Integrated, multiplexed high-frequency electronic analysis of DNA in nanopores

描述（由申请人提供）：对单分子、大规模并行和实时的第三代DNA测序系统有强烈的需求。然而，对于单分子光学技术，来自单个荧光团的信号通常< 2500光子/秒（相当于50 fA量级的电流水平）。这导致复杂的光学器件试图收集发射的每个光子，并使平台的缩放变得困难。此外，合成反应必须有意地减慢到1 Hz（或更慢），以允许这些弱的、有噪声的光学信号有足够的成像时间。 单分子的局限性 光学技术突出了电化学检测方法的关键优势，电化学检测方法具有显著更高的信号水平（通常高出三个数量级），允许通过换能器、检测器和放大器的适当协同设计进行高带宽检测的可能性。已经针对纳米孔技术作为一种潜在的生物电子转导机制的发展做出了重大努力。然而，已经证明纳米孔受到生物分子在孔的电荷敏感区域中花费的相对短的时间的极大限制。受现成电子器件使用的限制，纳米孔测量的噪声限制带宽通常小于100 kHz，限制了可用的感测和致动策略，并且阻碍了任何测序应用所需的多路集成。 在这四年的努力中，我们专注于显着提高纳米孔检测电子设备的噪声限制带宽，使其充分发挥潜力， 可以通过电子器件和孔的紧密集成来实现，同时支持同一检测衬底上的多个纳米孔的高水平平行性。我们考虑将固态（特定目标1）和生物孔（特定目标3）以大规模并行方式（特定目标2）集成到这些测量基板上的技术。我们提出的利用商品CMOS技术和共集成检测电子器件的技术是完全通用的，对所有其他单分子生物电子转导方法具有重要意义。这些高带宽集成电子器件还将实现“闭环”传感和驱动（具体目标4），允许在微秒（或更好）的时间尺度上动态操纵捕获和移位动力学。 公共卫生相关性：CMOS电子器件与纳米孔的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。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。

DESCRIPTION (provided by applicant): There is strong demand for third-generation DNA sequencing systems to be single-molecule, massively- parallel, and real-time. For single-molecule optical techniques, however, the signal from a single fluorophore is typically < 2500 photons/sec (equivalent to electrical current levels on the order of 50 fA). This leads to complex optics to try to collect every photon emitted and makes scaling of the platforms difficult. Additionally, synthesis reactions must be intentionally slowed to 1 Hz (or slower) to allow sufficient imaging times for these weak, noisy optical signals. The limitations of single-molecule optical techniques highlight key advantages of electrochemical detection approaches, which have significantly higher signal levels (typically three orders of magnitude higher), allowing for the possibility for high-bandwidth detection with the appropriate co-design of transducer, detector, and amplifier. Significant effort has been directed toward the development of nanopore technology as one potential bioelectronic transduction mechanism. Nanopores, however, have proved to be extremely limited by the relatively short time biomolecules spend in the charge-sensitive region of the pore. Restricted by the use of off- the-shelf electronics, the noise-limite bandwidth of nanopore measurements is typically less than 100 kHz, limiting the available sensing and actuation strategies and defying multiplexed integration which would be required for any sequencing application. In this four-year effort, we focus on improving significantly the noise-limited bandwidth of the detection electronics for nanopores allowing their full potential to be realized through close integration of the electronics and the pore while simultaneously supporting high levels of parallelism with multiple nanopores on the same detection substrate. We consider techniques for integrating both solid-state (Specific Aim 1) and biological pores (Specific Aim 3) onto these measurement substrates in a massively parallel manner (Specific Aim 2). The techniques we propose for leveraging commodity CMOS technology and co-integrating detection electronics are completely general and have significance to all other single-molecule bioelectronic transduction approaches. These high-bandwidth integrated electronics will also enable "closed-loop" sensing and actuation (Specific Aim 4), allowing dynamic manipulation of capture and translocation dynamics at microsecond (or better) timescales. PUBLIC HEALTH RELEVANCE: The integration of CMOS electronics with nanopores will address key obstacles that must be overcome to achieve nanopore-based low-cost high-speed sequencing of chromosomal length DNA molecules. 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.