Name: determination of zeta potential via nanoparticle translocation velocities through a tunable nanopore: using dna-modified particles as an example
Uploaded: 2016-10-26T14:00:00.000+00:00
Duration: 8 min 42 s
Description: Watch this Scientific Journal Video about Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example at JoVE.com

作者感谢IZON科技有限公司的支持。这项工作是由欧洲委员会研究（PCIG11-GA-2012-321836 Nano4Bio）的支持。

1.用吐温20（PBST）缓冲使磷酸盐缓冲液<ol><li>溶解1的PBS片剂（0.01M磷酸盐缓冲液，0.0027 1M氢氯化物，0.137氯化钠，pH值7.4）在200毫升的去离子水（18.2兆欧厘米）。 </li><li>加入100微升（0.05（V / V）％）吐温20至200毫升缓冲液溶液作为表面活性剂。 </li></ol> 2.准备羧基聚苯乙烯颗粒标准<ol><li>涡旋超声2分钟前30秒的校准颗粒在80瓦到创建粒子的单分散性。 </li><li>稀释校准颗粒在100 1至在PBST缓冲液和涡旋1×10 10个粒子/ ml的浓度为30秒。 </li></ol> 3.准备链亲和素包覆颗粒<ol><li>涡旋超声2分钟前30秒的颗粒在80瓦的单分散性保证。 </li><li>稀释链霉包衣颗粒1 100在PBST缓冲到交流hieve的1×10 9个粒子/ ml，30秒所得浓度和旋涡。 注:一个典型的样本量为200微升。例如，如果调查不同的DNA的浓度制备1ml适当稀释的链霉亲涂覆颗粒。 </li></ol> 4.寡核苷酸的制备<ol><li>重构用去离子水的寡核苷酸到所得100μM的浓度。 </li></ol> 5.加成捕获探针（CP）的DNA的链亲和素包被的颗粒<ol><li>之前DNA结合，涡流链霉包衣颗粒（200微升样品体积）30秒，随后通过一个2分钟超声处理在80瓦。 </li><li>基于由供应商（4352皮摩尔/毫克）所提供的结合能力，加DNA的适当的浓度在颗粒为10，20，30，40，47，95，140，和210纳米的DNA得到的浓度。 </li><li>涡旋10秒并置于样品上在室温下在旋转轮30分钟以允许将DNA通过链霉抗生物素相互作用结合到粒子表面。 </li><li>一旦捕获的DNA已被添加，并与链霉亲包衣颗粒孵育，通过将试样放在一个磁架30分钟除去在经由磁分离溶液中的过量的DNA。 </li><li>除去上清液，注意不要扰乱最接近磁铁粒子的新形成的群集，并用相同体积的新的PBST缓冲液的更换。 </li></ol> 6.杂交的互补DNA到CP颗粒<ol><li>添加所需的靶DNA（超过500纳米）的量，以确保最大可能的靶结合为止。 </li><li>涡旋样品10秒和地点上在室温下在旋转轮30分钟。 </li><li>一旦杂交完成后，通过将样品上的磁架30分钟除去通过磁分离过量的靶DNA。 </li><li>除去上清液，注意不要扰乱最接近磁铁粒子的新形成的群集，并用相同体积的新的PBST缓冲液的更换。 </li><li>重复步骤重复样本6.1至6.4，并放置这些样品，在旋转轮在室温下16小时，以研究DNA杂交时间。 </li></ol> 7. TRPS设置<ol><li>插入仪器到计算机系统中以代替软件。 </li><li>校准用卡尺的初始拉伸。 <ol><li>测量两个平行夹爪的外侧之间的距离。 </li><li>输入通过在"仪器设置"选项卡中输入在"拉伸"字段拉伸，然后点击"校准舒展"的标签下的软件。 </li></ol></li><li>横向适合分析适当大小的聚氨酯纳米孔膜上朝上纳米孔ID号钳口。然后，伸展钳口用于使用分析所需的拉伸在仪器的一侧伸展调节手柄。伸展43和48毫米的下颌。 注意:使校准微粒封锁是至少0.3 nA的尺寸是一起施加电压所确定的拉伸的精确值。拉伸已经输入到软件在步骤7.2和作为钳口被拉伸会自动调整。 </li><li>放置80微升PBST缓冲液中的下部流体细胞，纳米孔的下方，以确保不存在本气泡可能影响测量。如果有气泡看出，取出并更换缓冲。 </li><li>点击上方的流体细胞到位，并把40微升缓冲进去，从而也确保没有泡沫存在。如果气泡存在于上部流体细胞，通过更换液体移除。 </li><li>当可再现的基线电流已从用缓冲液更换上部流体细胞达到，通过点击"ST添加40微升样品到上部流体细胞和测量的"中的"数据采集"软件屏幕上标签的艺术。 注:数据采集完成后，在50千赫的0.05呐封锁幅度下限的频率，虽然可以使用通过"分析数据"选项卡中的软件进行更改（在"分析设置"和"电阻式封锁"） 。 </li><li>放置一个法拉第笼在流体电池系统的顶部，以减少对测量电背景噪声。 </li><li>使用一个可变压力模块（VPM）来的压力或真空施加到样品。 <ol><li>施加的外部压力，喷嘴连接到该上部流体细胞，然后旋转压力臂和卡入到位（取决于是否将被施加正压力（PRE）或真空（VAC））。 </li><li>在使用位于VPM顶部的压力级旋钮&#39;厘米&#39;或&#39;毫米&#39;刻度施加压力。按下旋钮，适用于"厘米"的规模压力，向上拉Ťo在"毫米"规模施加压力。 </li></ol></li></ol> 8.准备的样品分析TRPS <ol><li>涡旋样品在TRPS分析前80瓦特30秒，超声2分钟。 </li></ol> 9.校准纳米孔的泽塔分析<ol><li>将40微升校准颗粒（1×10粒/ ml）倒入上层液体电池后，完成TRPS测量（设置为在第7）在3施加的电压。 " - "按钮上的电压规模在"仪器设置"中的软件选项卡中点击"+"和改变电压。 </li><li>检查3电压回到约140，110背景电流和80 nA的。保证在中等电压的校准微粒产生至少0.3的平均阻滞大小nA的。 </li><li>施加压力，使所述校准微粒的平均半峰全宽（FWHM）的持续时间至少是0.15毫秒。为此使用连接到可变压力模块的压力臂手动。选择压力（PRE）或真空（VAC），直到它卡在期望的位置，并且在步骤7.8.2所适用相应以下设置指示所述臂旋转。一旦这些条件已经实现，通过点击"数据采集"选项卡中的软件&#39;开始&#39;开始运行。 </li><li>当至少有500个颗粒进行了测量（见"粒子计数"在软件屏幕在测量过程中底部），运行已超过30秒，由"数据采集"标签按下"停止"完成运行（参见"运行时间&#39;也朝向屏幕的底部）。 </li><li>通过描述每一种新的纳米孔引入时间或分析每一个新的一天完成步骤9.1-9.4完成校准运行校准系统。 </li></ol> 10.运行样品<ol><li>运行在最高的样品或第二高电压作为确保类似的（±10 NA）的校准样品，如果不相同，基线电流。 <ol><li>一旦适当的基准电流来实现，用40微升样品的替换在上部流体单元中的电解质。当引入的样品，封锁将看到的信号路径。开始通过点击"开始"中的"数据采集"选项卡中运行样品并记录最低500粒子（检查&#39;粒子计数"信号走线下位于），并确保运行时间最少30秒（见"运行时代"也位于信号迹线下方）。 </li><li>要完成测量，点击"停止"中的"数据采集卡"并保存数据文件。 </li></ol></li><li>要保存文件，输入如下格式的文件信息; &#39;调查&#39;是文件将保存在该文件夹，"纳米孔ID&#39;正在被使用，&#39;部分＃&#39;我毛孔的序列号š孔的类型（ 即 ，NP150 / NP200），&#39;样品ID&#39;是样品的名字，"校准或样品"的信息是否是一个校准或测量样品，"稀释"，如果样品稀释被使用（键入100如果样品稀释100倍），"压力"是所施加的压力到样品（以厘米 - 见第7.8节），&#39;电解质ID"是的缓冲样品中由姓名，和&#39;注意事项&#39;是有关样本或运行任何个人笔记。 </li><li>每个样品运行之间，通过将40微升的PBST缓冲液到上部流体细胞几次并施加各种压力（在-10通常，-5厘米（真空），以及5和10厘米（正压力）），直到洗系统没有更多的阻断事件都存在，确保有残留在系统中没有残留的粒子和样品之间，因此没有交叉污染。在这种洗涤步骤每个重复样品之间完成一式三份样品运行身份运行WELl由于不同样本之间。 </li></ol>

