作者感谢H2020资助项目的财政支持:ACEnano（赠款协议号720952）。这项工作还部分得到了韩国贸易、工业和能源部资助的国际合作研发方案（赠款号N05310009，"Horizon2020 Kor-EU合作研发ACEnano工具箱"），使韩国合作伙伴能够参与 Horizon 2020 ACEnano 项目财团。

此处描述的方法用于第三轮实验室间比较。
1. 样品准备
<ol><li>通过 0.02 μm 注射器过滤器过滤水。水过滤是必要的，以消除任何污染颗粒之前，使用它的样品稀释。</li>
	<li>要分析新鲜制备的样品，请在过滤的超纯水中稀释 60 nm 黄金胶体分散体积的 50 倍。NTA 分析的建议浓度为每 mL 1 x 107 - 1 x10 9 颗粒。</li>
</ol>2. 执行测量
<ol><li>打开系统<ol>
<li>连接 NTA 仪器、注射器泵和计算机。打开硬件和软件。相关软件（见 材料表）可确保所有硬件通信运行，并显示实时温度读出。</li>
		<li>从 NTA 中取出激光模块，使用组织和压缩空气完全干燥玻璃表面和低体积流动电池 （LVFC） 内部通道、管子和流体端口。</li>
	</ol>
</li>
	<li>撬动管子<ol>
<li>用超纯水冲洗入口流体管，以去除任何颗粒，减少气泡干扰测量的可能性。对于冲洗，仪器外壳内的进水管末端放置在一个废物容器中。</li>
		<li>将过滤水的 1 mL 注射器（无针头）插入 Luer 端口，并尽可能快地将约 900μL 的液体推入进水管。离开装有剩余液体的注射器，以防止任何回音。</li>
	</ol>
</li>
	<li>注射器泵管连接<ol>
<li>将LVFC组装到激光模块上，创建 图1中看到的样品室。将出口管连接到 LVFC 的右侧端口。 注:入口和出口管的直径不同，入口直径小于出口。交换入出口管连接可能会导致对流电池施加压力和泄漏。</li>
		<li>将注射器与入入口管断开，换上含有 1 mL 过滤水的新注射器，确保液体与液体的接触。将入口管连接到 LVFC 的左端口。慢慢将约500μL的液体引入样品室。请注意，确保装载过程中不会引入气泡。最终的管子配置显示在 图 2中。</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61741/61741fig1v2.jpg"> 图1:安装在激光模块上的低体积流细胞组件。 
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61741/61741fig02.jpg"> 图2:低体积流管配置。<a href="https://www.jove.com/files/ftp_upload/61741/61741fig02large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<ol start="4"><li>激光模块加载和系统检查<ol>
<li>将充满水的LVFC插入激光模块，然后锁定到位。</li>
		<li>将注射器放入注射器泵摇篮并牢固。通过单击软件界面中的 "启动相机"来 初始化摄像机。在界面的 硬件 选项卡中，单击 散射 以移动参考位置。</li>
		<li>将相机水平设置为 16，并手动调整对焦，以检查任何颗粒的 diluent。通过左键单击主查看窗口并使用鼠标上下拖动以检查任何粒子来调整视场位置。如果视野中有三个以上的颗粒，这意味着净水或清洁过程存在问题，因此，清洁过程需要重复，或者需要更换或过滤水。</li>
		<li>从仪器中取出激光模块。</li>
		<li>将注射器与入口管断开，仅用充满空气的注射器更换。慢慢地将空气引入样品室，以去除里面的液体。从激光模块中取出 LVFC 并断开管子。用水清洁激光模块的LVFC玻璃表面和光学玻璃，用纸巾和压缩空气干燥。用压缩空气干燥管子。将 LVFC 重新组装到激光模块上并连接管子，以便进行样品加载。 注:此步骤并不总是需要的，但是，在这种情况下，它被添加为额外的预防措施，以进一步减少任何可能的变化。</li>
	</ol>
</li>
	<li>加载样本<ol>
<li>重复步骤2.2.2。将第 1.1 步中分散的 60 nm 金纳米粒子中的 1 mL 的注射器连接到 Luer 端口。通过入口管将样品的 750 μL 缓慢注入 LVFC，并在仪器外观看激光模块，以确保不会引入气泡。</li>
		<li>将激光模块加载回 NTA 仪器，然后通过单击软件界面中的 "启动相机"来 初始化相机。在界面的 硬件 选项卡中，单击 散射 以移动到参考对焦位置，检查是否正确设置以提供粒子的清晰图像。</li>
		<li>检查视场是否与激光束位置集中设置。通过左键单击软件中的主要查看窗口和鼠标上下拖动来相应地进行调整。</li>
		<li>运行 自动设置 功能，自动优化对焦和摄像机水平，确保实现最佳图像质量。 注:自动摄像头和对焦参数允许不同实验室之间更加一致，因为这是用户独立。</li>
	</ol>
</li>
	<li>样本分析<ol>
<li>在标准测量（SOP选项卡）中创建一个测量脚本，以获得 5 个 60 年代慢速下的重复视频（粒子应在大约 10 s 时从屏幕的一侧传到另一侧）和恒定流（补充文件 1）。 注:建议流，以确保更好地表示整体样本的测量。当样品上传递缓慢的流时，浓度测量的精度和可重复性显著提高，以确保更多的新粒子流经测量区并在实验期间进行分析。视频长度取决于配置文件分布以及分析时间的可变性。60s 的 5 个视频被视为典型的测量持续时间。</li>
		<li>为数据设置实验文件名称和位置并开始运行。概述程序之后的分析是由地平线2020 ACEnano项目17的七个实验室进行的。</li>
	</ol>
</li>
</ol>3. 数据分析
注:所有数据分析均在 v 3.4 软件内完成（见 材料表），不使用其他手动转换或计算。粒子大小数据以原始形式作为直方图分布呈现，并使用斯托克斯-爱因斯坦方程从粒子位置的测量变化中计算。该软件确定 x 和 y 平面中每个粒子移动的平均距离。此值允许确定粒子扩散系数 （D），如果已知样品温度 T 和溶剂粘度η，则可以计算粒子的等效球形流动力学半径 RH。样本的温度由 NTA 自动记录。软件使用的默认样品粘度为水，并包含在上面所示的测量脚本中，但当在测量之前或之后使用不同的样品 diluent 时，用户可以修改粘度。
<ol><li>通过拖动滑块条或在检测阈值下单击软件中的 + 和 按钮 来设置 检测阈值（DT），这是在 2 到 20 之间对可视化粒子进行最佳跟踪的分析参数。确保所选的DT值识别和跟踪尽可能多的可见粒子（在软件图像屏幕上自动标记为红色十字）。<ol>
<li>作为设置检测阈值的指导，图像中已识别的粒子数应在大约 30-80 之间，其中不超过 10 个红十字应与观察者不被视为粒子的站点相对应。观察到的蓝色十字架不应超过 5 个（表示噪音）。</li>
	</ol>
</li>
</ol><img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61741/61741fig03.jpg"> 图3:阈值设置观察。坏（左）和好（右）检测阈值设置观察。 <a href="https://www.jove.com/files/ftp_upload/61741/61741fig03large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<ol start="2"><li>通过按下软件中的 "过程 "按钮自动处理粒子跟踪分析视频。将所有处理参数设置为自动并导出数据，作为.csv格式结果文件，并附上完整的粒子大小分布和描述测量设置的其他元数据。要验证测量质量，请查看软件中的 分析 选项卡或检查.csv输出文件以查看任何警告消息或警报。PDF 结果报告的示例显示在 补充文件 2 中。
</li>
	<li>阅读模式大小结果和与 PDF 报告相关的标准偏差。 注:模式大小结果用于比较在七个实验室中获得的大小，并在第 5 节中显示和讨论。</li>
</ol>4. 清洁和干燥
<ol><li>使用后，用干净的水彻底冲洗系统，以清除管道和光学表面的所有样品痕迹。通过观察视野中的颗粒量，可以监测清洁效果。</li>
	<li>从 NTA 仪器中取出激光模块。</li>
	<li>通过系统加载空气注射器以清空管道和 LVFC。</li>
</ol>

