作者要感谢整个ACEEnano财团在编写本议定书的所有阶段进行了富有成果的讨论。作者还感谢欧盟地平线2020方案（H2020）根据ACEnano项目框架下的赠款协议no 720952提供的资金。

1. AF4 系统设置
<ol><li>按照制造商手册中的说明，组装 AF4 墨盒并连接 AF4 系统的所有硬件组件和 UV-vis 探测器（材料表）。</li>
	<li>按照制造商手册中的说明安装所有必要的软件包，用于控制、数据采集、处理和评估。</li>
	<li>确保AF4系统和UV-vis探测器之间的所有必要信号连接已经建立。</li>
	<li>确保已建立的AF4-UV-vis连接紧密且无泄漏，通过用超纯水（UPW）冲洗设置15分钟（尖流速1 mL∙分钟-1，对焦流速1 mL∙min-1，交叉流速1.5 mL∙分钟-1）。为此，打开 AF4 控制软件，将流量率输入着陆页右上侧的相应面板。如有必要，拧紧相应的连接器（配件），并重复操作，直到无法观察到泄漏。 注:应监控所有测量过程中的内部系统压力，并且必须在 4 到 12 条以内。如果压力较高或更低，需要调整后压管。此外，在完整的测量时间内，通道压力趋势应保持不变。 注意:如果有通道烤箱，将其温度设置为 25 °C，以确保所有 AF4 实验的可比测量条件。</li>
</ol>2. 为AF4-UV-vis系统资格和样品分析准备解决方案和暂停
<ol><li>清洁解决方案<ol>
<li>将 8 克固体氢氧化钠 （NaOH） 和 2 克硫酸钠 （SDS） 加入 1 升 UPW，搅拌溶液直到完全溶解。</li>
	</ol>
</li>
	<li>埃伦特<ol>
<li>将 500 μL 的过滤表面活性剂混合物添加到 2 升过滤和去胶化 UPW 中，以获得 0.025% （v/v），pH 约为 9.4）。 注:表 1（ 也是 材料表）详细描述了表面活性混合物的化合物。</li>
	</ol>
</li>
	<li>用于大规模恢复确定的任意 AuNP 大小标准<ol>
<li>漩涡任意 AuNP 大小标准 （50 毫克∙L-1）2 分钟，并稀释它 1:4 与 UPW 获得 12.5 毫克∙L-1的最终质量浓度。涡流在稀释后额外 2 分钟，以使获得的悬架均匀化。 警告:在处理化学品（尤其是 NaOH 颗粒）时，需要必要的预防措施和适当的防护设备。 注:一般建议使用 0.1 μm 膜过滤器（亲水 PVDF 或类似）去气和过滤所有必要的溶液（清洁溶液除外），以确保 AF4-UV-vis 实验期间的低颗粒背景。这可以通过专用的真空过滤装置或使用注射器过滤器来建立。</li>
	</ol>
</li>
</ol>3. AF4-紫外线-维斯系统资格
<ol><li>使用步骤 1.4 中描述的软件设置，用清洁解决方案冲洗系统 30 分钟（提示流速 1 mL ∙分钟-1，焦点流速 1 mL ∙ 分钟-1，交叉流速 1.5 mL ∙ 分钟-1）。</li>
	<li>更换各自的发光瓶，用 UPW 冲洗系统 20 分钟（提示流速 1 mL ∙分钟-1，焦点流速 1 mL ∙分钟-1，交叉流速 1.5 mL ∙分钟-1）。</li>
	<li>更换各自的内联泵过滤器。</li>
	<li>打开 AF4 墨盒并更换 AF4 膜。重新组装AF4墨盒并将其与AF4-UV-维斯系统重新连接。</li>
	<li>将清洁的AF4-UV-vis系统与精灵擦洗至少30分钟，以平衡膜和稳定系统（提示流速1 mL∙分钟-1，焦点流速1 mL∙分钟-1，交叉流速1.5 mL∙最小-1）。再次检查是否有潜在泄漏（见步骤 1.4）。</li>
	<li>通过使用任意的 AuNP 尺寸标准确定质量恢复和保留时间的变化，从而对 AF4-UV-vis 系统进行资格验证。<ol>
<li>在不应用分离力的情况下进行直接注射运行。<ol>
<li>通过打开文件|创建新的测量 文件新|在 AF4控制软件中运行。</li>
			<li>在 "运行" 选项卡中定义样本和测量描述以及注塑量和样本名称。测量条件显示在 表2中。</li>
			<li>根据表 2设置第二个选项卡FFF 方法中的测量参数。</li>
			<li>单击 "运行" 按钮以开始测量。</li>
		</ol>
</li>
		<li>使用分离力（交叉流）执行分数运行。<ol>
<li>使用 表 3中指定的分数条件定义上一节中描述的分数法。</li>
			<li>单击 "运行" 按钮以开始测量。</li>
			<li>以四倍值执行测量。 注:第一次运行旨在对系统（即AF4膜）进行调节，并将排除在对系统资格结果的最终评估之外。 注:建议通过打开文件|保存所有生成的运行文件保存在AF4控制软件中。</li>
			<li>如果任意 AuNP 尺寸标准获得了 =80% 的质量恢复和保留时间的变化 [lt;2%"，则考虑 AF4-UV-vis 系统是否合格。</li>
			<li>当使用自动取样器作为注塑系统时，将自动取样器的针头清洗储液箱装满通过 AF4-UV-vis 系统泵送的相同溶液（例如清洁液、UPW 或相应的排污液），以确保最佳运行条件。在更改 eluent 时，通常建议通过监测紫外线对探测器信号，跟踪 AF4 系统的重新平衡，直到其基线在恒定水平上保持稳定。</li>
		</ol>
</li>
	</ol>
</li>
</ol>4. AF4-紫外线-对样本分析
<ol><li>通过调整各自的 AuNP 悬架（20 纳米、40 纳米、80 纳米、100 纳米、每个 50 毫克）来准备所有 AuNP 尺寸标准，以进行外部尺寸校准。L-1）2 分钟，用 UPW 稀释 1:4，最终质量浓度为 12.5 毫克∙L-1。涡流在稀释后额外 2 分钟，以使获得的悬架均质化。</li>
	<li>准备未知的 AuNP 样本，以便采用与步骤 4.1 中描述的校准标准相同的程序进行分析。</li>
	<li>使用 表 2中显示的 AF4 方法对所有 AuNP 尺寸标准进行直接注射测量。<ol>
<li>为此，在适当位置将 表 2 中总结的各自值输入制造商的软件，以定义分离和示例参数，并按 下"运行" 按钮开始实验。</li>
	</ol>
</li>
	<li>使用 表 3 中显示的 AF4 方法单独分馏每个 AuNP 大小标准，以建立外部大小校准功能。<ol>
<li>在适当位置将 表 3 中总结的各自值输入制造商的软件。分馏法由对焦步骤、几个分化步骤和冲洗步骤定义。设置方法后，按 运行 按钮开始实验。</li>
	</ol>
</li>
	<li>使用 表 2中显示的 AF4 方法对未知的 AuNP 样本进行直接注射测量。</li>
	<li>通过执行 表 3中列出的 AF4 方法，执行未知的 AuNP 样本的分馏。</li>
	<li>执行 第 3 节和第 4 节中提及的所有三重测量，除非另有说明，以确保有意义的和统计相关的结果。<ol>
<li>使用前将 50 毫克∙L-1 AuNP 股票暂停存放在 4-8 °C。稀释的 AuNP 悬架最好在申请前 30 分钟内准备就绪。 注意:涡流通常足够，无需超声波悬架。</li>
		<li>为了使未知 AuNP 样本的保留时间与 AuNP 大小标准获得的保留时间相关联，请使用相同的 AF4 方法测量所有样品。 注:为确保持续有效的分离条件，请在确定数量的样本测量（例如 10 次测量）之后，包括/重复系统资格部分中描述的分数步骤（见步骤 3.6.2）。此外，记录系统压力和紫外线探测器基线稳定性。它们应该保持稳定和恒定，沿着一个完整的AF4-UV-vis运行。 注:通常，当紫外线检测器（或多角度光散射 （MALS） 探测器（如果可用）显示噪声水平升高或未达到定义的系统资格标准（如恢复、样本峰值形状或可重复性）时，更换超滤膜（或 AF4-UV-vis 系统要经过彻底的清洁程序）。根据此处描述的条件，合格的 AF4-UV-vis 系统通常稳定，使用同一膜进行至少 50 次测量:但是，符合定义质量标准的可能连续测量的数量可能因样本、样本矩阵和精简成分而异。</li>
	</ol>
</li>
</ol>5. 数据评估
<ol><li>使用 AF4-UV-vis 系统制造商提供的数据评估软件执行大规模恢复计算，或在按照制造商手册中给出的说明从各自的数据采集软件导出所有必要的原始数据（即 UV-vis 峰值区域）后进行电子表格分析。<ol>
<li>使用以下方程比较分数测量（A分馏）和直接注射测量（直接注射）的相应紫外线峰值下的区域，计算 AuNP 质量恢复: <img alt="Equation 2" src="/files/ftp_upload/61757/61757equ02.jpg"> 注:在直接注射测量过程中，不应用分离力，因此可以忽略分析物种与堆积壁的潜在相互作用。使用 Beer-Lambert 定律，可使用 Beer-Lambert 定律将相应紫外线-顶峰下的区域与 AuNP 质量直接相关，假设样本中没有其他物种在各自的波长下吸收，并且/或 i） 在分馏条件下的另一个保留时间（ii）通过 AF4 膜去除。</li>
		<li>导入从直接注射和分数运行中获得的数据文件。</li>
		<li>在 "概述" 选项卡中选择紫外线检测器跟踪。</li>
		<li>定义所有测量的信号和基线视图中的感兴趣区域 （ROI） 和基线。</li>
		<li>通过插入插入插入直接注射校准。</li>
		<li>选择直接注塑 校准设置 视图中的所有直接注射运行，并输入紫外线灭绝系数。 注:在校准和分馏测量中使用相同的紫外线与灭绝系数非常重要。</li>
		<li>使用投资回报率内紫外线信号跟踪下的区域以及从输入浓度和注入体积计算的注入量来建立校准线。获得的校准将显示在单独的 直接注射校准功能 窗口中。</li>
		<li>将校准功能分配给相应的分数测量。 注:对于每个校准尺寸标准和未知的 AuNP 样本，由于 AuNP 的尺寸依赖紫外线吸收，需要建立单独的校准功能。紫外线探测器的这一缺点可以通过大规模敏感探测器（如 ICP-MS）来规避。</li>
		<li>通过插入 定量结果 计算来执行分析，结果将显示在右侧的表中，作为浓度和注入量值。</li>
	</ol></li>
	<li>使用 AF4 系统制造商提供的数据评估软件或电子表格分析，根据制造商手册中给出的说明，从各自的数据采集软件中输出所有必要的原始数据（即在各自的 UV-vis 峰值最大值和相应的空隙时间下，将 AuNP 校准标准的保留时间）导出后，计算保留时间的变化。<ol>
<li>打开 概述 窗口，显示所有导入测量的各自紫外线痕迹。</li>
		<li>峰值检测将自动执行:调整信号处理工具箱内的峰值检测参数，以优化性能。通过浏览所有测量文件，提取相应的峰值最大值。</li>
		<li>使用以下方程计算所有测量的相对标准偏差: <img alt="Equation 1" src="/files/ftp_upload/61757/61757equ01.jpg"> 也可以使用相应的电子表格软件进行计算。</li>
	</ol></li>
	<li>使用制造商提供的数据评估软件或电子表格分析，在按照制造商手册中给出的说明，从各自的数据采集软件中导出所有必要的原始数据（在 UV-vis 峰值分析的保留时间和相应的空隙时间）后执行大小确定。外部大小校准函数可以通过绘制 AuNP 大小标准（20 nm、40nm、80 nm、100 nm）的空隙时间校正保留时间（净保留时间，见表 5）来建立，其水动力学大小来自以前执行的 DLS 测量（见表 4）。 注:DLS 测量应与各自的分数测量结果在同一天进行，以确保可比的样本属性。<ol>
<li>导入.dat文件后，所有测量结果都显示在 "概述 "选项卡中。从覆盖窗口下方显示的探测器列表中选择 UV-vis 探测器信号。定义每个测量的 ROI 和基线，可在 信号和基线 视图中进行调整。使用右侧的 信号处理 工具箱来平滑噪声信号。将 分配处理参数用于其他运行 功能，以便将参数分配给其他测量信号。</li>
		<li>从"插入"选项卡中选择粒子大小校准。</li>
		<li>通过单击右上角校准表 的"选择参考" 中各自的测量结果，选择所有校准运行。所有选定的测量结果将显示在下表中。输入水动力学半径，以进行 表 4中指定的所有校准测量。该函数将显示在粒子大小校准-函数窗口和方程也将显示。 注:已确定大小校准函数的相关系数 （R2）必须≥0.990。</li>
		<li>通过选择 "分配选择运行" 列表中的各自分数，将校准功能分配到未知 AuNP 样本的测量中。</li>
		<li>通过在插入选项卡中打开粒子大小分布计算来显示结果。以前创建的粒子大小校准将被列为未知的 AuNP 样本测量的校准，该校准显示在正确的窗口设置中。计算大小将显示在标记为峰值最大值的大小分布窗口中。选择样本复选框的平均信号，以平均一个样本的所有测量结果，并在峰值最大标签中列出结果。</li>
		<li>此外，通过选择显示校准曲线复选框，在分形图上绘制 校准 线。通过选择 "显示"累计分发 复选框，可进行累积大小分布。 注:在使用制造商的软件进行数据评估时，建议将所有结果添加到报告中，该结果可以通过单击"插入"选项卡内的报告生成。报告按钮将所有结果、表格和图表添加到文档中。在"报告"选项卡下，可以通过在文档部分内打开报告设置来更改报告设置。</li>
	</ol></li>
</ol>