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<html>为ζ电势用于计算由Arjmandi 等人 21的工作有关的校准基础的方法。因为它们穿过纳米孔被测量作为所施加电压的函数，使用平均电场和粒子速度定期锥形孔的整体上颗粒的易位持续时间。的电泳迁移率是1 / T（其中T是封锁持续时间）相对于电压的导数，乘以感应区长度的平方， 湖在通过传感区域的多个基准点的平均速度测量以允许最小的误差在使用这种方法计算ζ电位。...

官能化的纳米颗粒变得如在医疗和环境领域的生物传感器日益流行。的能力，以改变纳米颗粒的表面化学，用DNA，例如，被证明是用于靶向药物递送系统1和监测DNA-蛋白质相互作用2-4是有用的。被利用在生物测定一个越来越普遍的纳米颗粒性质和在治疗剂的递送是超顺磁性5。超顺磁性粒子（SPP的）是在确定和从复杂混合物中去除特定分析物是非常有用的，可以用简单的使用单个磁体的这样做。一旦移除，所述分析物结合的颗粒可以被表征和分析适合目的。 用于纳米颗粒的检测和表征以前的方法包括光学技术，诸如动态光散射（DLS），或称为光子相关光谱法。虽然一喜GH可以通过技术，DLS被限定于一个平均为基础的技术，并没有加入专业软件分析多峰样品时，较大的颗粒将产生一个更主导信号，留下一些较小的颗粒完全被忽视6,7。粒子按颗粒表征技术，因此更利于分析和纳米功能化纳米粒子系统。 基于RPS技术基于周围施加电场到样品，并通过合成或生物纳米孔监测粒子的输送机构。根据RPS相对近期的纳米检测与表征技术是可调电阻脉冲感应（TRPS）8-16。 TRPS是由弹性，可调谐孔隙膜分开的两个电极系统。的可调孔径的方法允许在各种形状17和大小的分析物通过其反待测量通过毛孔口的机制。可调孔隙先前已经用于生产比较的结果，其他技术如透射电子光谱法（TEM）10检测的小颗粒（70-95纳米直径）。当施加电场时，离子电流被观察到并作为颗粒/分子穿过孔，它们暂时阻止孔径，导致在当前的减少，可以被定义为一个"封锁事件&#39;。每个封锁事件是代表单个颗粒，使得样本内的每个颗粒可以表征单独基于所述封锁幅度，Δ <img alt="式（1）" src="/files/ftp_upload/54577/54577eq1.jpg" /> 和全宽度半最大，FWHM，以及其他封锁性能。判断单个颗粒，因为他们通过纳米孔为多峰的样品有利的，因为TRPS可以成功地和有效区分的范围内的颗粒尺寸阿蒙GST一个样本。可调电阻脉冲感测完成大小10，同时ζ电位12,18和浓度15测量在单次运行，因此，仍可以区分的类似的样品，如果不通过表面电荷19相同的大小;在施胶替代技术的优势。 ζ电位是指在剪切20的平面上的静电势，并作为它们穿过的孔19从粒子速度计算。单个颗粒的ζ电位测量值从而给出洞察易位机制和在溶液中的纳米颗粒系统中，纳米颗粒测定设计未来的有价值的信息用于一系列应用的行为。粒子通过粒子这种性质的分析，还允许之间的样本人群中蔓延，zeta电位值的分布加以探讨，使邻更多信息要达到n个反应动力学（单链到双链DNA，例如）和颗粒稳定性在溶液中。 在这里，我们描述了检测和表征未修饰和DNA修饰SPP表面的技术。本文描述的协议是适用于一系列无机和生物纳米颗粒，但我们证明用DNA修饰表面的方法，由于其广泛的应用。该技术允许用户在纳米颗粒表面的单链和双链DNA靶区分基础上，通过一个孔体系粒子易位速度和因此它们的ζ电势。

