作者承认（1）从纳米生物医学科学与BK21 PLUS NBM全球研究再生医学中心的檀国大学，并系资金​​支持的重点由NRF，大韩民国，（资助研究中心计划（编号2009-0093829） 2）设施，科学和技术援助的澳大利亚中心显微镜和微量分析在悉尼大学。 HKC感谢澳大利亚研究理事会通过发现项目补助（DP0985367＆DP120102778）的财务支持。世界锦标赛是感谢澳大利亚研究理事会通过联动项目补助（LP120200489，LP110200316）的财务支持。

1，准备胶体探针和原子力显微镜基板<ol><li>为了准备胶体探针，使用由作者以前开发的方法。19 <ol><li>简而言之，使用45°角座贴上无针尖悬臂45°（ 图1A）的特定角度。 </li><li>通过涂抹一薄层的环氧树脂在显微镜载玻片上制备环氧滑动。用干净的抹刀或缓慢的氮气流，以确保环氧树脂加入到显微镜载玻片的层是最小的高度。 </li><li>加盖环氧幻灯片使用一个定制设计的支架（ 图1B）一个40X光学变倍显微镜镜头。然后使用悬臂接近环氧滑动并获取对悬臂少量的环氧树脂。 </li><li>重复上述步骤，还附上感兴趣的单个粒子在悬臂（ 图1C）的顶点。 </li></ol></li><li>通过粘贴胶体面值准备基板的AFM幕进行到使用热塑性胶粘剂安装的原子力显微镜的盖玻片。 <ol><li>热火35毫米的圆形盖玻片至120℃，并涂抹少量的粘合剂盖玻片。高温是必要的，以熔化热塑粘合剂应用。 </li><li>然后冷却，将盖玻片至40℃喷粉胶体粒子上胶前。注:在40℃下的胶被充分地设定，该粒子将不会成为嵌入到胶，但胶是足够粘性的，以确保颗粒将附着在基板上。 </li><li>进一步冷却盖玻片至室温，并用氮气吹干吹掉多余的独立的粒子。 </li><li>与将用于胶体探针测量液体介质洗涤所述基板几次，以确保所有未连接的颗粒从基片上除去。注意:这是重要的，以减少自由流动的颗粒的测量过程中的影响，这可以跨行事悬臂和结果引入误差。 </li></ol></li></ol> 2，安装的胶体探头，对准激光和平衡系统<ol><li>安装盖玻片与胶体颗粒进入底部的液态电池的一半，并确保该O型圈安装正确，以防止任何泄漏。 </li><li>放置的疏水性透明片材上的显微镜载物台上，以防止可能在实验过程中漏出的液体，特别是如果只使用一种液体的细胞进行测定，下半部分，然后将液晶单元上的显微镜载物台上。注意:为了简单起见可以使用一个液晶单元的唯一的下半区，鉴于该系统可以适当地平衡化;提示 - 蒸发改变了测量和影响结果/读取的情况。 </li><li>附加胶体探针的原子力显微镜扫描头和组装到原子力显微镜。随着对原子力显微镜仪器软件，请使用旋钮扫描头使悬臂尖端成为关注的焦点。注意:所有的程序步骤和测量使用的MFP-3D生物原子力显微镜与庇护研究软件完成。 </li><li>为了最大限度地提高强度，对准激光上使用适当的调节旋钮上的扫描头的悬臂的末端。 </li><li>允许系统达到平衡5-10分钟，或直至偏转值稳定。使用该偏转调节旋钮使偏转到零或稍负。 </li><li>当系统在空气中达到平衡，使用原子力显微镜的软件（在主面板窗口中热板），以热计算InvOLS（灵敏度）和胶体探针的弹簧常数。