<ol>
	<li>Thwala, M., Musee, N., Sikhwivhilu, L., Wepener, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oxidative+toxicity+of+Ag+and+ZnO+nanoparticles+towards+the+aquatic+plant+Spirodela+punctuta+and+the+role+of+testing+media+parameters.">The oxidative toxicity of Ag and ZnO nanoparticles towards the aquatic plant Spirodela punctuta and the role of testing media parameters.</a> Environmental Science-Processes and Impacts. 15 (10), 1830-1843 (2013).</li><li>Lowry, G. V., Gregory, K. B., Apte, S. C., Lead, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformations+of+nanomaterials+in+the+environment.">Transformations of nanomaterials in the environment.</a> Environmental Science &amp; Technology. 46 (13), 6893-6899 (2012).</li><li>Auffan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+definition+of+inorganic+nanoparticles+from+an+environmental,+health+and+safety+perspective.">Towards a definition of inorganic nanoparticles from an environmental, health and safety perspective.</a> Nature Nanotechnology. 4 (10), 634-641 (2009).</li><li>Valsami-Jones, E., Lynch, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+safe+are+nanomaterials.">How safe are nanomaterials.</a> Science. 350 (6259), 388-389 (2015).</li><li>Briffa, S. M., Nasser, F., Valsami-Jones, E., Lynch, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+and+impacts+of+polyvinylpyrrolidone+(PVP)+capped+metal+oxide+nanoparticles+on+Daphnia+magna:+role+of+core+composition+and+acquired+corona.">Uptake and impacts of polyvinylpyrrolidone (PVP) capped metal oxide nanoparticles on Daphnia magna: role of core composition and acquired corona.</a> Environmental Science: Nano. 5, 1745-1756 (2018).</li><li>Hassell&#246;v, M. a. R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analysis and Characterization of Manufactured Nanoparticles in Aquatic Environments. Environmental and Human Health Impacts of Nanotechnology. , 211-266 (2009).</li><li>Hoet, P., Br&#252;ske-hohlfeld, I., Salata, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticles+and+known+and+unknown+health+risks.">Nanoparticles and known and unknown health risks.</a> Journal of Nanobiotechnology. 2 (1), 12 (2004).</li><li>Briffa, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+transformations+of+manufactured+nanomaterials+as+a+proposed+proxy+for+ageing.">Thermal transformations of manufactured nanomaterials as a proposed proxy for ageing.</a> Environmental Science: Nano. 5, 1618-1627 (2018).</li><li>Kestens, V., Bozatzidis, V., De Temmerman, P. J., Ramaye, Y., Roebben, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+a+particle+tracking+analysis+method+for+the+size+determination+of+nano-+and+microparticles.">Validation of a particle tracking analysis method for the size determination of nano- and microparticles.</a> Journal of Nanoparticle Research. 19 (8), 271 (2017).</li><li>Walker, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+nano-particle+tracking+analysis.">Improved nano-particle tracking analysis.</a> Measurement Science and Technology. 23 (6), 065605 (2012).</li><li>Using NTA to Study Aggregation Behavior of Liposome-Protein Complexes. Malvern Panalytical Available from: <a target="_blank" href="https://www.news-medical.net/whitepaper/20161125/Using-NTA-to-Study-Aggregation-Behavior-of-Liposome-Protein-Complexes.aspx">https://www.news-medical.net/whitepaper/20161125/Using-NTA-to-Study-Aggregation-Behavior-of-Liposome-Protein-Complexes.aspx</a> (2019)</li><li>Filipe, V., Hawe, A., Jiskoot, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+evaluation+of+nanoparticle+tracking+analysis+(NTA)+by+NanoSight+for+the+measurement+of+nanoparticles+and+protein+aggregates.">Critical evaluation of nanoparticle tracking analysis (NTA) by NanoSight for the measurement of nanoparticles and protein aggregates.</a> Pharmaceutical Research. 27 (5), 796-810 (2010).</li><li>Roebben, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interlaboratory+comparison+of+size+and+surface+charge+measurements+on+nanoparticles+prior+to+biological+impact+assessment.">Interlaboratory comparison of size and surface charge measurements on nanoparticles prior to biological impact assessment.</a> Journal of Nanoparticle Research. 13 (7), 2675 (2011).</li><li>Hole, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interlaboratory+comparison+of+size+measurements+on+nanoparticles+using+nanoparticle+tracking+analysis+(NTA).">Interlaboratory comparison of size measurements on nanoparticles using nanoparticle tracking analysis (NTA).</a> Journal of Nanoparticle Research: An Interdisciplinary Forum for Nanoscale Science and Technology. 15 (12), 2101 (2013).</li><li>Analytical and Characterisation Excellence in nanomaterial risk assessment: A tiered approach. ACEnano Available from: <a target="_blank" href="https://cordis.europa.ue/project/id/720952">https://cordis.europa.ue/project/id/720952</a> (2019)</li><li>Soille, P., Vincent, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Determining watersheds in digital pictures via flooding simulations. 1360, (1990).</li></ol>

