DNA sequencing using nanopore-nanoelectrode devices for sensing and manipulation

描述（由申请人提供）： 这项研究工作的目的是利用我们最近开发的亚纳米精密透射电子束烧蚀光刻（TEBAL）的独特能力，证明固态纳米孔与纳米电极，纳米通道和其他纳米结构的精确集成将解决必须克服的关键障碍，以实现基于纳米孔的染色体长度DNA分子的低成本高速测序，以及由此产生的医疗和科学效益。快速和低成本的全基因组DNA测序将允许例如在理解、诊断、治疗和预防疾病方面的重大改进，以及在进化研究和对细胞操作的理解方面的重大进展。 该项目将建立在过去十年中基于纳米孔的DNA测序的显著进展的基础上，并计划继续分享结果的传统，技术和纳米孔装置与研究界，使所进行的工作将作出最大的贡献，以推进最先进的国家。预计纳米孔-所生产的纳米电极装置将为其他小组在各种横向电极传感方法和基于纳米电极的单DNA分子操纵方面的进一步工作提供种子，这将有助于实现基于纳米孔的“1000美元基因组测序仪”。 该开发（R21）项目将开始通过展示使用具有集成纳米电极和纳米通道的纳米孔装置可以实现的改进来实现上述长期目标。除了开发可靠的纳米孔-纳米电极装置之外，所提出的工作的独特方面包括几何上更复杂的电极图案的集成以操纵DNA运动，以及这些装置与微流体和荧光显微镜设置的集成以允许跟踪DNA分子，以便它们可以被主动运输到纳米孔。具体任务是要表明： * 利用纳米电极横向传感技术可以更精确地测量DNA分子长度 * 通过纳米电极施加力可以降低双链DNA的移位速度 * 通过约束DNA分子，长度测量分辨率提高了较长的分子 * 可以选择单个DNA分子，将其运输到纳米孔并通过纳米孔移位 这些目标将分几步实现。所需的纳米孔-纳米电极，纳米孔-纳米通道和微流体装置将首先被制造和表征（其中一些已经实现）。接下来，将对这些设备进行实验，以评估其性能并找出问题。最后，几个周期的器件改进和进一步的实验将解决这些问题，并提高器件性能的最佳水平，使目标的实现可以证明。 该研究旨在通过开发纳米技术传感器来实现更快和更低成本的DNA测序。这种传感器的工作原理就像通过手指来识别绳子上的结一样，只不过绳子要细一百万倍！它将使我们能够确定潜在的遗传原因和症状，在患者中快速准确地检测这些疾病，并适当地治疗它们，从而在疾病的理解，诊断，治疗和预防方面取得重大进展。

DESCRIPTION (provided by applicant): The aim of this research effort is to leverage the unique capabilities of our recently developed sub-nanometer precision transmission electron beam ablation lithography (TEBAL) to demonstrate that the precise integration of solid-state nanopores with nanoelectrodes, nanochannels and other nanostructures will address key obstacles that must be overcome to achieve nanopore-based low-cost high-speed sequencing of chromosomal length DNA molecules, and the resultant medical and scientific benefits enabled by this technology. Fast and low cost full genome DNA sequencing will allow, for example, major improvements in the understanding, diagnosis, treatment and prevention of disease, and significant advances in evolutionary research and the understanding of cellular operation. This project will build on the remarkable progress towards nanopore-based DNA sequencing over the past decade, and it is planned to continue the tradition of sharing results, techniques and nanopore devices with the research community so that the work carried out will make the maximal contribution to advancing the state of the art. It is expected that the nanopore-nanoelectrode devices produced will seed further work by other groups on a variety of transverse electrode sensing methodologies and nanoelectrode-based single DNA molecule manipulation, which will contribute to the achievement of a nanopore-based "$1000 genome sequencer". This development (R21) project will begin work on the long term goals described above by demonstrating the improvements that can be achieved using nanopore devices with integrated nanoelectrodes and nanochannels. Beyond developing reliable nanopore-nanoelectrode devices, the unique aspects of the proposed work include the integration of geometrically more complex electrode patterns to manipulate DNA motion, and the integration of these devices with microfluidics and a fluorescent microscope setup to allow tracking of DNA molecules, so that they can be actively transported to the nanopore. The specific tasks are to show that: * DNA molecule length can be measured more accurately by transverse sensing with nanoelectrodes * the translocation speed of double stranded DNA can be reduced by applying forces via nanoelectrodes * by constraining DNA molecules, length measurement resolution improves for longer molecules * individual DNA molecules can be selected, transported to a nanopore and translocated through it These objectives will be accomplished in several steps. The required nanopore-nanoelectrode, nanopore-nanochannel and microfluidics devices will first be fabricated and characterized (some of this has already been achieved). Next, experiments with these devices will be conducted to evaluate their performance and identify problems. Finally, several cycles of device refinement and further experiments will resolve these problems and improve device performance to optimal levels, so that achievement of the objectives can be demonstrated. PROJECT HEALTH RELEVANCE This research aims to achieve much faster and lower-cost DNA sequencing by developing a nanotechnological sensor. This sensor works just like picking out the knots on a string by running it through one's fingers, except the string is a million times thinner! It will enable major improvements in the understanding, diagnosis, treatment and prevention of disease, by allowing us to determine the underlying genetic causes and symptoms, detect these rapidly and accurately in patients, and treat them appropriately.