注意:这种敏感性会暂时使用，直到真正的灵敏度的测量是在测量完成（见第4步）。 <ol><li>选择"校准弹簧常数"或"校准InvOLS"，然后点击"捕获热数据"。 </li><li>一旦一个突出的峰值很明显，停止捕获数据，然后单击放大了主峰。 </li><li>点击"初始化飞度"，其次是"适合热数据"，以获得自动计算弹簧常数或InvOLS值。 </li></ol></li><li>慢慢加入2毫升液体培养基来使用注射器将液体细胞，并确保没有气泡周围的悬臂本。重新对准激光器中，由于介质的折射率现在已经改变，并再次平衡系统允许挠度值，以调整所述偏转回零之前稳定下来。注意:如果环境和液体之间的温差较大存在，平衡将需要更长的时间。 </li></ol> 3，成像和数据采集<ol><li>设置初始扫描大小为20微米，扫描速度为1赫兹，扫描角度90°时，设置点到0.2 V，将获得的样品的扫描。如需要获得重叠跟踪调整增益而折回的曲线。 </li><li>一旦感兴趣的粒子被发现，立刻放大到该粒子限制与获得力量体积测量前基底延伸探针的相互作用。 </li><li>一旦放大，获得单个粒子的单个粒子的或部分的足够的图像。然后切换到部队面板中的软件。把红色位置栏到最高位置，设置强制距离5微米，扫描速率为0.1赫兹，触发通道为none，并进行单测力。确保探头不接触基板。 </li><li>从所获得的单次测量图，计算出虚拟的偏转线通过右键单击图形窗口，并选择"计算虚拟高清线"选项。这将自动计算虚拟偏转，并根据需要在软件中更新值。 </li><li>改变触发通道偏转，并设置触发点到20纳米。将力区ANCE到1微米和根据感兴趣的测力需要调节扫描速度。 </li><li> 2-3进行初步的连续单力测量后手动调整数值的审查小组委员会在偏转逆光杠杆灵敏度（InvOLS）。 <ol><li>进行单测力，然后点击在部队面板的"审阅"按钮，打开了一个法师部队面板。 </li><li>突出显示最近完成的测力。在"轴"的标题确保只有"DeflV"被选中。以"日"使用下拉菜单更改"X轴"输入字段，并单击"制作图表。" </li><li>点击主面板部队的"帕姆"选项卡上，并调整"InvOLS"的值，直到图形的接触区域是完全垂直的。然后填充这个值在"DEFL InvOLS"栏位于下加州60，子选项卡中的强制选项卡位于主主面板窗口上。 </li><li>重复这个2-3次，以确保InvOLS值不显著改变。 </li></ol></li><li>现在，所有的参数都已经设置，确保液体介质水平仍然足够，偏转仍然是稳定的。注意:在这个时候，可以得到单力曲线或力地图。如果停留力的测量需要，驻留选项可强制面板中进行访问。 </li></ol> 4，后调整灵敏度的分析<ol><li>在完成测量数据的收集之后，测量胶体探针的真实感光度。要做到这一点，使用相对大挠度/力与胶体探针在相同的液体培养基中对"无限"硬表面如云母进行测力。实验结束后，获得灵敏度，因为大挠度/力可能会损坏胶体:注意新娘探针制备多孔或脆弱的胶体。 </li><li>接触区域的斜率是所使用的软件，自动计算出的灵敏度（ 图2）。在所有使用该特定胶体探针得到的曲线数据分析使用灵敏度这个真正的价值。 </li></ol>

