An innovative label-free dual-nanopore TOF sensor for detection and identification of single molecules

标题：精准医学生物模块化多尺度系统（CBM 2）生物技术资源中心 TR&D 2：用于检测和识别单分子的创新无标记双纳米孔TOF传感器 摘要 纳米流体装置为分析单分子提供了有前途的和高度创新的方法， 获得由于缩放问题而不能使用微流体实现的生物物理信息。的能力 提供可靠，快速，定量和低成本的单分子鉴定将提供令人兴奋的新 广泛的生物医学应用的机会。生物技术资源TR& D2的目标 生物模块化多尺度系统中心（CBM 2）的精准医学是生产一种创新的无标签 纳米流体传感器不仅可以检测单个分子，还可以识别它们。背后的假设 我们的纳米传感器是，“通过二维纳米管电动移动的单个分子将经历时间- 飞行时间（TOF）取决于它们的分子身份。我们已经在我们的 活性P41与荧光标记的脱氧核苷酸、核糖核苷酸单磷酸的基线分离 和寡核苷酸通过它们的飞行时间（TOF）通过基于聚合物的纳米柱。TR&D 2旨在扩展 将单个分子的TOF分化转化为无标记的方法。分子的无标记读出- 使用双纳米孔TOF传感器实现依赖性TOF，其中两个或更多个面内纳米孔 放置在纳米柱的两端，用于纳米级电泳。分子飞行时间 由两个连续的电流瞬态信号之间的时间延迟测量提供了一个签名， 单个分子的鉴定。在现役的P41中心，初步数据已经证明了这一能力。 该TR&D将开发高通量无标记TOF传感所需的硬件和软件。高 具有亚5 nm平面内纳米孔的纳米传感器的速率制造将通过使用 我们在P41中开发的制造协议。这将与发展相结合， 数据处理电子学和基于机器学习的单分子识别算法， 识别精度。在单个芯片上构建多个纳米传感器（>100个传感器）所需的技术 每个芯片）将开发，其中包括大面积成型工具，复制工艺，可靠的 电/流体连接和电子/软件。这将允许高吞吐量处理单个 分子。作为本TR&D中开发的技术的演示，我们将使用双纳米孔TOF 传感器来评估DNA的表观遗传修饰。通过将固态纳米反应器（TR&D 1）与 通过优化平面内纳米孔和纳米柱的尺寸，该传感器可以被配置为提供 来自未扩增目标（DNA、RNA和蛋白质）的分子信息，具有前所未有的识别能力 准确度（>95%）。这将改变单分子处理，以支持广泛的生物医学服务 社区的广泛应用。

TITLE: Biotechnology Resource Center of BioModular Multi-scale Systems (CBM2) for Precision Medicine TR&D 2: An innovative label-free dual-nanopore TOF sensor for detection and identification of single molecules Abstract Nanofluidic devices offer promising and highly innovative approaches for analyzing single molecules and obtaining biophysical information that cannot be realized using microfluidics due to scaling issues. The ability to provide reliable, rapid, quantitative, and low-cost identification of single molecules will offer exciting new opportunities for a broad range of biomedical applications. The goal of TR&D 2 of the Biotechnology Resource Center of BioModular Multi-scale Systems (CBM2) for Precision Medicine is to produce an innovative label-free nanofluidic sensor for not only detecting single molecules, but identifying them as well. The hypothesis behind our nanosensor is, “individual molecules moving electrokinetically through a 2D nanotube will experience time- of-flight (TOF) that are dependent upon their molecular identity.” We have demonstrated this concept in our active P41 with baseline separations of fluorescently labeled deoxynucleotide, ribonucleotide monophosphates and oligonucleotides via their time-of-flight (TOF) through a polymer-based nano-column. TR&D 2 aims to extend the TOF differentiation of single molecules to a label-free approach. Label-free readout of the molecular- dependent TOF is achieved using dual-nanopore TOF sensors, where two or more in-plane nanopores are placed at either end of a nano-column, which is used for nanoscale electrophoresis. The molecular TOF measured by the time delay between two consecutive current transient signals provides a signature to allow for identification of single molecules. In the active P41 Center, preliminary data have demonstrated this capability. This TR&D will develop the hardware and software required for high throughput label-free TOF sensing. High rate manufacturing of the nanosensor with sub-5 nm in-plane nanopores will be achieved via NIL using the manufacturing protocols that we developed in our active P41. This will be combined with the development of data processing electronics and single-molecule identification algorithms based on machine learning to increase identification accuracies. Technologies required to build multiple nanosensors on a single chip (>100 sensors per chip) will be developed, which include large area molding tools, replication processes, reliable electrical/fluidic connections, and electronics/software. This will allow for high throughput processing of single molecules. As a demonstration of the technology developed in this TR&D, we will use the dual-nanopore TOF sensor to assess epigenetic modifications of DNA. By integrating solid-state nano-reactors (TR&D 1) with optimized dimensions of the in-plane nanopores and nano-columns, this sensor can be configured to provide molecular information from unamplified targets (DNA, RNA, and proteins) with unprecedented identification accuracies (>95%). This will transform single-molecule processing to allow servicing a broad biomedical community for a wide range of applications.