该项目已根据第720952号赠款协议（ACEnano）获得了欧盟地平线2020计划（H2020）的资助。作者要感谢Sigrid Benemann的SEM测量，Markus Schneider的ToF-SIMS测量和PCA，以及Philipp Reichardt的拍摄协助。

注意：纳米颗粒的毒理学性质仍在研究中;由于它们的大小，即使它们由本质上无害的材料组成，它们也会在人类和环境中产生独特的危害。在对纳米颗粒进行任何工作之前，应完成适当的风险评估，并根据要研究的材料的危险水平，适当的工程控制，实验室程序和PPE（个人防护设备）到位29，30，31，32。
1. 硅晶圆的制备
注意：这些步骤对于去除不需要的（有机）污染物并增加表面润湿性是必要的。所有使用的溶剂应至少为ACS级。标准超声处理浴（35 kH 和 120 瓦）是合适的。
<ol><li>硅晶圆的湿化学清洗<ol>
<li>将Si晶片放入装有异丙醇的烧杯中，并超声处理5分钟。</li>
		<li>将Si晶圆转移到带有碱玻璃清洁溶液的烧杯中，并超声处理10分钟。</li>
		<li>将威化片放入装有超纯水的烧杯中。通过倒出水并重新填充烧杯来更换水10次;由于毛细管效应，Si晶圆将保持在底部。</li>
		<li>用干净的N2 气体干燥晶圆。 注意：用N2 干燥可防止水干燥形成"咖啡环"和其他伪影。</li>
		<li>将晶圆放入第二个烧杯中，用异丙醇和超声波灭菌10分钟。</li>
		<li>用干净的N2 气体干燥晶圆。</li>
		<li>将晶圆放入装有乙醇的烧杯中，并超声处理10分钟。</li>
		<li>用干净的N2 气体干燥晶圆。该协议可以在此处暂停。</li>
	</ol>
</li>
	<li>硅晶圆的等离子或紫外线/臭氧清洗<ol>
<li>将硅晶圆引入等离子体或紫外线/臭氧清洁剂中，然后打开30分钟。 注：晶圆在使用前应立即进行等离子或紫外线/臭氧清洗。</li>
	</ol>
</li>
</ol>2. 悬浮液沉积纳米颗粒
注意：纳米颗粒最常见的暴露途径是吸入。使用悬架可以最大限度地减少暴露危险。
<ol><li>粉末纳米颗粒悬浮液的制备 注：此处描述的所有数量均为示例。该方法应针对每种情况下使用的特定纳米颗粒进行优化。<ol>
<li>将15mg纳米颗粒粉末（±10%）准确地称量到10mL管中。</li>
		<li>在约 8 mL 超纯水中准确称量。</li>
		<li>关闭管，用纸巾包装在50 mL离心管中，以3，000rpm的速度放入涡旋器中15分钟。</li>
	</ol>
</li>
	<li>从水悬浮液中滴铸导电纳米颗粒<ol>
<li>将晶圆放入紫外线/臭氧清洁剂中30分钟。</li>
		<li>将晶圆置于晶圆支架的一半中，并在环的中心放置3μL纳米颗粒悬浮液。</li>
		<li>将直径为6.07 mm的Viton O形圈安装在液滴周围的晶圆上。注意戒指不要接触液滴。</li>
		<li>将晶圆置于真空干燥器中，在4 mbar的真空下干燥15分钟以干燥晶圆。</li>
		<li>从干燥器中取出晶片，并使用光学显微镜和XPS进行检查，以确定颗粒层是均匀和封闭的。重复步骤 2.2.1 和 2.2.2，直到分析显示一个封闭且均匀的层。该协议可以在此处暂停。</li>
	</ol>
</li>
	<li>水悬浮液中电非导电纳米颗粒的旋涂<ol>
<li>将晶圆放入紫外线/臭氧清洁剂中30分钟。 注意：通过使用相同的方案对不同浓度的悬浮液进行旋涂，可以实现不同水平的表面覆盖。</li>
		<li>对旋转涂布机进行编程。一个合适的示例程序是：步骤1：500 rpm / s斜坡到1，000 rpm（5 s）;第 2 步：1，000 转/秒斜坡至 2，000 转/分（3 分钟）;第 3 步：以 2，000 rpm/s 至 0 rpm 的速度减速。</li>