<ol>
	<li>Sindel, U., Zimmermann, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+interaction+forces+between+individual+powder+particles+using+an+atomic+force+microscope.">Measurement of interaction forces between individual powder particles using an atomic force microscope.</a> Powder Technology. 117, 247-254 (2001).</li><li>Ducker, W. A., Senden, T. J., Pashley, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+colloidal+forces+using+an+atomic+force+microscope.">Direct measurement of colloidal forces using an atomic force microscope.</a> Nature. 353, 239-241 (1991).</li><li>Israelachvili, J. N., Adams, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+forces+between+two+mica+surfaces+in+aqueous+electrolyte+solutions+in+the+range+0&#8211;100+nm.">Measurement of forces between two mica surfaces in aqueous electrolyte solutions in the range 0&#8211;100 nm.</a> Journal of the Chemical Society, Faraday Transactions. 1, 975-1001 (1978).</li><li>Upadhyay, D., 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=Magnetised+thermo+responsive+lipid+vehicles+for+targeted+and+controlled+lung+drug+delivery.">Magnetised thermo responsive lipid vehicles for targeted and controlled lung drug delivery.</a> Pharmaceutical Research. 29, 2456-2467 (2012).</li><li>Chrzanowski, W., 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=Biointerface:+protein+enhanced+stem+cells+binding+to+implant+surface.">Biointerface: protein enhanced stem cells binding to implant surface.</a> Journal of Materials Science: Materials in Medicine. 23, 2203-2215 (2012).</li><li>Chrzanowski, W., 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=Nanomechanical+evaluation+of+nickel&#8211;titanium+surface+properties+after+alkali+and+electrochemical+treatments.">Nanomechanical evaluation of nickel&#8211;titanium surface properties after alkali and electrochemical treatments.</a> Journal of The Royal Society Interface. 5, 1009-1022 (2008).</li><li>Tran, C. T., Kondyurin, A., Chrzanowski, W., Bilek, M. M., McKenzie, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+pH+on+yeast+immobilization+on+polystyrene+surfaces+modified+by+energetic+ion+bombardment.">Influence of pH on yeast immobilization on polystyrene surfaces modified by energetic ion bombardment.</a> Colloids and Surfaces B: Biointerfaces. 104, 145-152 (2013).</li><li>Page, K., 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=Study+of+the+adhesion+of+Staphylococcus+aureus+to+coated+glass+substrates.">Study of the adhesion of Staphylococcus aureus to coated glass substrates.</a> Journal of materials science. 46, 6355-6363 (2011).</li><li>Binnig, G., Quate, C. F., Gerber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+force+microscope.">Atomic force microscope.</a> Physical Review Letters. 56, 930-933 (1103).</li><li>Butt, H. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+electrostatic,+van+der+Waals,+and+hydration+forces+in+electrolyte+solutions+with+an+atomic+force+microscope.">Measuring electrostatic, van der Waals, and hydration forces in electrolyte solutions with an atomic force microscope.</a> Biophysical Journal. 60, 1438-1444 (1991).</li><li>Johnson, K., Kendall, K., Roberts, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+energy+and+the+contact+of+elastic+solids.">Surface energy and the contact of elastic solids.</a> Proceedings of the Royal Society of London. A. Mathematical and Physical Sciences. 324, 301-313 (1971).</li><li>Deraguin, B., Landau, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+of+the+stability+of+strongly+charged+lyophobic+sols+and+of+the+adhesion+of+strongly+charged+particles+in+solution+of+electrolytes.">Theory of the stability of strongly charged lyophobic sols and of the adhesion of strongly charged particles in solution of electrolytes.</a> Acta Physicochim: USSR. 14, 633-662 (1941).</li><li>Derjaguin, B., Muller, V., Toporov, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+contact+deformations+on+the+adhesion+of+particles.">Effect of contact deformations on the adhesion of particles.</a> Journal of Colloid and Interface Science. 53, 314-326 (1975).</li><li>Verwey, E. J. W., Overbeek, J. T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+of+the+stability+of+lyophobic+colloids.">Theory of the stability of lyophobic colloids.</a> DoverPublications.com, doi:10.1021/j150453a001. , (1999).</li><li>Kappl, M., Butt, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+colloidal+probe+technique+and+its+application+to+adhesion+force+measurements.">The colloidal probe technique and its application to adhesion force measurements.</a> Particle &amp; Particle Systems Characterization. 19, 129-143 (2002).</li><li>Tran, C. T., Kondyurin, A., Chrzanowski, W., Bilek, M. M., McKenzie, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+pH+on+yeast+immobilization+on+polystyrene+surfaces+modified+by+energetic+ion+bombardment.">Influence of pH on yeast immobilization on polystyrene surfaces modified by energetic ion bombardment.</a> Colloids and Surfaces B: Biointerfaces. , (2012).</li><li>Sa, D. J., de Juan Pardo, E. M., de Las Rivas Astiz, R., Sen, S., Kumar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+indentational+elasticity+measurements+of+hydrogel+extracellular+matrix+substrates.">High-throughput indentational elasticity measurements of hydrogel extracellular matrix substrates.</a> Applied Physics Letters. 95, 063701-063701 (2009).</li><li>Zauscher, S., Klingenberg, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Friction+between+cellulose+surfaces+measured+with+colloidal+probe+microscopy.">Friction between cellulose surfaces measured with colloidal probe microscopy.</a> Colloids and Surfaces A: Physicochemical and Engineering Aspects. 178, 213-229 (2001).</li><li>Sa, D., Chan, H. -. K., Chrzanowski, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attachment+of+Micro-+and+Nano-particles+on+Tipless+Cantilevers+for+Colloidal+Probe+Microscopy.">Attachment of Micro- and Nano-particles on Tipless Cantilevers for Colloidal Probe Microscopy.</a> International Journal of Colloid and Interface. , (2014).</li></ol>

