Nanofluidic Devices for Studying Assembly of Single Virus Particles

用于研究单一病毒颗粒组装的纳米流体装置

• 批准号: 8791699
• 负责人: Stephen C Jacobson
• 金额: $ 29.36万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8791699
• 关键词: Antiviral Agents; Antiviral Therapy; Architecture; Behavior; Capsid; Chronic Hepatitis B; Computer Simulation; Core Protein; Coupled; Data; Development; Devices; Dimensions; Event; Evolution; Foundations; Geometry; Hepatitis B Virus; Kinetics; Knowledge; Label; Liver diseases; Measurement; Measures; Methods; Microfluidic Microchips; Modeling; Monitor; Pathway interactions; Physiologic pulse; Play; Population; Process; Property; Reaction; Recruitment Activity; Role; Series; Shapes; Solutions; Structure; Surface Properties; System; Techniques; Testing; Time; Viral; Virion; Virus; Virus Assembly; Work; design; dimer; improved; in vivo; mutant; nanochannel; nanofluidic; nanoparticle; nanopore; nanoscale; particle; simulation software; stem; two-dimensional; virus core

DESCRIPTION (provided by applicant): We will establish general methods to determine the assembly pathways of viruses and self- assembling nanoparticles. We will demonstrate these methods using Hepatitis B virus (HBV) capsid assembly. Resistive-pulse sensing allows real time monitoring of assembly by identifying single particles and assembly intermediates. Understanding the mechanism of virus assembly requires not only knowledge of precursors and final product structures, but also access to intermediates. Where many rare intermediates are involved, ensemble methods obscure them so that virus assembly resembles a two-state reaction. HBV has acutely infected more than 2B people; about 360M people have chronic HBV; every year nearly 1M will die of HBV-related liver disease. Assembly of HBV's icosahedral capsid has been identified as a new target for antiviral therapies. HBV assembly and the behavior (and development) of antiviral assembly effectors are relatively well understood, largely stemming from work in the Zlotnick lab, but there has been no direct observation and characterization of critical early intermediates in solution. Computational models of assembly suggest that the observed kinetics reflect early establishment of a constellation of intermediates needed to support capsid formation. The nucleation step and early intermediates are believed to play a role in recruiting viral components in vivo. Antiviral assembly effectors over-stimulate nucleation, distorting the distribution of intermediates and very often their structure. In our previous work, we established HBV assembly as a well-defined experimental system for nanofluidics and now have the foundation to interrogate assembly and antiviral assembly effectors. Nanofluidic components integrated with microfluidic devices offer a unique platform for answering these outstanding questions. Resistive-pulse sensing on these devices permits a real time, label-free approach to monitoring assembly at biologically relevant concentrations (nM to mM). More specifically, we will develop devices and methods to study particle transport properties through nanochannel networks. These coupled nanochannels can be arranged in virtually any two-dimensional format and operated with modest applied potentials. How particle transport is influenced depends strongly on the dimensions and geometries of the nanoscale conduits, applied waveforms, surface properties of the conduit, and composition of the transport medium, and particle shape and composition. We will optimize these device parameters in order to develop a fundamental understanding of capsid formation. The Specific Aims for this application are to: (1) characterize capsid assembly under various reaction conditions; (2) fabricate and test in-plane nanochannels with single pores and multiple pores in series for improved resistive-pulse sensing; (3) computationally simulate (i) assembly and (ii) particle transport in nanofluidic devices; and (4) develop coupled nanochannels to sense particles of different sizes and to perform reactions with single capsids.

