This work has been financially supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (No. 25248027 to M.S.).

1.样品制备<ol><li> 温度响应单体的纯化 <ol><li>在100毫升甲苯中溶解20g的N- -isopropylacrylamide的（NIPA）。 </li><li>过滤下抽吸该溶液，以消除粉尘。 </li><li>混合用500ml石油醚滤液。 </li><li>放置在反应容器在冰 - 水浴中。 </li><li>搅拌溶液，直到单体被沉淀（通常为30分钟）。 </li><li>过滤下抽吸该溶液，得到沉淀的单体。 </li><li>干燥在减压下（100帕）过夜单体。 </li></ol></li><li> 温度响应性聚合物溶液的制备 <ol><li>脱气20毫升去离子水，使用隔膜泵1.0分钟。 </li><li>溶解780.8毫克的纯化NIPA的在9.5毫升脱气和去离子水。 </li><li>放置在反应容器在冰 - 水浴中。 </li><li>屏蔽从反应由覆盖用铝箔装置的光。 </li><li>轻轻搅拌该溶液10分钟，同时通过连接于气瓶用管枪头轻轻引入Ar气的适度流动。 </li><li>添加11.9微升的N，N，N&#39;，N&#39;四甲基乙二胺经由微量溶液。 </li><li>搅拌该溶液1.0分钟，同时引入Ar气，如在步骤1.2.5提及。 </li><li>在搅拌的同时将样品，溶解4.0毫克过硫酸铵在0.5毫升脱气和去离子水。 </li><li>混合样品溶液（从步骤1.2.7）和过硫酸铵溶液（来自步骤1.2.8）。 </li><li>搅拌该溶液30秒，同时引入Ar气，如在步骤1.2.5提及。 </li><li>用铝箔覆盖该溶液，并保持它在冰箱（4℃）过夜。 </li></ol></li><li> 坐骑样品的制备 <ol><li>将样品溶液的60微升（从步1.2.11）的一腔幻灯片。 </li><li>封面用圆形玻璃盖的解决方案。要小心，不要陷气泡。 </li><li>除去使用微量和实验室湿巾过量的溶液。 </li><li>密封胶水样品。让在室温（通常为6小时）的胶干燥。 </li><li>通过以下步骤1.3.1-1.3.4制备填充有0.1重量％的聚苯乙烯胶乳（100nm的粒径）悬浮另一个滑动。该滑动用作标准。 </li></ol></li></ol>用动态光散射显微镜2.粒度测量<ol><li> 该仪器的优化 <ol><li>放置聚苯乙烯胶乳悬浮滑动（从步骤1.3.5）在倒置显微镜的阶段。玻璃盖面应朝下。 </li><li>放置在检测器的前面的光束阻尼器（雪崩光电二极管和自相关）。 </li><li>施加激光束（固态激光器，λ= 488纳米，30毫瓦，连续波）到样品通过物镜（10×）。的反射光的一部分穿过显微镜的发射镜，并通过CCD照相机观察到安装在显微镜（ 图1）的侧端口。 </li><li>调整物镜的高度由从低到高的位置移动物镜的高度，以设定在样品悬浮液的焦点。在此过程中，被反射的图像被聚焦三次:在盖玻璃的表面上，在玻璃盖和样品之间的界面，并在样品和空穴载玻片之间的接口。设置第二个和第三个点之间的焦点。 </li><li>通过改变激光功率衰减的散射光强度。 </li><li>通过在检测器的前面取出光束阻尼器引进的散射光进入检测器。此单元测量的光强的时间相关性。 </li><li>设置一个针孔（φ= 50微米）betwEEN显微镜和检测器来实现共焦的效果。调节针孔的位置在检测器以最大化光强度。 </li><li>通过经由计算机启动所述相关器的操作测量30秒散射光强度的时间相关函数。测得的相关函数通常表示为g（2）（T） - 1，其中t是相关时间4和<img alt="式（1）" src="/files/ftp_upload/54885/54885eq1.jpg" /> 。这里，I（t）是在时间t和（•••）T是时间平均散射光强度。衰减时间将约为0.1毫秒。 </li><li>调整着力点，获得了广泛的时间相关函数的初始幅度（ 克 （2）（T = 0） - 1）。 注:初始振幅强烈影响的反射光的量。通过移动焦点拖ARD玻璃盖和样品，反射光的量增加之间的接口。对于强光散射，如聚苯乙烯胶乳，初始振幅可以从0改变为1。然而，这是难以设置接近1为更常见的聚合物溶液的初始振幅，因为反射光的强度远高于散射光的。 </li><li>应用Laplace逆变换（使用约束正计划CONTIN 13,14）所获得的时间相关的函数来获取大小分布函数。在将初始振幅设定为小于0.2的情况下，水动力半径的分布函数会显示约100纳米，这是实际的半径（详见讨论）两次的尖峰。 </li></ol></li><li> 样品测量 <ol><li>设置了阶段温度至25℃。 </li><li>将带有聚NIPA（PNIPA）SOLUT准备了幻灯片离子显微镜的阶段（步骤1.3.4）。 </li><li>通过以下步骤2.1.4-2.1.8测量散射光强度的时间相关函数。如果初始振幅是大于0.2，通过以下步骤2.1.9调整焦点，以使时间相关函数的初始振幅小于0.2。一个小幅度的初始简化了分析。 </li><li>将舞台温度35℃，等到溶液变浑浊。的PNIPA溶液的低临界溶液温度（LCST）是32℃下15。 </li><li>通过以下步骤测量时间相关函数2.1.4-2.1.8。如果可行的话，调整焦点的位置，使时间相关函数小于0.2的初始振幅。为混浊的解决方案，它们的初始振幅趋于增加，因为散射光的强度增大而反射光的保持不变。 </li><li>应用拉普拉斯逆transformati上所获得的时间相关函数，得到的粒度分布的功能。请注意，实际的大小为一半的情况下所获得的值，其中初始振幅是小于0.2。 </li></ol></li></ol>

