作者想约翰Grazul（康奈尔大学），安德烈Marziali（在温哥华不列颠哥伦比亚大学）和外袍，科萨文森特（渥太华大学），以感谢他们的意见。这项工作是由美国国家科学基金会（DMR - 0706517和DMR - 1006332）和国立卫生研究院（R01 GM088403）资助部分经费。纳米孔钻在康奈尔大学材料研究中心（CCMR）从国家科学基金会支持的电子显微镜机制 - 材料研究科学和工程中心（MRSEC）程序（DMR 0520404）。氮化硅膜的制备是在康奈尔大学的纳米基金，国家纳米技术基础设施网络的成员，这是由国家科学基金会（格兰特ECS - 0335765）的支持。

1。氮化硅膜固态纳米孔的制造<ol><li>把费Tecnai F20的S /透射电镜一个200千伏的加速电压。如果使用不同的S /透射电子显微镜，加速电压应大于或等于200千伏9 </li><li> TEM样品架装入一个20纳米厚的SPI氮化硅窗口电网和清洁除去持有​​人的任何污染物与氧气30秒的等离子体。 </li><li>装入的S / TEM样品，并允许为真空抽空。一旦S /透射电镜抽空真空，发现在明场TEM模式通过寻找光明广场上的Ronchigram的氮化硅窗口。确保透射电镜正确对齐，然后调整和重点样本。 </li><li>切换成干模式的S / TEM。使用HAADF（或同等学历）探测器的图像样本，并确保它是正确对齐。 </li><li>的单色设置为较低的值。一个单色低的值允许为一个更高的CURRENT为钻探提供的电子。 S /透射电镜如果没有一个单色，忽略这一步。 </li><li>选择适当的位置钻纳米孔大小。与此对应的电子探针的直径。 3纳米探头为钻孔直径为5 nm的毛孔。一个较大的探头（5 nm）的钻更快速地创建一个较大的孔隙。一个较小的探头（1纳米）将需要更长的时间进行钻孔，创建一个较小的孔隙。 </li><li>调整干线路走向，如果必要的话。 </li><li>焦点氮化物膜，并把它放大的x1.3M。微调焦应通过监测Ronchigram。 </li><li>如果有任何运动的样品，然后等待样品稳定。这可能需要长达一个小时。而等待，以免破坏氮化物空白的电子束。 </li><li>电子探针和样品开始钻探。 </li><li>定期检查与HAADF探测器扫描样品，既要确保纳米孔是被DRIlled（它看起来像一个暗斑），并检查有没有样品的运动。如果样品略有漂移，调整电子探针在纳米孔的位置。 </li><li>观看Ronchigram确定何时已形成的孔隙。当焦点，氮化物膜有一个闪闪发光的外观。这会消失的纳米孔形式时。把重点Ronchigram略有允许一看菲涅尔纳米孔的边缘。 </li><li>以纳米孔与HAADF形象。 </li><li>做一个纳米孔图像的强度分布。纳米孔的直径可估计的剖面上最黑暗的地区。 </li><li>扩大纳米孔可通过移动电子探针沿孔隙边缘。 </li><li>一旦所需的直径是纳米孔，放入TEM模式的S / TEM。 </li><li>其标准的制定和图像使用明场透射电子显微镜确认大小的纳米孔带来的单色<strong（图1）。 </li></ol> 2。润湿的固态纳米孔<ol><li>德气体的去离子水，含有氯化钾，1米10 mM磷酸钾，pH值7.4。这可以通过在真空状态下的解决方案，并放置在20分钟洗澡sonicator。 </li><li>纳米孔含氮化硅芯片放入10毫升耐热烧杯。请注意不要打破氮化窗口，因为它是非常微妙的。烧杯放在一个烤盘在通风柜设置为100 ° C。 </li><li>清洁食人鱼纳米孔的芯片解决方案，使用非常谨慎。首先加入3毫升硫酸的容器，使用的玻璃吸管。接下来，仔细加1 ml双氧水，硫酸，使食人鱼解决方案14。请采取一切预防措施。 </li><li>允许的纳米孔的芯片，在食人鱼溶液浸泡10分钟。 </li><li>从烧杯中使用的玻璃吸管取出食人鱼解决方案。它放到一个适当的存储插座。 </li><li>连接LL与瓦斯的使用干净的玻璃吸管，去离子水的烧杯。空烧杯的水和重复至少5次。 </li><li>纳米孔的芯片，并用干净的镊子取出干轻吸</li><li>随即，密封进入会议厅（图2A，2B）纳米孔的芯片 。O形圈或硅酮密封胶，该芯片可在会议厅内担保。 </li><li>填写KCl溶液室</li><li>银/氯化银电极连接到室（图2b）。电极可浸泡在漂白隔夜银线。 </li><li>适用于整个电极跨膜电位和监测使用在电压钳模式轴突200B膜片钳放大器的电流响应。 </li><li>构造一个从测量的I / V（电流 - 电压）曲线。 I / V曲线高度线性的，当使用1米氯化钾（图3）。纳米孔电导应与它的直径。如果纳米孔不显示线性I / V曲线，电导过低，或不太稳定的纳米孔的开放电流（图4a），这意味着纳米孔是不正确的润湿和食人鱼洗应反复。 </li></ol> 3。监控蛋白质吸附<ol><li>这是非常重要的，执行与控制的单纳米孔膜或平行的纳米孔阵列（见下文），监察目前缺乏蛋白质样品与缓冲条件实验。我们建议以下两个步骤:（一）使用高纯度级缓冲（纯度&gt; 99.9％;色谱级别），及（ii）使用一个缓冲区，其互动的固态纳米孔不产生单通道事件。此外，我们建议获得高纯度的蛋白质样品，使用标准的蛋白质化学程序。蛋白质样品的纯度应检查十二烷基硫酸钠聚丙烯酰胺（SDS - PAGE）凝胶电泳分析和高分辨率的马SS法15。 </li><li>添加到接地浴室研究的蛋白质纯化样品。允许样品弥漫整个洗澡的时间。典型的蛋白质，可测浓度纳米几十μm范围7,15-21。 </li><li>套用一个跨膜电位偏压极性相反的被研究的蛋白质的总负责。例如，BSA在pH 7.4的一个有效的净负电荷，因此应适用于一个正电压偏置。外加电压相反电荷的纳米孔内部和蛋白质内创建电位梯度将被相反的力量驱动。只有驱动的蛋白质纳米孔的电压极性将建立可测量的信号。 </li><li>一个应用潜力为200 mV的幅度应该足够大，观察大多数蛋白质分析物。事件将出现从基线（图4b）的瞬态电流挠度 。如果没有信号，提高蛋白的浓度，在会议厅内。更高的电压，提高信噪比。氮化物纳米孔可以承受几伏。 </li><li>蛋白质吸附在纳米孔（图4c）的基准电流长寿命的变化会出现 。 </li></ol> 4。功能化的纳米孔的可能性功能团体申请氮化硅22,23存在的几种方法。大多数的氮化物薄膜有一个在外面，暴露在臭氧的一层薄氧化膜可以创建一个附加的氧化层，如果有必要的。可以使用不同的有机硅烷自组装到这样的层次。特别感兴趣的是氨基硅烷，自我组装并可以进一步修改（即，羧酸和醛类），允许检查范围的有机表面。在纳米孔已洗与食人鱼在chambe担保R，胺可以直接加入0.5 M的TBACl（四丁基）和用作溶剂的无水甲醇（甲醇）与支持电解质室洗澡。 400 mV的电压偏置和测量电流下降 23的应用可以监测单分子膜的形成。 5。表观一级反应速率常数的测定和Langmuir吸附常数使用并行纳米孔阵列 5.1表观一级反应吸附和解吸的速率常数我们强调积极的一面，这种方法是在单分子水平上观察个别吸附能力。到无机表面蛋白质的吸附单分子的测量，可以按比例增加由用人固态纳米孔平行阵列。并行阵列的孔洞是必要的，因为许多毛孔检测需要得到可靠的统计第为此，使用了纳米孔阵列将允许个别吸附的监测以及测量多个事件作进一步的分析。在样品上钻多个孔，只需通过上述协议，可能会形成氮化硅纳米孔阵列。每个纳米孔应大小相似。应有足够的6x6或7X7纳米孔阵列。除了在会议厅内的蛋白质分析物后，电流会以指数方式衰减与下面的表达式: I T =∞ - （我∞ -我0）EXP（- K"T） 在这里，I T，表示当前处于试验时间 t， 我 0是原来的电流通过纳米孔阵列。 我 ∞表示饱和程度的电流（即无穷大）。K&#39;是明显的一阶反应速率不变，这可以从日决定发送合适的实验曲线。更大K&#39;可以理解为一个更快的吸附率。比我 ∞/ I 0，也称为归饱和电流，是一个无量纲数0和1之间。此参数是衡量吸附的蛋白质分析物的占用。因此，每个不同的实验条件下，应与两个特定的输出参数 我 ∞/ 我 0和k&#39; 。 应该指出的是明显的一阶吸附速率常数和规范化的电流是由蛋白质所花的孔洞内的有效时间的影响。这一次是依赖于散装水相中的蛋白质 24分析物浓度。因此，需要根据由蛋白质所花的有效时间内的纳米孔内部实施额外的校正这些数字。我们建议测量频率的FAST（易位）事件和乘以平均停留时间等事件。这将使单位时间内的蛋白质在纳米孔的内部花的平均时间。 解吸速率常数可确定使用了纳米孔以及并行阵列。只要目前的水平达到饱和（∞），电压应颠倒过来，所以没有更多的蛋白质被困入孔洞。更改记录的电流走向更大的价值将伴随着个别蛋白的解吸。从目前的水平上升速率常数，然后将提取。 5.2 Langmuir吸附常数到孔洞的无机表面的蛋白质分析物的吸收是依赖于在水相中的蛋白浓度。这种现象已经观察到，在单分子水平 13 。如果θ代表<html>的正常化饱和电流（ 我 ∞/ 我 0），然后是一个典型的Langmuir等温方程由下式: <img alt="图5.2" src="/files/ftp_upload/3560/3560fig5_2.jpg" /> 其中 C是在水相中的蛋白浓度。α是Langmuir吸附常数。吸附结合能增加，温度下降，这不断增加。 θ的数据点的集合将采用并行阵列在各种蛋白质在水相中的浓度进行测量。分析数据应与其他技术相结合，以验证拟合Langmuir吸附常数的大小。此外，全面的原子分子动力学模拟25,26可能也被用于帮助所获得的实验数据的解释。 题目是"&gt; 6代表性的结果。 固态纳米孔的典型结果将会如下。开放孔隙当前应高度稳定， 如图所示。 4A。纳米孔的I / V特性，应高度1米氯化钾，磷酸钾，pH值7.4， 如图所示的线性 。 2。 I / V曲线的线性拟合的斜率将提供单一的纳米孔电导。电导有直接的关系，纳米孔直径应符合公式: <img alt="图方程" src="/files/ftp_upload/3560/3560figeqn.jpg" /> ， 其中 G是电导的纳米孔，D是其直径，l是其长度，σ是导电性的解决方案在会议厅内 14 。这个值应该匹配到30％以内。如果实在是太小，你的毛孔很可能不湿。此外蛋白质，快速的事件应该随之而来， 如图所示。 4B。普罗特EIN吸附是一个长期存在的电流图显示下降。 4C。一些蛋白质是高度不稳定，并进行内部孔隙内的结构转型。在这种情况下，长寿命的压降将伴随着快速波动。 <img alt="图1" src="/files/ftp_upload/3560/3560fig1.jpg" /> 图1。固态纳米孔采用一个Tecnai F20的S / TEM。