The authors declare that they have no competing financial interests.

系统不稳定时液体胶体探针纳米显微目前的几个来源可以很容易地通过适当的平衡过程来缓解。所讨论的不稳定性先前导致错误的结果和力曲线更难以客观分析。如果不稳定的所有来源已趋向和图形相似，在图4所示的仍然存在，另一次测量的参数可以是原因。这是要考虑的重要过程中的胶体探针纳米显微其他测量参数包括在该悬臂啮合并从样品回缩的速度和力测量的触发点。另外，?...

原子力显微镜（AFM）是一种技术，它使定性和定量成像和探测的材料的表面。4-6传统AFM用于表面形貌，形貌和多相位材料的结构的评估。 AFM具有定量评价纳米尺度相互作用，如在空气和液体介质的特定探针和基板之间的电荷，吸引，排斥和粘附力的能力。7,8最初由分级，第四纪和Gerber 9的用途开发的原子力显微镜探测已知的/确定的灵敏度和弹性系数接近和/或扫描样本。由于探针与样品之间的物理相互作用，悬臂接触或接近时偏转，这取决于操作的模式下，该偏转可以被转换为获得本探针和样品之间的样品或测量力的地形。修改的原子力显微镜TECHNI阙，如胶体探针纳米显微，10已允许科学家直接评价存在于感兴趣的胶体系统，两种材料间的纳米相互作用力。 在胶体探针纳米显微，选择的球形颗粒附着到悬臂的先端，取代了传统的圆锥形和棱锥形的提示。球形粒子是理想的，允许与理论模型，如强生，肯德尔，罗伯茨（JKR）11和Derjaguin，朗多，Vervwey，Overbeek（DLVO）12-14理论，并尽量减少表面粗糙度对测量的影响比较。 15这些理论用于定义接触力学和颗粒间力预期的胶体系统中。 DLVO理论结合了有吸引力的范德华力和静电排斥力（由于双电层）来定量解释含水的胶体体系的聚集行为，而歼KR理论结合的接触压力和粘附到模型两个组件之间的弹性接触的效果。一旦适当的探针产生的，它被用来逼近的任何其它材料/颗粒，以评估在两个部件之间的力。使用制造的小费标准之一将是能够测量的尖端和选择的材料之间的相互作用力，但使用一个定制的胶体探针的利益使得目前所研究的系统中存在材料之间的力的测量。可测量相互作用包括:粘合剂，有吸引力，排斥力，电荷和存在于粒子之间甚至静电力16此外，胶体探针技术可以用来探索本颗粒和弹性材料之间的切向力17,18 进行测量，在各种媒体的能力是胶体探针纳米显微的主要优点之一。环境条件，液态间EDIA，或湿度可控的条件下都可以被用来模仿所研究的系统的环境条件。进行测量，在液体环境中的能力，使胶体系统中，它天然存​​在的环境中的研究;因此，能够定量地采集数据是直接翻译的系统在其自然状态。例如，定量吸入器（MDI）中存在粒子的相互作用可以用一个模型液体推进剂具有类似属性的计量吸入器所用的推进剂进行研究。在空气中测得相同的相互作用并不能代表在吸入器系统存在的。此外，液体介质可以被修改来评价水分侵入，二次表面活性剂，或温度上在MDI粒子相互作用的影响。以控制温度的能力，可用于模拟在胶体系统的制造某些步骤以评估温度无论是在制造或存储胶体系统可能对粒子的相互作用产生影响。 可以使用胶体探针来获得测量包括;地形扫描，个人的力量 - 距离曲线，力 - 距离粘连的地图里，住力距离测量。正在使用本文介绍的胶体探针纳米显微方法测量关键参数包括管理单元，最大负荷和分离能量值。管理单元是引力的测量，最大负载的最大附着力的价值​​，以及分离能传达到从接触撤回粒子所需的能量。这些值可以通过瞬时或停留力的测量来测量。两种不同类型的停留测量包括变形和压痕。驻留测量的长度和类型可以专门选择模仿所关心的系统中存在特异性相互作用。一个例子是使用偏转停留 - 持有来评估发展中形成的聚集体分散体胶粘剂债券 - 在希望的弯沉值在接触样品。所形成的粘合剂粘结可以测量作为时间的函数，并且可以提供深入了解再分散后的长期储存的聚集体所需的力。数据可以使用这种方法获得的大量证明了该方法的通用性。