这份手稿的所有作者都是波斯特诺瓦分析有限公司的员工，其产品在本协议中使用。

AF4 与紫外线-维斯探测器一起，使用定义明确的 AuNP 尺寸标准，准确评估了未知 AuNP 的水动力学尺寸，范围从 20 nm 到 100 nm 不等。开发的 AF4 方法使用恒定的横流轮廓进行优化，以便在测量的保留时间和 AuNP 大小之间建立线性关系，从而允许从线性回归分析中直接确定大小。特别注重实现足够高的恢复率，表明在分馏过程中没有显著的样本损失，并且开发的AF4方法，包括应用的精英和膜与所有分馏的AuNP样品非常匹配。
方法开发可以说是 AF4 中最关键的一步，必须考虑几个参数，包括通道尺寸、流量参数以及流体、膜、间隔高度，甚至样品属性，以便在给定的分流时间窗口内提高分数。本段的目的是引导读者完成优化的关键步骤，以成功确定此处讨论的未知 AuNP 样本的大小。为了更详细地描述如何一般开发AF4方法，读者将参考AF4部分的"ISO/TS21362:2018 - 纳米技术 - 纳米物体分析使用不对称流和离心场流分数"25。仔细观察表 3中的应用分数条件，第一个关键步骤是在 AF4 通道中引入和放松 AuNP 样本。此步骤受注水流、对焦流和交叉流的约束，其相互作用迫使样本定位在靠近膜表面的地方，并将其浓缩在靠近AF4通道喷射端口的窄带中，基本上定义了分馏的起点。必须充分放松样品，因为在此步骤中，不同大小的样本成分位于AF4通道的不同高度，从而为成功的尺寸分馏提供了基础。不完整的样品松弛通常由未实现的（即非放松）样本成分引起的空隙峰值区域增加可见。这种影响可以通过增加注射时间和/或应用的交叉流速来缓解。然而，这两个参数都需要仔细优化，特别是对于容易聚集和吸附到AF4膜上的样品，并且可以通过为不同的参数设置36，37获得的各自的恢复率进行监测。应用注射时间为 5 分钟，交叉流速为 1.0 mL ∙min-1，可显示所有 AuNP 样本的恢复率 +80%，以及表示接近最佳放松条件的可忽略不计的空隙峰值区域。在充分放松 AuNP 样本后，焦点流停止，沿 AF4 通道长度向相应的紫外线检测器传输样品，这是第二个关键步骤。为了在合理的分析时间确保足够高的分馏功率，应用了 1 . 0 mL ∙最小- 1代表 30 - 50 分钟（取决于相应的分馏 AuNP 大小标准），然后以 0 . 5 mL . min - 1 的探测器流速进行了10分钟的线叉流量衰变。使用所有 AuNP 大小标准分离的恒定交叉流剖面，揭示了 FFF-理论22之后保留时间与 AuNP 大小之间的线性关系，从而通过简单的线性回归分析实现未知 AuNP 样本的大小确定。然而，除了恒定的交叉流外，轮廓也被利用来大小纳米粒子，最终导致保留时间和粒子大小38，39之间的非线性关系。此外，使用定义明确的尺寸标准在AF4中确定尺寸不限于AuNP，还可以应用于具有其他尺寸和元素成分的纳米粒子（如银38、40或硅纳米粒子41、42）。此外，在处理稀释样品时，ICP-MS 是一种高度敏感的元素探测器，可与 AF4 配合，从而增加了这种分析方法的多功能性，用于在悬架中对多种纳米粒子进行尺寸调整。
尽管其应用广泛，但在 AF4 中使用定义明确的尺寸标准进行外部尺寸校准具有一些特殊性，在使用它精确大小未知样本时需要考虑这些特性。首先，在分馏各自的尺寸标准和实际样本时，它在很大程度上依赖于可比条件的应用。因此，在此处介绍的案例中，必须使用相同的 AF4 方法以及相同的流体和相同的膜对 AuNP 大小标准以及未知的 AuNP 样本进行分馏，使这种方法变得相当不灵活。此外，由于手头没有大小敏感探测器，例如光散射（MALS 和 DLS），因此很难确定使用尺寸标准的相应 AF4 方法是否工作得足够好。对于显示非常宽幅分布的未知样本尤其如此，目前尚不清楚所有样本成分是否遵循正常的分化模式:从较小的颗粒到较大的颗粒的分馏，或者较大的样本成分是否已经在超层模式下分离，从而有可能与较小的样本成分43、44共同分离。此外， 尽管FF理论强调AF4完全基于水动力学大小的差异而分离，粒子被认为是点质量，而与它们的环境没有任何相互作用，但现实却用粒子粒子和粒子膜讲述了一个不同的故事 相互作用（如静电吸引/排斥或范德瓦尔斯吸引）可能发挥相当大的作用，并可能引入一个可衡量的偏差到大小确定通过外部大小校准45，46。因此，建议使用与感兴趣的粒子的组成和表面特性（Zeta潜力）理想匹配的尺寸标准 40、42 或， 如果没有这些，至少使用具有良好特征的粒子大小标准（如聚苯乙烯乳胶颗粒），并仔细评估其可比性与感兴趣的粒子，特别是在其表面Zeta潜力在各自的环境中，其中分析应进行41，47。
AF4的多功能性通常被认为是它最大的优势，因为它提供了一个应用范围，超越了这个领域的大多数其他常见的大小技术22，48，49。同时，由于其相关的复杂性，它也可能被视为其最大的缺点，特别是对快速和表面上易于使用的大小技术，如DLS，纳米粒子跟踪分析，或单粒子ICP-MS。尽管如此，当将AF4与这些流行的尺寸技术结合在一起时，很明显，所有技术都有其优点和缺点，但所有这些技术都有助于更全面地了解纳米粒子的物理化学性质，因此应被视为互补的，而不是具有竞争力的。
此处介绍的标准操作程序 （SOP）， 突出了AF4-UV-vis的出色适用性，外部尺寸校准用于悬架中未知的AuNP样本的大小，并最终被应用为国际实验室间比较（ILC）中对未知的AuNP样本进行AF4分析的推荐指南，该比较是在Horizan 2020项目ACEnano（本ILC的结果将成为未来出版物的主题）框架内进行的。因此，该议定书加在一起，鼓励和正在进行的国际努力，以验证和标准化AF4方法25，50，51，52强调AF4在纳米粒子定性领域的有希望的潜力。