		<li>将晶圆插入旋涂机，打开真空进行固定。</li>
		<li>将80μL悬浮液沉积在晶圆上并开始程序。</li>
		<li>从旋涂机中取出晶圆。</li>
		<li>将样品储存在新的、干净的晶圆托盘中。该协议可以在此处暂停。</li>
		<li>使用SEM分析样品，以确认基板的无间隙覆盖。</li>
	</ol>
</li>
</ol>3. 从粉末中沉积纳米颗粒
<ol><li>纳米颗粒沉积在双面胶带上（"粘住就走"）<ol>
<li>将双面胶粘剂固定在样品架上，然后取下衬垫。</li>
		<li>取纳米颗粒粉末的刮刀尖端，并将其浸入粘合剂上。</li>
		<li>将样品铺在粘合剂上，并用刮刀压入粘合剂中，直到尽可能多的粉末粘附。</li>
		<li>通过倒置和敲击样品架，并通过吹过样品架的气体流（例如氮气）来检查粉末是否固定在水龙头上。该协议可以在此处暂停。 注意：或者，可以将少量粉末放在清洁的表面上（铝箔或载玻片），然后用粘合剂和双面样品架从上方压制。</li>
		<li>将粉末的刮刀尖端放在清洁的表面上。将带有粘合剂的样品架从上方压到粉末上。</li>
		<li>通过倒置和敲击样品架，并通过吹过样品架的气体流（例如氮气）来检查粉末是否固定在水龙头上。该协议可以在此处暂停。</li>
	</ol>
</li>
	<li>
压制粉末颗粒的制备
	<ol>
<li>彻底清洁颗粒模具的所有部分，注意不要划伤抛光表面。</li>
		<li>倒置沉淀模具并放在一个小垫片上。</li>
		<li>插入柱塞和一个不锈钢颗粒，抛光面朝上，并将柱塞拉过，直到有足够的空间填充粉末样品。</li>
		<li>用少量样品（1个大铲头）填充模具，然后插入第二个不锈钢沉淀，抛光面朝向样品。</li>
		<li>将底座放在身体上，小心地倒置。如果需要真空并且可用，请将真空泵连接到颗粒模具的底部。</li>
		<li>将模具放入压力机中，确保其居中。</li>
		<li>施加轻负荷（2 kN）约20秒并释放。</li>
		<li>施加较重的负载（6 kN）2分钟并释放。</li>
		<li>释放负载后，释放真空泵。 注意：由于各种纳米颗粒的材料性质不同，制备一系列具有不同载荷和载荷时间的颗粒以确定最佳颗粒压制条件可能是有利的。</li>
		<li>倒置模具，将提取环放置到位，并在柱塞和提取环之间放置轻负载（最大1 kN）。</li>
		<li>从压机上取出模具部件，并用镊子小心地提取样品沉淀。</li>
		<li>使用双面粘合剂将样品轻轻地安装在清洁的Si晶圆上。该协议可以在此处暂停。</li>
	</ol>
</li>
</ol>4. 纳米颗粒悬浮液的冷冻固定
<ol><li>用液氮填充快速冷冻装置的主腔。</li>
	<li>用冷冻剂（丙烷）填充冷却的快冻室。</li>
	<li>让快速冷冻设备冷却至其工作温度。 注：在样品制备之前，快速冷冻装置需要一些时间才能达到工作温度，因此冷冻固定样品需要合理的时间范围（几个小时）。</li>
	<li>用移液器将10-20μLNP悬浮液滴铸到清洁的Si晶圆上。</li>
	<li>用固定镊子握住硅晶圆，将其放入陷入式冷冻装置内。</li>
	<li>将固定镊子移动到切入位置。</li>
	<li>按下按钮将样品放入制冷剂内。</li>
	<li>等待几秒钟，直到样品完全冻结。</li>
	<li>将冷冻样品尽可能快地转移到冷却环境中。</li>
	<li>将冷冻固定的样品（硅晶圆）放入样品架中，并将其转移到仪器内。 注意：对于运输，建议使用干冰，并且可以短期储存样品。样品可以在冷冻状态下使用冷却仪器或在稳定后通过冷冻干燥样品来测量样品。</li>
</ol>