<table border="0" cellpadding="0" cellspacing="0" width="782">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Name of Material/ Equipment</td>
			<td style="width:179px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Double-Bubble Epoxy</td>
			<td style="width:179px;">Hardman</td>
			<td style="width:119px;">4004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Veeco Tipless Probes</td>
			<td style="width:179px;">Veeco</td>
			<td style="width:119px;">NP-O10&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Porous Particles</td>
			<td style="width:179px;">Pearl Therapeutics</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Atomic Force Microscope (MFP)</td>
			<td style="width:179px;">Asylum&#160;</td>
			<td style="width:119px;">MFP-3D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">SPIP Scanning Probe Image Processor Software</td>
			<td style="width:179px;">NanoScience&#160; Instruments</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">35 mm Coverslips</td>
			<td style="width:179px;">Asylum</td>
			<td style="width:119px;">504.003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Tempfix</td>
			<td style="width:179px;">Ted Pella. Inc.</td>
			<td style="width:119px;">16030</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative and qualitative examination of particle-particle interactions using colloidal probe nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

液体胶体系统用于多种药物给药系统。用于吸入给药，常见的胶体系统是悬浮加压计量剂量吸入器（pMDI的）。在的pMDI中存在粒子的相互作用配方的物理稳定性，存储和药物输送均匀性起到了至关重要的作用。在这个手稿，多孔基于脂质的颗粒之间的颗粒间力（〜2微米的光学平均粒径）在一个模型中的推进剂（2H，3H-全氟戊烷），在室温下进行了评价，以传达的功能，并且与呈现相关的可能的错?...

Watch this Scientific Journal Video about Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy at JoVE.com

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

粒子粒子相互作用采用胶体探针纳米显微定量和定性检查

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

quantitative-qualitative-examination-particle-particle-interactions

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

11.0K 次观看.  University of Sydney. Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems. 

视频: Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Qualitative Identification of Carboxylic Acids, Boronic Acids, and Amines Using Cruciform Fluorophores

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with electron-donors, and the other with electron-acceptors, cruciforms&#39; HOMO will localize along the electron-rich and LUMO along the electron-poor axis. This spatial isolation of cruciforms&#39; frontier molecular orbitals (FMOs) is essential to their use as sensors, since analyte binding to the cruciform invariably changes its HOMO-LUMO gap and the associated optical properties. Using this principle, Bunz and Miljani&#263; groups developed 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole cruciforms, respectively, which act as fluorescent sensors for metal ions, carboxylic acids, boronic acids, phenols, amines, and anions. The emission colors observed when these cruciform are mixed with analytes are highly sensitive to the details of analyte&#39;s structure and - because of cruciforms&#39; charge-separated excited states - to the solvent in which emission is observed. Structurally closely related species can be qualitatively distinguished within several analyte classes: (a) carboxylic acids; (b) boronic acids, and (c) metals. Using a hybrid sensing system composed from benzobisoxazole cruciforms and boronic acid additives, we were also able to discern among structurally similar: (d) small organic and inorganic anions, (e) amines, and (f) phenols. The method used for this qualitative distinction is exceedingly simple. Dilute solutions (typically 10-6 M) of cruciforms in several off-the-shelf solvents are placed in UV/Vis vials. Then, analytes of interest are added, either directly as solids or in concentrated solution. Fluorescence changes occur virtually instantaneously and can be recorded through standard digital photography using a semi-professional digital camera in a dark room. With minimal graphic manipulation, representative cut-outs of emission color photographs can be arranged into panels which permit quick naked-eye distinction among analytes. For quantification purposes, Red/Green/Blue values can be extracted from these photographs and the obtained numeric data can be statistically processed.

Cross-conjugated cruciform fluorophores based on 1,4-distyryl-2,5-bis(arylethynyl)benzene and benzobisoxazole nuclei can be used to qualitatively identify diverse Lewis acidic and Lewis basic analytes. This method relies on the differences in emission colors of the cruciforms that are observed upon analyte addition. Structurally closely related species can be distinguished from each other.

使用十字型荧光基团的羧酸，硼酸，胺的定性鉴别

Molecular cruciforms are X-shaped systems in which two conjugation axes intersect at a central core. If one axis of these molecules is substituted with ...

科研

化学

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type.

