1，标准工作协议 1.1仪器设置和样品制备<ol><li>将仪器连接到一台计算机安装了IZON控制套件软件。 </li><li>选择要使用的纳米孔尺寸:对电动汽车ÂNP150（目标尺寸范围85 - 300毫微米）的大小和浓度测量或NP200（目标尺寸范围为100 - 400纳米）是最常用的。当与已使用的协议，涉及搬迁的大型电动车，例如通过使样品通过220纳米过滤器分离出电动汽车的工作中，NP100孔（目标尺寸范围70 - 200纳米）都可以使用。当与电动汽车在生物样品或电动汽车使用不同的协议离体工作时，NP200可以使用，因为它会堵塞较少。其它纳米孔如NP300（目标尺寸范围150 - 600纳米）或NP400（目标尺寸范围200 - 800纳米），或者甚至更大的孔洞，可用于较大型电动汽车。 </li><li>选择聚苯乙烯CALIBR通报BULLETIN颗粒配合步骤1.1.2选择的纳米孔。对于NP100，NP150和NP200纳米孔使用CPC100，CP100及CPC200颗粒，分别为。对于尺寸准确估计，保证校验颗粒具有类似大小的未知粒子。 </li><li>以确保均匀的校准微粒，涡流短时间（30秒）。任选地，应用超声处理以除去聚集物。 </li><li>在至少40微升的体积稀释的校准微粒在PBS中的目标浓度。注意:目标浓度变化的基础上，在步骤1.1.2中选择的纳米孔。靶浓度附带的纳米孔。 </li><li>应用，直接在下层液体电池取出78微升PBS;施加电解质的下部流体细胞时，当纳米孔是在位置这润湿的下部流体细胞的减少纳米孔下的气泡形成的危险。 </li><li>将纳米孔到4臂仪器。使用数字测径器测量两个相对臂之间的距离，并进入在毫米的距离变成了"拉伸"的输入字段，并单击"校准信号展宽"来校准纳米孔伸展。 </li><li>伸展纳米孔到47毫米，通过转动侧轮和因此增加了仪器的相对的臂之间的距离，重新应用78微升PBS中，以较低的流体单元之前。 注:电气干扰会大大影响测量结果的质量。当使用笔记本电脑运行IZON控制套件软件，请确保笔记本电脑使用接地插座和插头连接到电网。手机保持在接近仪器也可以是电干扰源。电气干扰，观察基线电流不断重复的峰，常与均方根（RMS）噪声&gt; 10 pA的。 注:几乎所有的缓冲区可以用来稀释校准颗粒和电动汽车的跨国激进党characteri矩阵特殊积。盐的存在是为建立一个电流的一个先决条件。对于电动车的测量使用PBS作为缓冲。校准颗粒应该总是在相同的缓冲液稀释作为电动汽车，以确保精确的测量。 </li></ol> 1.2。确定测量的最佳设置注:拍摄前，它建立最佳的测量设置是非常重要的。造成的颗粒穿过纳米孔封锁大小是依赖于所施加的拉伸和施加的电压。对于可靠的测量RMS噪声应&lt;10 PA和模式封锁幅度应&gt; 0.1呐。 <ol><li>将上液细胞和屏蔽笼上的纳米孔，并在上部流体细胞引入40微升稀释的校准微粒。使用VPM申请≥0.8千帕的正压。 </li><li>减少应用舒展慢慢地向44毫米同时分析造成校准封锁事件颗粒。注意:当通过纳米孔减小孔径的颗粒的运动将不太可能，因此，粒子速度将减小。然而，由于孔隙的增加相对的封锁将发生较大的封锁事件导致改进的信号与噪声的比值。增加的电压可进一步提高封锁大小也可以增加RMS噪声。 </li><li>减少拉伸，直到相应的阻断事件（ 图1c）在"信号跟踪"面板中观察到。 （模式&gt; 0.1 NA）和相应的粒子速度&gt; 100 /分钟。