以胶体金为形式的金纳米粒子（AuNP）早在人们了解纳米粒子是什么之前，在纳米粒子进入当代科学词汇之前，就已经是人类文化的一部分。由于对其纳米尺度外观有明显了解，悬挂的AuNP在公元前1世纪的V-VI世纪已经用于中国古代、阿拉伯和印度的医疗和其他目的，古罗马人还利用其红宝石红色在大英博物馆2号的莱库格斯杯展览中著名的陶器污渍。在西方世界，从中世纪到现代的几个世纪里，悬浮的AuNP主要用作玻璃和釉质（卡西乌斯的紫色）3的着色剂，以及治疗各种疾病（可饮用的黄金），特别是梅毒4。
然而，所有这些研究主要侧重于暂停的AuNP的应用，这是由迈克尔法拉第在1857年引入第一个理性的方法，以调查其形成，其性质，以及他们的属性5。虽然法拉第已经意识到，这些AuNP必须有非常微小的尺寸，直到电子显微镜的发展时，关于其大小分布的明确信息是可访问6，7，最终使大小和其他AuNP属性之间的相关性。
如今，由于其相当简单明了的合成，显着的光学特性（表面质子共振），良好的化学稳定性，因此毒性较小，以及其在可用尺寸和表面修饰方面的高多功能性，AuNP已经发现广泛应用领域，如纳米电子8，诊断9，癌症治疗10，或药物输送11。显然，对于这些应用，准确了解应用的 AuNP 的大小和大小分布是确保最佳功效12的基本先决条件，并且需要大量可靠和可靠的工具来确定这一关键的物理化学参数。今天，有大量的分析技术，能够大小AuNP悬架，包括， 例如，紫外线光谱（UV-vis）13、动态光散射（DLS）14或单粒子感应耦合等离子体质谱仪（spICP-MS）15，场流分馏（FFF）是该领域16、17、18、19、20的关键播放器。
FFF于1966年由J.卡尔文·吉丁斯21号首次概念化，它包括一系列基于精英的分馏技术，分离发生在一个薄薄的丝带状通道内，没有固定的第22、23阶段。在FF中，分离是由样品与外力场的相互作用引起的，外力场的作用垂直于层压通道流的方向，其中样品通常向下游输送到各自的内联探测器。在这些相关的FF技术中，不对称流场流分数（AF4），其中第二个流（交叉流）作为力场，已成为最广泛使用的亚型24。在 AF4 中，通道底部（堆积墙）配备了一个半透水超滤膜，能够保留样品，同时允许交叉流通过膜并通过额外的插座离开通道。通过这种方式，交叉流可以将样品推向堆积壁，从而抵消其扩散引起的通量（布朗运动）。在由此产生的场和扩散引起的通量平衡中:表现出较高扩散系数的较小样本成分更接近通道中心，而具有较低扩散系数的较大样本成分则位于靠近堆积壁的地方。由于通道内的抛物线流剖面，因此较小的样本成分在通道流的较快层压中运输，并在较大的样本成分之前进行椭然运输。使用FF保留参数和斯托克斯-爱因斯坦扩散系数方程，AF4样品的流出时间和分别流出体积可以直接转换为其水动力学大小22。这里描述的精英行为是指正常的精英模式，通常适用于AF4在粒子大小范围内约1-500纳米（有时高达2000纳米取决于粒子属性和分数参数），而石阶超层精英通常发生在这个大小阈值25以上。
通过 FFF 分离后，有三种常见方法可以获取大小信息。由于FF是一种模块化仪器，它可以结合下游多个探测器，如大小敏感光散射探测器基于多角度光散射（MALS）26，27，动态光散射（DLS）28，29，甚至两者的组合，以获得额外的形状信息30，31。但是，由于 FFF 通道中样品的保留行为通常受定义明确的物理力控制，因此大小也可以使用数学方法（FFF 理论）进行计算，其中简单的浓度探测器（例如 UV-vis 探测器）足以表示存在32、33个引出样本。
作为第三个选项，我们在这里报告应用外部大小校准34，35使用定义明确的 AuNP 标准在大小范围为 20-100 nm， 以大小未知的金纳米粒子样品悬架使用 AF4 加上紫外线- 维斯检测.这种简单的实验设置是故意选择的，目的是让尽可能多的实验室加入国际实验室间比较（ILC），该比较后来根据此处提出的议定书在欧洲联盟地平线2020项目ACEnano的框架下进行。