描述（由申请人提供）：我们将建立一般方法来确定病毒和自组装纳米颗粒的组装途径。我们将使用丙型肝炎病毒（HBV）衣壳组件来证明这些方法。电阻脉冲传感可以通过识别单个颗粒和组装中间体来实时监测组件。了解病毒装配的机制不仅需要前体和最终产品结构的知识，而且还需要进入中间体。如果涉及许多罕见的中间体，合奏方法掩盖了它们，使病毒组装类似于两态反应。 HBV急性感染了2B多人。大约36000万人患有慢性HBV；每年将近1M死于与HBV相关的肝病。 HBV的二十面体capsid的组装已被确定为抗病毒疗法的新靶标。 HBV组装和抗病毒组装效应子的行为（以及开发）相对众所周知，这在很大程度上源于Zlotnick Lab的工作，但是没有直接观察和表征溶液中关键的早期中间体。组装的计算模型表明，观察到的动力学反映了早期建立支持衣壳形成所需的中间体的星座。据信，成核步骤和早期中间体在体内募集病毒成分中起作用。抗病毒装配效应子过度刺激成核，使中间体的分布及其结构扭曲。在我们以前的工作中，我们建立了HBV组装作为纳米流体学的明确定义的实验系统，现在有基础来询问组装和抗病毒装配效应子。 与微流体设备集成的纳米流体组件为回答这些出色的问题提供了一个独特的平台。这些设备上的电阻脉冲传感允许以生物学相关浓度（NM至mm）进行实时，无标签的方法来监测组装。更具体地说，我们将开发通过纳米通道网络研究颗粒传输性能的设备和方法。这些耦合的纳米通道几乎可以以任何二维格式排列，并具有适度的应用电势。颗粒传输的影响如何在很大程度上取决于纳米级导管的尺寸和几何形状，应用波形，导管的表面特性以及传输介质的组成以及粒子的形状和组成。我们将优化这些设备参数，以便对Capsid形成产生基本了解。此应用的具体目的是：（1）在各种反应条件下表征capsid组装； （2）制造和测试具有单个孔和多个毛孔的平面内纳米通道，以改善电阻脉冲传感； （3）计算模拟（i）组装和（ii）纳米流体设备中的粒子传输； （4）开发耦合的纳米通道，以感知不同大小的颗粒并与单个衣壳进行反应。

项目成果

Fundamental studies of nanofluidics: nanopores, nanochannels, and nanopipets.

• DOI: 10.1021/ac504180h
• 发表时间: 2015-01-06
• 期刊: ANALYTICAL CHEMISTRY
• 影响因子: 7.4
• 作者: Haywood, Daniel G.;Saha-Shah, Anumita;Baker, Lane A.;Jacobson, Stephen C.
• 通讯作者: Jacobson, Stephen C.

Nanofluidic Devices with 8 Pores in Series for Real-Time, Resistive-Pulse Analysis of Hepatitis B Virus Capsid Assembly.

• DOI: 10.1021/acs.analchem.6b04491
• 发表时间: 2017-05-02
• 期刊: Analytical chemistry
• 影响因子: 7.4
• 作者: Kondylis P;Zhou J;Harms ZD;Kneller AR;Lee LS;Zlotnick A;Jacobson SC
• 通讯作者: Jacobson SC

Characterization of Virus Capsids and Their Assembly Intermediates by Multicycle Resistive-Pulse Sensing with Four Pores in Series.

通过四孔串联多周期电阻脉冲传感表征病毒衣壳及其组装中间体。

• DOI: 10.1021/acs.analchem.8b00452
• 发表时间: 2018
• 期刊: Analytical chemistry
• 影响因子: 7.4
• 作者: Zhou,Jinsheng;Kondylis,Panagiotis;Haywood,DanielG;Harms,ZacharyD;Lee,LyeSiang;Zlotnick,Adam;Jacobson,StephenC
• 通讯作者: Jacobson,StephenC

Electroosmotic flow in nanofluidic channels.

• DOI: 10.1021/ac502596m
• 发表时间: 2014-11-18
• 期刊: ANALYTICAL CHEMISTRY
• 影响因子: 7.4
• 作者: Haywood, Daniel G.;Harms, Zachary D.;Jacobson, Stephen C.
• 通讯作者: Jacobson, Stephen C.

Conductivity-based detection techniques in nanofluidic devices.

• DOI: 10.1039/c5an00075k
• 发表时间: 2015-07-21
• 期刊: The Analyst
• 作者: Harms ZD;Haywood DG;Kneller AR;Jacobson SC
• 通讯作者: Jacobson SC