定量蛋白质组学使用还原二甲基化的稳定同位素标记

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

Synthesis and Purification of Iodoaziridines Involving Quantitative Selection of the Optimal Stationary Phase for Chromatography

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. Diiodomethyllithium is prepared by the deprotonation of diiodomethane with LiHMDS, in a THF/diethyl ether mixture, at -78 &#176;C in the dark. These conditions are essential for the stability of the LiCHI2 reagent generated. The subsequent dropwise addition of N-Ts aldimines to the preformed diiodomethyllithium solution affords an amino-diiodide intermediate, which is not isolated. Rapid warming of the reaction mixture to 0 &#176;C promotes cyclization to afford iodoaziridines with exclusive cis-diastereoselectivity. The addition and cyclization stages of the reaction are mediated in one reaction flask by careful temperature control.
Due to the sensitivity of the iodoaziridines to purification, assessment of suitable methods of purification is required. A protocol to assess the stability of sensitive compounds to stationary phases for column chromatography is described. This method is suitable to apply to new iodoaziridines, or other potentially sensitive novel compounds. Consequently this method may find application in range of synthetic projects. The procedure involves firstly the assessment of the reaction yield, prior to purification, by 1H NMR spectroscopy with comparison to an internal standard. Portions of impure product mixture are then exposed to slurries of various stationary phases appropriate for chromatography, in a solvent system suitable as the eluent in flash chromatography. After stirring for 30 min to mimic chromatography, followed by filtering, the samples are analyzed by 1H NMR spectroscopy. Calculated yields for each stationary phase are then compared to that initially obtained from the crude reaction mixture. The results obtained provide a quantitative assessment of the stability of the compound to the different stationary phases; hence the optimal can be selected. The choice of basic alumina, modified to activity IV, as a suitable stationary phase has allowed isolation of certain iodoaziridines in excellent yield and purity.

A protocol for the diastereoselective one-pot preparation of cis-N-Ts-iodoaziridines is described. The generation of diiodomethyllithium, addition to N-Ts aldimines and cyclization of the amino gem-diiodide intermediate to iodoaziridines is demonstrated. Also included is a protocol to rapidly and quantitatively assess the most appropriate stationary phase for purification by chromatography.

合成和Iodoaziridines纯化涉及定量选择最优固定相的色谱

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. ...

Synthesis and Exfoliation of Discotic Zirconium Phosphates to Obtain Colloidal Liquid Crystals

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and engineers. Growth of such anisotropic nanocrystals through a simple chemical method is a challenging task. In this study, we fabricate discotic nanodisks of zirconium phosphate [Zr(HPO4)2&#183;H2O] as a model material using hydrothermal, reflux and microwave-assisted methods. Growth of crystals is controlled by duration time, temperature, and concentration of reacting species. The novelty of the adopted methods is that discotic crystals of size ranging from hundred nanometers to few micrometers can be obtained while keeping the polydispersity well within control. The layered discotic crystals are converted to monolayers by exfoliation with tetra-(n)-butyl ammonium hydroxide [(C4H9)4NOH, TBAOH]. Exfoliated disks show isotropic and nematic liquid crystal phases. Size and polydispersity of disk suspensions is highly important in deciding their phase behavior.

A two dimensional model material of discotic zirconium phosphate was developed. The inorganic crystal with lamellar structure was synthesized by hydrothermal, reflux, and microwave-assisted methods. On exfoliation with organic molecules, layered crystals can be converted to monolayers, and nematic liquid crystal phase was formed at sufficient concentration of monolayers.

合成与盘状磷酸盐锆剥落获取胶体液晶

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and ...

A Quantitative Glycomics and Proteomics Combined Purification Strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

定量糖组学和蛋白质组学组合式净化策略

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

用尺寸排阻ICP-MS标准定量分析Metalloproteomic

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

洞察氨基酸和多肽与无机材料的使用相互作用的单分子力谱

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Facet-to-facet Linking of Shape-anisotropic Colloidal Cadmium Chalcogenide Nanostructures

Here, we describe a protocol that allows for shape-anisotropic cadmium chalcogenide nanocrystals (NCs), such as nanorods (NRs) and tetrapods (TPs), to be covalently and site-specifically linked via their end facets, resulting in polymer-like linear or branched chains. The linking procedure begins with a cation-exchange process in which the end facets of the cadmium chalcogenide NCs are first converted to silver chalcogenide. This is followed by the selective removal of ligands at their surface. This results in cadmium chalcogenide NCs with highly reactive silver chalcogenide end facets that spontaneously fuse upon contact with each other, thereby establishing an interparticle facet-to-facet attachment. Through the judicious choice of precursor concentrations, an extensive network of linked NCs can be produced. Structural characterization of the linked NCs is carried out via low- and high-resolution transmission electron microscopy (TEM), as well as energy-dispersive X-ray spectroscopy, which confirm the presence of silver chalcogenide domains between chains of cadmium chalcogenide NCs.