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The authors have nothing to disclose.

时间相关函数的初始振幅在很大程度上依赖于焦点， 如图2（a）中 。这看似矛盾的事实，即溶液是均质的（除了在界面处的薄层）8。在最初的振幅的这种变化是由于在反射光的量的变化。偏差理论16预测的初始振幅，A中 ，散射光强度，I s和反射光强度，I R，满足下式1 <img alt="公式（2）" src="/files/ftp_upload/54885/54885eq2.jpg" />这个公式表明，I R变得越大，越小A变得。因此，A通过设置靠近接口的焦点位置降低。表观扩散常数D A CAÑ通过在单分散的解决方案的情况下，拟合时间相关函数来获得: <img alt="公式3" src="/files/ftp_upload/54885/54885eq3.jpg" />哪里<img alt="公式4" src="/files/ftp_upload/54885/54885eq4.jpg" /> 。这里，n是溶剂（水，1.33）的折射率，θ是散射角（180°），和λ是光（514.5nm的）的波长。因为我们施加散射几何学，q的值是固定的。然而，这点是通过使用不同波长的光解决。请注意，任何类型的连续波激光源的可用来构造对DLS显微镜。由于小照射量，相干因子17估计为0.99以上，是可以忽略不计。对于多分散溶液，由逆拉普拉斯变换获得的D A的分布函数。偏差次eory还预测，D A是不一样的实际扩散常数D。这两个扩散常数满足以下公式: <img alt="公式5" src="/files/ftp_upload/54885/54885eq5.jpg" />扩散常数D被转换成使用爱因斯坦-Stokes方程4中的流体动力学半径R H。当A = 1，这种关系变得D A = D。在这种情况下，数据转换过程是相同的，对于共同的动态光散射。在图2（b）中所示的红色线对应于这种情况。与此相反，这种关系变为D A = 0.5 D钮 A的极限→0，因此，大小估计为两倍的实际大小为大时，A是小的（实际上，小于0.2），如图所示由图2（b）的蓝线</s仲&gt;。如果我们知道A是显著小，横轴可以移动， 如图2（c）所示 。原则上，我们可以D A转换成D代表A的任意值。在实践中，然而，最好是设定为比0.2的初始振幅小，由于简单的近似D A〜0.5ð成立。 动态光散射显微镜技术的突出特点是使用PNIPA解决方案演示。 PNIPA的下面和高于LCST构象已经使用小角中子散射15,18被广泛地研究。相比之下，动态光散射尚未用于PNIPA的LCST以上的分析，因为它的浊度19。这个问题是由动态光散射显微镜解决， 如图3（a）和（b）中 。这些聚集体的尺寸为几＆＃181;米，这不能由任一小角X射线/中子散射或常规的光散射技术获得。使用该系统的时间分辨的测量的温度变化过程中得到的聚集过程的信息。 动态光散射显微镜的缺点也被在图3中示出。为低于LCST的结果，时间相关函数强烈地受到本灰尘的极少量的（黑线在图3中）的影响。例如，该时间相关函数不完全衰减，即使在1.0秒的数量级相关时间。这是因为该装置（约1.0微米）的照射体积比照射与通常的动态光散射装置（大约100微米）显著小。在的情况下的散射光的强度较弱，信号被噪音掩盖，比如导致由s商场的量在溶液中的灰尘。因此，在如图3（b）中所示的三个峰可能不具有定量的重要性虽然尺寸的一般顺序是有意义的。另外，这样的弱散射体可以通过常规的动态光散射装置进行测定。 我们已经证明，在动态光散射显微镜使我们能够用相同的设置测量透明和混浊样本。由于样品中的光路长度短，该技术可以适用于强的光吸收的样品，如碳纳米管悬浮液20。此外，由于其高的空间分辨率，该技术可以适用于生物细胞。其应用到生物学，这种方法也可与其它成像技术，如荧光和拉曼成像相结合。因此，我们认为，在动态光散射显微镜为广泛研究领域的有力工具。

