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: 10683967
• 负责人: Marija Drndic
• 金额: $ 75万
• 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/10683967
• 关键词: Address; Amplifiers; Biological; Boron; Buffers; Complex; Custom; DNA; DNA sequencing; Detection; Development; Diagnosis; Diameter; 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; Signal Transduction; Speed; Stochastic Processes; Structure; System; Techniques; Technology; Temperature; Time; Transition Elements; Work; base; cost; design; detection platform; disorder prevention; electric field; experience; fabrication; fluorophore; graphene; improved; 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，受控的静电架 人们对第三代DNA测序系统的需求很强 并行和实时，同时还降低运营成本并支持长长的读取长度。没有技术 尚未满足这一挑战，但最成功的尝试是基于方法 跟踪在DNA链上运行的单个酶分子的实时操作。光学方法 单聚合酶成像受到弱光子发射的低信噪比比率。 单个荧光团（<2500个光子/秒），因此需要复杂的光学元件和故意还原碱 合并率（〜1 Hz）。基于纳米孔的检测方法可提供更快的检测，并且已经 证明以较高的掺入速率（〜10-100 Hz）跟踪聚合酶活性，但与 可靠性问题和与流行蛋白产生的静止信号水平相关的高错误率 纳米孔。 这些斗争表明，基于纳米的单分子测序技术不会 对活性酶的实时成像进行剪辑将具有几个重要的优势。首先， 他们可以比自由运行的聚合酶分子更快地提供测序速度。第二，删除 来自检测平台的活动酶提供了更大的自由来优化关键参数，例如缓冲区 天然酶工作范围以外的条件和温度。第三，测序平台 如果没有活性酶，则可以证明可以更简单，更便宜地运营，运输和存储。最后，纳米孔，特别 大部分生物学，面临电子设备作为电子设备的可靠性挑战，在使用过程中经历降解。 在这四年的努力中，我们专注于开发多重的固态纳米孔平台ENA- 将每孔测序速率至少为105个碱基/秒，利用集成电子设备和最新 基于分层二维材料的超薄膜并提供的固态纳米孔 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 信噪比低于50 pa，大于8，带宽优于2 MHz，使可能的高 速度自由运行的单分子电泳测序，如果易位速率和扩散运动的运动 可以控制易位的DNA。这是通过静电控制通过门电子来完成的 孔本身和闭环反馈中的曲线。通过三个特定目标来实现这一目标： 基于分层的二维材料的固态纳米孔的符号包括硝化醇（H-） BN）和石墨烯或过渡金属二核苷以及这些毛孔的应用在易位DNA （特定目标1）;针对这些纳米孔的高速多路复用检测优化的电子产品的设计 和孔内门的闭环电子控制（特定目标2）；和该系统的应用 控制测序的易位速率（特定目标3）。