A protocol detailing how shape-anisotropic colloidal cadmium chalcogenide nanocrystals can be covalently linked via their end facets is presented here.

形状-各向异性胶体镉硫纳米结构的 Facet-to-facet 连接

Here, we describe a protocol that allows for shape-anisotropic cadmium chalcogenide nanocrystals (NCs), such as nanorods (NRs) and tetrapods (TPs), to be ...

Photodeposition of Pd onto Colloidal Au Nanorods by Surface Plasmon Excitation

A protocol is described to photocatalytically guide Pd deposition onto Au nanorods (AuNR) using surface plasmon resonance (SPR). Excited plasmonic hot electrons upon SPR irradiation drive reductive deposition of Pd on colloidal AuNR in the presence of [PdCl4]2-. Plasmon-driven reduction of secondary metals potentiates covalent, sub-wavelength deposition at targeted locations coinciding with electric field &#8220;hot-spots&#8221; of the plasmonic substrate using an external field (e.g., laser). The process described herein details a solution-phase deposition of a catalytically-active noble metal (Pd) from a transition metal halide salt (H2PdCl4) onto aqueously-suspended, anisotropic plasmonic structures (AuNR). The solution-phase process is amenable to making other bimetallic architectures. Transmission UV-vis monitoring of the photochemical reaction, coupled with ex situ XPS and statistical TEM analysis, provide immediate experimental feedback to evaluate properties of the bimetallic structures as they evolve during the photocatalytic reaction. Resonant plasmon irradiation of AuNR in the presence of [PdCl4]2- creates a thin, covalently-bound Pd0 shell without any significant dampening effect on its plasmonic behavior in this representative experiment/batch. Overall, plasmonic photodeposition offers an alternative route for high-volume, economical synthesis of optoelectronic materials with sub-5 nm features (e.g., heterometallic photocatalysts or optoelectronic interconnects).

A protocol for anisotropic photodeposition of Pd onto aqueously-suspended Au nanorods via localized surface plasmon excitation is presented.

通过表面等离子体激发将 Pd 光沉积到胶体 Au 纳米棒上

A protocol is described to photocatalytically guide Pd deposition onto Au nanorods (AuNR) using surface plasmon resonance (SPR). Excited plasmonic hot ...

Facile Synthesis of Colloidal Lead Halide Perovskite Nanoplatelets via Ligand-Assisted Reprecipitation

In this work, we demonstrate a facile method for colloidal lead halide perovskite nanoplatelet synthesis (Chemical formula: L2[ABX3]n-1BX4, L: butylammonium and octylammonium, A: methylammonium or formamidinium, B: lead, X: bromide and iodide, n: number of [BX6]4- octahedral layers in the direction of nanoplatelet thickness) via ligand-assisted reprecipitation. Individual perovskite precursor solutions are prepared by dissolving each nanoplatelet constituent salt in N,N-dimethylformamide (DMF), which is a polar organic solvent, and then mixing in specific ratios for targeted nanoplatelet thickness and composition. Once the mixed precursor solution is dropped into nonpolar toluene, the abrupt change in the solubility induces the instantaneous crystallization of nanoplatelets with surface-bound alkylammonium halide ligands providing colloidal stability. Photoluminescence and absorption spectra reveal emissive and strongly quantum-confined features. X-ray diffraction and transmission electron microscopy confirm the two-dimensional structure of the nanoplatelets. Furthermore, we demonstrate that the band gap of perovskite nanoplatelets can be continuously tuned in the visible range by varying the stoichiometry of the halide ion(s). Lastly, we demonstrate the flexibility of the ligand-assisted reprecipitation method by introducing multiple species as surface-capping ligands. This methodology represents a simple procedure for preparing dispersions of emissive 2D colloidal semiconductors.

This work demonstrates facile room-temperature synthesis of colloidal quantum-confined lead halide perovskite nanoplatelets by ligand-assisted reprecipitation method. Synthesized nanoplatelets show spectrally narrow optical features and continuous spectral tunability throughout the visible range by varying the composition and thicknesses.