注意:粒子速度是一个不太严格的截止，但是作为至少500个颗粒的测量是理想的，&lt;100 / min的粒子速率将导致记录的至少5分钟的持续时间。粒子速率比2000 /分高可导致不准确的测量值（如果存在的话，样品稀释液，应执行）。 </li></ol> 1.3测量校准颗粒的UPER流体单元和采样测量洗衣机<ol><li>在从胶质母细胞瘤细胞系U87-MG / EGFRvIII的的细胞培养物上清液本条电动车的特征。这些电动车辆的分离和制备以前已描述和可视化18。 </li><li>将校准颗粒在上部流体细胞。申请使用VPM和记录&gt; 500个颗粒的压力（例如0.8千帕）。 </li><li>如果执行多测量压力，增加所施加的压力（例如，以1.0千帕）和记录的第二校准文件。注:至少0.2千帕的差异是必需的。 </li><li>除去从上液细胞的校准样品。洗上部流体细胞3次，用100μl的PBS中，以除去残留的颗粒。引入样品进入上部液细胞前，用无绒组织，从上液细胞除去任何残余的PBS中。 </li><li>将样品引入到上部流体细胞。确保基线电流在测量校准颗粒时所观察到的基线电流的3％。如果未在3％以内，适用如下所述，以稳定的基线电流的策略。适用的确切压力作为施加到校准微粒和记录样品的文件。 注意:粒子速度曲线应显示恒定的粒子检测（ 图2a）。如果粒子探测，在基准电流突然下降，或RMS噪声突然增大的突然中断，孔可能堵塞;因此，暂停录制。为了恢复基准，水龙头或扭曲的屏蔽罩，适用于活塞，或者完全删除纳米孔，并用去离子水清洗，并更换仪器。 注意:可替换地，增加纳米孔伸展到47毫米，与从虚拟池主控者的最大压力的组合，时间约为5分钟。 </li></ol> 1.4数据分析<ol><li>点击";分析数据"选项卡，进入该软件的分析部分。右键单击"未处理的文件"并选择"流程文件"过程中的校准和示例文件。 </li><li>点击旁边的复选框样品中的"校准"列夫妇样本文件来校准记录。选择相应的样品和校准文件，并单击"确定"。注意:当使用多压力校准选项中选择就留给夫妻多个样品到多个校准文件"多压力校准"选项卡。 </li><li>一旦成功地耦接时，IZON控制Suite软件将显示不同样品的特征，例如大小分布（ 图2b），基线的持续时间，半峰全宽（FWHM）和浓度分析。可选:对于每个样本，各个数据点可以导出为逗号分隔的文件。 </li></ol> 2。lternative协议 - 尖峰样品与校准珠请注意:一般来说，标准操作程序可以与分离的电动车辆工作时使用。当与非隔离的电动车生物样品，或沾染大量蛋白质聚集体分离的EV制剂，工作操作仪器是具有挑战性的。这些挑战主要包括纳米孔阻塞（在基准电流突然下降），无法在校准测量，或在相同的样品（ 图3a）之间的粒子速度显著差异的3％恢复基准电流率高。样本显示这些困难替代协议进行量化电动车开发17。这种方法依赖于引进大聚苯乙烯校准珠入息（ 图3b）的样品。对于该备选方案的详细过程将在下面讨论。 2.1。样片准备注意:当使用另一种方法制备的样品中，期望建立的周围1同样的EV至珠的比例，它是必不可少的，以包括一个"校准珠只有&#39;样品，以允许的精确"门控"校准珠和确定背景粒子（例如蛋白质聚集体）存在于该缓冲区的数目。 <ol><li>离心机100微升细胞培养物上清液为7分钟，在300 XG </li><li>加入20μl上清液至20微升PBS和10μl的75倍稀释的335纳米的聚苯乙烯珠（股票7e10 /毫升）。 </li></ol> 2.2。