<table>
	<tbody>
		<tr>
			<td>0.1 &#181;m Membrane Filters (hydrophilic PVDF)</td>
			<td>Postnova Analytics GmbH</td>
			<td>Z-FIL-TEF-002</td>
			<td>Used for filtration of aqueous solutions</td>
		</tr>
		<tr>
			<td>0.22 &#181;m PVDF Syringe Filter (d = 33 mm)</td>
			<td>Merck Millipore</td>
			<td>Durapore Millex</td>
			<td>Used for filtration of NovaChem100</td>
		</tr>
		<tr>
			<td>Adjustable Volume Pipettes (1000 &#181;L)</td>
			<td>Eppendorf AG</td>
			<td>Research Plus</td>
			<td>Used to prepare diluted AuNP suspensions</td>
		</tr>
		<tr>
			<td>AF4 cartridge</td>
			<td>Postnova Analytics GmbH</td>
			<td>AF2000 MF - AF4 Analytical Channel</td>
			<td>Component of the AF2000 MF - MultiFlow FFF setup, which is described as AF4-system in the manuscript</td>
		</tr>
		<tr>
			<td>AF4 Membrane - Regenerated Cellulose (10 kDa MWCO)</td>
			<td>Postnova Analytics GmbH</td>
			<td>Z-AF4-MEM-612-10KD</td>
			<td>Component of the AF2000 MF - MultiFlow FFF setup, which is described as AF4-system in the manuscript</td>
		</tr>
		<tr>
			<td>Analytical Balance (0.1 mg precision)</td>
			<td>Sartorius</td>
			<td>ENTRIS124I-1S</td>
			<td>Used to weigh SDS and NaOH pellets for preparation of cleaning solution</td>
		</tr>
		<tr>
			<td>Autosampler</td>
			<td>Postnova Analytics GmbH</td>
			<td>PN5300</td>
			<td>Component of the AF2000 MF - MultiFlow FFF setup, which is described as AF4-system in the manuscript</td>
		</tr>
		<tr>
			<td>Channel Oven</td>
			<td>Postnova Analytics GmbH</td>
			<td>PN4020</td>
			<td>Component of the AF2000 MF - MultiFlow FFF setup, which is described as AF4-system in the manuscript</td>
		</tr>
		<tr>
			<td>Crossflow Module</td>
			<td>Postnova Analytics GmbH</td>
			<td>AF2000 MF Control Module</td>
			<td>Component of the AF2000 MF - MultiFlow FFF setup, which is described as AF4-system in the manuscript</td>
		</tr>
		<tr>
			<td>Disposable Pipette Tips (1000 &#181;L)</td>
			<td>Eppendorf AG</td>
			<td>ep T.I.P.S</td>
			<td>Used to prepare diluted AuNP suspensions</td>
		</tr>
		<tr>
			<td>Flasks (e.g. 2 liter volume)</td>
			<td>neoLab</td>
			<td>1-0199</td>
			<td>Used for eluent storage</td>
		</tr>
		<tr>
			<td>Focus Pump</td>
			<td>Postnova Analytics GmbH</td>
			<td>PN1131</td>
			<td>Component of the AF2000 MF - MultiFlow FFF setup, which is described as AF4-system in the manuscript</td>
		</tr>
		<tr>
			<td>Glass Vials (e.g. 1.5 mL volume)</td>
			<td>Postnova Analytics GmbH</td>
			<td>VIA-002</td>
			<td>Used for sample storage</td>
		</tr>
		<tr>
			<td>Gold Nanoparticle Size Standards (20 nm, 40 nm, 80 nm, 100 nm)</td>
			<td>Postnova Analytics GmbH</td>
			<td>NovaCal Gold</td>
			<td>50 mg L-1 each, used to establish the size calibration function</td>
		</tr>
		<tr>
			<td>Magnetic Stirrer</td>
			<td>IKA</td>
			<td>VIBRAX-VXR</td>
			<td>Used to accelerate dissolution of SDS and NaOH pellets in UPW</td>
		</tr>
		<tr>
			<td>Personal Computer (PC)</td>
			<td>Dell Technologies</td>
			<td>/</td>
			<td>Unit to control AF4 runs, record and evaluate collected data, for necessary hardware and software requirements the reader is referred to the Postnova AF2000 manual</td>
		</tr>
		<tr>
			<td>Personal protection gear (gloves, lab coat, glasses etc.)</td>
			<td>/</td>
			<td>/</td>
			<td>In accordance with respective laboratory&#8217;s safety rules for working with chemicals including engineered nanomaterials</td>
		</tr>
		<tr>
			<td>Screw Top for Glass Vials (e.g. 1.5 mL volume)</td>
			<td>Postnova Analytics GmbH</td>
			<td>Z-VIA-09150868</td>
			<td>Used for sample storage</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate (SDS), &#8805;99 %, Blotting Grade</td>
			<td>Carl Roth GmbH &#38; Co KG</td>
			<td>2326.1</td>
			<td>Used for the preparation of the cleaning solution</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide (NaOH) Pellets, &#8805;98 %, p.a</td>
			<td>Carl Roth GmbH &#38; Co KG</td>
			<td>6771.1</td>
			<td>Used for the preparation of the cleaning solution</td>
		</tr>
		<tr>
			<td>Software Package for Control and Data Acquisition</td>
			<td>Postnova Analytics GmbH</td>
			<td>NovaFFF AF2000 Software</td>
			<td>Software for performing Af4 runs and data aquisition, for necessary hardware and software requirements the reader is referred to the Postnova AF2000 manual</td>
		</tr>
		<tr>
			<td>Software Package for Data Evaluation</td>
			<td>Postnova Analytics GmbH</td>
			<td>NovaAnalysis Software</td>
			<td>Software for AF4 data evaluation, for necessary hardware and software requirements the reader is referred to the Postnova NovaAnalysis manual</td>
		</tr>
		<tr>
			<td>Software Package for final Data Processing</td>
			<td>OriginLab Corporation</td>
			<td>Origin 2019</td>
			<td>Used for final data processing</td>
		</tr>
		<tr>
			<td>Solvent Degasser</td>
			<td>Postnova Analytics GmbH</td>
			<td>PN7520</td>
			<td>Component of the AF2000 MF - MultiFlow FFF setup, which is described as AF4-system in the manuscript</td>
		</tr>
		<tr>
			<td>Solvent Selector</td>
			<td>Postnova Analytics GmbH</td>
			<td>PN7310</td>
			<td>Component of the AF2000 MF - MultiFlow FFF setup, which is described as AF4-system in the manuscript</td>
		</tr>
		<tr>
			<td>Solvent Organizer</td>
			<td>Postnova Analytics GmbH</td>
			<td>PN7140</td>
			<td>Component of the AF2000 MF - MultiFlow FFF setup, which is described as AF4-system in the manuscript</td>
		</tr>
		<tr>
			<td>Surfactant Mixture</td>
			<td>Postnova Analytics GmbH</td>
			<td>NovaChem100</td>
			<td>Mixture of different surfactants and salts used for eluent preparation</td>
		</tr>
		<tr>
			<td>Tip Pump</td>
			<td>Postnova Analytics GmbH</td>
			<td>PN1130</td>
			<td>Component of the AF2000 MF - MultiFlow FFF setup, which is described as AF4-system in the manuscript</td>
		</tr>
		<tr>
			<td>Unknown AuNP sample</td>
			<td>BBI Solutions</td>
			<td>EM.GC60</td>
			<td>60 nm AuNP sample used for size determination via size calibration function</td>
		</tr>
		<tr>
			<td>UV-vis Detector</td>
			<td>Postnova Analytics GmbH</td>
			<td>PN3211</td>
			<td>UV-vis detector For downstream coupling with the AF4 system</td>
		</tr>
		<tr>
			<td>Vacuum Filtration Unit</td>
			<td>Postnova Analytics GmbH</td>
			<td>Eluent Filtration System</td>
			<td>Used to ensure low particle backgrounds and removal of dissolved air in the used eluents to ensure optimum AF4 fractionation conditions</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>IKA</td>
			<td>Vortex Genie 2</td>
			<td>Used for homogenization of diluted AuNP suspensions</td>
		</tr>
		<tr>
			<td>Water Purification System</td>
			<td>Merck Millipore</td>
			<td>Milli-Q Integral 5</td>
			<td>Used to generate ultrapure water (UPW, 18.2 M&#937;cm resistivity) for preparation of cleaning solution, eluents and dilution of AuNP suspensions</td>
		</tr>
	</tbody>
</table>

asymmetrical flow field-flow fractionation for sizing of gold nanoparticles in suspension

