Enzymeless, controlled electrostatic ratcheting in solid-state nanopores

固态纳米孔中的无酶、可控静电棘轮 对第三代DNA测序系统的强烈需求是单分子、大规模... 并行、实时，同时还降低了运营成本并支持长读取长度。没有技术 还没有迎接这一挑战，但迄今为止最成功的尝试是基于 跟踪在DNA链上操作的单酶分子的实时操作。光学方法来实现 单聚合酶成像的低信噪比是由弱光子发射引起的 单个荧光团(2500光子/秒)，因此既需要复杂的光学元件，又需要特意减少的基团 参入率(~1赫兹)。基于纳米孔的检测方法提供了更快的检测速度，并已 被证明以更高的参入率(~10-100赫兹)跟踪聚合酶活性，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。 这些挣扎表明，基于纳米孔的单分子测序技术不会破坏... Pend对活性酶的实时成像将有几个重要的优势。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。第二，移除 来自检测平台的活性酶为优化缓冲液等关键参数提供了更大的自由度 在天然酶作用范围之外的条件和温度。第三，测序平台 没有活性的酶可能会证明操作、运输和储存更简单、更便宜。最后，纳米孔，粒子-- 作为电子设备，大型生物设备面临着可靠性挑战，在使用过程中会出现退化。 在这四年的努力中，我们专注于开发多路复用型固态纳米孔平台ENA- 每孔测序速率至少为105个碱基/秒，利用集成电子学和最新技术-- 基于层状二维材料的超薄薄膜和输送的ART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如下信号电平 在信噪比大于8，带宽优于2 MHz时低至50 pA，使高性能成为可能 快速自由运行单分子电泳法测序 DNA的移位是可以控制的。这是通过门电子通过静电控制来实现的。 在毛孔本身的踏板和闭环反馈。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卤代化合物以及这些孔在DNA移位中的应用 (具体目标1)；为高速多路检测这些纳米孔而优化的电子学设计 和气孔内浇口的闭环电子控制(具体目标2)；以及该系统的应用 控制测序的易位率(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).