3月27日 机械楼410室 司伟 Recent progress in nanopore DNA sequencing technology
伊利诺伊州大学香槟分校 2016年10月16日 物理楼106F 司伟 Discrimination of the folding states of proteins by nanopore ionic current
伊利诺伊州大学香槟分校 2017年10月2日 物理楼106F 司伟 Fingerprinting of Protein Molecules by Ionic Current
     
学术会议
会议名称时间地点本人报告本人报告题目
美国机械工程师协会(ASME) 2013年11月15日至21日 SANDIEGO,CA, USA Molecular Dynamics Study of DNA Transport through Graphene Nanopores
The Ohio State University 2017年8月16日至19日 Columbus, OH, USA. Nanopore Sensing of Protein Shape and Folding-Unfolding
     
代表作
论文名称
Detecting DNA Using a Single Graphene Pore by Molecular Dynamics Simulations
Effect of nanopore size on poly(dT)30 translocation through silicon nitride membrane
The molecular dynamics study for detection of ssDNA by monolayer graphene nanopore
Electrophoresis of poly(dT)20 through alpha-hemolysin nanopore in high concentration potassium chlor
Investigation on the interaction length and access resistance of a nanopore with an atomic force mic
 
答辩委员会组成信息
姓名职称导师类别工作单位是否主席备注
龙亿涛 正高 教授 博导 华东理工大学
毛庆和 正高 研究员 博导 中国科学院合肥物质科学研究院
倪中华 正高 教授 博导 东南大学
毕可东 正高 博导 东南大学
沙菁洁 副高 副教授 博导 东南大学
      
答辩秘书信息
姓名职称工作单位备注
张艳 副高 副教授 东南大学