样品测​​量<ol><li>使用1.2节中描述的策略，以确定最佳的仪器设置。 </li><li>样品首先测量"仅校准珠"。确保该背景检测小非珠颗粒的是尽可能地低（&lt;珠粒10％）。 </li><li>测量每个单独的SAmple一次之前的记录复制以在纳米孔的条件分布的波动同样在不同的样品。测量每个样品至少重复3次。 </li><li>完成所有样品的记录后，重新测量的"校准珠只有&#39;样本。 </li></ol> 2.3。数据分析注意:当使用替代协议，独家采用IZON控制套件软件不足够的浓度计算。附加的电子表格软件。 如表1所示浓度的计算在图3所示的样品的一个例子。 <ol><li>打开"校准珠只有&#39;样品和一个或多个示例文件。 </li><li>确定封锁事件大小（NA），可作为截止的电动汽车和聚苯乙烯小球之间的区别。确定对应于所述聚苯乙烯珠粒种群的左基部封锁值（NA）（ <st荣&gt; 图3b）。注意:确保滨尺寸测量所有的设定相同（可在"ViewSettings&#39;，这是通过点击下面的"个人跟踪封锁"中的"弹出"按钮，进入调整）。 </li><li>检索的总粒子数的值，通过点击该样品的"粒子分析概要"选项卡中的每个样本。 </li><li>筛选使用在步骤2.3.2中确定的截止电平的数据组。通过选择"数据过滤"弹出。仅显示粒子比截止较小。 </li><li>检索EV计数的值从"粒子分析摘要"每个样本。 </li><li>减去电动汽车的量从总的颗粒，以确定的校准珠的量。 </li><li>确定电动汽车到珠比除以电动汽车数量由校准珠计数。 </li><li>通过平均确定每个与比率确定该平均背景比率＃8220;珠唯一的"校准样品。减去从每个样品此值。 </li><li>乘以调整后的EV-到-珠比通过校准珠的浓度来确定电动车辆的各样品的浓度。 </li><li>乘以在步骤2.3.9由通过加入校准珠向EV样品引入的EV稀释因子中发现的浓度。注意:在这个例子中的样品的设置，总稀释在PBS中，校准珠样品的2.5倍，因此在步骤2.3.9中发现的浓度应为2.5相乘，以确定原始EV样品浓度。 </li><li>计算统计值，如平均值，标准偏差，及平均每个组重复的标准误差。 注:在某些情况下，电动汽车和尖刺的聚苯乙烯小球之间的重叠是观察。如果需要进行修正的EV浓度的低估，没有尖刺聚苯乙烯珠粒的样品也应进行测量。使用相同的cut-断所确定的步骤2.3.2确定"胎圈到EV"的比例，来计算落下的尖刺聚苯乙烯珠粒的性能范围内的电动车辆的比率。此珠至EV比率应添加到EV至珠比在步骤2.3.8确定。 </li></ol> 2.4。可选:EV粒度分布使用替代方法。 <ol><li>在Control Suite软件中打开一个样本记录的两倍。 </li><li>置的样品，仅显示颗粒大于上面所确定的截止之一的过滤器选项。这将只显示校准颗粒。 </li><li>将过滤后的样品为"校准文件"，进入校准珠的模式大小。 </li><li>夫妇的样本文件，在1.4.2中所述的步骤2.4.3创建的"校准文件"。示例文件现在会显示两个电动汽车和标定球的基础上，加标校准珠的粒径分布。 注:标准歌剧鼎协议将最常足以满足电动汽车的尺寸分布的测定。有时然而，确切的缓冲组分是未知的（例如在血浆或尿液），这使得它不可能制备的校准珠的样品在相同的缓冲液作为感兴趣的电动汽车。掺有校准颗粒的EV样品可用于电动汽车的大小，估计在这些特定的条件。 </li></ol>