作者乔·沙利文、阿格涅什卡·西乌帕、宝琳·卡内尔-莫里斯和米歇尔·卡比尼是马尔文泛光有限公司的员工，该公司生产本文中使用的仪器。

从第 1 轮 ILC 获得的结果不一致，突出表明需要对较旧的系统进行仪器健康检查，以及开发更详细的 SOP、需要进行实践培训以及更好地了解测量和分析设置，以确保不同实验室的结果更加一致。事实上，Hole等人发现，缺乏关于如何使用NTA系统和准备样品的共享指南，导致实验室的变异性，即使是相对单分散的样品16。因此，所有ILC参与者都参加了一个培训讲习班，讲习班涵盖系统操作和测量条件的最佳做法，以及特定NTA仪器的清洁和维护指导。所有参与者还在自己的实验室中用同一仪器进行测量，以进行随后的ILC回合。该过程首先涉及在每个实验室中通过在乳胶标准样品（ILC 第 2 轮）上运行 ILC 对系统进行本地测试的一轮测试，然后由合作伙伴用于重复黄金测量（ILC 第 3 轮）。通过 NTA 测量这些黄金样品的目的是在纳米材料风险评估方法和做法中引入信心和清晰度，以影响纳米安全指导协议。
NTA是一种技术，可以测量粒子的流体球当量直径，并可用于粒子粒子，实时视觉分析的多分散系统范围从10 nm - 50nm，到约1000nm的大小（取决于样本属性和仪器配置）。需要最少的样品准备。尽管样品准备最少，但这一步骤对协议至关重要，在稀释样品和选择稀释剂时应小心谨慎。形状可能是NTA的一个限制因素，因为球形等效大小测量获得，非球形粒子将有一个不太准确的大小值。
对于 NTA 技术，由于仅从整个样本中观察到具有代表性的样本，因此始终预期会有一些结果变异。无论如何，所有结果都符合ISO 19430粒子尺寸标准。提供的最佳浓度通常在 30-60 秒分析时间内约为 108 颗粒/毫升。对于颗粒浓度较低的样品，需要更长的分析时间，以确保可重复的结果。对于颗粒浓度大于 109 颗粒/mL 的样品，跟踪问题的可能性更大，需要稀释到适合 NTA 测量的范围。
总体而言，第三轮ILC的结果表明，通过NTA，金纳米粒子测量具有良好的可重复性，提高了准确性和可重复性。所有 NTA 测量均使用自动摄像机级别和对焦设置进行，以调整图像，这是由软件中的自动设置功能选择的。该软件设置的摄像机级别非常一致，在所有情况下设置的摄像机级别为 10 或 11，这表明，正如预期的那样，过程包含的自动化程度越高，实现的一致性就越高。尺寸结果与制造商通过 TEM 获得的量身定做结果相当，表明其结果可重复，但由于 TEM 无法确定水动力直径，预计不同技术的差异将微乎其微。结果一致性显著提高，表明仪器维护、详细 SOP、最佳实践更新、用户知识和 NTA 应用测量自动化的重要性和益处。最后，ILC 验证了这一特定的 NTA SOP，从而为这种特定的纳米材料风险评估方法带来了信心和清晰度。

