三维微纳米网格和纳米孔垂直集成结构在单分子检测中的应用

发布时间：2018-05-19 04:30

本文选题：基因测序 + 固态纳米孔　；参考：《吉林大学》2017年硕士论文

【摘要】：DNA(Deoxyribonucleic Acid,脱氧核糖核酸)蕴含了整个生物体的遗传信息。能够快速和准确获取DNA序列的信息对于对探究生命奥秘,疾病诊断,药物研发,育种等领域的技术发展起到不可忽视的推动作用。高通量、低成本的纳米孔(直径为几个纳米的小孔)测序技术由于其能直接检测无标记的单分子,是下一代DNA测序技术的重要候选者。其原理是通过检测DNA分子易位穿过纳米孔时引起的特征离子电流来分辨DNA链上的四种不同类型的碱基,从而读取整个DNA长链的信息。根据其制作材料的不同,纳米孔大致可被分为两类:生物纳米孔和固态纳米孔。固态纳米孔杰出的热学、力学、化学稳定性和大规模集成性的优势,使其被广泛应用。然而固态纳米孔的制备受到传统制备方法复杂、高成本、低产量的限制。另一个制约固态纳米孔测序发展的难题是:纳米孔器件承载膜太厚和DNA穿孔的易位速率太快,导致在空间分辨率和时间分辨率上都达不到分辨单碱基的目的。因此,如何有效控制DNA分子在纳米孔内的传输行为,减慢DNA分子的易位速率,对纳米孔测序技术突破具有重要意义。首先,本文介绍了一种快速、易操作的制备纳米孔方法,在电解质溶液中的,通过在绝缘薄膜上施加可调电流脉冲,使薄膜发生电介质击穿形成具有纳米精度的单个固态纳米孔,脉冲参数可以调整而适用于不同厚度和材料的薄膜。相比于其他制备纳米孔的微纳加工工艺,此方法大大降低了操作复杂性和工艺成本,可实现批量化生产。我们利用LabVIEW虚拟仪器控制可编程的数字源表Keithley 2450实现电介质击穿过程的测量与数据记录。此法制备出的氮化硅和石墨烯纳米孔稳定性良好,在后续的DNA检测应用中表现出了优异的传感性能。其次,提出微纳米网格和氮化硅纳米孔垂直集成的新结构,一定电压下,聚合物纳米纤维网格把缠绕的DNA并解开成线性状态,克服纳米孔对较长DNA分子形成的熵势垒,从实验上观测到了减慢长链DNA分子易位速率的现象,DNA易位速率最长可减缓至136.77/(7。利用MATLAB仿真软件建立了微纳米网格-纳米孔结构的物理模型,从理论上计算了加入纳米纤维网格前后,纳米孔附近离子浓度和电场分布的变化,解释了微纳米网格影响DNA易位行为的物理机制。另外,本文还尝试制备了纳米孔集成氧化锌纳米线网格结构,为其今后应用于DNA测序领域验证了可行性。值得欣喜的是,本文还利用厚度接近于碱基间距的石墨烯纳米孔首次探测到了单碱基的信号,并采用增加电解质粘度的办法,实验中利用有机离子液体BMIMCl将单碱基的易位速率减缓至毫秒量级,并尝试区分出不同的碱基种类。研究成果将为基于纳米孔的DNA测序技术研究、蛋白质和长链聚合物检测等领域的实际应用提供必要的理论依据、基础实验数据和技术储备。
[Abstract]:DNA(Deoxyribonucleic acid (deoxyribonucleic acid) contains genetic information for the whole organism. The rapid and accurate acquisition of DNA sequences plays an important role in exploring the mystery of life, disease diagnosis, drug research and development, breeding and other fields of technological development. High-throughput and low-cost nano-pore sequencing (several nanometers in diameter) is an important candidate for the next generation of DNA sequencing because of its ability to directly detect unlabeled monolayers. The principle is to detect the characteristic ion currents caused by the translocation of DNA molecules through the nanopores to distinguish four different types of bases in the DNA chain, so as to read the information of the whole DNA long chain. According to the different materials, nano-pores can be divided into two types: biological nano-pores and solid nano-pores. The outstanding thermal, mechanical, chemical stability and large-scale integration advantages of solid-state nano-pore make it widely used. However, the preparation of solid nanoparticles is limited by the traditional preparation methods, such as complex, high cost and low production. Another difficult problem that restricts the development of solid-state nano-pore sequencing is that the carrier film thickness of nano-hole device and the translocation rate of DNA perforation are too fast, resulting in the spatial resolution and time resolution can not achieve the purpose of single base resolution. Therefore, how to effectively control the transport behavior of DNA molecules in nano-pores and slow down the translocation rate of DNA molecules is of great significance for the breakthrough of nano-pore sequencing technology. First of all, this paper introduces a fast and easy to operate method for the preparation of nano-pores in electrolyte solution by applying adjustable current pulses to the insulating film. A single solid nanometer pore with nanometer precision can be formed by dielectric breakdown of the film. The pulse parameters can be adjusted and applied to thin films with different thickness and material. Compared with other micro-nano fabrication processes, this method greatly reduces the operation complexity and process cost, and can realize batch production. We use LabVIEW virtual instrument to control the programmable digital source meter Keithley 2450 to realize the measurement and data recording of dielectric breakdown process. The nano-pore of silicon nitride and graphene prepared by this method has good stability and excellent sensing performance in the subsequent application of DNA. Secondly, a new structure of vertical integration of nanoscale meshes and nano-pores of silicon nitride is proposed. At a certain voltage, polymer nanofilament meshes winding DNA and unwrapping it into a linear state to overcome the entropy barrier formed by nano-pores on longer DNA molecules. The phenomenon of slowing down the rate of translocation of long-stranded DNA molecules has been observed experimentally. The longest rate of DNA translocation can be reduced to 136.77 / 7. The physical model of micro-nano mesh-nano-pore structure was established by using MATLAB simulation software. The changes of ion concentration and electric field distribution near nano-pore before and after the addition of nano-fiber mesh were calculated theoretically. The physical mechanism of the effect of micro-nano-mesh on DNA translocation behavior is explained. In addition, this paper also attempted to prepare nano-pore integrated ZnO nanowire grid structure, which proved the feasibility of its application in the field of DNA sequencing in the future. It is gratifying to note that the signal of a single base is detected for the first time by using graphene nanorods with thickness close to base spacing, and the method of increasing electrolyte viscosity is adopted. In the experiment, the translocation rate of single base was reduced to millisecond by using organic ionic liquid (BMIMCl), and different base species were identified. The research results will provide the necessary theoretical basis, basic experimental data and technical reserve for the research of DNA sequencing technology based on nano-pore, protein and long chain polymer detection and other practical applications.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q503

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