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Vis. Exp. (3037), (2012).</li><li>Yang, L., Broom, M. F., Tucker, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+nanoparticulate+drug+delivery+system+using+scanning+ion+occlusion+sensing.">Characterization of a nanoparticulate drug delivery system using scanning ion occlusion sensing.</a> Pharmaceutical research. 29, 2578-2586 (2012).</li></ol>

概述的协议，这份手稿的写作的发展一直资助，部分由荷兰大脑基金会，舒马赫克莱默基金会和Bohnenn基金。制作这个视频文章部分被IZON sponsorted。

在这个手稿的报价方法进行定量分析，采用的TRP电动汽车的规模特征描述的协议。这些TRP平台的主要优点是样本量小，相对短的测量时间，并没有所需的样品操纵。 前提条件进行精确测量的TRP是保持条件的校准和样品测量之间是相同的。这包括相同的缓冲液的使用量以及相同的仪器设定，例如纳米孔尺寸，电压和施加压力。原VPM缺少一种机制，用于在施加压力的精确设定，从而使在样品之间施加的压力的微小差异。另外，吸液在VPM蒸发可以在不同时间点测量诱发轻微的压力差和VPM因此，应该经常被重新灌注。这些限制可能已经解决了引进VPM2，哪有一个基于点击的比例，并基于空气压力。 在这个手稿中描述的备选方案是特别适用于电动车辆的测量非纯化的生物样品17中。我们相信，缓冲成分，如糖，脂质，蛋白质和其它较大的碎片，可以在某些情况下影响测量条件太多的标准协议是适用的。另外的校准珠的样品，而不是比较两个独立的测量值引入"实时校准"。比较具有不同的和/或未知流体的背景含量样品（不同供体的例如血浆）时，这个方法是特别合适的。尽管差异电动汽车和聚苯乙烯颗粒（ 例如颗粒密度和表面电荷），理论模型和实验数据之间存在突出的聚苯乙烯小珠的可用性量化和电动汽车的尺寸分析，的前提下，该显著施加压力15,19。为了尽量减少电动力的影响，相对较大的NP150 / NP200纳米孔和显著正压的使用建议。 电动汽车和校准珠粒按大小区分。因此，纳米孔具有通过施加拉伸，制成直径在那里检测两个电动汽车和较大的校准微粒的观察被打开。自开业以来的孔隙会降低对较小颗粒的敏感性，只比电动车具有一定规模的记录（使用335 nm的校准珠时，往往电动车&gt; 120 nm）的大。最低检出限为电动汽车可以降低到大约90纳米，用203纳米校准珠上的NP150纳米孔。然而，这种设置可能是不可行的，当较大的电动车引起的纳米孔经常堵塞。这些阻碍电动车的存在可以强制设置的利用率，其中人口电动车，太小意图CH中的检测阈值，将不会被检测到。 难度，试图测量颗粒比大小100nm以下时，操作该系统增加。在这种情况下，检测可以通过增加电解质的盐的浓度可以提高。离子浓度的增加将导致相对增加的封锁量值为小颗粒（较大的信号 - 噪声比）。这种技术对于电动车辆的测量的生存能力有虽然被验证，如增加盐的浓度可影响电动车辆的体积。 总之，这些TRP平台，可用于直接量化和电动车的尺寸表征。因为没有隔离或电动操纵（抗体结合或荧光标记）是必需的，该平台适用于在生物流体直接EV定量。一种替代方案是提供一种能够有利于样品，其中缓冲液组分诱导显著孔隙cloggin克事件，使得标准协议不可行的可靠利用。