纳米材料（NMs）在物理和化学特性上都可能有所不同，进而影响其行为、稳定性和毒性1、2、3、4、5。在深入了解NM特性、危害和行为时，主要困难之一是能够获得有关物理和化学纳米材料特征的可重复信息。这些物理特性的例子包括粒子大小和大小分布6，7，8。这些都是重要的参数，因为它们是欧盟委员会（EC）对"纳米"9一词定义的一个关键方面。
除了了解NMs6、10的命运和毒性影响外，实现精确的粒子尺寸测量对于许多不同的工业和研究应用和过程也至关重要。重要的是要有成熟的方法，能够准确，可靠和可重复地测量NM的大小。此外，所报告的信息应深入了解所使用的技术，例如，指示大小参数的类型（例如实际大小或水动力学大小）以及样本条件，例如存在 NM 的特定介质，以及用于在不同介质中可靠执行的方法。为了测量尺寸，可以使用许多技术，包括电子显微镜（EM）、动态光散射（DLS）、单粒子感应耦合等离子体质谱仪（SPICP-MS）、差分离心沉降（DCS）、扫描探针显微镜（SPM）、小角度X射线散射（SAXS）和纳米粒子跟踪分析（NTA）。
NTA 是一项相对较新的技术，近年来取得了长足的进步，并已被证明能够可靠地测量复杂水态介质（如与环境相关的技术，例如淡水系统）中球形 NM 的水动力学直径。水动力学直径是"一个假想的硬球体的大小，以与所测粒子相同的方式扩散"11:在实际操作中，在水介质中，这描述了一个直径大于粒子本身的直径，其中还包括一层分子（主要是水），由弱静电力保持在粒子表面。粒子的水动力学直径会因不同介质而变化，随着测量粒子的离子强度升高而变小。
NTA 技术的另一个重要特点是，它允许分析员实现数字加权大小测量，这是 EC 纳米材料定义所要求的。高分辨率、逐粒子分析使这种技术在粒子吞吐量为10、12的异质试验样品中出现时，更容易受到聚合物或较大颗粒的干扰。
测量过程包括准备一个合适的悬浮样品，这往往需要样品稀释，然后视频记录粒子的布朗运动行为和视频分析。从样品室中，激光束经过，激光束路径中的悬浮粒子通过装有摄像头的光学显微镜散射光，导致其可视化。摄像机从在布朗运动下移动的粒子中捕捉散射激光的视频文件。许多粒子可以单独跟踪以确定其扩散系数，其水动力学直径可以使用斯托克斯-爱因斯坦方程计算:d = kT/3πηD d 是水动力学直径，k 是博尔茨曼常数，T 是温度，η是粘度，D 是扩散系数10。NTA还可用于跟踪通常胶体不稳定的粒子的聚集行为（然而，粒子必须在测量时间尺度上胶体稳定）13，14。NTA 是一种绝对方法，在这项工作中使用的仪器上不需要系统校准。如果用户想要检查系统性能，可以通过按需要频繁测量尺寸标准材料来轻松完成此任务。
NTA 仪器易于操作，分析时间快（每个样品不到 10 分钟）。对于具有良好数据可重复性和可重复性的高质量测量，在样品准备和仪器操作中应考虑若干因素。如果不仔细考虑这些因素，不同实验室和操作员对同一材料的测量可能会受到未知或未量化的不确定性的影响。在 NP 定性中，使用内部开发的 SOP 的最佳实践并不总是保证与其他实验室的一致性，如 Roebben 等人对 DLS 技术15所示。
事实上，不同实验室、用户和仪器之间的早期（第一轮）NTA ILC显示的结果不一致。其中一个主要问题是使用各种没有定期服务或校准检查的旧遗留文书，以及方法解释的差异。霍尔等人的NTA ILC研究发现，由于缺乏关于如何使用系统和准备样品的共同指南，即使对于相对单一分散的样品16，实验室的变异性也会很大。这以及第一轮 ILC 的结果突出了良好的仪器维护以及方法培训和发达的标准操作程序 （SOP） 的必要性。后者是描述和记录遵守良好做法的有力工具。如果详细，标准操作程序 （SOP） 可以提供清晰、解释、理解、标准化和质量保证。
因此，采用ILC研究的建议非常适合开发和测试协议16。ILC 演习旨在验证这一特定的 NTA SOP，从而为这种特定的纳米材料风险评估方法带来了信心和清晰度。它涉及三轮。第 1 轮在训练前分析了每个参与者自己的仪器上的 60 纳米金纳米粒子。第 2 轮涉及使用具有常见配置的新仪器分析 100 nm 乳胶作为简单测试，以确定仪器设置正确，用户对如何使用仪器有良好的了解。第3轮涉及在训练后对新仪器上60纳米金纳米粒子进行分析，并具有共同的配置。ILC的参与者来自七个不同的实验室，都是地平线2020 ACEnano项目17的财团成员。
本文的目的是讨论 NTA 技术第三轮基准测试的方法和结果，在详细培训和 SOP 开发之后，7 个合作伙伴重新分析了 60 nm 黄金 NP。还将比较和参考在ILC第一轮中取得的结果。ILC 第 3 轮的所有分析均使用配备 405 nm 激光器和高灵敏度 sCMOS 摄像机的相同配置的相同仪器（见 材料表）进行。基准评估技术在样品上的性能，从而导致"最佳实践"协议的制定。因此，本文还分享并使本ILC中使用的仪器的NTA方法可供科学界使用，因为它通过按照国际标准对ILC进行和评估而得到协调。