Particle size is one of the most fundamental properties of colloidal and polymer solutions. Numerous techniques are used to measure the particle size. Particle sizes of 1.0 &#181;m or larger can be measured directly using an optical microscope. For smaller particles, alternative techniques, such as laser diffraction, electron microscopy, or atomic force microscopy, are used2,3. Dynamic light scattering is a commonly-used technique for the measurement of particle size distributions in solutions4. The results obtained using this technique are not derived from images of the particles but from the characteristic time of the fluctuations in scattered light intensity. These fluctuations originate from Brownian motion, which is characterized by a diffusion constant. The size distribution is obtained from the distribution of diffusion constants using the Einstein-Stokes equation. Due to its simplicity, dynamic light scattering is widely used for the routine assessment of solutions, such as paints and food colloids.
Pretreatment is required for most of the techniques used for the particle size measurement of solution samples. In the case of electron microscopy and atomic force microscopy, the sample must be analyzed under vacuum conditions. Therefore, it is difficult to observe the samples in their native forms. Furthermore, for laser diffraction and dynamic light scattering, only diluted samples that are free from multiple scattering and light absorption can be measured. To overcome this difficulty, several new techniques have been proposed for the measurement of dynamic light scattering from undiluted, concentrated solutions, such as cross-correlation spectroscopy5,6, low-coherence dynamic light scattering7,8, diffusing-wave spectroscopy9,10, and differential dynamic microscopy11,12.
We have developed a new apparatus called a dynamic light scattering microscope1. This apparatus enables us to measure turbid samples without dilution by means of a confocal optical system in which multiple scattering is eliminated using a pinhole. However, the measurement procedure and data analysis are slightly more complicated than those of commercially-available instruments. This video explains the measurement procedure and data analysis in detail using the analysis of the temperature-responsive polymer, poly(N-isopropylacrylamide), as an example.

