DNA sequencing using nanopore-nanoelectrode devices for sensing and manipulation

描述(申请人提供)：这项研究的目的是利用我们最近开发的亚纳米精密透射式电子束消融光刻(TEBAL)的独特能力，展示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低成本高速染色体长度DNA分子测序，以及这项技术所带来的医疗和科学效益。例如，快速和低成本的全基因组DNA测序将使人们在理解、诊断、治疗和预防疾病方面取得重大进展，并在进化研究和对细胞运作的理解方面取得重大进展。 该项目将建立在过去十年基于纳米孔的DNA测序的显著进展的基础上，并计划继续与研究界分享结果、技术和纳米孔设备的传统，以便所开展的工作将对推动最先进的技术做出最大贡献。预计所生产的纳米孔-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”。 这一开发(R21)项目将通过展示使用集成纳米电极和纳米通道的纳米孔装置可以实现的改进，开始上述长期目标的工作。除了开发可靠的纳米孔-纳米电极设备外，拟议工作的独特方面包括集成几何上更复杂的电极图案来操纵DNA运动，以及将这些设备与微流体和荧光显微镜设置相结合，以允许跟踪DNA分子，从而使它们能够被主动输送到纳米孔。具体任务是表明： *用纳米电极横向传感可以更准确地测量DNA分子长度 *通过纳米电极施力可以降低双链DNA的移位速度 *通过限制DNA分子，提高了较长分子的长度测量分辨率 *可以选择单个DNA分子，将其运输到纳米孔中，并通过它进行转移 这些目标将分几步实现。将首先制造和表征所需的纳米孔-纳米电极、纳米孔-纳米通道和微流体装置(其中一些已经实现)。接下来，将对这些设备进行实验，以评估它们的性能并找出问题。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，并将器件性能提高到最佳水平，从而证明目标的实现。这项研究旨在通过开发一种纳米技术传感器来实现更快、更低成本的DNA测序。这个传感器的工作原理就像是通过手指来识别绳子上的结，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，快速准确地在患者身上检测到这些原因和症状，并对他们进行适当的治疗，从而大大改进对疾病的理解、诊断、治疗和预防。

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.