从细胞起源囊泡体液1非常丰富。这些所谓的胞外囊泡（电动车）（50 - 在大小为1000nm）由任一融合多泡体与细胞膜或细胞膜的直接向外出芽形成。近年来，在电动汽车科研的兴趣大大增加，从而导致过多的电动汽车为重点的出版物，其中新的功能和电动车的特征进行了描述1。现在电动车被认为是参与生理和病理过程，如信号转导，免疫调节等血液凝固1-4浩如烟海。在癌症，电动车似乎在转移前龛5,6，转移亲癌变内容7,8血管8的和刺激的形成的作用。除此之外，电动车进行了探讨，作为治疗药物9投递代理。 尽管有这些德velopments，电动汽车的可靠的定量仍然具有挑战性。传统上，间接量化方法被使用，它依赖于总蛋白含量或特定蛋白质的定量。尽管广泛使用，这些技术不占蛋白质每EV的差异，也污染蛋白聚集体和蛋白质电动车之间造成歧视。此外，这些技术都需要隔离电动车辆，这在许多情况下，使EV浓度的生物样品中的比较是不可能的。 因此，努力着手开发新的方法，让更多的精确和直接的EV测量10。这份报告描述了可调电阻脉冲感应（跨国激进党）的可靠的定量和电动汽车的规模纹。 目前，qNano仪（ 图1a）是跨国激进党市面上唯一平台。中的TRP，非导电性弹性膜间断无线个纳米大小的孔被分隔两个流体细胞。一流体细胞填充有感兴趣的样品，而其他细胞填充有颗粒的无电解质。通过施加电压，离子流/电流被建立，其在颗粒的通孔的转移改变（ 图1b）。这个电流阻断（&#39;电阻脉冲&#39;）的幅度正比于粒子11（ 图1c）的量。封锁时间可以用来评估的ζ-电势的颗粒，这依赖于颗粒的特性，如电荷或形状12。未知粒子的大小仿形可以通过比较所引起的未知粒子与具有已知直径所造成的校准微粒的电阻脉冲电阻脉冲来进行。除了封锁事件的严重程度，这其中发生率进行测量。这计数率热力ES上的粒子浓度。由于浓度和封锁的速率是线性比例13中 ，使用单个校准样品与已知浓度和粒径的颗粒允许浓度14和未知样品的粒度分布11的测定。 颗粒通过纳米孔的运动由电kinetic-（电泳和电渗透）和流体力15确定。通过使用可变压力模块（VPM）的流体单元之间的压力差可以被诱导作为一个额外的力。施加正压力增加粒子的流动速率，这可能是有益的，当粒子浓度低。此外，压力可被施加以减少电动力学力的作用。使用纳米孔具有相对小的孔隙直径时，这一点尤为重要（NP100，NP150和NP200可能），为经常用于电动汽车的检测。这些纳米孔，施加显著压力即使当，电动力学的力可以根据粒子的表面电荷，仍不可忽视16。通过测量颗粒速度在多重压力，一个电 - 动力学校正，从而更准确地，EV浓度可以计算出来。 在这里，提供详细的协议来确定电动车的尺寸分布和浓度。旁边的常规操作协议，另一种方法被描述，其中样品加入已知大小和浓度17的聚苯乙烯珠粒。这种实时校准技术可用于克服一些技术挑战直接在生物体液，如尿，血浆和细胞培养上清，或测量电动汽车时遇到当纳米孔的比的测量很长一段时间的稳定性不能保证。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td><a href="http://www.izon.com/trps-with-qnano/">qNano instrument</a></td>
			<td>Izon Science Ltd.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td><a href="http://www.izon.com/products/components-and-consumables/vpm/">Variable pressure module</a></td>
			<td>Izon Science Ltd.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td><a href="http://www.izon.com/products/components-and-consumables/nanopores/">Nanopore</a></td>
			<td>Izon Science Ltd.</td>
			<td>NP100, NP200</td>
			<td>Choice of nanopore varies based on target particle. Different nanopores are available for different target sizes.</td>
		</tr>
		<tr>
			<td>Calibration Particles</td>
			<td>Izon Science Ltd.</td>
			<td>CPC100, CPC200, CPC400</td>
			<td>Calibration particles are available in different sizes.</td>
		</tr>
		<tr>
			<td>Sonication bath</td>
			<td>Multiple available</td>
			<td></td>
			<td>Basic sonication bath is sufficient</td>
		</tr>
		<tr>
			<td>(Mini) vortexer</td>
			<td>Multiple available</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lift-free tissues</td>
			<td>Multiple available</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Multiple available</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Windows based computer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Izon Control Suite 2.2</td>
			<td>Izon Science Ltd.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Spreadsheet Software</td>
			<td>Multiple available</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification and size-profiling of extracellular vesicles using tunable resistive pulse sensing

