Enzymeless, controlled electrostatic ratcheting in solid-state nanopores

固态纳米孔中的无酶、受控静电棘轮

• 批准号: 10439291
• 负责人: Marija Drndic
• 金额: $ 79.5万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10439291
• 关键词: Address; Amplifiers; Biological; Boron; Buffers; Caliber; Complex; Custom; DNA; DNA sequencing; Detection; Development; Diagnosis; Diffuse; Electric Capacitance; Electrodes; Electronics; Electrostatics; Entropy; Enzymes; Face; Feedback; Freedom; Generations; Goals; Image; Length; Measurement; Measures; Mechanics; Membrane; Methods; Motion; Noise; Optics; Performance; Photons; Polymerase; Proteins; Public Health; Research; Resolution; Running; Ships; Signal Transduction; Speed; Stochastic Processes; Structure; System; Techniques; Technology; Temperature; Thinness; Time; Transition Elements; Work; base; cost; design; detection platform; disorder prevention; electric field; experience; fluorophore; graphene; multiplex detection; nanopore; operation; real-time images; sequencing platform; single molecule; solid state; temporal measurement; two-dimensional; voltage; voltage clamp; whole genome

Enzymeless, controlled electrostatic ratcheting in solid-state nanopores There is strong demand for third-generation DNA sequencing systems to be single-molecule, massively- parallel, and real-time, while also reducing operating costs and supporting long read lengths. No technologies have yet met this challenge, but the most successful attempts to date have been based on methods which track the real-time operation of single enzyme molecules operating on a strand of DNA. Optical approaches to single-polymerase imaging suffer from low signal-to-noise ratios deriving from the weak photon emission from single fluorophores (< 2500 photons/sec), and thus demand both complex optics and purposely-reduced base incorporation rates (~1 Hz). Nanopore-based detection approaches offer faster detection and have been demonstrated to track polymerase activity at higher incorporation rates (~10-100 Hz), but have struggled with reliability issues and high error rates associated with the still-weak signal levels produced by popular protein nanopores. These struggles suggest that nanopore-based single-molecule sequencing techniques which do not de- pend on real-time imaging of active enzymes would have several important advantages. First and foremost, they could offer sequencing speeds even faster than a free-running polymerase molecule. Second, removing active enzymes from the detection platform offers more freedom to optimize key parameters such as buffer conditions and temperatures outside the operating range of natural enzymes. Third, sequencing platforms without active enzymes may prove simpler and cheaper to operate, ship, and store. Lastly, nanopores, particu- larly biological ones, face reliability challenges as electronic devices, experiencing degradation during use. In this four-year effort, we focus on the development of a multiplexed solid-state nanopore platform ena- bling a per-pore sequencing rate of at least 105 bases/sec, leveraging integrated electronics and state-of-the- art solid-state nanopores based on ultra-thin membranes of layered two-dimensional materials and delivering useful signal bandwidths in excess of 10 MHz when required. We expect to be able to detect signal levels as low as 50 pA at signal-to-noise ratios greater than 8 and bandwidth better than 2 MHz, making possible high- speed free-running single-molecule electrophoretic sequencing if the translocation rate and diffusive motion of the translocating DNA can be controlled. This is accomplished through electrostatic control through gate elec- trodes in the pore itself and closed-loop feedback. This goal is pursued through three Specific Aims: the de- sign of solid-state nanopores based on layered two-dimensional materials include hexagonal boron nitride (h- BN) and graphene or transition metal dichalcogenides and application of these pores to translocating DNA (Specific Aim 1); the design of electronics optimized for high-speed multiplexed detection of these nanopores and closed-loop electronic control of the gates within the pore (Specific Aim 2); and application of this system to controlling translocation rates for sequencing (Specific Aim 3).

固态纳米孔中的无酶、受控静电棘轮 人们对第三代 DNA 测序系统的强烈需求是单分子、大规模- 并行和实时，同时还降低了运营成本并支持长读取长度。没有技术 尚未遇到这一挑战，但迄今为止最成功的尝试都是基于以下方法： 跟踪 DNA 链上单个酶分子的实时操作。光学方法 单聚合酶成像的信噪比较低，这是由于微弱的光子发射造成的 单荧光团（< 2500 光子/秒），因此需要复杂的光学器件和特意减少的碱基 掺入率（~1 Hz）。基于纳米孔的检测方法可提供更快的检测速度，并且已被 被证明可以以较高的掺入率（~10-100 Hz）追踪聚合酶活性，但一直在努力 与流行蛋白质产生的仍然微弱的信号水平相关的可靠性问题和高错误率 纳米孔。 这些斗争表明，基于纳米孔的单分子测序技术并不能解决问题。 对活性酶的实时成像进行研究将具有几个重要的优势。首先也是最重要的， 它们可以提供比自由运行的聚合酶分子更快的测序速度。二、去除 检测平台中的活性酶提供了更大的自由度来优化缓冲液等关键参数 条件和温度超出天然酶的工作范围。三、测序平台 没有活性酶的操作、运输和储存可能会更简单、更便宜。最后，纳米孔，特别是 大多数生物设备都面临着电子设备的可靠性挑战，在使用过程中会出现退化。 在这四年的努力中，我们专注于开发多重固态纳米孔平台 利用集成电子设备和最先进的技术，每孔测序速率至少为 105 个碱基/秒 基于层状二维材料超薄膜的艺术固态纳米孔并提供 需要时，有用信号带宽超过 10 MHz。我们期望能够检测信号水平 信噪比大于 8 且带宽优于 2 MHz 时电流低至 50 pA，从而使高 加速自由运行的单分子电泳测序，如果易位率和扩散运动 DNA的易位是可以控制的。这是通过栅极电子的静电控制来完成的 微孔本身的踏步和闭环反馈。这一目标是通过三个具体目标来实现的： 基于层状二维材料的固态纳米孔的标志包括六方氮化硼（h- BN）和石墨烯或过渡金属二硫属化物以及这些孔在 DNA 易位中的应用 （具体目标1）；针对这些纳米孔的高速多重检测而优化的电子器件设计 以及孔内闸门的闭环电子控制（具体目标 2）；以及本系统的应用 控制测序的易位率（具体目标 3）。