<table><tbody><tr><td>60 nm gold colloid dispersion</td><td>BBI Solutions OEM Ltd.</td><td>Product EM. GC60, Batch number 024650</td><td></td></tr><tr><td>0.02 &amp;micro;m syringe filter - Whatman Anotop 25 Sterile Syringe Filters</td><td>Sigma Aldrich</td><td>WHA68092102</td><td></td></tr><tr><td>NanoSight</td><td>Malvern Panalytical Ltd.</td><td>NS300</td><td></td></tr><tr><td>NanoSight NTA Software v3.4</td><td>Malvern Panalytical Ltd.</td><td>v3.4</td><td></td></tr><tr><td>Syringe PP/PE without needle luer slip tip, centered, capacity 1&amp;nbsp;mL, graduated, 0.01&amp;nbsp;mL, sterile</td><td>Sigma Aldrich</td><td>Z230723</td><td></td></tr></tbody></table>

nanoparticle tracking analysis of gold nanoparticles in aqueous media through an inter-laboratory comparison

在纳米技术领域，分析性特征在理解纳米材料（NMs）的行为和毒性方面起着至关重要的作用。特征化需要彻底，所选择的技术应非常适合要确定的属性、所分析的材料和存在的介质。此外，仪器操作和方法需要得到用户的充分开发和明确理解，以避免数据收集错误。应用方法或程序的任何差异都可能导致已获得数据的差异和可重复性差。本文旨在通过纳米粒子跟踪分析（NTA）来阐明测量金纳米粒子水动力学直径的方法。这项研究是在七个不同的实验室之间作为实验室间比较（ILC）进行的，以验证标准操作程序的性能和可重复性。ILC 研究的结果揭示了详细标准操作程序 （SOP）、最佳实践更新、用户知识和测量自动化的重要性和益处。