外囊泡（EVS），其中包括"微泡"和"外来"，是体液非常丰富。近年来，两国在电动汽车的兴趣大幅增加。电动车辆已经显示出在各种生理和病理过程，包括凝血，免疫应答和肿瘤起着重要的作用。此外，电动汽车具有潜在的治疗剂，例如作为药物递送载体或作为再生医学。由于其体积小（50〜1000纳米）的准确定量和电动车的尺寸分析在技术上具有挑战性。 这个协议描述了如何可调电阻脉冲感测（的TRP）技术，采用了qNano系统，可以被用于确定电动车辆的浓度和大小。的方法，该方法通过一种纳米尺寸的细孔依赖于它们的传输电动车辆的检测，是比较快的，足以使用小体积的样品，并且不需要的PURification和电动汽车的浓度。旁边的正常运行协议的另一种方法是使用描述样品加入已知大小和浓度的聚苯乙烯珠粒。直接在生物流体测量电动汽车时，这种实时校准技术可用于克服技术障碍遇到。

使用的TRP仪器，非导电纳米孔必须被放置在4臂的机器（ 图1a）和一个电压（ 图1b）已被应用。一旦电基线电流确立，造成颗粒穿过孔的电阻的脉冲将被检测到， 如图1c所示 。 电动车是由成胶质细胞瘤细胞系U87-MG / EGFRvIII的通过超速离心的细胞培养物上清液纯化。在NP100纳米孔测量隔离电动车（ 图2a），稳定的粒子速率情节观察。这种稳定的粒子速率剧情需要一个可靠的EV浓度测量。配对的EV-样品记录到的115纳米的聚苯乙烯校准珠，尺寸分布（ 图2b）和EV-样品的浓度估计的记录后可以得到的（数据未示出）。 （Figrue 3A）中断和/或波动。这会导致不准确的EV浓度估计。由尖峰与已知浓度和大小的聚苯乙烯小珠的样本中，EV-到-珠比可以被确定。 图3b示出尖峰细胞培养物上清液与在大小335纳米的聚苯乙烯珠后得到的结果。两个明显的群体得到遵守。诱导小于0.46 nA的封锁颗粒决心电动车，较大的颗粒被确定聚苯乙烯珠粒。电动汽车到聚苯乙烯珠的比例来计算电动汽车（ 表1）的原始浓度。 图3c示出了根据尖刺聚苯乙烯珠粒的两个种群的大小估计。纳米孔的设置重新使用导致了这样的电动车辆的&gt; 140nm的尺寸的检测。这可以通过降低纳米孔开口被降低，然而，这也将导致更多的堵塞事件。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/51623/51623fig1highres.jpg" width="600" /> 图1:qNano仪器和操作模式。 （一）摄影仪器。纳米孔被放置在仪器上，从上流体细胞分离较低的流体单元。流体细胞免受由屏蔽盖的环境电磁干扰（B）图解概述可调电阻脉冲感测（的TRP）。的非导电性弹性纳米孔被分隔两个流体细胞。通过施加电压，使电流通过打孔在纳米孔的孔成立。由于外囊泡移动通过纳米孔，离子流发生改变，并检测为电阻式脉冲。中的TRP纳米孔的开口尺寸可以被调整（减少或增加），由通过增加距离的仪器的相对的臂之间，或减少该距离拉伸的纳米孔（℃）电阻脉冲的说明性示例。一个电阻脉冲的幅值正比于颗粒体积:较大的脉冲表示较大的颗粒<a href="https://www.jove.com/files/ftp_upload/51623/51623fig1highres.jpg" target="_blank">，请点击这里查看该图的放大版本。</a> <img alt="图2" fo:content-width="6in" src="/files/ftp_upload/51623/51623fig2highres.jpg" width="600" /> 图2:颗粒计数的情节和测量隔离电动车从U87-MG / EGFRvIII的细胞培养上清液得到大小分布（A）粒子计数的情节表明总体不变粒子探测。简要减少partic的80和100秒记录的之间观察勒检测。暂停记录和攻丝的屏蔽盖后，粒子速度稳定已恢复的记录之后，（B）隔离的电动汽车的尺寸分布校准未知样品（电动车）至115纳米的聚苯乙烯校准珠后作图。 （5 nm的垃圾桶大小）。 <a href="https://www.jove.com/files/ftp_upload/51623/51623fig2highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/51623/51623fig3highres.jpg" width="600" /> 图3:使用另一种协议的细胞培养上清电动车的TRP量化。 （一）直接在生物流体测量电动车的时候得到的典型粒子速率曲线。堵塞毛孔造成短暂中断和波动的粒子检测率。每曲线表示的相同样品的重复测量。扣球细胞培养物上清液与335纳米的聚苯乙烯校准珠后得到的（B）的三个重复的大小分布图。所有颗粒诱导的小于0.46 nA的电阻脉冲被选择作为电动汽车（C）的加标聚苯乙烯珠粒可用于获得样品的尺寸分布。 （5 nm的垃圾桶大小）。 <a href="https://www.jove.com/files/ftp_upload/51623/51623fig3highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 测量 </td><td> 校准仅＃1 </td><td> 校准仅＃2 </td><td> 上清＃1 </td><td> 上清＃2 </td><td> 上清＃3 </td></tr><tr><td>平均电流（NA） </td><td> 117 </td><td> 120 </td> &lt;TD&gt; 116 </td><td> 118 </td><td> 120 </td></tr><tr><td>粒子速度</td><td> 172 </td><td> 194 </td><td> 250 </td><td> 246 </td><td> 196 </td></tr><tr><td>使用截止（NA） </td><td> 0.46 </td><td> 0.46 </td><td> 0.46 </td><td> 0.46 </td><td> 0.46 </td></tr><tr><td>总颗粒</td><td> 303 </td><td> 317 </td><td> 489 </td><td> 488 </td><td> 454 </td></tr><tr><td>外囊泡</td><td> 3 </td><td> 1 </td><td> 213 </td><td> 215 </td><td> 213 </td></tr><tr><td>尖刺校准珠</td><td> 300 </td><td> 316 </td><td> 276 </td><td> 273 </td><td> 241 </td></tr><tr><td>电动车/校准珠</td><td> 0.01 </td><td> 0.003 </td><td> 0.772 </td><td> 0.788 </td><td> 0.884 </td></tr><tr><td>样品 - 背景</td><td></td><td></td><td> 0.765 </td><td> 0.781 </td><td> 0.877 </td></tr><tr><td> Extracelullar囊泡（10 7）/毫升</td><td></td><td></td><td> 7.14 </td><td> 7.29 </td><td> 8.18 </td></tr><tr><td>样品稀释2.5倍</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>原料浓度电动车（10 7）/毫升</td><td></td><td></td><td> 17.85 </td><td> 18.22 </td><td> 20.46 </td></tr></tbody></table> 表1:使用另一种协议的示例计算EV浓度的临界值被确定为从校准珠区分电动车。接着，可以检索到电动汽车和珠子的总数。对于每个测量的EV-到-珠比率的计算。在电解质（例如蛋白质聚集体）的背景颗粒的量是通过计算EV至珠比为"仅校准珠"样品的各个测量值计算。各样品的背景比被从所获得的比值中减去。这adjusted比乘以校准珠的样品中的浓度（在此示例中:9.33e7 /毫升）。来确定电动车辆的原始浓度，所得到的浓度乘以总电动汽车的稀释因子（在本例中:2.5）。

Watch this Scientific Journal Video about Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing at JoVE.com

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

外囊泡发挥生理和病理过程，包括凝血，免疫应答和肿瘤或在药物递送或再生医学作为潜在治疗剂的重要作用。该协议提出了在采用可调电阻脉冲检测各种流体隔离和非隔离外囊泡的定量和大小的表征方法。

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 ...

quantification-size-profiling-extracellular-vesicles-using-tunable

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JoVE Journal

Bioengineering

18.3K 次观看.  University Medical Center Utrecht. 外囊泡发挥生理和病理过程，包括凝血，免疫应答和肿瘤或在药物递送或再生医学作为潜在治疗剂的重要作用。该协议提出了在采用可调电阻脉冲检测各种流体隔离和非隔离外囊泡的定量和大小的表征方法。 