粒子大小可以说是与纳米粒子概念相关的最重要的物理化学参数。准确了解纳米粒子的大小和大小分布对于各种应用至关重要。尺寸范围也很重要，因为它定义了纳米粒子剂量中最"活跃"的成分。
不对称流场流分馏 （AF4） 是一种强大的技术，用于在大约 1-1000 nm 的大小范围内对悬架中的粒子进行大小大小测定。有几种方法可以从 AF4 实验中获取大小信息。除了根据多角度光散射或动态光散射原理在线将 AF4 与大小敏感探测器耦合之外，还有可能使用成熟的理论方法（FFF 理论）或将其与定义明确的粒子大小标准（外部大小校准）的保留时间进行比较，将样品的大小与其保留时间相关联。
我们在这里描述开发和内部验证的标准操作程序 （SOP） 的大小未知的金纳米粒子样本由 AF4 加上紫外线 - 维斯检测使用外部大小校准与黄金纳米粒子标准在 20-100 nm 的大小范围内。该程序详细描述了已开发的工作流程，包括样品制备、AF4仪器设置和鉴定、AF4方法开发和未知金纳米粒子样品的分馏，以及所获得的结果与既定外部尺寸校准的相关性。此处描述的 SOP 最终在实验室间比较研究的框架中得到了成功验证，该研究强调了 AF4 在悬架中纳米粒子样品尺寸的卓越坚固性和可靠性。