<table><tbody><tr><td>&lt;em&gt;N&lt;/em&gt;-isopropylacrylamide, 98%</td><td>Tokyo Chemical Industry Co., Ltd.</td><td>I0401</td><td></td></tr><tr><td>toluene, 99%</td><td>Wako Pure Chemical Industries, Ltd.</td><td>201-01876</td><td></td></tr><tr><td>petroleum ether, distillation temperature 30 ~ 60 &amp;deg;C</td><td>Wako Pure Chemical Industries, Ltd.</td><td>169-22565</td><td></td></tr><tr><td>&lt;em&gt;N,N,N&#039;,N&#039;&lt;/em&gt;-tetramethylethylenediamine, 99%</td><td>Sigma</td><td>T9281</td><td></td></tr><tr><td>ammonium persulfate, 98%</td><td>Sigma</td><td>248614</td><td></td></tr><tr><td>polystyrene latex suspension, 1 wt%</td><td>Duke Scientific Corporation</td><td>3500A</td><td></td></tr><tr><td>argon</td><td>Koike Sanso Kogyo Co., Ltd.</td><td></td><td>purity &gt; 99.999 vol.%</td></tr><tr><td>cavity slide</td><td>Matsunami Glass Ind.,Ltd.</td><td>83-0336</td><td></td></tr><tr><td>inverted microscope</td><td>Nikon Instech Co., Ltd.</td><td>ECLIPSE Ti-U</td><td></td></tr><tr><td>Thermo Plate</td><td>Tokai Hit CO.,Ltd</td><td>TP-108R-C</td><td></td></tr><tr><td>Solid-state laser</td><td>Coherent</td><td>OBIS 488LX</td><td></td></tr><tr><td>avalanche photodiode</td><td>ALV-GmbH</td><td>ALV-High Q.E. Avalanche Photo Diode</td><td></td></tr><tr><td>correlator</td><td>ALV-GmbH</td><td>ALV-5000/EPP</td><td></td></tr></tbody></table>

measurement of particle size distribution in turbid solutions by dynamic light scattering microscopy

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a technique used to measure the size distribution of polymer solutions or colloidal particles. Although this technique is widely used for the assessment of polymer solutions, it is difficult to measure the particle size in concentrated solutions due to the multiple scattering effect or strong light absorption. Therefore, the concentrated solutions should be diluted before measurement. Implementation of the confocal optical component in a dynamic light scattering microscope1 helps to overcome this barrier. Using such a microscopic system, both transparent and turbid systems can be analyzed under the same experimental setup without a dilution. As a representative example, a size distribution measurement of a temperature-responsive polymer solution was performed. The sizes of the polymer chains in an aqueous solution were several tens of nanometers at a temperature below the lower critical solution temperature (LCST). In contrast, the sizes increased to more than 1.0 &#181;m when above the LCST. This result is consistent with the observation that the solution turned turbid above the LCST.

在不同的焦点进行测定，示于图2（a）:为聚苯乙烯胶乳悬浮液散射光强度的时间相关函数（50纳米粒子半径）。这些相关的功能被转换成由逆拉普拉斯变换的流体力学半径的分布函数（参照图2（b）和（c）项 ）。采用相同的步骤，在25℃下分别得到的PNIPA溶液的流体动力学半径的时间相关函数和分发功能和35℃。 图3（a）和（b）示出的散射光强度的时间相关函数和下面的PNIPA溶液（25℃）和上述（35℃）的LCST的相应尺寸分布函数。大小分布函数是由逆拉普拉斯变换之后所获得的偏差校正。低于LCST的平均流体动力学半径为几十纳米，这是典型的聚合物溶液。与此相反，在高于LCST的流体动力学半径为约1.0微米。这一结果与以下事实的溶液是高于LCST混浊一致。红色和蓝色线条图3表示后立即得到的，该溶液后20分钟分别变为混浊，PNIPA溶液的粒度分布。 如图3（b）清楚地表明，聚集的生长。 <img alt="图1" src="/files/ftp_upload/54885/54885fig1.jpg" /> 图1.动态光散射显微镜的示意图。针孔（PH），光束分离器（BS），偏振器（POL）和雪崩光电二极管（APD）。 <a href="http://ecsource.jove.com/files/ftp_upload/54885/54885fig1large.jpg" target="_blank">请点击这里查看</a>一个更大的版本这个数字。 <img alt="图2" src="/files/ftp_upload/54885/54885fig2.jpg" /> 图2.为聚苯乙烯胶乳悬浮液的代表性结果。 （a）用于在聚苯乙烯胶乳悬浮液中的散射光强度的时间相关函数。标称半径为50nm，浓度为0.1％（重量）。两个数据集分别从不同的散射点获得。 （ 二 ），（ 三 ）相应大小分布为聚苯乙烯胶乳悬浮液的逆拉普拉斯变换获得的功能（一个）。红线对应的时间相关函数的初始振幅是约1.0，和蓝线对应于一个初始幅值即约0.2。横轴没有（B）和（c）考虑部分外差（PHD）的影响，计算的时候A &lt;&lt;<html> ; 1. <a href="http://ecsource.jove.com/files/ftp_upload/54885/54885fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54885/54885fig3.jpg" /> 图3为一个PNIPA溶液代表性的结果。 （a）用于所述PNIPA溶液散射光强度的时间相关函数。 （b）在通过（a）的逆拉普拉斯变换获得的PNIPA溶液通讯尺寸分布函数。横轴计算考虑局部外差的每个数据集的效果。黑线表示在25℃下获得的数据。红线代表刚后溶液变混浊而获得的数据（35℃）。蓝线代表红色线的20分钟测量后获得的数据。/54885/54885fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy at JoVE.com

