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

纳米孔中 DNA 的集成、多重高频电子分析

• 批准号: 8365334
• 负责人: Kenneth L Shepard
• 金额: $ 50万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-14 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365334
• 关键词: Address; Amplifiers; Biological; Caliber; Charge; Complex; Custom; DNA; DNA Sequence; DNA analysis; DNA-Directed DNA Polymerase; Detection; Development; Devices; Diagnosis; Diffusion; Electronics; Frequencies; Generations; Genome; Image; Length; Lipid Bilayers; Measurement; Measures; Membrane; Noise; Operating System; Optics; Photons; Post Technic; Process; Reaction; Reading; Research; Semiconductors; Signal Transduction; Sodium Chloride; Speed; System; Techniques; Technology; Testing; Time; Transducers; base; cost; design; detector; disorder prevention; fluorophore; improved; instrumentation; millisecond; nanofabrication; nanopore; operation; silicon nitride; single molecule; solid state

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.

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）。这导致了复杂的光学系统，试图收集每一个发射的光子，并使平台的缩放变得困难。此外，合成反应必须有意减慢到1hz（或更慢），以便为这些微弱的、有噪声的光信号提供足够的成像时间。单分子的局限性