使用各种 NTA 仪器配置的第 1 轮 ILC 结果显示在 图 4中。除实验室 6 外，5 个捕获重复之间的可重复性良好，但多个实验室记录的模式大小高于预期。实验室 6 结果表明，可重复性差，测量的模式大小要高得多。调查后发现，报告最大尺寸变化的系统要么没有按照建议进行维护，要么分析受到样品制备不一致的影响，即稀释步骤可能因不同的管道设备、用户操作和技术以及/或测量设置（包括流细胞不干净、使用错误的摄像机水平、图像未正确对焦点以及设置分析检测阈值）而产生变异。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61741/61741fig4v2.jpg"> 图4:ILC第1轮模式大小结果。模式大小来自所有 NTA 基准合作伙伴在不同的 NTA 仪器上进行的第 1 轮 60 nm 金纳米粒子分散（如 x 轴的缩写）。
实施相同 SOP 和仪器设置的所有实验室都提高了第 3 轮的 NTA 结果精度。此 ILC 第 3 轮获得的模式大小结果可在 图 5中看到。所有实验室的平均模式为 62.02 ± 1.97 nm。第 3 轮的所有测量结果都比第一阶段的结果更一致，结果远远低于制造商所述批次 60.5 nm 平均尺寸的 10%。制造商陈述的黄金样品变异系数为≤8%。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61741/61741fig5v2.jpg"> 图5:ILC第3轮模式大小结果。模式大小来自所有 NTA 基准合作伙伴的结果，用于在同一 NTA 仪器上分析的 60 nm 黄金 ILC 第 3 轮。所有实验室的平均模式为 62.02 ± 1.97 nm。
为了验证粒子大小，根据制造商提供，传输电子显微镜 （TEM） 分析了少量 （N=82） 粒子。大约 10μl 的未浸出分散物被投在碳涂层 Cu TEM 网格上，在 200 kV 的分析 TEM 成像之前在空气中干燥。像 补充图1 这样的图像取自粒子重叠最小的区域，并使用半自动图像分析过程进行分析。一种自动分水岭方法应用于分离粒子，这个过程的人工制品被排除在外，以及边缘粒子18。平均直径的计算方法是主轴和次小轴的平均值（61±7 nm），或假设球形粒子从测量区域（62±6nm）的转换。粒子似乎大部分是球形的，平均纵横比为1.1。TEM 结果显示直径略高于制造商值 （60.5 nm），但处于公差水平。此外，与 NTA 的流体动力学直径推导值有非常好的协议。
补充图1:60纳米金纳米粒子的TEM图像。<a href="https://cloudflare.jove.com/files/ftp_upload/61741/61741supfig01.jpg" target="_blank">请点击这里下载此数字。</a>
补充文件1:测量脚本。<a href="https://cloudflare.jove.com/files/ftp_upload/61741/Measurement_script.docx" target="_blank">请点击这里下载此文件。</a>
补充文件2:PDF结果报告示例。<a href="https://cloudflare.jove.com/files/ftp_upload/61741/PDF_results_report.pdf" target="_blank">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Nanoparticle Tracking Analysis of Gold Nanoparticles in Aqueous Media through an Inter-Laboratory Comparison at JoVE.com

Nanoparticle Tracking Analysis of Gold Nanoparticles in Aqueous Media through an Inter-Laboratory Comparison

此处描述的协议旨在通过纳米粒子跟踪分析 （NTA） 在水介质中测量球形纳米粒子（更具体地说是金纳米粒子）的水动力学直径。后者包括跟踪由于布朗运动而产生的粒子的运动，并实施斯托克斯-爱因斯坦方程来获得水动力学直径。

通过实验室间比较，对液态介质中的金纳米粒子进行纳米粒子跟踪分析

In the field of nanotechnology, analytical characterization plays a vital role in understanding the behavior and toxicity of nanomaterials (NMs). ...

nanoparticle-tracking-analysis-gold-nanoparticles-aqueous-media

Research

JoVE Journal

Chemistry

7.2K 次观看.  University of Birmingham. 此处描述的协议旨在通过纳米粒子跟踪分析 （NTA） 在水介质中测量球形纳米粒子（更具体地说是金纳米粒子）的水动力学直径。后者包括跟踪由于布朗运动而产生的粒子的运动，并实施斯托克斯-爱因斯坦方程来获得水动力学直径。

视频: Nanoparticle Tracking Analysis of Gold Nanoparticles in Aqueous Media through an Inter-Laboratory Comparison

Analyzing the Movement of the Nauplius 'Artemia salina' by Optical Tracking of Plasmonic Nanoparticles

We demonstrate how optical tweezers may provide a sensitive tool to analyze the fluidic vibrations generated by the movement of small aquatic organisms. A single gold nanoparticle held by an optical tweezer is used as a sensor to quantify the rhythmic motion of a Nauplius larva (Artemia salina) in a water sample. This is achieved by monitoring the time dependent displacement of the trapped nanoparticle as a consequence of the Nauplius activity. A Fourier analysis of the nanoparticle&#39;s position then yields a frequency spectrum that is characteristic to the motion of the observed species. This experiment demonstrates the capability of this method to measure and characterize the activity of small aquatic larvae without the requirement to observe them directly and to gain information about the position of the larvae with respect to the trapped particle. Overall, this approach could give an insight on the vitality of certain species found in an aquatic ecosystem and could expand the range of conventional methods for analyzing water samples.

Analyzing the Movement of the Nauplius 'Artemia salina' by Optical Tracking of Plasmonic Nanoparticles

We use optical tracking of plasmonic nanoparticles to probe and characterize the frequency movements of aquatic organisms.

分析无节幼体的运动"<em&gt;卤虫</em&gt;&#39;由电浆纳米粒子的光学跟踪

We demonstrate how optical tweezers may provide a sensitive tool to analyze the fluidic vibrations generated by the movement of small aquatic organisms. A ...

科研

工程学

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 ...

生物工程

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and provides a theoretical basis for the rational design of nano-medicine delivery. Single particle tracking (SPT) analysis could probe the position and orientation of individual nanoparticles on cell membrane, and reveal their translational and rotational states. Here, we show how to use traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on live cell membrane. We also show how to extract the location and orientation of AuNRs using ImageJ and MATLAB, and how to characterize the diffusive states of AuNRs. Statistical analysis of hundreds of particles show that single AuNRs perform Brownian motion on the surface of U87 MG cell membrane. However, individual long trajectory analysis shows that AuNRs have two distinctly different types of motion states on the membrane, namely long-range transport and limited-area confinement. Our SPT methods can be potentially used to study the surface or intracellular particle diffusion in different biological cells and can become a powerful tool for investigations of complex cellular mechanisms.

Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis.

使用单纳米粒子暗场显微镜可视化细胞膜上金纳米棒的扩散动力学

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and ...

生物化学

UV-Vis Spectroscopic Characterization of Nanomaterials in Aqueous Media

The physicochemical characterization of nanomaterials (NMs) is often an analytical challenge, due to their small size (at least one dimension in the nanoscale, i.e. 1&#8211;100 nm), dynamic nature, and diverse properties. At the same time, reliable and repeatable characterization is paramount to ensure safety and quality in the manufacturing of NM-bearing products. There are several methods available to monitor and achieve reliable measurement of nanoscale-related properties, one example of which is Ultraviolet-Visible Spectroscopy (UV-Vis). This is a well-established, simple, and inexpensive technique that provides non-invasive and fast real-time screening evaluation of NM size, concentration, and aggregation state. Such features make UV-Vis an ideal methodology to assess the proficiency testing schemes (PTS) of a validated standard operating procedure (SOP) intended to evaluate the performance and reproducibility of a characterization method. In this paper, the PTS of six partner laboratories from the H2020 project ACEnano were assessed through an interlaboratory comparison (ILC). Standard gold (Au) colloid suspensions of different sizes (ranging 5&#8211;100 nm) were characterized by UV-Vis at the different institutions to develop an implementable and robust protocol for NM size characterization.

This study presents the benchmarking results for an interlaboratory comparison (ILC) designed to test the standard operating procedure (SOP) developed for gold (Au) colloid dispersions characterized by ultraviolet-visible Spectroscopy (UV-Vis), amongst six partners from the H2020 ACEnano project for sample preparation, measurement, and analysis of the results.

水介质中纳米材料的紫外-可见光谱表征

The physicochemical characterization of nanomaterials (NMs) is often an analytical challenge, due to their small size (at least one dimension in the ...

Gold Nanoparticle Synthesis

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of tetrachloroauric acid in 3.0 g (3.7 mmol, 3.6 mL) of oleylamine (technical grade) and 3.0 mL of toluene into a boiling solution of 5.1 g (6.4 mmol, 8.7 mL) of oleylamine in 147 mL of toluene. While boiling and mixing the reaction solution for 2 hours, the color of the reaction mixture changed from clear, to light yellow, to light pink, and then slowly to dark red. The heat was then turned off, and the solution was allowed to gradually cool down to room temperature for 1 hour. The gold nanoparticles were then collected and separated from the solution using a centrifuge and washed three times; by vortexing and dispersing the gold nanoparticles in 10 mL portions of toluene, and then precipitating the gold nanoparticles by adding 40 mL portions of methanol and spinning them in a centrifuge. The solution was then decanted to remove any remaining byproducts and unreacted starting materials. Drying the gold nanoparticles in a vacuum environment produced a solid black pellet; which could be stored for long periods of time (up to one year) for later use, and then redissolved in organic solvents such as toluene.

A protocol for synthesizing ~12 nm diameter gold nanoparticles (Au nanoparticles) in an organic solvent is presented. The gold nanoparticles are capped with oleylamine ligands to prevent agglomeration. The gold nanoparticles are soluble in organic solvents such as toluene.

金纳米粒子合成

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of ...

化学

Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting and drug delivery are some of the applications of these AuNPs, but endothelial extracellular matrices&#39; defensive properties hamper particle uptake. To address this issue, we describe a synthesis method for ultrasmall gold nanoparticles to improve vascular delivery, with customizable functional groups and polymer lengths for further adjustments. The protocol yields 2.5 nm AuNPs that are capped with tetrakis(hydroxymethyl)phosphonium chloride (THPC). The replacement of THPC with hetero-functional polyethylene glycol (PEG) on the surface of the AuNP increases the hydrodynamic radius to 10.5 nm while providing various functional groups on the surface. The last part of the protocol includes an optional addition of a fluorophore to allow the AuNPs to be visualized under fluorescence to track nanoparticle uptake. Dialysis and lyophilization were used to purify and isolate the AuNPs. These fluorescent nanoparticles can be visualized in both in vitro and in vivo experiments due to the biocompatible PEG coating and fluorescent probes. Additionally, the size range of these nanoparticles render them an ideal candidate for probing the glycocalyx without disrupting normal vasculature function, which may lead to improved delivery and therapeutics.

We describe a method of synthesizing biocompatible 10-nm gold nanoparticles, functionalized by coating poly-ethylene glycol onto the surface. These particles can be used in vitro and in vivo for delivering therapeutics to nanoscale cellular and extracellular spaces that are difficult to access with conventional nanoparticle sizes.

用于内皮靶向和药物传递的功能化10纳米高分子涂层金粒子的合成

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting ...

