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: 10676761
• 负责人: Marija Drndic
• 金额: $ 15.08万
• 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 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10676761
• 关键词: 3-Dimensional; Advanced Development; Architecture; Biological; Buffers; Calibration; Complex; Custom; DNA; DNA Microarray Chip; DNA Sequence; DNA sequencing; Data Analyses; Detection; Development; Devices; Diagnosis; Diameter; 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; Rationalization; Reader; Research; Resistance; Route; Running; 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; rapid detection; 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

项目摘要 为了提高DNA和RNA测序的准确性，鲁棒性和速度，NIH R21项目专注于使用具有两个平行固态SiN-2D纳米孔的双层设计 在低噪音全玻璃芯片上，朝向DNA测序和直接RNA测序。基本 纳米孔背后的概念涉及使用施加的电压来驱动单链DNA 分子通过狭窄的纳米孔，将电解质溶液的腔室分开。这 电压还驱动电解质离子流通过孔，以电流测量。 当分子通过纳米孔时，它们改变了离子的流动， 可以通过分析所产生的电流的持续时间和幅度来提取信息 减少。纳米孔的氮化硅薄膜，以及二维膜，改善信号- 因为对离子流通过的阻力 纳米孔随着纳米孔厚度线性增加，因此离子的大小 电流和具有移位分子的阻断电流随着纳米孔的减小而增加 高度具体来说，我们寻求使固态离子电流为基础的纳米孔测序 可能通过结合几个重要组成部分：我们建议演示一个两层上- 芯片固态SiN-2D孔系统，通过两个平行的限制DNA运动的范围， 可电独立寻址的近端孔。我们创建包含一个 具有一个氮化硅（SiN）孔的第二层，其平行于包含所述氮化硅（SiN）孔的主层; 原子级薄的2D孔，将ssDNA限制在设备内的受限几何形状，但允许 盐离子的自由运动以保持高信噪比。我们提出了一个具体的两层 概念，其中两个层非常接近，具有两个独立的电连接， 以及相应的芯片器件结构来实现这一目标。在这种方法中，有一个中心， 高度敏感的2D孔，我们称之为主要的传感/测序2D纳米孔。一 第二层具有与感测孔共享相同电极对的第二孔，而且 具有其自己的独立电极对以被分别探测。虽然我们有两个毛孔， 由于它们的接近性，它们可以作为连续系统操作。我们概述了三维有限元 分析建模和实际实施（两个版本），这些概念与硅为基础的 技术，包括DNA（和生物分子）测序的优势和挑战 （分析）在溶液中。我们的方法消除了对任何酶的需要，使DNA和 生物分子被引导通过坚固和持久的纳米孔，由定制的便利， 设计的芯片结合了SiN和2D孔隙目前可以提供的最佳功能。 说明1：拟议的双层 NIH R21的设备概念 建议，依靠最小化 DNA熵运动用两个 近端平行SiN-2D孔， 在电学上独立地 接触：引导的SiN孔 可变直径和优化的 传感2D材料孔隙。的 两层之间的间隔是 可调节到几nm （通过单一纳米控制， RIE或TEM蚀刻）。这个Si平台 是通用的，兼容任何 2D材质（显示为绿色 三角形）。 1