Enzymeless, controlled electrostatic ratcheting in solid-state nanopores

固态纳米孔中的无酶受控静电棘轮效应 对于第三代DNA测序系统的强烈需求是单分子的，大规模的- 并行和实时，同时还降低了运营成本并支持长读取长度。无技术 但迄今为止最成功的尝试是基于以下方法， 跟踪单酶分子在DNA链上的实时操作。光学方法 单聚合酶成像的缺点是低信噪比， 单个荧光团（< 2500光子/秒），因此需要复杂的光学器件和降低成本的基底 掺入率（~1 Hz）。基于纳米孔的检测方法提供了更快的检测，并且已经被广泛应用。 已经证明在较高的掺入率（~10-100 Hz）下跟踪聚合酶活性，但一直在努力 可靠性问题和与流行蛋白质产生的仍然微弱的信号水平相关的高错误率 纳米孔 这些努力表明，基于纳米孔的单分子测序技术，不去- 致力于活性酶的实时成像将具有几个重要的优点。首先， 它们可以提供比自由运行的聚合酶分子更快的测序速度。第二，去除 来自检测平台的活性酶提供了更大的自由度来优化关键参数， 天然酶的操作范围之外的条件和温度。三、测序平台 没有活性酶，可以证明操作、运输和储存更简单和更便宜。最后，纳米孔，特别是- 主要是生物学的，面临着电子设备的可靠性挑战，在使用过程中经历退化。 在这四年的努力中，我们专注于开发一种多路固态纳米孔平台， 利用集成电子设备和最新技术，每个孔的测序速率至少为105个碱基/秒 基于层状二维材料的超薄膜的固态纳米孔， 当需要时，有用的信号带宽超过10 MHz。我们希望能够检测到信号水平， 在信噪比大于8和带宽优于2 MHz时，低至50 pA， 速度自由运行的单分子电泳测序，如果易位率和扩散运动的 可以控制易位的DNA。这是通过静电控制，通过栅极整流器实现的。 在孔隙中的电极和闭环反馈。这一目标是通过三个具体目标实现的： 基于层状二维材料的固态纳米孔的迹象包括六方氮化硼（H- BN）和石墨烯或过渡金属二硫属化物以及这些孔在易位DNA中的应用 （具体目标1）;针对这些纳米孔的高速多重检测优化的电子器件的设计 和孔内闸门的闭环电子控制（具体目标2），以及该系统的应用 控制易位率进行测序（具体目标3）。

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).