Asymmetrical Flow Field-Flow Fractionation for Sizing of Gold Nanoparticles in Suspension

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and size distribution of nanoparticles is of utmost importance for various applications. The size range is also important, as it defines the most &#8220;active&#8221; component of a nanoparticle dose.
Asymmetrical Flow Field-Flow Fractionation (AF4) is a powerful technique for sizing of particles in suspension in the size range of approximately 1&#8211;1000 nm. There are several ways to derive size information from an AF4 experiment. Besides coupling AF4 online with size-sensitive detectors based on the principles of Multi-Angle Light Scattering or Dynamic Light Scattering, there is also the possibility to correlate the size of a sample with its retention time using a well-established theoretical approach (FFF theory) or by comparing it with the retention times of well-defined particle size standards (external size calibration).
We here describe the development and in-house validation of a standard operating procedure (SOP) for sizing of an unknown gold nanoparticle sample by AF4 coupled with UV-vis detection using external size calibration with gold nanoparticle standards in the size range of 20&#8211;100 nm. This procedure provides a detailed description of the developed workflow including sample preparation, AF4 instrument setup and qualification, AF4 method development and fractionation of the unknown gold nanoparticle sample, as well as the correlation of the obtained results with the established external size calibration. The SOP described here was eventually successfully validated in the frame of an interlaboratory comparison study highlighting the excellent robustness and reliability of AF4 for sizing of nanoparticulate samples in suspension.

This protocol describes the use of Asymmetrical Flow Field-Flow Fractionation coupled with UV-vis detection for the determination of the size of an unknown gold nanoparticle sample.

悬浮中金纳米粒子尺寸的不对称流场-流分数

Particle size is arguably the most important physico-chemical parameter associated with the notion of a nanoparticle. Precise knowledge of the size and ...

Improving Reproducibility to Meet Minimal Information for Studies of Extracellular Vesicles 2018 Guidelines in Nanoparticle Tracking Analysis

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many consider that NTA instruments and their software packages can be easily utilized following minimal training and that size calibration is feasible in-house. As both NTA acquisition and software analysis constitute EV characterization, they are addressed in Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018). In addition, they have been monitored by Transparent Reporting and Centralizing Knowledge in Extracellular Vesicle Research (EV-TRACK) to improve the robustness of EV experiments (e.g., minimize experimental variation due to uncontrolled factors).
Despite efforts to encourage the reporting of methods and controls, many published research papers fail to report critical settings needed to reproduce the original NTA observations. Few papers report the NTA characterization of negative controls or diluents, evidently assuming that commercially available products, such as phosphate-buffered saline or ultrapure distilled water, are particulate-free. Similarly, positive controls or size standards are seldom reported by researchers to verify particle sizing. The Stokes-Einstein equation incorporates sample viscosity and temperature variables to determine particle displacement. Reporting the stable laser chamber temperature during the entire sample video collection is, therefore, an essential control measure for accurate replication. The filtration of samples or diluents is also not routinely reported, and if so, the specifics of the filter (manufacturer, membrane material, pore size) and storage conditions are seldom included. The International Society for Extracellular Vesicle (ISEV)&#39;s minimal standards of acceptable experimental detail should include a well-documented NTA protocol for the characterization of EVs. The following experiment provides evidence that an NTA analysis protocol needs to be established by the individual researcher and included in the methods of publications that use NTA characterization as one of the options to fulfill MISEV2018 requirements for single vesicle characterization.

Nanoparticle tracking analysis (NTA) is a widely used method to characterize extracellular vesicles. This paper highlights NTA experimental parameters and controls plus a uniform method of analysis and characterization of samples and diluents necessary to supplement the guidelines proposed by MISEV2018 and EV-TRACK for reproducibility between laboratories.

提高可重复性以满足细胞外囊泡研究的最少信息 2018纳米颗粒跟踪分析指南

Nanoparticle tracking analysis (NTA) has been one of several characterization methods used for extracellular vesicle (EV) research since 2006. Many ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

适用于比色金纳米粒子检测方法的选择结构切换适配方法

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

生物学

Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving area of research. There are many different methods for EV isolation, each with distinct advantages and disadvantages that affect the downstream yield and purity of EVs. Thus, characterizing the EV prep isolated from a given source by a chosen method is important for interpretation of downstream results and comparison of results across laboratories. Various methods exist for determining the size and quantity of EVs, which can be altered by disease states or in response to external conditions. Nanoparticle tracking analysis (NTA) is one of the prominent technologies used for high-throughput analysis of individual EVs. Here, we present a detailed protocol for quantification and size determination of EVs isolated from mouse perigonadal adipose tissue and human plasma using a breakthrough technology for NTA representing major advances in the field. The results demonstrate that this method can deliver reproducible and valid total particle concentration and size distribution data for EVs isolated from different sources using different methods, as confirmed by transmission electron microscopy. The adaptation of this instrument for NTA will address the need for standardization in NTA methods to increase rigor and reproducibility in EV research.

We demonstrate how to use a novel nanoparticle tracking analysis instrument to estimate the size distribution and total particle concentration of extracellular vesicles isolated from mouse perigonadal adipose tissue and human plasma.

细胞外囊泡的量化和尺寸测定纳米粒子跟踪分析

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving ...