Electronic DNA Sequencing with Ion Channel Research

这份SGER提案描述了对制造“纳米孔”(例如，5到100纳米)所需的技术和技术的探索性研究。这些纳米孔将成为一个独特的平面纳米管的一部分，用于研究生物细胞中的单离子通道--生物细胞需要围绕纳米孔的开口形成一个巨型细胞。在未来，我们将在多井芯片中形成的这些纳米孔开口几毫米处定位低噪声放大器和传感器。在单个离子通道的单通道记录领域中，记录噪声是表征药物存在下的离子特定通道行为的重要限制因素。我们计划研究纳米孔技术，如电子束、聚焦离子束(FIB)和扫描电子显微镜的场发射。我们的离子通道研究结果可以应用于在外加电场下将多核苷酸、单链DNA分子穿过纳米孔。当DNA通过一个开放的纳米孔的限制孔径时，纳米孔中的离子流动应该以一种指示多核苷酸链中每个核苷酸的分子大小和化学性质的方式被“阻止”。因此，我们希望利用单通道记录来以电子方式确定单链DNA分子中单个碱基的直接序列，从而在几小时内为人类基因组测序打开大门，而不是像毛细管电泳法(CE)和超薄平板凝胶技术那样需要数年时间。离子通道研究将在了解心血管系统和中枢神经系统的众多疾病和过程中发挥越来越重要的作用。偏头痛、癫痫、阿尔茨海默氏症、心律失常、囊性纤维化和高血压等疾病都被发现与离子通道有关，针对这些和其他疾病的药物治疗主要集中在离子通道上。

This SGER proposal describes exploratory research into the technology and techniques required for fabrication of 'nanopores' (e.g. 5 to 100 nm). These nanopores will become part of a unique planar nanopipette for the study of single ion channels in biological cells - cells that need to form a gigaseal around the opening of the nanopore. In the future we will locate low-noise amplifiers and sensors millimeters from these nanopore openings formed in a multi-well chip. In the field of single channel recordings from individual ion channels the recording noise is an important limiting factor in characterizing the behavior of ion-specific channels in the presence of drugs. We plan to investigate nanopore techniques, such as E-beam, focus-ion beam (FIB) and field emission from a scanning transmission electron microscop. The results of our ion-channel research may be applied to the 'threading' of a polynucleotide, single-strand, DNA molecule through a nanopore under an applied electric field. As the DNA passes through the limiting aperture of an open nanopore, the flow of ions in the nanopore should be 'blocked' in a manner indicative of the molecular size and chemical properties of each nucleotide in the polynucleotide chain. Thus, we hope to utilize single channel recordings to determine electronically the direct sequence of the individual bases in a single-stranded DNA molecule, thereby opening the door to sequencing the human genome in hours rather than years as with capillary electrophoresis (CE) and ultra-thin slab gel techniques. Ion channel research will play an increasingly important role in understanding numerous diseases and processes in the cardiovascular system and the central nervous system. Diseases and disorders such as migraine, epilepsy, Alzheimer's, cardiac arrhythmias, cystic fibrosis, and hypertension have all been found to have ion channel involvement and drug therapies directed at these and other disorders are focused on ion channels.