作者没有竞争利益可披露。

已经提出了许多使用XPS和ToF-SIMS制备用于表面分析的纳米颗粒的方法。我们在 表2中总结了这些方法的优缺点，以及误差的可能来源和不同材料的适用性。如代表性结果所示，纳米颗粒的制备可以强烈影响所得表面分析的成功。此外，由于信号干扰基板或安装材料，非导电厚膜中的充电效应，纳米颗粒作为粉末或悬浮液的状态，敏感外层的潜在损坏，生物结构的破坏以及聚集和界面上的信息，或敏感的超高真空仪器对自由纳米颗粒的脆弱性等因素，并非所有方法都适用于所有颗粒类型。
由于XPS和ToF-SIMS测量在一个区域内是平均的，而不是测量单个颗粒，因此只能从均匀的层中获得可重复的结果;因此，应避免颗粒在基板上聚集或团聚。此外，太厚的非导电材料层在分析过程中会引起充电效应，这可能导致光谱中出现不需要的伪影，特别是无法用泛光枪补偿的部分电荷。另一方面，不完整的薄膜显示来自基板或安装材料（例如粘合剂）的强信号，这可能会干扰来自颗粒表面的敏感峰。薄膜的理想厚度取决于材料，应通过对不同厚度薄膜的分析通过实验确定。特别是，使用旋涂制备的样品应使用SEM进行分析，以确保涂层的完整性。
与使用NP粉末相比，使用NP悬浮液的暴露危险和安全要求更少。液滴铸是一种相对简单的方法，设备要求较低，特别适用于不考虑薄膜厚度的悬浮液中的导电纳米颗粒。虽然样品可以在大气条件下轻松干燥，但真空干燥器用于减少液滴的干燥时间，并保护晶圆免受污染。氟橡胶环用于改变液滴的蒸发模式，从而最大限度地减少咖啡环的形成。蒸发模式也可能受到使用清洁方案改变基材亲水性或通过应用替代涂层51，52，在溶剂气氛中蒸发53，甚至通过加热基质54的影响。对于悬浮液中非导电纳米颗粒的悬浮液，建议使用旋涂，因为它能够产生足够薄的均匀颗粒层以避免充电效应，但仍然足够厚以防止Si衬底对XPS和ToF-SIMS光谱做出贡献。对于每个单独的NP系统和浓度，离心机和旋涂参数都必须进行优化，但即使在不同的仪器上也可以非常可靠地再现。由于旋涂液滴始终位于晶圆的中间，因此旋转半径无关紧要，可以使用"每分钟转数"（rpm）单位。启动程序后，悬浮液也可以沉积在晶圆上;然而，这将需要不同的旋涂参数和更多的悬浮液来获得更厚的涂层。
由于其极小的尺寸，纳米颗粒可能从基板上脱落，并在受到离子或X射线束撞击时在超高真空室内自由移动。对于用粉末制备的样品来说，这是一个特殊的问题。在某些情况下，纳米颗粒可以渗透到仪器的敏感组件中，需要昂贵且耗时的维护。由于施加了加速电压，使用ToF-SIMS损坏敏感部件的危险比使用XPS更大。粉末状样品，特别是使用"粘即走"方法制备的粉末样品，应仔细检查，以确保粉末足够牢固地固定，特别是对于ToF-SIMS分析。例如，可以通过将样品倒置并吹过其上的气体流（例如N2）来证实这一点。在分析之前，样品也可以在仪器的气闸或其他初始样品进入室中过夜，其中稳定的真空可以表明样品中没有松散的颗粒。然而，作为颗粒制备的纳米颗粒甚至可以溅射（在低加速度电压下）而不会损坏仪器;这种方法可以消除从压机引入的污染物，特别是碳氢化合物，还可以对颗粒进行批量分析。
在样品架存根中制备NP粉末可以制备具有定义几何形状和宏观平坦表面的样品。关键点是压制样品的工具的清洁度，以及使用低压以避免由于该过程而导致纳米颗粒表面的变化。它的缺点是需要相对大量的材料，以及高真空仪器中材料损失的潜在问题。我们不建议将此方法用于ToF-SIMS分析，因为颗粒不会以任何方式压缩或固定。
关于NP材料，样品制备的首要考虑因素是消除或尽量减少NP与类似材料的基板之间的干扰;例如，硅晶圆不适合使用XPS和ToF-SIMS分析SiO2 NPs，即使有足够的样品覆盖率。由于纳米颗粒和双面粘合剂之间缺乏信号干扰，金属或无机纳米颗粒可以很容易地分析为粘合剂上的粉末（假设它们不包含有机层或涂层），这种制备方法不适合聚合物NP。 并且可能是用相对较少的设备进行直接铸造的;然而，它们可能含有来自其合成的大量杂质和稳定剂，必须小心地去除而不损坏颗粒。聚合物纳米颗粒可能更容易被模压损坏，但也可能更容易在颗粒中保持在一起，这取决于所使用的压力。NP表面上的颗粒或柔软的有机涂层也可能对损伤敏感。溶液中的直接沉积有可能通过悬浮液或干燥过程损坏敏感涂层，但对于分析悬浮液中已经存在的NP是有利的。冷冻固定是分析悬浮液中化学结构、表面或界面的合适方法，这些结构、表面或界面会被各种其他样品制备技术损坏或破坏，但需要XPS和ToF-SIMS46'47的专用冷冻设备。
虽然本文描述了几种可用于样品制备的示例性方法，但在每种情况下，都应使用替代分析方法对该方法进行优化和验证。最近发表了不同因素影响的详细概述22。除了开发和验证合适的制备方法外，这些步骤的记录也至关重要40。本出版物介绍了一些易于处理的方法，并指导您根据特定任务的要求修改或开发新方法。