首先，AuNP 尺寸标准由 AF4 分馏，并通过 UV-vis 检测到，该标准的吸收波长为 532 nm（AuNP 的表面质子共振）。获得的分形图的叠加在 图1中。 表5列出了每个 AuNP 在各自的紫外线与峰值最大值上的保留时间，这些保留时间来自三次测量。所有保留时间的相对标准偏差低于 1.1%，测量差异随着尺寸的增大而缩小。总体而言，实现了出色的可重复性。应用了恒定的分离力，从而产生了排出时间和水动力学大小的线性关系。外部大小校准线是通过绘制指定的水动力学半径与校正排空时间（净保留时间）的空隙来建立的。线性回归分析可产生线性校准功能，拦截为 -3.373 nm ± 1.716 nm，坡度 b = 1.209 nm ∙min-1 ± 0.055 nm ∙min-1。以0.9958的平方相关系数R2 确认了流出的线性行为。图 2中可直观地显示相应的校准功能。
第二部分涉及对未知的 AuNP 样本的分析。根据协议部分（第4.2节）中描述的程序编写了样品的三个名曲。使用同样适用于 AuNP 大小标准的 AF4 分馏方法，对三个名曲逐一进行了三次调查。从未知的 AuNP 样本中获得的 9 个 AF4-UV-UV-vis 分形图均在 图 3 中显示，并在表 6中汇总了各自的评估结果。各保留时间的相对标准偏差显著较低，在 0.1% 到 0.5% 之间。利用从 AuNP 大小标准的分馏中获得的粒子大小校准功能，并将其与 UV-vis 峰值最大值未知 AuNP 样本获得的保留时间相关联，可以计算出总体平均水动力学半径为 29.4 nm ± 0.2 nm。此外，获得83.1%±1.2%的合理质量回收表明，AuNP样品没有显著聚集或溶解，或颗粒大量吸附到膜表面。 图 4 显示获得的粒子大小分布，所有 9 个 UV-vis 信号痕迹均值突出应用 AF4 方法的优秀稳健性。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61757/61757fig01.jpg"> 图1: AF4-UV-维斯分形图从四个单独的 AuNP 大小校准标准的三重分析中获得，具有标准化的信号强度并应用了恒定的交叉流速（黑线）。空峰在 5.9 分钟左右以灰色突出显示， <a href="https://www.jove.com/files/ftp_upload/61757/61757fig01large.jpg" target="_blank">请单击此处查看此数字的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61757/61757fig02.jpg"> 图2: 获得外部大小校准功能，包括从 DLS 测量 （表 4）的相应标准偏差中得出的误差条，以及获得 AF4 保留时间 （表 5）中的差异，在根据每个 AuNP 大小校准标准的保留时间绘制相应的峰值最大值后绘制指定的水动力学半径。线性校准函数与标准误差的形式 y = a + bx 与 = 3.373 nm ± 1.716 nm 和 b = 1.209 nm\min-1 ± 0.055 nm\min-1 是根据线性回归分析计算的。确定了与 R2 = 0.9958 的平方相关系数，表示线性关系。 <a href="https://www.jove.com/files/ftp_upload/61757/61757fig02large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61757/61757fig03.jpg"> 图3: AF4-UV-维斯三重测量的分形图，显示未知的AuNP。测量时间内应用的恒定横流速率被说明为一条黑线。大约 5.9 分钟的空峰以灰色突出显示。 <a href="https://www.jove.com/files/ftp_upload/61757/61757fig03large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61757/61757fig04.jpg"> 图4: 未知的 AuNP 样品获得的平均颗粒大小分布（红色）和应用的线性校准功能（虚线）的叠加。 <a href="https://www.jove.com/files/ftp_upload/61757/61757fig04large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="2">组件</td>
			<td>中科院-否</td>
			<td>重量 （%）</td>
		</tr>
<tr>
<td colspan="2">水</td>
			<td>7732-18-5</td>
			<td>88.8</td>
		</tr>
<tr>
<td colspan="2">9-八氯乙烯酸（Z），化合物，2，2'-硝基醇[乙醇]（1:1）</td>
			<td>2717-15-9</td>
			<td>3.8</td>
		</tr>
<tr>
<td colspan="2">碳酸钠</td>
			<td>497-19-8</td>
			<td>2.7</td>
		</tr>
<tr>
<td colspan="2">酒精， C12-14 次， 乙醇</td>
			<td>84133-50-6</td>
			<td>1.8</td>
		</tr>
<tr>
<td colspan="2">四钠 EDTA</td>
			<td>64-02-8</td>
			<td>1.4</td>
		</tr>
<tr>
<td colspan="2">聚乙烯乙二醇</td>
			<td>25322-68-3</td>
			<td>0.9</td>
		</tr>
<tr>
<td colspan="2">油酸钠</td>
			<td>143-19-1</td>
			<td>0.5</td>
		</tr>
<tr>
<td colspan="2">碳酸氢钠</td>
			<td>144-55-8</td>
			<td>0.1</td>
		</tr>
</tbody></table>表1: 用于制备精灵的表面活性混合物的成分列表（另见 材料表）。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>AF4-紫外线-视参数</td>
			<td>单位</td>
			<td>价值</td>
		</tr>
<tr>
<td>间隔厚度</td>
			<td>微米</td>
			<td>350</td>
		</tr>
<tr>
<td>探测器流速</td>
			<td>mL最小-1
</td>
			<td>0.5</td>
		</tr>
<tr>
<td>交叉流速</td>
			<td>mL最小-1
</td>
			<td>0（恒定8分钟）</td>
		</tr>
<tr>
<td>焦点流速率</td>
			<td>mL最小-1
</td>
			<td>0</td>
		</tr>
<tr>
<td>延迟时间/稳定时间</td>
			<td>分钟</td>
			<td>0</td>
		</tr>
<tr>
<td>注塑流速率</td>
			<td>mL最小-1
</td>
			<td>0.5</td>
		</tr>
<tr>
<td>过渡时间</td>
			<td>分钟</td>
			<td>0</td>
		</tr>
<tr>
<td>注射时间</td>
			<td>分钟</td>
			<td>0.1</td>
		</tr>
<tr>
<td>埃卢蒂翁步骤</td>
			<td>分钟</td>
			<td>8</td>
		</tr>
<tr>
<td>冲洗步骤时间</td>
			<td>分钟</td>
			<td>0.1</td>
		</tr>
<tr>
<td>冲洗步速率</td>
			<td>mL最小-1
</td>
			<td>0.1</td>
		</tr>
<tr>
<td>注射量</td>
			<td>μl</td>
			<td>10</td>
		</tr>
<tr>
<td>样本浓度</td>
			<td>毫克L-1
</td>
			<td>12.5</td>
		</tr>
<tr>
<td>膜类型</td>
			<td></td>
			<td>再生纤维素</td>
		</tr>
<tr>
<td>膜分子量截止</td>
			<td>Kda</td>
			<td>10</td>
		</tr>
<tr>
<td>埃伦特</td>
			<td></td>
			<td>0.025% （v/v） 表面活性混合物</td>
		</tr>
<tr>
<td>紫外线波长</td>
			<td>纳米</td>
			<td>532</td>
		</tr>
<tr>
<td>紫外线灵敏度</td>
			<td>-</td>
			<td>0.001</td>
		</tr>
</tbody></table>表2: 在不应用分离力的情况下执行直接喷射运行的 AF4-UV-vis 分馏方法参数的摘要。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>AF4-紫外线-视参数</td>
			<td>单位</td>
			<td>价值</td>
		</tr>
<tr>
<td>间隔厚度</td>
			<td>微米</td>
			<td>350</td>
		</tr>
<tr>
<td>探测器流速</td>
			<td>mL最小-1
</td>
			<td>0.5</td>
		</tr>
<tr>
<td>交叉流速</td>
			<td>mL最小-1
</td>
			<td>1 （60 分钟常数， 10 分钟线性）</td>
		</tr>
<tr>
<td>焦点流速率</td>
			<td>mL最小-1
</td>
			<td>1.3</td>
		</tr>
<tr>
<td>延迟时间/稳定时间</td>
			<td>分钟</td>
			<td>2</td>
		</tr>
<tr>
<td>注塑流速率</td>
			<td>mL最小-1
</td>
			<td>0.2</td>
		</tr>
<tr>
<td>过渡时间</td>
			<td>分钟</td>
			<td>0.2</td>
		</tr>
<tr>
<td>注射时间</td>
			<td>分钟</td>
			<td>5</td>
		</tr>
<tr>
<td>埃卢蒂翁步骤</td>
			<td>分钟</td>
			<td>70 （60 分钟常数， 10 分钟线性）</td>
