Name: high resolution physical characterization of single metallic nanoparticles
Uploaded: 2019-06-28T11:00:00
Description: Watch this Scientific Journal Video about High Resolution Physical Characterization of Single Metallic Nanoparticles at JoVE.com

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。

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
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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...

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。

<table>
	<tbody>
		<tr>
			<td>Nanopatch DC System</td>
			<td>Electronic Biosciences, Inc., EBS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Millipore LC-PAK</td>
			<td>Millipore vacuum filter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-Diphytanoyl-sn- Glycero-3-Phosphocholine (DPhPC)</td>
			<td>Avanti Polar Lipids, Alabaster, AL</td>
			<td>850356P</td>
			<td></td>
		</tr>
		<tr>
			<td>Decane, ReagentPlus, &#8805;99%,</td>
			<td>Sigma-Aldrich</td>
			<td>D901</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;HL</td>
			<td>List Biological Laboratories, Campbell, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ag wire</td>
			<td>Alfa Aesar</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2 mm Ag/AgCl disk electrode</td>
			<td>In Vivo Metric</td>
			<td>E202</td>
			<td></td>
		</tr>
		<tr>
			<td>High-impedance amplifier system</td>
			<td>Electronic Biosciences, San Diego, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>quartz capillaries</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>custom polycarbonate test cell</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Data Processing and Analysis MOSAIC</td>
			<td>https://pages.nist.gov/mosaic/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphotungstic acid hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>455970</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium phosphate monobasic monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>71507</td>
			<td></td>
		</tr>
	</tbody>
</table>

high resolution physical characterization of single metallic nanoparticles

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。

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...

Watch this Scientific Journal Video about High Resolution Physical Characterization of Single Metallic Nanoparticles at JoVE.com

High Resolution Physical Characterization of Single Metallic Nanoparticles

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。

单金属纳米粒子的高分辨率物理特性

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single ...