纳米材料被定义为具有1 nm至100 nm之间的任何外部尺寸或在此尺度上具有内部或表面结构的材料1。由于其小规模和相应大表面积（以及其他因素）所产生的独特性能，它们在农业，化学，汽车制造，化妆品，环境，医药，印刷，能源和纺织等广泛领域中得到越来越多的应用。这种使用的增加意味着人类和环境都将以前所未有的规模暴露于这些材料，这些材料的毒理学性质尚不完全清楚，其尺寸使其能够轻松集成到生物或环境系统中2。
在表面积和粒径/粒径分布的基本性质之后，表面化学和涂层被确定为纳米材料最关键的特性3;较小的颗粒具有更高的单位质量表面积，因此表面与体积原子的比例更高。实际上，对于1nm大小的纳米颗粒，可以在角落或边缘找到超过70%的原子;这强烈影响了表面性质，例如化学吸附，其高度依赖于原子尺度的表面形态4。涉及纳米材料的法规要求有关物理化学性质的准确数据以及对这些材料的毒理学性质的可靠估计。为了从纳米材料的物理和化学性质中有效地估计毒理学性质，纳米材料界需要可靠，标准化和经过验证的分析程序。ACEnano5等项目旨在收集和关联来自纳米颗粒的准确和可验证的物理数据，以便更好地调节和表征纳米材料。ACS Nano的编辑们也支持这种对标准化分析程序的推动，希望"巩固并商定表征方法和材料的最低分析水平6"。此外，XPS和ToF-SIMS为阐明核壳纳米粒子的粒子结构提供了新的可能性7，8。
与表1中比较的X射线光电子能谱（XPS）和飞行时间二次离子质谱（ToF-SIMS）是研究表面原子的成熟方法。在XPS中，样品被能量在1到2 keV之间的X射线照射，由于光电效应导致电子发射。这些发射的电子具有相同范围内的动能，与固体中电子的结合能相关;因此，在这些定义的结合能和可测量的强度下，光电子的外观允许对成分进行定量分析。由于这些光电子的平均游离途径低于10 nm，因此XPS是一种用于定量分析的高表面敏感技术。此外，对高分辨光谱中结合能的详细分析使得能够定量测定这些电子的价态。
在ToF-SIMS中，表面用聚焦离子束（初级离子）溅射，从材料中喷射的离子（二次离子）在飞行时间质谱仪中收集和分析。获得的质量/电荷图允许测定元素，同位素或分子组成。由于二次离子的平均自由通路，该技术也具有高度的表面敏感性，并且具有1-2nm的信息深度，但由于基质效应，二次离子的电离概率（因此产量）受到其周围基质的强烈影响，因此充其量是半定量的。ToF-SIMS可以在静态或动态模式下运行;两者之间的区别在于影响表面的主要离子通量。静态SIMS将初级离子通量保持在影响（即碎片）最多1%-10%表面的水平;表面保持相对不受干扰，这允许分析材料的顶层原子层。由于即使是静态SIMS也会对表面造成一些破坏，因此这两种方法被认为"非破坏性"较小。
这些表面敏感技术允许分析材料的前几纳米，包括有意或无意的涂层，对于纳米材料，这些涂层可以显着影响材料性能。有意涂层的例子包括：在量子点上封顶层以提高光致发光量子产率并降低环境反应性9，氧化铝或二氧化硅涂层用于防止防晒剂中二氧化钛纳米颗粒的光催化活性10，表面功能化以实现生物结合和随后的生物活性11，用于诊断和药物递送应用的涂层12和碳氟化合物涂层在磁性颗粒上，用于铁磁流体和核壳金属体系，以提高催化剂性能13。生物系统中的无意涂层，如氧化，表面污染或蛋白质电晕对纳米颗粒性质具有同样强烈的影响，并且至关重要的是，实验制备程序确保涂层以及更普遍的纳米材料的表面化学不被破坏或转化。评估纳米颗粒的性质也至关重要，因为它们是原位的，因为它们的性质可以通过变化而发生巨大变化2，14，15。此外，纳米颗粒悬浮液中稳定剂的浓度可以显着影响纳米颗粒的分析和结构完整性;稳定剂的存在可能导致分析中出现大的不需要的信号（例如，C，H，O和Na），而其去除可能导致纳米颗粒的损坏或团聚。
由于它们的尺寸和表面积，纳米颗粒的储存条件也会影响它们的行为，无论是作为储存的粉末/悬浮液还是作为制备的样品。各种研究表明，次优储存条件的影响，特别是室温储存和暴露在光下，会导致纳米颗粒降解，这已被证明会改变颗粒的物理，化学和/或毒理学性质14，15，16，17，18.较小的纳米颗粒已被证明比较大的纳米颗粒氧化更快，其氧化/降解速率取决于储存条件15以及表面化学14。储存过程中纳米颗粒降解的影响已被证明会显着影响物理化学性质，包括毒性14，而氧化生长可以以牺牲核心为代价向内进行15。
因此，仔细储存和制备纳米材料对于准确的表面分析至关重要，并且应仔细考虑可能影响样品表面和/或测量质量的任何因素。应该注意的是，由于XPS（在μm范围内）和ToF-SIMS（几百nm）的空间分辨率相对较低，因此只能研究纳米颗粒的一小部分;这些方法在一个区域内平均，并且不具备像电子显微镜等技术那样对单个粒子进行成像的能力。因此，任何分析都需要将纳米颗粒沉积在连续层中，以确保没有来自基板的干扰。因此，电子显微镜和XPS / ToF-SIMS通常一起用作纳米材料分析的补充方法。
除了表面化学的变化之外，制备用于XPS和ToF-SIMS分析的纳米颗粒样品的主要挑战是制备一层：均匀的，以提高可重复性;无间隙，以尽量减少基板对光谱的贡献;足够薄以避免充电效应（对于非导电样品）;并牢固地固定在基板上，以避免自由纳米颗粒进入并损坏超高真空仪器
纳米颗粒可以从悬浮液或粉末沉积到基板上。首先，我们将讨论从悬浮液中沉积纳米颗粒的不同方法。硅晶片是悬浮沉积常用的衬底，因为它们相对便宜，很容易作为由纯硅或掺杂硅组成的高纯度产品（掺杂避免充电效应），并且对于大多数纳米颗粒，光谱峰不与纳米颗粒的典型峰重叠。这最后一点是重要的。在分析之前，应确保衬底峰与纳米颗粒的预期峰值很好地分离，否则光谱的解释是复杂或不可能的，并且无法验证纳米颗粒对衬底的连续覆盖。在使用硅晶圆之前，需要广泛的清洁程序（如本出版物所述）以去除（有机）污染物并提高表面润湿性。其他合适的基材，如金膜，高序热解石墨（HOPG）或铟箔已被成功使用，但关于其制备的讨论超出了本工作的范围19，20，21，22。
其次，我们提出了在基板上沉积纳米颗粒粉末的方法，用于XPS和ToF-SIMS分析，并介绍了每种方法的优缺点，使该技术的研究人员能够找到适合其目的的最佳制备方法。第三，我们讨论了冷冻固定，这是一种合适的制备方法，可以保存诸如团聚行为，有机电晕，固体/水界面23，24 或NPs生物介质25 中的分布等特征。该过程不会导致冰晶形成，但会形成无定形冰，使膜以及细胞和组织结构保持其天然生物状态，避免水结晶过程造成的损害，并使所有细胞代谢物和细胞膜化合物的精确化学分布得以维持26，27，28.该制备方法对于呈现实际NP附聚物或杂凝聚物的精确化学图谱，在悬浮液中直接在纳米颗粒附近可视化精确化学空间，或关联NP附聚物或杂聚物内的细胞组织特异性特征或细胞内区室可能特别感兴趣。
通过本工作中呈现的结果所示，在特定情况下最合适的程序取决于各种参数，例如纳米颗粒的亲水性，稳定性，导电性，状态（例如，粉末或悬浮液）和手头的分析问题（例如，尺寸，体积性质或表面涂层）。这里介绍了可用于制备用于表面分析的NP的各种方法，以及其优缺点的比较。