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

一种用于使用动态光散射显微镜浓缩溶液的粒度分布的直接测量协议提出。

通过动态光散射显微镜混浊解决方案粒度分布的测定

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

measurement-particle-size-distribution-turbid-solutions-dynamic-light

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14.3K 次观看.  University of Tokyo. 一种用于使用动态光散射显微镜浓缩溶液的粒度分布的直接测量协议提出。 

视频: Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

恒压控制的挤压方法制备纳米脂质体

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

科研

生物工程

Dispersion of Nanomaterials in Aqueous Media: Towards Protocol Optimization

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and stability of the suspension. In this study, a systematic step-wise approach is carried out to identify optimal sonication conditions in order to achieve a stable dispersion. This approach has been adopted and shown to be suitable for several nanomaterials (cerium oxide, zinc oxide, and carbon nanotubes) dispersed in deionized (DI) water. However, with any change in either the nanomaterial type or dispersing medium, there needs to be optimization of the basic protocol by adjusting various factors such as sonication time, power, and sonicator type as well as temperature rise during the process. The approach records the dispersion process in detail. This is necessary to identify the time points as well as other above-mentioned conditions during the sonication process in which there may be undesirable changes, such as damage to the particle surface thus affecting surface properties. Our goal is to offer a harmonized approach that can control the quality of the final, produced dispersion. Such a guideline is instrumental in ensuring dispersion quality repeatability in the nanoscience community, particularly in the field of nanotoxicology.

Here, we present a step-wise protocol for the dispersion of nanomaterials in aqueous media with real-time characterization to identify the optimal sonication conditions, intensity, and duration for improved stability and uniformity of nanoparticle dispersions without impacting the sample integrity.

纳米材料在水介质中的分散: 对协议优化的研究

The sonication process is commonly used for de-agglomerating and dispersing nanomaterials in aqueous based media, necessary to improve homogeneity and ...

环境科学

Controlled Synthesis and Fluorescence Tracking of Highly Uniform Poly(N-isopropylacrylamide) Microgels

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this work, we use single particle tracking of fluorescently labeled PNIPAM microgels as a showcase for tuning microgel size by a rapid non-stirred precipitation polymerization procedure. This approach is well suited for prototyping new reaction compositions and conditions or for applications that do not require large amounts of product. Microgel synthesis, particle size and structure determination by dynamic and static light scattering are detailed in the protocol. It is shown that the addition of functional comonomers can have a large influence on the particle nucleation and structure. Single particle tracking by wide-field fluorescence microscopy allows for an investigation of the diffusion of labeled tracer microgels in a concentrated matrix of non-labeled microgels, a system not easily investigated by other methods such as dynamic light scattering.