这种基于纳米孔的检测方法证明了对单个分子进行物理特征化和识别的潜力。与金属纳米粒子（本视频的目的）合作，挑战了我们对测量精度和精度限制范围的理解。该技术的主要优点是，该装置比传统的脂质双层存储器具有更大的带宽，这使我们能够在纳米孔中看到更快的结果和更多的反应。这项技术之所以成为可能，是因为在20世纪90年代初，它表明蛋白质纳米孔的浇注，即在不同的状态之间切换，可以控制，分子可以与孔壁结合。这项技术是为商业DNA测序应用开发的，因为它使用最少的样本。它不需要荧光标签，读取长度比传统方法长得多。与凝胶电泳和色谱仪不同，该技术还以基于其尺寸的尺寸来区分聚合物，其精度和速度更高。首先，在超纯水中准备500毫升的氯化钠溶液和10毫升磷酸钠作为电解质。最好使用新的解决方案和样品与这个特定的设置。此外，我们发现，高质量的去电水对于成功形成膜非常重要。接下来，将一毫克冻干野生型单体S.aureusα血脂素粉与一毫升超纯水混合。不要忘记，使用金黄色葡萄球菌α血红素毒素蛋白可能是危险的。执行此过程时，请遵循 MSDS 中的注意事项。接下来，在玻璃闪烁小瓶中，将四毫升五毫克的DPhyPC制成末因。用聚四氟乙烯衬里盖上小瓶，并在四摄氏度下存放长达一个月。之后，在电解质的10毫升中溶解57.6毫克12磷酸水合物，制造两毫摩尔多氧金属酸溶液。将 POM 溶液分成两个五毫升。使用三摩尔氢氧化钠将一个 POM 溶液的 pH 调至 5.5。要开始准备测试，请组装平面脂质双层电生理学仪器的测试单元。首先，用600砂纸磨一条银丝，在市售的次氯酸钠漂白剂中浸泡10分钟，使银氯化银线电极。在将电线电极插入仪器的石英毛细管之前，请冲洗并干燥。将电极放在石英纳米孔膜（QNM）内，分离毛细管和储液罐。然后将一个圆柱形银氯化银颗粒电极安装在储液罐中的银丝上。将两个电极连接到放大器电路板。接下来，打开数据采集程序，以零毫伏启动电源。确认空单元中的电极之间未检测到直流电流。在演示中，观察跨膜压力如何影响膜形成和随后的纳米孔形成非常重要。如果不正确执行这两个步骤，实验将不起作用。将大约一毫升电解质加载到注射器中，然后将其连接到连接到储液罐的流体管路。向储液罐中加入电解质，直到 QNM 表面被淹没，如电极之间的离子电流所示，该电流使放大器饱和。如果放大器不饱和，则通过施加正负一伏的弹出式电压或增加约 300 毫米汞的毛细管压力来解开 QNM。如有必要，请使用这两种技术。一旦放大器饱和，从储液罐中提取电解质，直到溶液液位降至 QNM 以下，如电流返回零所示。要开始脂质双层层形成过程，请将电解质溶液液水平远远高于 QNM 的面，并注意需要多少溶液。然后将 10 微升移液器尖端浸入 DPHyPC 溶液中，将所有可见的脂质浸入尖端。将移液器的尖端触摸到电解质溶液的表面，让脂质从移液器流过空气水接口。等待两到五分钟，使脂质均匀地分布在溶液中。接下来，慢慢提取电解质，直到溶液表面低于QNM，然后缓慢地添加足够的电解质，将溶液提升至QNM上方，形成绝缘脂质双层膜。如果三次尝试后没有形成双层，请应用前面描述的另一部分脂质，然后重复此过程。一旦双层层似乎已经形成，增加压力毛细管弹出双层，然后通过慢慢降低和提高溶液水平，以相同的方式，改革它。重复此过程多次，以确保 QNM 未堵塞且已形成双层。接下来，在冰上或室温下解冻一种α血红素蛋白。用250纳米的单体α血红素蛋白和约250微升的超纯水填充储液罐。然后，应用200至400毫伏偏置，诱导孔形成。为了促进孔隙的形成，将毛细管压力增加 40 至 200 毫米汞，以从 QNM 中扩展双层。一旦纳米孔形成，减少对测量电压的施加偏差，并降低压力到插入压力的一半。要开始测试，请重新调整直流偏移电压，以确保当应用电位设置为零时没有测量电流。接下来，获取相对于储层应用电位为 120 到负 120 毫伏的离子电流轨迹。确认没有污染物，这可以通过自发电流封锁来表示。向储层添加 1 至 6 微升 pH 5.5 双毫摩尔 POM 聚类溶液。在施加的负 120 毫伏电压下应用离子电流时间序列测量。单个心离子磷酸分子通过α血红素通道的运动产生瞬态封锁，使平均开孔离子电流降低约80%，持续时间和封锁电流都与粒子孔相互作用直接相关，使离子物种在相对封锁深度比的直方图中得到区分。在pH5.5时，观察到两个峰值，深度比约为0.06和0.16。基于磷核磁共振，小峰为六减磷吨位物种，主要峰顶为七减磷态物种。在pH7.5时，相对浓度转移到6-减物种与7-减物种的比率较高。与pH 5.5相比，封锁事件减少了20倍，归因于无磷酸盐和钨态离子的降解。适应居住时间分布需要多个指数函数，指示每个离子物种内多种类型的粒子孔相互作用。pH 7.5 的较短的居住时间表明，颗粒孔径相互作用比 pH 5.5 弱，与先前的研究表明，在α血红素通道流明中或附近固定电荷的相对数量发生依赖性变化。高纯度去水对于这种特殊设置至关重要。一个已花的有机物过滤器盒可能是我们实验室几个月来无法在QNM上形成膜的原因。对于这种特定版本的方法，新进入的人也可能难以为之奋斗，因为膜非常小，而且需要让纳米孔形成。这种方法是几十年前开始采用的，已被证明对分析离子、核酸、合成聚合物以及DNA测序非常有用。它还可以在生物物理学和基本细胞生物学中找到应用。例如，此过程可用于研究合成聚合物和生物聚合物的物理特性，以研究其大小、与其他分子的相互作用以及其他重要问题。

Research

JoVE Journal

Chemistry

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

不相关的代谢组学，生物源使用超高效液相色谱 - 高分辨质谱（UPLC-HRMS）

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

科研

化学

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

Preparation and Characterization of SDF-1ÃƒÅ½Ã‚Â±-Chitosan-Dextran Sulfate Nanoparticles

SDF-1α - 壳聚糖硫酸葡聚糖纳米粒子的制备与表征

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under ...

Synthesis, Characterization, and Functionalization of Hybrid Au/CdS and Au/ZnS Core/Shell Nanoparticles

合成，表征和混合金/硫化镉和Au / ZnS核/壳纳米颗粒功能化

Plasmonic nanoparticles are an attractive material for light harvesting applications due to their easily modified surface, high surface area and large ...

Synthesis and Characterization of Supramolecular Colloids

超分子胶体的合成与表征

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

植物材料的快速高通量物种鉴定实时高分辨质谱法直接分析

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, ...

Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level

环状聚合物的合成及其在熔融状态扩散运动的表征在单分子水平

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single ...

High Pressure Single Crystal Diffraction at PX^2

高压单晶衍射PX ^ 2

In this report we describe detailed procedures for carrying out single crystal X-ray diffraction experiments with a diamond anvil cell (DAC) at the ...

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

原位金属氧化物纳米粒子的核探针检测与单细胞定量研究

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

利用二次 Nanoelectrospray 电离耦合高分辨质谱的实时呼吸分析

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Synthesis and Characterization of Amphiphilic Gold Nanoparticles

两栖金纳米粒子的合成与表征

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of ...