Large-Scale Nanopore Arrays for DNA Sequencing

描述（由申请人提供）：如今，合成纳米孔装置是作为用于高速非酶促询问个体生物大分子的有趣候选装置的深入研究的主题。例如，为了以高速对未知组成的单个DNA片段进行测序，需要这种功能。 虽然目前由几个NIH资助的小组正在研究关于大分子易位物理学和新型读出方案的许多理论方面，但测序系统中纳米孔的实际利用还需要并行工作的大型纳米孔阵列，每个纳米孔都连接到高灵敏度电子放大器/检测器。 为了实现与当今最先进的通过合成方案（SBS）的并行酶促测序（例如由454 Life Sciences提供的那些，其报道了以20 Mb/天对细菌基因组的高保真测序）相比的通量的显著改进，这种并行度和系统集成是必要的。为了说明对阵列的需要，可以考虑例如具有100 b/ms/孔的平均纳米孔移位速率的100纳米孔阵列原则上可以以10 Gb/天的速率（高保真度，100次重复）对片段进行测序，从而允许在一天内对整个人类基因组进行测序，比SBS快500倍。 该提案的目的是（a）开发高度集成的纳米孔阵列，其可以通过光刻方法沿着芯片上基于硅的电子电路和电路技术来制造，所述电路技术放大和隔离其各种电信号，以及（B）探索偶极感测方法，其原则上可以区分来自每个DNA碱基的信号。正交电容传感器将被纳入一些制造的纳米孔。 纳米孔阵列将使用自对准组合方法在硅衬底上构建。 正交偶极矩检测器将被构造成产生独立于DNA分子相对于电极的旋转的信号。

DESCRIPTION (provided by applicant): Today, synthetic nanopore devices are the subject of intense investigation as intriguing candidate devices for high-speed non-enzymatic interrogation of individual biological macromolecules. Such functionality is required for example in order to sequence individual DNA fragments of unknown composition at high speeds. While many theoretical aspects regarding macromolecule translocation physics and novel readout schemes are presently under investigation by several NIH-funded groups, the practical utilization of nanopores in sequencing systems also requires large arrays of nanopores working in parallel, each of these connected to high-sensitivity electronic amplifiers/detectors. Such degree of parallelism and systems integration is necessary in order to achieve significant improvements in throughput compared with today's state-of-the-art massively-parallel enzymatic sequencing by synthesis schemes (SBS) (such as those offered by 454 Life Sciences which reports high-fidelity sequencing of bacterial genomes at 20 Mb/day). To illustrate the need for arrays one may consider for example, a 100- nanopore array with average nanopore translocation rate of 100b/ms/pore could in principle sequence fragments at (high-fidelity, 100 repeats) rates of 10 Gb/day, allowing an entire human genome to be sequenced in a day, 500 times faster than SBS. The aims of this proposal are (a) the development of highly-integrated arrays of nanopores that can be fabricated by lithographic methods along with on-chip silicon-based electronic circuits and circuit techniques that amplify and isolate their various electrical signals, and (b) the exploration of a dipole sensing methodology which in principle can distinguish signals from each of the DNA bases. A quadrature capacitance sensor will be incorporated in some of the fabricated nanopores. Arrays of nanopores will be constructed on silicon substrates using a self-aligned compositional approach. Quadrature dipole moment detectors will be constructed that yield a signal independent of the rotation of the DNA molecule relative to the electrodes.