		</tr>
<tr>
<td>冲洗步骤</td>
			<td>分钟</td>
			<td>9</td>
		</tr>
<tr>
<td>冲洗步速率</td>
			<td>mL最小-1
</td>
			<td>0.5</td>
		</tr>
<tr>
<td>注射量</td>
			<td>μl</td>
			<td>50</td>
		</tr>
<tr>
<td>样本浓度</td>
			<td>毫克L-1
</td>
			<td>12.5</td>
		</tr>
<tr>
<td>膜类型</td>
			<td></td>
			<td>再生纤维素</td>
		</tr>
<tr>
<td>膜分子量截止</td>
			<td>Kda</td>
			<td>10</td>
		</tr>
<tr>
<td>埃伦特</td>
			<td></td>
			<td>0.025% （v/v） 表面活性混合物</td>
		</tr>
<tr>
<td>紫外线波长</td>
			<td>纳米</td>
			<td>532</td>
		</tr>
<tr>
<td>紫外线灵敏度</td>
			<td>-</td>
			<td>0.001</td>
		</tr>
</tbody></table>表3: AF4-UV-vis 分馏法参数的摘要，以应用交叉流作为分离力执行分馏运行。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>校准标准</td>
			<td>封顶代理</td>
			<td>平均尺寸 （TEM） （nm）</td>
			<td>简历（平均尺寸） （%）</td>
			<td>泽塔潜力 （mV）</td>
			<td>SD （泽塔潜力） （mV）</td>
			<td>水动力学半径 （DLS） （nm）</td>
			<td>SD（水动力学半径） （nm）</td>
			<td>Pdi</td>
			<td>SD （PDI）</td>
		</tr>
<tr>
<td>奥恩普 20 纳米</td>
			<td>柠檬酸</td>
			<td>20.1</td>
			<td>≤ 8</td>
			<td>-48.9</td>
			<td>1.5</td>
			<td>10.95</td>
			<td>0.12</td>
			<td>0.082</td>
			<td>0.009</td>
		</tr>
<tr>
<td>奥恩普 40 纳米</td>
			<td>柠檬酸</td>
			<td>40.8</td>
			<td>≤ 8</td>
			<td>-30.4</td>
			<td>1.0</td>
			<td>20.30</td>
			<td>0.13</td>
			<td>0.127</td>
			<td>0.006</td>
		</tr>
<tr>
<td>奥恩普 80 纳米</td>
			<td>柠檬酸</td>
			<td>79.2</td>
			<td>≤ 8</td>
			<td>-51.5</td>
			<td>1.3</td>
			<td>38.85</td>
			<td>0.23</td>
			<td>0.138</td>
			<td>0.013</td>
		</tr>
<tr>
<td>奥恩普 100 纳米</td>
			<td>柠檬酸</td>
			<td>102.2</td>
			<td>≤ 8</td>
			<td>-50.9</td>
			<td>0.9</td>
			<td>52.30</td>
			<td>0.37</td>
			<td>0.078</td>
			<td>0.009</td>
		</tr>
</tbody></table>表4: 应用的 AuNP 校准标准的物理化学参数摘要，包括封顶剂、TEM 平均尺寸、在原生悬架中确定的 Zeta 潜力以及 DLS 水动力学半径，以及在 eluent 中确定的多分散度指数 （PDI）。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>校准标准</td>
			<td>运行</td>
			<td>最高保留时间（分钟）</td>
			<td>最高值（分钟）的净保留时间</td>
			<td>平均净留存时间（分钟）</td>
			<td>SD （%）（净保留时间）</td>
			<td>SD（分钟） （净保留时间）</td>
		</tr>
<tr>
<td rowspan="3">奥恩普 20 纳米</td>
			<td>1</td>
			<td>17.368</td>
			<td>11.468</td>
			<td rowspan="3">11.56</td>
			<td rowspan="3">1.02</td>
			<td rowspan="3">0.12</td>
		</tr>
<tr>
<td>2</td>
			<td>17.409</td>
			<td>11.509</td>
		</tr>
<tr>
<td>3</td>
			<td>17.589</td>
			<td>11.689</td>
		</tr>
<tr>
<td rowspan="3">奥恩普 40 纳米</td>
			<td>1</td>
			<td>25.316</td>
			<td>19.416</td>
			<td rowspan="3">19.49</td>
			<td rowspan="3">0.68</td>
			<td rowspan="3">0.13</td>
		</tr>
<tr>
<td>2</td>
			<td>25.32</td>
			<td>19.42</td>
		</tr>
<tr>
<td>3</td>
			<td>25.548</td>
			<td>19.648</td>
		</tr>
<tr>
<td rowspan="3">奥恩普 80 纳米</td>
			<td>1</td>
			<td>42.095</td>
			<td>36.195</td>
			<td rowspan="3">36.29</td>
			<td rowspan="3">0.23</td>
			<td rowspan="3">0.08</td>
		</tr>
<tr>
<td>2</td>
			<td>42.219</td>
			<td>36.319</td>
		</tr>
<tr>
<td>3</td>
			<td>42.257</td>
			<td>36.357</td>
		</tr>
<tr>
<td rowspan="3">奥恩普 100 纳米</td>
			<td>1</td>
			<td>50.975</td>
			<td>45.075</td>
			<td rowspan="3">45.06</td>
			<td rowspan="3">0.07</td>
			<td rowspan="3">0.03</td>
		</tr>
<tr>
<td>2</td>
			<td>50.924</td>
			<td>45.024</td>
		</tr>
<tr>
<td>3</td>
			<td>50.986</td>
			<td>45.086</td>
		</tr>
</tbody></table>表5: AuNP校准标准的保留时间在各自的UV-Vis峰值最大，从各自的AF4-UV-维斯分形图中使用 表3中描述的方法。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>阿利奎特</td>
			<td>运行</td>
			<td>保留时间峰值最大值（分钟）</td>
			<td>最高值的平均保留时间（分钟）</td>
			<td>最高值（分钟）的净保留时间</td>
			<td>SD （%）保留时间</td>
			<td>水动力学半径（nm）</td>
			<td>恢复 （%）</td>
		</tr>
<tr>
<td rowspan="3">1</td>
			<td>1</td>
			<td>32.689</td>
			<td rowspan="3">32.70</td>
			<td>26.789</td>
			<td rowspan="3">0.07</td>
			<td rowspan="3">29.03</td>
			<td rowspan="3">85.34</td>
		</tr>
<tr>
<td>2</td>
			<td>32.687</td>
			<td>26.787</td>
		</tr>
<tr>
<td>3</td>
			<td>32.719</td>
			<td>26.819</td>
		</tr>
<tr>
<td rowspan="3">2</td>
			<td>1</td>
			<td>32.989</td>
			<td rowspan="3">33.08</td>
			<td>27.089</td>
			<td rowspan="3">0.37</td>
			<td rowspan="3">29.49</td>
			<td rowspan="3">81.73</td>
		</tr>
<tr>
<td>2</td>
			<td>33.073</td>
			<td>27.173</td>
		</tr>
<tr>
<td>3</td>
			<td>33.187</td>
			<td>27.287</td>
		</tr>
<tr>
<td rowspan="3">3</td>
			<td>1</td>
			<td>33.053</td>
			<td rowspan="3">33.14</td>
			<td>27.153</td>
			<td rowspan="3">0.49</td>
			<td rowspan="3">29.56</td>
			<td rowspan="3">82.14</td>
		</tr>
<tr>
<td>2</td>
			<td>33.071</td>
			<td>27.171</td>
		</tr>
<tr>
<td>3</td>
			<td>33.291</td>
			<td>27.391</td>
		</tr>
</tbody></table>表6: 从外部大小校准（图 2）计算出的 UV-Vis 峰值最大值的保留时间摘要，以及从 AF4-UV-vis 分析中获得的未知 AuNP 样本的恢复率。