<table>
	<tbody>
		<tr>
			<td>4-figure Laboratory balance</td>
			<td>Kern &#38; Sohn GmbH</td>
			<td>ADB200-4A</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mm Pellet die</td>
			<td>Specac</td>
			<td>GS03060</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkali glass cleaning solution</td>
			<td>Sigma-Aldrich</td>
			<td>Hellmanex&#8482; III Z805939</td>
			<td>Special cleaning solution for cuvettes</td>
		</tr>
		<tr>
			<td>Carbon adhesive tabs</td>
			<td>Plano</td>
			<td>&#34;Leit-Tabs&#34; G3347</td>
			<td></td>
		</tr>
		<tr>
			<td>Clean laboratory beakers</td>
			<td>any</td>
			<td></td>
			<td>e.g. 300 mL</td>
		</tr>
		<tr>
			<td>Cryo-freezer</td>
			<td>Electron Microscopy Sciences</td>
			<td>EMS-002 Cryo Workstation</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tube with fasteners</td>
			<td>Medicell Membranees Ltd</td>
			<td>DTV12000.06.30</td>
			<td>Molecular weight cut-off (MWCO) 12-14 kDa</td>
		</tr>
		<tr>
			<td>Die press</td>
			<td>any</td>
			<td></td>
			<td>Capable of 2 kN force</td>
		</tr>
		<tr>
			<td>Disposable syringe, 1 mL, Luer-slip</td>
			<td>TH Geyer</td>
			<td>Labsolute 7657545</td>
			<td>Any appropriate volume can be used</td>
		</tr>
		<tr>
			<td>Double-sided adhesive</td>
			<td>3M</td>
			<td>Removable Repositionable Tape 665</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry ice</td>
			<td>Linde AG</td>
			<td>ICEBITZZZ&#174;</td>
			<td>For short term storage/cooling</td>
		</tr>
		<tr>
			<td>Eppendorf transfer pipette and tips</td>
			<td>Eppendorf</td>
			<td>various</td>
			<td>Check correct size for planned pipetting volume</td>
		</tr>
		<tr>
			<td>Ethanol, ACS grade</td>
			<td>Merck KGaA</td>
			<td>1009832500</td>
			<td></td>
		</tr>
		<tr>
			<td>FFP2 or FFP3 mask</td>
			<td>various</td>
			<td></td>
			<td>For working with nanoparticles from non-hazardous materials, when not in a fume hood or glove box</td>
		</tr>
		<tr>
			<td>Isopropanol, ACS grade</td>
			<td>Merck KGaA</td>
			<td>1096342500</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab coat, gloves and goggles</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory centrifuge</td>
			<td>Eppendorf</td>
			<td>Centrifuge 5430</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory fume hood</td>
			<td>any</td>
			<td></td>
			<td>necessary for working with nanoparticles</td>
		</tr>
		<tr>
			<td>Laboratory stirrer &#38; stirrer bar</td>
			<td>NeoLab</td>
			<td>D-6010</td>
			<td></td>
		</tr>
		<tr>
			<td>Lint-free wipes</td>
			<td>Kimberley Clark Professional</td>
			<td>Kimtech Science Precision wipes</td>
			<td>Recommended for working with Si wafers</td>
		</tr>
		<tr>
			<td>Liquid Nitrogen</td>
			<td>Linde AG</td>
			<td>Stickstoff fl&#252;ssig 5.0</td>
			<td>Only for cooling of the cryogen.</td>
		</tr>
		<tr>
			<td>Microtube/centrifuge tube 1,5 mL</td>
			<td>T.H. Geyer GmbH &#38; Co. KG</td>
			<td>Labsolute 7696751</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen 5.0</td>
			<td>any</td>
			<td></td>
			<td>99.999% purity</td>
		</tr>
		<tr>
			<td>Pasteur pipette, PE, plastic 3 mL</td>
			<td>TH Geyer</td>
			<td>Labsolute 7 691 203</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette, PE, plastic 3 mL</td>
			<td>TH Geyer</td>
			<td>Labsolute 7 691 203</td>
			<td></td>
		</tr>
		<tr>
			<td>Powder sample holder</td>
			<td>BAM workshop</td>
			<td></td>
			<td>&#34;Home-made&#34; sample holder</td>
		</tr>
		<tr>
			<td>Propane</td>
			<td>Sigma-Aldrich</td>
			<td>769037</td>
			<td>The cryogen should be of highest possible purity.</td>
		</tr>
		<tr>
			<td>Sample vial or centrifuge tube 1 mL</td>
			<td>Greiner Bio-One GmbH</td>
			<td>Cellstar 188 261</td>
			<td>Should be capable of being fixed in the Vortexer</td>
		</tr>
		<tr>
			<td>Silicon wafers</td>
			<td>any</td>
			<td></td>
			<td>ideally 1cm2 pre-cut</td>
		</tr>
		<tr>
			<td>Spin-coater</td>
			<td>SPS Europe</td>
			<td>SPIN150i-NPP</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter 0,45 &#181;m</td>
			<td>Th Geyer</td>
			<td>Labsolute 7699803</td>
			<td>For smaller samples; larger versions exist for larger sample volumes</td>
		</tr>
		<tr>
			<td>ToF-SIMS</td>
			<td>IONTOF GmbH</td>
			<td>ToF-SIMS IV or V, equipped with Bi LMIG and flood gun</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers for handling Si wafers</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ultrapure water</td>
			<td>TKA</td>
			<td>MicroPure 08.1202</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonicator</td>
			<td>Bandelin</td>
			<td>Sonorex Super</td>
			<td></td>
		</tr>
		<tr>
			<td>UV/Ozone cleaner</td>
			<td>NanoBioAnalytics</td>
			<td>UVC-1014</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum dessicator</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum pump (membrane/diaphragm)</td>
			<td>Vacuubrand GmbH</td>
			<td>Type MD-4T</td>
			<td></td>
		</tr>
		<tr>
			<td>Viton O-ring 6.07 x 1.78 mm</td>
			<td>Betech GmbH</td>
			<td>2-010, FKM 80</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>Heathrow Scientific</td>
			<td>Vortexer HS120212</td>
			<td></td>
		</tr>
		<tr>
			<td>Wafer Holder 25mm coin style</td>
			<td>Semiconductor Production Systems Europe</td>
			<td>eWB0091-ASSY-1</td>
			<td></td>
		</tr>
		<tr>
			<td>XPS</td>
			<td>Kratos</td>
			<td>Kratos Axis Ultra DLD</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of nanoparticles for tof-sims and xps analysis