<table><tbody><tr><td>Phosphate buffered Saline (PBS)</td><td>Sigma Aldrich, UK</td><td>P4417</td><td>1 tablet dissolved in 200&amp;nbsp;ml deionized water to make buffer solution.</td></tr><tr><td>Tween-20</td><td>Sigma Aldrich, UK</td><td>P1379</td><td>0.05% (v/v) in PBS buffer as a surfactant</td></tr><tr><td>Carboxyl polystyrene nanoparticles</td><td>Bangs Laboratories, US</td><td>CPC200</td><td>Nominal diamter of 220&amp;nbsp;nm, raw concentration of 1 x 10&lt;sup&gt;12&lt;/sup&gt; particles/ml, specific surface charge of 86 &amp;micro;eq/g (equivalent to a surface charge density of 3.2 x 10&lt;sup&gt;19&lt;/sup&gt; C/nm&lt;sup&gt;2&lt;/sup&gt;.</td></tr><tr><td>Streptavidin coated nanoparticles</td><td>Ademtech, France</td><td>3121</td><td>Batch had binding capacity of 4,352 pmol/mg (188 nM theoretical DNA binding capacity) at a raw concentration of 1.1 x 10&lt;sup&gt;11&lt;/sup&gt; particles/ml.</td></tr><tr><td>Biotinylated oligonucleotides</td><td>Sigma Aldrich, UK</td><td>VC00001</td><td>Supplier spec: Reverse Phase 1 purification (0.05 Scale); Biotin modification at 3&#039; end; Lyophilized powders reconstituted to 100&amp;nbsp;&amp;micro;M using deionized water, and diluted as required. Sequences: CP 5&#039;ATGGTTAAACCTCACTAC
GCGTGGC[Btn]3&#039;</td></tr><tr><td>Standard olignonucleotides</td><td>Sigma Aldrich, UK</td><td>VC00001</td><td>Supplier spec: Reverse Phase 1 purification (0.05 Scale); Lyophilized powders reconstituted to 100 &amp;micro;M using deionized water, and diluted as required. Sequences of DNA targets: Fully complementary - 5&#039;GCCACGCGTAGTGA
GGTTTAACCAT3&#039;, Middle binding - 5&#039;GTAGTGAGGT3&#039;, End binding - 5&#039;GTTTAACCAT3&#039;, Partially complementary overhanging - 5&#039;GTGAGGTTTAACCAT
TTTTTTTTTTTTTTT3&#039;.</td></tr><tr><td>Izon qNano</td><td>Izon Science, NZ</td><td></td><td>Inherent pressure on system of 47&amp;nbsp;Pa</td></tr><tr><td>Izon Variable Pressure Module (VPM)</td><td>Izon Science, NZ</td><td></td><td>Each &#039;cm&#039; of pressure is equivalent to approximately 100 Pa.</td></tr><tr><td>Polyurethane nanopore membranes</td><td>Izon Science, NZ</td><td>NP150</td><td>Analyte size range 60-480 nm, pore diameter of calculated to be 799 nm at a 45 mm stretch.&amp;nbsp;</td></tr><tr><td>Magrack 6</td><td>GE Healthcare, UK</td><td>28-9489-64</td><td></td></tr><tr><td>Sonic Bath</td><td>Fisher Scientific, UK</td><td>10692353</td><td>80 Watts</td></tr><tr><td>Vortexer</td><td>IKA, Germany</td><td>0003365000</td><td></td></tr><tr><td>Rotary Wheel&amp;nbsp;</td><td>Labnet International, US</td><td>H5500-230 V</td><td></td></tr></tbody></table>

determination of zeta potential via nanoparticle translocation velocities through a tunable nanopore: using dna-modified particles as an example

