Advanced Parallel Readers for DNA Sequencing Through a 2D Nanopore

用于通过 2D 纳米孔进行 DNA 测序的高级并行读取器

• 批准号: 10437327
• 负责人: Marija Drndic
• 金额: $ 27.33万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-04 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10437327
• 关键词: 3-Dimensional; Advanced Development; Architecture; Biological; Buffers; Caliber; Calibration; Complex; Custom; DNA; DNA Microarray Chip; DNA Sequence; DNA sequencing; Data Analyses; Detection; Development; Devices; Diagnosis; Electrodes; Electrolytes; Entropy; Enzymes; Face; Finite Element Analysis; Freedom; Genetic; Geometry; Glass; Goals; Grant; Height; Image; Ions; Machine Learning; Measurement; Measures; Membrane; Methods; Modeling; Molecular; Motion; Noise; Nucleotides; Optics; Patients; Photons; Polymerase; Procedures; Reader; Research; Resistance; Route; Running; Ships; Signal Transduction; Silicon; Single-Stranded DNA; Sodium Chloride; Speed; Stochastic Processes; Symptoms; System; Techniques; Technology; Temperature; Testing; Thick; Thinness; Time; United States National Institutes of Health; base; cost; design; detection platform; disorder prevention; ds-DNA; electronic sensor; experience; fluorophore; improved; nanometer; nanopore; nanoscale; novel strategies; operation; pragmatic implementation; rational design; sequencing platform; silicon nitride; single molecule; solid state; temporal measurement; transcriptome sequencing; voltage

Project Summary To improve DNA and RNA sequencing with respect to accuracy, robustness and speed, this NIH R21 project focuses on using a two-layer design with two parallel solid-state SiN-2D nanopores on low-noise all-glass chips, towards DNA sequencing and direct RNA sequencing. The basic concept behind nanopores involves using an applied voltage to drive single-stranded DNA molecules through a narrow nanopore, which separates chambers of electrolyte solution. This voltage also drives a flow of electrolyte ions through the pore, measured as an electric current. When molecules pass through the nanopore they modify the flow of ions, and structural information can be extracted by analysis of the duration and magnitude of the resulting current reductions. Nanopore in ultrathin SiN membranes, as well as 2D membranes, improve the signal- to-noise ratio for molecular detection and analysis because the resistance to the ionic flow through a nanopore increases linearly with the nanopore thickness, so both the magnitudes of the ionic current and the blocked current with a translocating molecule increase with decreasing nanopore height. Specifically, we seek to make solid-state ionic-current based nanopore sequencing possible by combining several important components: we propose to demonstrate a two-layer on- chip solid-state SiN-2D-pore system that limits the range of DNA motion through two parallel proximal pores that are electrically independently addressable. We create devices containing a second layer with one silicon nitride (SiN) pore, parallel to a primary layer containing the atomically-thin 2D pore that confine ssDNA within a device to a restricted geometry, yet allow the free motion of salt ions to maintain a high signal-to-noise ratio. We propose a specific two-layer concept, where the two layers are in close proximity, with two independent electrical connections, and corresponding chip device architecture to achieve this goal. In this method, there is a central, highly sensitive 2D pore which we refer to as the main sensing/sequencing 2D nanopore. A secondary layer has a second pore sharing the same electrode pair as the sensing pore, but also having its own independent electrode pair to be probed separately. Although we have two pores, they can operate as a continuous system due to their proximity. We outline the 3D finite element analysis modeling and practical implementation (two versions) of these concepts with Si-based technology, including advantages and challenges involved for DNA (and biomolecule) sequencing (analysis) in solution. Our approach eliminates the need for any enzymes and enables DNA and biomolecules to be guided through robust and long-lasting nanopores, facilitated by the custom- designed chip combining the best of what the SiN and 2D pores can currently offer. Illustration 1: Proposed two-layer device concept for this NIH R21 proposal, relying on minimization of DNA entropic motion using two proximal, parallel SiN-2D pores that are electrically independently contacted: a guiding SiN pore of variable diameter and an optimized sensing 2D materials pore. The spacing between the two layers is adjustable down to a few nm (facilitated by the single nm control of RIE or TEM etching). This Si platform is versatile and compatible with any 2D materials (shown as green triangle). 1

项目概要 为了提高 DNA 和 RNA 测序的准确性、稳健性和速度，NIH R21项目侧重于使用具有两个平行固态SiN-2D纳米孔的两层设计 在低噪声全玻璃芯片上进行 DNA 测序和直接 RNA 测序。基本的 纳米孔背后的概念涉及使用外加电压来驱动单链 DNA 分子通过狭窄的纳米孔，该纳米孔分隔电解质溶液室。这 电压还驱动电解质离子流过孔隙，以电流的形式测量。 当分子穿过纳米孔时，它们会改变离子的流动和结构 可以通过分析产生的电流的持续时间和幅度来提取信息 减少。超薄 SiN 膜以及 2D 膜中的纳米孔可改善信号 分子检测和分析的信噪比，因为离子流经的阻力 纳米孔随着纳米孔厚度线性增加，因此离子的大小 随着纳米孔的减少，电流和易位分子阻挡的电流增加 高度。具体来说，我们寻求基于固态离子电流的纳米孔测序 通过组合几个重要的组件可以实现：我们建议演示一个两层的on- 芯片固态 SiN-2D 孔系统，通过两个平行的通道限制 DNA 运动范围 可独立电寻址的近端孔。我们创建的设备包含 第二层具有一个氮化硅 (SiN) 孔，与包含 原子级薄的 2D 孔，将装置内的 ssDNA 限制在有限的几何形状内，但允许 盐离子的自由运动以保持高信噪比。我们提出了一个特定的两层 概念，其中两层非常接近，具有两个独立的电气连接， 以及相应的芯片器件架构来实现这一目标。在这个方法中，有一个中心， 高灵敏度的二维孔，我们称之为主要传感/测序二维纳米孔。一个 第二层有一个第二个孔，与传感孔共享相同的电极对，而且 有自己独立的电极对，可以单独探测。虽然我们有两个毛孔， 由于距离较近，它们可以作为一个连续的系统运行。我们概述了 3D 有限元 基于 Si 的这些概念的分析建模和实际实现（两个版本） 技术，包括 DNA（和生物分子）测序所涉及的优势和挑战 （分析）溶液中。我们的方法消除了对任何酶的需求，使 DNA 和 在定制的促进下，生物分子被引导通过坚固且持久的纳米孔 设计的芯片结合了 SiN 和 2D 孔目前所能提供的最佳功能。 图 1：建议的两层结构 NIH R21 的设备概念 提案，依赖于最小化 DNA 熵运动使用两个 近端平行的 SiN-2D 孔 电气上独立 接触：SiN引导孔 可变直径和优化的 传感二维材料孔隙。这 两层之间的间距为 可调节至几纳米 （通过单纳米控制促进 RIE 或 TEM 蚀刻）。这个Si平台 用途广泛且兼容任何 2D 材料（显示为绿色 三角形）。 1