近年来，纳米颗粒由于其在医学，化妆品，化学等不同领域的潜力和应用以及它们使先进材料成为可能而受到越来越多的关注。为了有效地理解和调节纳米颗粒的物理化学性质和潜在的不利影响，需要开发经过验证的纳米颗粒各种性质的测量程序。虽然测量纳米颗粒尺寸和尺寸分布的程序已经建立，但分析其表面化学性质的标准化方法尚未到位，尽管表面化学对纳米颗粒性质的影响是无可争议的。特别是，用于表面分析的纳米颗粒的储存和制备强烈影响各种方法的分析结果，为了获得一致的结果，样品制备必须既优化又标准化。在本文中，我们详细介绍了一些制备用于表面分析的纳米颗粒的标准程序。原则上，纳米颗粒可以从悬浮液或粉末沉积在合适的基板上。硅（Si）晶圆通常用作基板，但是，它们的清洁对工艺至关重要。对于悬浮液的样品制备，我们将讨论滴铸和旋涂，其中不仅基质的清洁度和悬浮液的纯度，而且其浓度对制备方法的成功起着重要作用。对于具有敏感配体壳或涂层的纳米颗粒，沉积为粉末更合适，尽管这种方法在固定样品时需要特别注意。

Watch this Scientific Journal Video about Preparation of Nanoparticles for ToF-SIMS and XPS Analysis at JoVE.com

Preparation of Nanoparticles for ToF-SIMS and XPS Analysis

介绍了制备用于表面分析的纳米颗粒的多种不同程序（滴铸，旋涂，粉末沉积和冷冻固定）。我们讨论了每种方法的挑战，机遇和可能的应用，特别是关于不同制备方法引起的表面性能变化。

用于ToF-SIMS和XPS分析的纳米颗粒的制备

Nanoparticles have gained increasing attention in recent years due to their potential and application in different fields including medicine, cosmetics, ...

preparation-of-nanoparticles-for-tof-sims-and-xps-analysis

Research

JoVE Journal

Chemistry

7.7K 次观看.  Federal Institute for Material Research and Testing (BAM). 介绍了制备用于表面分析的纳米颗粒的多种不同程序（滴铸，旋涂，粉末沉积和冷冻固定）。我们讨论了每种方法的挑战，机遇和可能的应用，特别是关于不同制备方法引起的表面性能变化。

视频: Preparation of Nanoparticles for ToF-SIMS and XPS Analysis

Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle diameters ranging from 30-250 nm by varying the reaction conditions. Through the selection of a microwave compatible solvent, silicic acid precursor, catalyst, and microwave irradiation time, these microwave-assisted methods were capable of overcoming the previously reported shortcomings associated with synthesis of silica nanoparticles using microwave reactors. The siloxane precursor was hydrolyzed using the acid catalyst, HCl. Acetone, a low-tan &#948; solvent, mediates the condensation reactions and has minimal interaction with the electromagnetic field. Condensation reactions begin when the silicic acid precursor couples with the microwave radiation, leading to silica nanoparticle sol formation. The silica nanoparticles were characterized by dynamic light scattering data and scanning electron microscopy, which show the materials&#39; morphology and size to be dependent on the reaction conditions. Microwave-assisted reactions produce silica nanoparticles with roughened textured surfaces that are atypical for silica sols produced by St&#246;ber&#39;s methods, which have smooth surfaces.