Controlled Synthesis and Fluorescence Tracking of Highly Uniform PolyN-isopropylacrylamide Microgels

Non-stirred precipitation polymerization provides a rapid, reproducible prototyping approach to the synthesis of stimuli-sensitive poly(N-isopropylacrylamide) microgels of narrow size distribution. In this protocol synthesis, light scattering characterization and single particle fluorescence tracking of these microgels in a wide-field microscopy setup are demonstrated.

可控合成和高度统一聚荧光追踪（<em&gt;ñ</em&gt; -isopropylacrylamide）微凝胶

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this ...

化学

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 ...

Application of Voltage in Dynamic Light Scattering Particle Size Analysis

Dynamic light scattering (DLS) is a common method for characterizing the size distribution of polymers, proteins, and other nano- and microparticles. Modern instrumentation permits measurement of particle size as a function of time and/or temperature, but currently there is no simple method for performing DLS particle size distribution measurements in the presence of applied voltage. The ability to perform such measurements would be useful in the development of electroactive, stimuli-responsive polymers for applications such as sensing, soft robotics, and energy storage. Here, a technique using applied voltage coupled with DLS and a temperature ramp to observe changes in aggregation and particle size in thermoresponsive polymers with and without electroactive monomers is presented. The changes in aggregation behavior observed in these experiments were only possible through the combined application of voltage and temperature control. To obtain these results, a potentiostat was connected to a modified cuvette in order to apply voltage to a solution. Changes in polymer particle size were monitored using DLS in the presence of constant voltage. Simultaneously, current data were produced, which could be compared with particle size data, to understand the relationship between current and particle behavior. The polymer poly(N-isopropylacrylamide) (pNIPAM) served as a test polymer for this technique, as pNIPAM's response to temperature is well-studied. Changes in the lower-critical solution temperature (LCST) aggregation behavior of pNIPAM and poly(N-isopropylacrylamide)-block-poly(ferrocenylmethyl methacrylate), an electrochemically active block-copolymer, in the presence of applied voltage are observed. Understanding the mechanisms behind such changes will be important when trying to achieve reversible polymer structures in the presence of applied voltage.

Here, a protocol to apply voltage to solution during dynamic light scattering particle size measurements with the intent to explore the effect of voltage and temperature changes on polymer aggregation is presented.

电压在动态光散射粒子尺寸分析中的应用

Dynamic light scattering (DLS) is a common method for characterizing the size distribution of polymers, proteins, and other nano- and microparticles. ...

Assembly and Characterization of Polyelectrolyte Complex Micelles

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful platform for exploring the physics of polyelectrolyte interactions and also offer a promising solution to the pressing problem of delivering therapeutic oligonucleotides in vivo. Developing predictive structure-property relationships for PCMs has proven difficult, in part due to the presence of strong kinetic traps during nanoparticle self-assembly. This article discusses criteria for choosing polymers for PCM construction and provides protocols based on salt annealing that enable assembly of repeatable, low-polydispersity nanoparticles. We also discuss PCM characterization using light scattering, small-angle X-ray scattering, and electron microscopy.

We provide protocols and representative data for designing, assembling, and characterizing polyelectrolyte complex micelles, core-shell nanoparticles formed by polyelectrolytes and hydrophilic charged-uncharged block copolymers.

聚电解质复合微晶体的组装与特性

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful ...

Routine Screening Method for Microparticles in Platelet Transfusions

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood banks. Cells fragment off microparticles (MP) when they are stressed. Blood and blood components may contain cellular fragments from a variety of cells, most notably from activated platelets. When performing their roles as innate immune cells and major players in coagulation and hemostasis, platelets change shape and generate microparticles. With dynamic light scattering (DLS)-based microparticle detection, it is possible to differentiate activated (high microparticle) from non-activated (low microparticle) platelets in transfusions, and optimize the use of this scarce blood product. Previous research suggests that providing non-activated platelets for prophylactic use in hematology-oncology patients could reduce their risk of becoming refractory and improve patient care. The goal of this screening method is to routinely differentiate activated from non-activated platelets. The method described here outlines the steps to be performed for routine platelet inventory management in a hospital blood bank: obtaining a sample from a platelet transfusion, loading the sample into the capillary for DLS measurement, performing the DLS test to identify microparticles, and using the reported microparticle content to identify activated platelets.

