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Introduction
Protocol
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Chimie

单金属纳米粒子的高分辨率物理特性

Published: June 28, 2019
doi:
10.3791/58257
, , ,
1Physical Measurement Laboratory,National Institute of Standards and Technology, 2Department of Chemical Engineering,Columbia University, 3Department of Applied Physics and Applied Math,Columbia University

Summary

在这里,我们提出了一个协议,使用基于生物纳米孔的电子平台,在单分子极限处检测离散金属氧簇、多氧金属酸盐(POM)。该方法为研究这些分子的传统分析化学工具提供了补充方法。

Abstract

单个分子可以通过测量它们减少流经单个纳米刻度孔的离子电流的程度来检测和特征。信号是分子的物理化学性质及其与孔隙相互作用的特征。我们证明,由细菌蛋白外毒素金黄色葡萄球菌α溶酶(αHL)形成的纳米孔可以在单分子极限处检测多氧金属酸盐(POM、离子金属氧簇)。此外,同时测量溶液中12磷酸POM(PTA,H3PW12O40)的多种降解产物。纳米孔法的单分子灵敏度使POM的显著浓度显著低于核磁共振(NMR)光谱。该技术可作为化学家研究多氧金属酸盐或其他金属簇的分子特性,更好地了解POM合成工艺,并可能提高其产量的新工具。假设,可以使用这种方法研究给定原子的位置或分子中片段的旋转以及金属氧化状态。此外,这项新技术的优点是能够实时监测溶液中的分子。

Introduction

使用纳米孔和测量离子电流调制,可以在单分子水平上检测生物分子分析物。通常,纳米孢子根据其制造分为两类:生物(由蛋白质或DNA折纸自行组装)1、2、3或固态(例如,由半导体加工工具)4,5.虽然固态纳米孔被认为具有潜在的物理上更坚固,可用于广泛的溶液条件,但迄今为止,蛋白质纳米孔具有更高的灵敏度、更强的抗结垢性、更大的带宽、更好的化学性选择性,以及更大的信噪比。

各种蛋白质离子通道,如金黄色葡萄球菌α-溶酶(αHL)形成的通道,可用于检测单个分子,包括离子(例如,H+和D=2,3,多核苷酸(DNA)和RNA)6,7,8,损坏的DNA9,多肽10,蛋白质 (折叠和展开)11,聚合物 (聚乙烯乙二醇等)12,13,14、金纳米粒子15、16、17、18、19等合成分子20。

我们最近证明,αHL纳米孔还可以轻松地在单分子水平上检测和表征金属簇,多氧金属酸盐(POM)。POM 是 1826年发现的离散纳米级电子金属氧簇,自那时以来,已经合成了更多类型。现在可用的多氧金属酸盐的不同尺寸、结构和元素组合物具有广泛的特性和应用,包括化学 22、23、催化24、材料科学25 ,26和生物医学研究27,28,29。

POM 合成是一种自组装过程,通常通过混合所需的单体金属盐量来在水中进行。一旦形成,POM 表现出巨大的大小和形状的多样性。例如,Keggin聚苯乙烯结构,XM12O40q-由一个异构体(X)组成,四氧包围形成四面体(q是电荷)。异构体位于由12个八面体MO6单位(其中M = 在高氧化状态下过渡金属)形成的保持架内,由相邻的共享氧原子相互连接。虽然钨多氧金属结构在酸性条件下稳定,氢氧化离子导致金属氧(M-O)键30的水解裂解。这种复杂的过程导致一个或多个MO6八面体亚单位的损失,导致形成单空和三空物种,并最终完全分解POM。我们在这里的讨论将仅限于pH5.5和7.5的12磷酸的部分分解产物。

该协议的目的是使用基于生物纳米孔的电子平台在单分子极限处检测离散金属氧团。此方法允许检测溶液中的金属簇。溶液中的多个物种可以比传统的分析方法更灵敏地进行区分33。通过它,可以阐明POM结构的细微差异,其浓度明显低于NMR光谱所需的浓度。重要的是,这种方法甚至允许对Na8HPW9O341的等同形式进行区分。

Protocol

注意:以下协议特定于电子生物科学 (EBS) 纳米片直流系统。然而,它可以很容易地适应其他电生理学仪器,用于测量电流通过平面脂双层膜(标准脂质双层膜室,U管几何,拉微毛细血管等)。给出了商业材料及其来源的鉴定,以描述实验结果。在任何情况下,这种鉴定都并不意味着国家标准与技术研究所的建议,也不意味着材料是最好的。 1. 溶液和分析剂制备 ?…

Representative Results

在过去的二十年中,膜结合蛋白纳米级孔被证明为多功能的单分子传感器。基于纳米孔的测量相对简单。 两个充满电解质溶液的腔室由嵌入在电绝缘脂质膜中的纳米孔隔开。修补夹放大器或外部电源通过浸入电解质储液罐中的 Ag/AgCl电极,在整个纳米孔中提供静电电位。电场将单个带电粒子驱动到孔隙中,从而根据粒子的大小、形状和电荷,产生离子电流的瞬态减小。计…

Discussion

由于其离子电荷,POM 可能通过静电相互作用与有机计数器阳离子相关联。因此,确定适当的溶液条件和正确的电解质环境(尤其是溶液中的阳离子)以避免与 POM 形成复杂情况非常重要。在缓冲液选择中需要特别小心。例如,使用三联甲酸甲烷和柠檬酸缓冲溶液的POM的捕获率明显低于磷酸盐缓冲溶液中的捕获率,这可能是因为前两个缓冲液中的任何一个与POM形成复合物,而POM不会与纳米孔的强相互作用。此…

Divulgations

The authors have nothing to disclose.

Acknowledgements

我们感谢欧洲分子生物学组织为博士后奖学金(J.E.)和NIH NHGRI(J.J.K.)提供的赠款提供财政支助。我们感谢朱景岳教授和谢尔盖·卡拉奇科夫教授(哥伦比亚大学)提供半球 +HL 的帮助,以及与弗吉尼亚联邦大学约瑟夫·赖纳教授的鼓舞人心的讨论。

Materials

Nanopatch DC System Electronic Biosciences, Inc., EBS
Millipore LC-PAK Millipore vacuum filter
1,2-Diphytanoyl-sn- Glycero-3-Phosphocholine (DPhPC) Avanti Polar Lipids, Alabaster, AL 850356P
Decane, ReagentPlus, ≥99%, Sigma-Aldrich D901
αHL List Biological Laboratories, Campbell, CA
Ag wire Alfa Aesar
2 mm Ag/AgCl disk electrode In Vivo Metric E202
High-impedance amplifier system Electronic Biosciences, San Diego, CA
quartz capillaries
custom polycarbonate test cell
Data Processing and Analysis MOSAIC https://pages.nist.gov/mosaic/
Phosphotungstic acid hydrate Sigma-Aldrich 455970
Sodium Chloride Sigma-Aldrich S3014
sodium phosphate monobasic monohydrate Sigma-Aldrich 71507
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Tags

Keywords: Metallic NanoparticlesNanopore DetectionPhysical CharacterizationSingle Molecule AnalysisProtein NanoporesDNA SequencingPolymer DiscriminationLipid BilayerElectrophysiologyPolyoxometalate Solution
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Ettedgui, J., Forstater, J., Robertson, J. W., Kasianowicz, J. J. High Resolution Physical Characterization of Single Metallic Nanoparticles. J. Vis. Exp. (148), e58257, doi:10.3791/58257 (2019).
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