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

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

科研

生物工程

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

复透皮电化学传感的空心微针为基础的传感器

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

阻隔组织破坏与有机电化学晶体管传感

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

电动细胞基质阻抗传感的内皮细胞增殖，屏障功能的定量和能动性

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain nucleic acid and protein cargo and are increasingly recognized as a means of intercellular communication utilized by both eukaryote and prokaryote cells. However, due to their small size, current protocols for isolation of EVs are often time consuming, cumbersome, and require large sample volumes and expensive equipment, such as an ultracentrifuge. To address these limitations, we developed a paper-based immunoaffinity platform for separating subgroups of EVs that is easy, efficient, and requires sample volumes as low as 10 &#956;l. Biological samples can be pipetted directly onto paper test zones that have been chemically modified with capture molecules that have high affinity to specific EV surface markers. We validate the assay by using scanning electron microscopy (SEM), paper-based enzyme-linked immunosorbent assays (P-ELISA), and transcriptome analysis. These paper-based devices will enable the study of EVs in the clinic and the research setting to help advance our understanding of EV functions in health and disease.

This protocol details a method to isolate extracellular vesicles (EVs), small membranous particles released from cells, from as little as 10 &#956;l serum samples. This approach circumvents the need for ultracentrifugation, requires only a few minutes of assay time, and enables the isolation of EVs from samples of limited volumes.

基于纸张的设备进行隔离外囊泡与表征

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

加工机械可调和生物活性金属支架的生物医学应用

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

微RNA循环使用定量DNA结合染料化学和液滴的数字PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

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

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and nanoparticles. Tunable resistive pulse sensing (TRPS) is a relatively recent adaptation to RPS that incorporates a tunable pore that can be altered in real time. Here, we use TRPS to monitor the translocation times of DNA-modified nanoparticles as they traverse the tunable pore membrane as a function of DNA concentration and structure (i.e., single-stranded to double-stranded DNA).
TRPS is based on two Ag/AgCl electrodes, separated by an elastomeric pore membrane that establishes a stable ionic current upon an applied electric field. Unlike various optical-based particle characterization technologies, TRPS can characterize individual particles amongst a sample population, allowing for multimodal samples to be analyzed with ease. Here, we demonstrate zeta potential measurements via particle translocation velocities of known standards and apply these to sample analyte translocation times, thus resulting in measuring the zeta potential of those analytes.
As well as acquiring mean zeta potential values, the samples are all measured using a particle-by-particle perspective exhibiting more information on a given sample through sample population distributions, for example. Of such, this method demonstrates potential within sensing applications for both medical and environmental fields.

Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

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 ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Evaluation of the Storage Stability of Extracellular Vesicles

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical development, not only their pharmaceutical activity is important but also their production needs to be evaluated. In this context, research focuses on the isolation of EVs, their characterization, and their storage. The present manuscript aims at providing a facile procedure for the assessment of the effect of different storage conditions on EVs, without genetic manipulation or specific functional assays. This makes it possible to quickly get a first impression of the stability of EVs under a given storage condition, and EVs derived from different cell sources can be compared easily. The stability measurement is based on the physicochemical parameters of the EVs (size, particle concentration, and morphology) and the preservation of the activity of their cargo. The latter is assessed by the saponin-mediated encapsulation of the enzyme beta-glucuronidase into the EVs. Glucuronidase acts as a surrogate and allows for an easy quantification via the cleavage of a fluorescent reporter molecule. The present protocol could be a tool for researchers in the search for storage conditions that optimally retain EV properties to advance EV research toward clinical application.

Here we present a readily applicable protocol to assess the storage stability of extracellular vesicles, a group of naturally occurring nanoparticles produced by cells. The vesicles are loaded with glucuronidase as a model enzyme and stored under different conditions. After storage, their physicochemical parameters and the activity of the encapsulated enzyme are evaluated.