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative in hospital blood banks. The goal is to differentiate activated from non-activated platelets to optimize the use of platelets. Providing non-activated platelets to hematology-oncology patients might reduce their high risk to become refractory.

血小板输血中微粒的常规筛选方法

Platelet inventory management based on screening microparticle content in platelet concentrates is a new quality improvement initiative for hospital blood ...

医学

Quantifying Cytoskeleton Dynamics Using Differential Dynamic Microscopy

Cells can crawl, self-heal, and tune their stiffness due to their remarkably dynamic cytoskeleton. As such, reconstituting networks of cytoskeletal biopolymers may lead to a host of active and adaptable materials. However, engineering such materials with precisely tuned properties requires measuring how the dynamics depend on the network composition and synthesis methods. Quantifying such dynamics is challenged by variations across the time, space, and formulation space of composite networks. The protocol here describes how the Fourier analysis technique, differential dynamic microscopy (DDM), can quantify the dynamics of biopolymer networks and is particularly well suited for studies of cytoskeleton networks. DDM works on time sequences of images acquired using a range of microscopy modalities, including laser-scanning confocal, widefield fluorescence, and brightfield imaging. From such image sequences, one can extract characteristic decorrelation times of density fluctuations across a span of wave vectors. A user-friendly, open-source Python package to perform DDM analysis is also developed. With this package, one can measure the dynamics of labeled cytoskeleton components or of embedded tracer particles, as demonstrated here with data of intermediate filament (vimentin) networks and active actin-microtubule networks. Users with no prior programming or image processing experience will be able to perform DDM using this software package and associated documentation.

Differential dynamic microscopy (DDM) combines features of dynamic light scattering and microscopy. Here, the process of using DDM to characterize reconstituted cytoskeleton networks by quantifying the subdiffusive and caged dynamics of particles in vimentin networks and the ballistic motion of active myosin-driven actin-microtubule composites is presented.

使用差分动力学显微镜定量细胞骨架动力学

Cells can crawl, self-heal, and tune their stiffness due to their remarkably dynamic cytoskeleton. As such, reconstituting networks of cytoskeletal ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

对于外来体定量和尺寸测量新方法研究

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

生物学

Dynamic Light Scattering Analysis for the Determination of the Particle Size of Iron-Carbohydrate Complexes

Intravenously administered iron-carbohydrate nanoparticle complexes are widely used to treat iron deficiency. This class includes several structurally heterogeneous nanoparticle complexes, which exhibit varying sensitivity to the conditions required for the methodologies available to physicochemically characterize these agents. Currently, the critical quality attributes of iron-carbohydrate complexes have not been fully established. Dynamic light scattering (DLS) has emerged as a fundamental method to determine intact particle size and distribution. However, challenges still remain regarding the standardization of methodologies across laboratories, specific modifications required for individual iron-carbohydrate products, and how the size distribution can be best described. Importantly, the diluent and serial dilutions used must be standardized. The wide variance in approaches for sample preparation and data reporting limit the use of DLS for the comparison of iron-carbohydrate agents. Herein, we detail a robust and easily reproducible protocol to measure the size and size distribution of the iron-carbohydrate complex, iron sucrose, using the Z-average and polydispersity index.

Dynamic light scattering (DLS) has emerged as a suitable assay for evaluating the particle size and distribution of intravenously administered iron-carbohydrate complexes. However, the protocols lack standardization and need to be modified for each iron-carbohydrate complex analyzed. The present protocol describes the application and special considerations for the analysis of iron sucrose.

动态光散射分析用于测定铁-碳水化合物复合物的粒径

Intravenously administered iron-carbohydrate nanoparticle complexes are widely used to treat iron deficiency. This class includes several structurally ...

通过动态光散射显微镜混浊解决方案粒度分布的测定

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