Silica nanoparticles were prepared using acid-catalysis of a siloxane precursor and microwave-assisted synthetic techniques resulting in the controlled growth of nanomaterials ranging from 30-250 nm in diameter. The growth dynamics can be controlled by varying the initial silicic acid concentration, time of the reaction, and temperature of reaction.

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle ...

科研

化学

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan&#8217;s central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

通过超性能液相色谱对细菌细胞壁进行隔离和制备，进行组成分析

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the ...

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under appropriate conditions. The glycan nanoparticles are useful carriers for protein factors, which facilitate the in vivo delivery of the proteins and sustain their retention in the targeted tissue. The glycan polyelectrolyte complexes are also ideal for protein delivery, as the incorporation is carried out in aqueous solution, which reduces the likelihood of inactivation of the proteins. Proteins with a heparin-binding site adhere to dextran sulfate readily, and are, in turn, stabilized by the binding. These particles are also less inflammatory and toxic when delivered in vivo. In the protocol described below, SDF-1&#945; (Stromal cell-derived factor-1&#945;), a stem cell homing factor, is first mixed and incubated with dextran sulfate. Chitosan is added to the mixture to form polyelectrolyte complexes, followed by zinc sulfate to stabilize the complexes with zinc bridges. The resultant SDF-1&#945;-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1&#945; is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.

Preparation and Characterization of SDF-1&amp;#945;-Chitosan-Dextran Sulfate Nanoparticles

The objective of this protocol is to incorporate SDF-1&#945;, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1&#945; from the nanoparticles.

SDF-1α - 壳聚糖硫酸葡聚糖纳米粒子的制备与表征

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under ...

Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of these applications require interaction with and adhesion of DNA nanostructures to a substrate. Due to its atomically flat and easily cleaned nature, mica has been the substrate of choice for DNA origami experiments. However, the practical applications of mica are relatively limited compared to those of semiconductor substrates. For this reason, a straightforward, stable, and repeatable process for DNA origami adhesion on derivatized silicon oxide is presented here. To promote the adhesion of DNA nanostructures to silicon oxide surface, a self-assembled monolayer of 3-aminopropyltriethoxysilane (APTES) is deposited from an aqueous solution that is compatible with many photoresists. The substrate must be cleaned of all organic and metal contaminants using Radio Corporation of America (RCA) cleaning processes and the native oxide layer must be etched to ensure a flat, functionalizable surface. Cleanrooms are equipped with facilities for silicon cleaning, however many components of DNA origami buffers and solutions are often not allowed in them due to contamination concerns. This manuscript describes the set-up and protocol for in-lab, small-scale silicon cleaning for researchers who do not have access to a cleanroom or would like to incorporate processes that could cause contamination of a cleanroom CMOS clean bench. Additionally, variables for regulating coverage are discussed and how to recognize and avoid common sample preparation problems is described.

Reproducible cleaning processes for substrates used in DNA origami research are described, including bench-top RCA cleaning and derivatization of silicon oxide. Protocols for surface preparation, DNA origami deposition, drying parameters, and simple experimental set-ups are illustrated.

对于DNA折纸分析和实验准备云母和硅衬底

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of ...

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time.

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work presents a protocol for liquid handling and sample introduction to a microchannel for in situ time-of-flight secondary ion mass spectrometry analysis of protein biomolecules in an aqueous solution.

在水中水合蛋白原位表征SALVI和TOF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. They offer new possibilities for the characterization of living systems and are particularly appropriate for detecting, localizing and quantifying the presence of metal oxide nanoparticles both in biological specimens and the environment. Indeed, these techniques all meet relevant requirements in terms of (i) sensitivity (from 1 up to 10 &#181;g.g-1 of dry mass), (ii) micrometer range spatial resolution, and (iii) multi-element detection. Given these characteristics, microbeam chemical element imaging can powerfully complement routine imaging techniques such as optical and fluorescence microscopy. This protocol describes how to perform a nuclear microprobe analysis on cultured cells (U2OS) exposed to titanium dioxide nanoparticles. Cells must grow on and be exposed directly in a specially designed sample holder used on the optical microscope and in the nuclear microprobe analysis stages. Plunge-freeze cryogenic fixation of the samples preserves both the cellular organization and the chemical element distribution. Simultaneous nuclear microprobe analysis (scanning transmission ion microscopy, Rutherford backscattering spectrometry and particle induced X-ray emission) performed on the sample provides information about the cellular density, the local distribution of the chemical elements, as well as the cellular content of nanoparticles. There is a growing need for such analytical tools within biology, especially in the emerging context of Nanotoxicology and Nanomedicine for which our comprehension of the interactions between nanoparticles and biological samples must be deepened. In particular, as nuclear microprobe analysis does not require nanoparticles to be labelled, nanoparticle abundances are quantifiable down to the individual cell level in a cell population, independently of their surface state.

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

We describe a procedure for the detection of chemical elements present in situ in human cells as well as their in vitro quantification. The method is well-suited to any cell type and is particularly useful for quantitative chemical analyses in single cells following in vitro metal oxide nanoparticles exposure.

原位金属氧化物纳米粒子的核探针检测与单细胞定量研究

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. ...

Determining the Chemical Composition of Corrosion Inhibitor/Metal Interfaces with XPS: Minimizing Post Immersion Oxidation

An approach for acquiring more reliable X-ray photoelectron spectroscopy data from corrosion inhibitor/metal interfaces is described. More specifically, the focus is on metallic substrates immersed in acidic solutions containing organic corrosion inhibitors, as these systems can be particularly sensitive to oxidation following removal from solution. To minimize the likelihood of such degradation, samples are removed from solution within a glove box purged with inert gas, either N2 or Ar. The glove box is directly attached to the load-lock of the ultra-high vacuum X-ray photoelectron spectroscopy instrument, avoiding any exposure to the ambient laboratory atmosphere, and thus reducing the possibility of post immersion substrate oxidation. On this basis, one can be more certain that the X-ray photoelectron spectroscopy features observed are likely to be representative of the in situ submerged scenario, e.g. the oxidation state of the metal is not modified.

A protocol to avoid the oxidation of metallic substrates during sample transfer from an inhibited acidic solution to an X-ray photoelectron spectrometer is presented.

确定缓蚀剂/金属界面的化学成分与XPS：最小化后浸入氧化

An approach for acquiring more reliable X-ray photoelectron spectroscopy data from corrosion inhibitor/metal interfaces is described. More specifically, ...

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) and their roles in environmental microbiology. This study outlines a method to cultivate biofilm attachment to the System for Analysis at the Liquid Vacuum Interface (SALVI) and achieve in situ chemical mapping of a living biofilm by time-of-flight secondary ion mass spectrometry (ToF-SIMS). This is done through the culturing of bacteria both outside and within the SALVI channel with our specialized setup, as well as through optical imaging techniques to detect the biofilm presence and thickness before ToF-SIMS analysis. Our results show the characteristic peaks of the Shewanella biofilm in its natural hydrated state, highlighting upon its localized water cluster environment, as well as EPS fragments, which are drastically different from the same biofilm's dehydrated state. These results demonstrate the breakthrough capability of SALVI that allows for in situ biofilm imaging with a vacuum-based chemical imaging instrument.

In Situ Characterization of Shewanella oneidensis MR1 Biofilms by SALVI and ToF-SIMS

This article presents a method for growing a biofilm for in situ time-of-flight secondary ion mass spectrometry for chemical mapping in its hydrated state, enabled by a microfluidic reactor, System for Analysis at the liquid Vacuum Interface. The Shewanella oneidensis MR-1 with green fluorescence protein was used as a model.

原位萨尔和 TOF-SIMS希瓦氏菌 oneidensis MR1 生物膜的表征

Bacterial biofilms are surface-associated communities that are vastly studied to understand their self-produced extracellular polymeric substances (EPS) ...

Ligand-Mediated Nucleation and Growth of Palladium Metal Nanoparticles

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution of capping ligands during the synthesis reaction, their role in regulating the nucleation and growth rates of colloidal nanoparticles is not well understood. In this work, we demonstrate a mechanistic investigation of the role of trioctylphosphine (TOP) in Pd nanoparticles in different solvents (toluene and pyridine) using in situ SAXS and ligand-based kinetic modeling. Our results under different synthetic conditions reveal the overlap of nucleation and growth of Pd nanoparticles during the reaction, which contradicts the LaMer-type nucleation and growth model. The model accounts for the kinetics of Pd-TOP binding for both, the precursor and the particle surface, which is essential to capture the size evolution as well as the concentration of particles in situ. In addition, we illustrate the predictive power of our ligand-based model through designing the synthetic conditions to obtain nanoparticles with desired sizes. The proposed methodology can be applied to other synthesis systems and therefore serves as an effective strategy for predictive synthesis of colloidal nanoparticles.

The main goal of this work is to elucidate the role of capping agents in regulating the size of palladium nanoparticles by combining in situ small angle x-ray scattering (SAXS) and ligand-based kinetic modeling.

配体介导成核与钯金属纳米粒子的生长

The size, size distribution and stability of colloidal nanoparticles are greatly affected by the presence of capping ligands. Despite the key contribution ...

Synthesis and Characterization of Amphiphilic Gold Nanoparticles

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of their interactions with cell membranes, lipid bilayers, and viruses. The hydrophilic ligands make these particles colloidally stable in aqueous solutions and the combination with hydrophobic ligands creates an amphiphilic particle that can be loaded with hydrophobic drugs, fuse with the lipid membranes, and resist nonspecific protein adsorption. Many of these properties depend on nanoparticle size and the composition of the ligand shell. It is, therefore, crucial to have a reproducible synthetic method and reliable characterization techniques that allow the determination of nanoparticle properties and the ligand shell composition. Here, a one-phase chemical reduction, followed by a thorough purification to synthesize these nanoparticles with diameters below 5 nm, is presented. The ratio between the two ligands on the surface of the nanoparticle can be tuned through their stoichiometric ratio used during synthesis. We demonstrate how various routine techniques, such as transmission electron microscopy (TEM), nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), and ultraviolet-visible (UV-Vis) spectrometry, are combined to comprehensively characterize the physicochemical parameters of the nanoparticles.

Amphiphilic gold nanoparticles can be used in many biological applications. A protocol to synthesize gold nanoparticles coated by a binary mixture of ligands and a detailed characterization of these particles is presented.

两栖金纳米粒子的合成与表征

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of ...