纳米孔技术，统称为电阻脉冲传感器（RPS），被用来检测，量化和表征的蛋白质，分子和纳米颗粒。可调电阻脉冲感测（TRPS）是一个相对较新的适应RPS并入，可以实时地改变的可调孔径。这里，我们使用TRPS监测DNA改性的纳米粒子的易位倍，因为它们穿过可调孔膜作为DNA浓度和结构的一个功能（ 即 ，单链到双链DNA）。 TRPS是基于两个的Ag / AgCl电极，通过它建立在所施加的电场产生稳定的离子电流的弹性体孔膜分离。与各种基于光学颗粒表征技术，TRPS可以表征单个颗粒之间的样本人口，允许能够轻松进行分析多式联运样品。这里，我们证明zeta电位测量通过已知的标准粒子易位速度和应用这些样品的分析物易位倍，从而导致测量这些分析物的Zeta电位。 以及获得的平均ζ电位的值，将样品全部采用的粒子由粒子立体显示通过样品人口分布的给定样品的详细信息，例如测量的。这样，这种方法演示了医疗和环境等领域的传感应用中的潜力。

<img alt="图1" src="/files/ftp_upload/54577/54577fig1.jpg" /> 图1. 磁性纯化和TRPS测量过程的示意图。 A）的开始与含有样品过量，未结合的捕获探针的DNA。B）的样本的磁性纯化的实施例的TRP测定例ⅰ）粒子穿过纳米孔和ii）从在孔粒子暂时阻塞离子封锁事件产生使暂时降低在目前的;从被用来计算粒子易位速度的信息。 <a href="https://www.jove.com/fi...

Watch this Scientific Journal Video about Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example at JoVE.com

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

在这里，我们使用集成到一个电阻脉冲感测技术经由粒子易位速度的测量，这可以被用来确定各个纳米粒子的ζ电位来表征纳米颗粒表面化学的聚氨酯可调谐纳米孔。

通过通过纳米孔可调纳米易位流速Zeta电位测定:利用DNA修饰颗粒为例

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

determination-zeta-potential-via-nanoparticle-translocation

Research

JoVE Journal

Bioengineering

12.1K 次观看.  Loughborough University. 在这里，我们使用集成到一个电阻脉冲感测技术经由粒子易位速度的测量，这可以被用来确定各个纳米粒子的ζ电位来表征纳米颗粒表面化学的聚氨酯可调谐纳米孔。 

视频: Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Fine-tuning the Size and Minimizing the Noise of Solid-state Nanopores

Solid-state nanopores have emerged as a versatile tool for the characterization of single biomolecules such as nucleic acids and proteins1. However, the creation of a nanopore in a thin insulating membrane remains challenging. Fabrication methods involving specialized focused electron beam systems can produce well-defined nanopores, but yield of reliable and low-noise nanopores in commercially available membranes remains low2,3 and size control is nontrivial4,5. Here, the application of high electric fields to fine-tune the size of the nanopore while ensuring optimal low-noise performance is demonstrated. These short pulses of high electric field are used to produce a pristine electrical signal and allow for enlarging of nanopores with subnanometer precision upon prolonged exposure. This method is performed in situ in an aqueous environment using standard laboratory equipment, improving the yield and reproducibility of solid-state nanopore fabrication.

A methodology for preparing solid-state nanopores in solution for biomolecular translocation experiments is presented. By applying short pulses of high electric fields, the nanopore diameter can be controllably enlarged with subnanometer precision and its electrical noise characteristics significantly improved. This procedure is performed in situ using standard laboratory equipment under experimental conditions.

微调尺寸和最小化固态纳米孔的噪音

Solid-state nanopores have emerged as a versatile tool for the characterization of single biomolecules such as nucleic acids and proteins1. However, the ...

科研

工程学

Monitoring Protein Adsorption with Solid-state Nanopores

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids 1-6, the unfolding of proteins 7, and binding affinity of different ligands 8. By coupling these nanopores to the resistive-pulse technique 9-12, such measurements can be done under a wide variety of conditions and without the need for labeling 3. In the resistive-pulse technique, an ionic salt solution is introduced on both sides of the nanopore. Therefore, ions are driven from one side of the chamber to the other by an applied transmembrane potential, resulting in a steady current. The partitioning of an analyte into the nanopore causes a well-defined deflection in this current, which can be analyzed to extract single-molecule information. Using this technique, the adsorption of single proteins to the nanopore walls can be monitored under a wide range of conditions 13. Protein adsorption is growing in importance, because as microfluidic devices shrink in size, the interaction of these systems with single proteins becomes a concern. This protocol describes a rapid assay for protein binding to nitride films, which can readily be extended to other thin films amenable to nanopore drilling, or to functionalized nitride surfaces. A variety of proteins may be explored under a wide range of solutions and denaturing conditions. Additionally, this protocol may be used to explore more basic problems using nanopore spectroscopy.

A method of using solid-state nanopores to monitor the non-specific adsorption of proteins onto an inorganic surface is described. The method employs the resistive-pulse principle, allowing for the adsorption to be probed in real-time and at the single-molecule level. Because the process of single protein adsorption is far from equilibrium, we propose the employment of parallel arrays of synthetic nanopores, enabling for the quantitative determination of the apparent first-order reaction rate constant of protein adsorption as well as and the Langmuir adsorption constant.

监测与固态纳米孔蛋白质吸附

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ...

生物工程

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its transparency. Due to the softness of the PDMS material, however, it is challenging to use PDMS for building nanochannels. The channels tend to collapse easily during plasma bonding. In this paper, we present an evaporation-driven self-assembly method of silica colloidal nanoparticles to create nanofluidic junctions with sub-50 nm pores between two microchannels. The pore size as well as the surface charge of the nanofluidic junction is tunable simply by changing the colloidal silica bead size and surface functionalization outside of the assembled microfluidic device in a vial before the self-assembly process. Using the self-assembly of nanoparticles with a bead size of 300 nm, 500 nm, and 900 nm, it was possible to fabricate a porous membrane with a pore size of ~45 nm, ~75 nm and ~135 nm, respectively. Under electrical potential, this nanoporous membrane initiated ion concentration polarization (ICP) acting as a cation-selective membrane to concentrate DNA by ~1,700 times within 15 min. This non-lithographic nanofabrication process opens up a new opportunity to build a tunable nanofluidic junction for the study of nanoscale transport processes of ions and molecules inside a PDMS microfluidic chip.

We propose a simple self-assembly technique of silica colloidal nanoparticles to create a nanofluidic junction between two microchannels in polydimethylsiloxane (PDMS). Using this technique, a nanoporous bead membrane with a pore size down to ~45 nm was built inside a microchannel and applied to electrokinetic preconcentration of DNA samples.

通过胶体粒子自组装过程创建在PDMS微流控芯片分50纳米的纳米流体结

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

动态和电泳光散射的 DNA-磁性颗粒结合分析

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

化学

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging.
This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.

Extracellular vesicles play important roles in physiological and pathological processes, including coagulation, immune responses, and cancer or as potential therapeutic agents in drug delivery or regenerative medicine. This protocol presents methods for the quantification and size characterization of isolated and non-isolated extracellular vesicles in various fluids using tunable resistive pulse sensing.

定量和大小分析外囊泡的使用可调电阻脉冲检测

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

High Resolution Physical Characterization of Single Metallic Nanoparticles

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single nanometer-scale pore. The signal is characteristic of the molecule&#39;s physicochemical properties and its interactions with the pore. We demonstrate that the nanopore formed by the bacterial protein exotoxin Staphylococcus aureus alpha hemolysin (&#945;HL) can detect polyoxometalates (POMs, anionic metal oxygen clusters), at the single molecule limit. Moreover, multiple degradation products of 12-phosphotungstic acid POM (PTA, H3PW12O40) in solution are simultaneously measured. The single molecule sensitivity of the nanopore method allows for POMs to be characterized at significantly lower concentrations than required for nuclear magnetic resonance (NMR) spectroscopy. This technique could serve as a new tool for chemists to study the molecular properties of polyoxometalates or other metallic clusters, to better understand POM synthetic processes, and possibly improve their yield. Hypothetically, the location of a given atom, or the rotation of a fragment in the molecule, and the metal oxidation state could be investigated with this method. In addition, this new technique has the advantage of allowing the real-time monitoring of molecules in solution.&#160;

Here, we present a protocol to detect discrete metal oxygen clusters, polyoxometalates (POMs), at the single molecule limit using a biological nanopore-based electronic platform. The method provides a complementary approach to traditional analytical chemistry tools used in the study of these molecules.

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 ...

A Closed-Type Wireless Nanopore Electrode for Analyzing Single Nanoparticles

Measuring the intrinsic features of single nanoparticles by nanoelectrochemistry holds deep fundamental importance and has potential impacts in nanoscience. However, electrochemically analyzing single nanoparticles is challenging, as the sensing nanointerface is uncontrollable. To address this challenge, we describe here the fabrication and characterization of a closed-type wireless nanopore electrode (WNE) that exhibits a highly controllable morphology and outstanding reproducibility. The facile fabrication of WNE enables the preparation of well-defined nanoelectrodes in a general chemistry laboratory without the use of a clean room and expensive equipment. One application of a 30 nm closed-type WNE in analysis of single gold nanoparticles in the mixture is also highlighted, which shows a high current resolution of 0.6 pA and high temporal resolution of 0.01 ms. Accompanied by their excellent morphology and small diameters, more potential applications of closed-type WNEs can be expanded from nanoparticle characterization to single molecule/ion detection and single-cell probing.

Here, we present a protocol for fabrication of a closed-type wireless nanopore electrode and subsequent electrochemical measurement of single nanoparticle collisions.

一种用于分析单纳米粒子的闭式无线纳米粒子电极

Measuring the intrinsic features of single nanoparticles by nanoelectrochemistry holds deep fundamental importance and has potential impacts in ...

High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are therefore poised to be useful in the field of mechanobiology. Since the method commonly relies on image-based tracking of magnetic beads, the speed limit in recording and analyzing images, as well as the thermal fluctuations of the beads, has long hampered its application in observing small and fast structural changes in target molecules. This article describes detailed methods for the construction and operation of a high-resolution MT setup that can resolve nanoscale, millisecond dynamics of biomolecules and their complexes. As application examples, experiments with DNA hairpins and SNARE complexes (membrane-fusion machinery) are demonstrated, focusing on how their transient states and transitions can be detected in the presence of piconewton-scale forces. We expect that high-speed MTs will continue to enable high-precision nanomechanical measurements on molecules that sense, transmit, and generate forces in cells, and thereby deepen our molecular-level understanding of mechanobiology.

Here, we describe a high-speed magnetic tweezer setup that performs nanomechanical measurements on force-sensitive biomolecules at the maximum rate of 1.2 kHz. We introduce its application to DNA hairpins and SNARE complexes as model systems, but it will be also applicable to other molecules involved in mechanobiological events.

高速磁镊，用于力敏元件的纳米力学测量

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are ...

生物化学

Monitoring Conformational Dynamics of Single Unmodified Proteins using Plasmonic Nanotweezers

Current single-molecule techniques to characterize proteins typically require labels, tethers, or the use of non-native solution conditions. Such changes can alter protein biophysics and reduce the usefulness of the data acquired. Plasmonic nanotweezers is a technique that uses localized surface plasmon resonance (LSPR) on gold nanostructures to enhance the electric field within a confined hotspot region. This field enhancement allows for the use of low laser powers to trap single nanoparticles far smaller than conventional optical tweezers, down to only a few nanometers in diameter, such as single proteins. Trapping of single protein molecules within the hotspot region induces a shift in the local refractive index (nprotein &#62; nwater), altering light scattering as a product of the molecule&#39;s polarisability, which is affected by its volume, shape anisotropy, and refractive index. An avalanche photodiode (APD) collects the subsequent changes in light scattering. These alterations can then be analyzed to determine changes in the trapped molecule, including its size, global conformation, and dynamics of conformational change over time. The incorporation of microfluidics within the system allows for controlled environmental changes and real-time monitoring of their subsequent effects on the molecule. In this protocol, we demonstrate the steps to trap single protein molecules, alter their environmental solution conditions, and monitor their corresponding conformational changes using a plasmonic nanotweezers system.

Plasmonic nanotweezers use localized surface plasmon resonance in gold nanostructures to trap single nanoparticles, including proteins, within a nanometer-scale optical field. Changes in the scattered signal reveal protein presence and conformational dynamics, enabling monitoring without fluorophore modifications or surface tethering.

使用等离子体纳米镊子监测单个未修饰蛋白质的构象动力学

Current single-molecule techniques to characterize proteins typically require labels, tethers, or the use of non-native solution conditions. Such changes ...