Watch this Scientific Journal Video about Asymmetrical Flow Field-Flow Fractionation for Sizing of Gold Nanoparticles in Suspension at JoVE.com

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

此协议描述了使用不对称流场流分数加上紫外线检测来确定未知金纳米粒子样本的大小。

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 ...

asymmetrical-flow-field-flow-fractionation-for-sizing-gold

Research

JoVE Journal

Bioengineering

6.1K 次观看.  Postnova Analytics GmbH. 此协议描述了使用不对称流场流分数加上紫外线检测来确定未知金纳米粒子样本的大小。

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

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

在生物系统中的离子浓度测量荧光纳米粒子

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

科研

生物工程

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

由多光谱成像流式细胞仪分析细胞内化纳米粒子和细菌

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

病毒粒子为<em在体内</em&gt;肿瘤显像

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

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 ...

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

细胞物质相互作用研究微细的纳米多孔金模式

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

纳米谐波再生研究

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

全球舒张功能通过静脉流的通过参数化舒张形式主义运动学建模为基础的定量分析

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

固体脂质纳米粒（前哨淋巴结）细胞内定位的应用

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

液体中脉冲激光烧蚀制备金属纳米粒子:研究纳米粒子抗菌性能的工具

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a cell-penetrating peptide, TAT, for intracellular delivery. However, TAT has shown non-specific biological activities. Gold nanoparticles (GNPs) can be used as drug delivery carriers without recognized toxicity. Therefore, we have used a GNP/peptide hybrid to deliver PKC&#948;i. Two short peptides (P2: CAAAAE and P4: CAAAAW), at a 95:5 ratio, were used to modify the surface properties of GNP. GNPs conjugated with PKC&#948;i (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and phosphate-buffered saline (PBS) containing bovine serum albumin or fetal bovine serum. Intravenous injection of GNP-PKCi was previously shown to prevent ischemia-reperfusion injury of the lung. This article outlines a protocol to formulate GNP/PKCi and assess the physiochemical properties of GNP/PKCi. We have used similar methods to formulate other peptide-based drugs with GNP. This article will hopefully draw more attention to this novel intracellular drug delivery technology and its applications in vivo.

We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly.

金纳米粒子的蛋白质激酶 c-三角洲抑制剂肽配方

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a ...