在STEM模式下钻一个直径为20 nm的孔。图像拍摄于明场TEM模式。氮化物是30纳米厚。 <img alt="图2" src="/files/ftp_upload/3560/3560fig2.jpg" /> 图2。商会用于住房电阻脉冲测量一个单一的固态纳米孔A）商会和自由站立氮化窗口（TEM网格）的硅芯片。纳米孔钻成氮化物，装载前。红色O形圈，形成一个良好的小号EAL有关的芯片，它分隔两个浴场离子溶液。二）安置方案浴缸和电极与纳米孔分示意图。 <img alt="图3" src="/files/ftp_upload/3560/3560fig3.jpg" /> 图3典型的I / V不同直径的固态纳米孔的痕迹 。单通道电气痕迹1米氯化钾，20毫米的Tris，pH值8.5 。 30 nm厚的氮化硅纳米孔钻。注意氮化物的厚度会影响单一的纳米孔电导。 <img alt="图4" src="/files/ftp_upload/3560/3560fig4.jpg" /> 图4。测量单通道电流的痕迹和蛋白质吸附检测A）的单通道电气跟踪显示当前打开一个直径为10纳米的纳米孔。二）当前挠度代表分区间，牛血清白蛋白（BSA）IOR的纳米孔。 c）对BSA吸附的表面氮化。从1米氯化钾，10 mM磷酸钾，pH值7.4纳米孔的所有痕迹。应用的跨膜电位为40 mV和BSA是添加浓度在120 nm的接地室洗澡。

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S., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+characterization+of+protein+molecules+by+a+solid-state+nanopore.">Electrical characterization of protein molecules by a solid-state nanopore.</a> Appl. Phys. Lett. 91, (2007).</li><li>Firnkes, M., Pedone, D., Knezevic, J., Doblinger, M., Rant, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrically+Facilitated+Translocations+of+Proteins+through+Silicon+Nitride+Nanopores:+Conjoint+and+Competitive+Action+of+Diffusion,+Electrophoresis,+and+Electroosmosis.">Electrically Facilitated Translocations of Proteins through Silicon Nitride Nanopores: Conjoint and Competitive Action of Diffusion, Electrophoresis, and Electroosmosis.</a> Nano. Lett. , (2010).</li><li>Pedone, D., Firnkes, M., Rant, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+analysis+of+translocation+events+in+nanopore+experiments.">Data analysis of translocation events in nanopore experiments.</a> Anal. Chem. 81, 9689-9694 (2009).</li><li>Oukhaled, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+Completely+Unfolded+and+Native+Proteins+through+Solid-State+Nanopores+as+a+Function+of+Electric+Driving+Force.">Dynamics of Completely Unfolded and Native Proteins through Solid-State Nanopores as a Function of Electric Driving Force.</a> ACS. Nano.. , (2011).</li><li>Yusko, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+protein+translocation+through+nanopores+with+bio-inspired+fluid+walls.">Controlling protein translocation through nanopores with bio-inspired fluid walls.</a> Nat. Nanotechnol. 6, 253-260 (2011).</li><li>Arafat, A., Schroen, K., de Smet, L. C., Sudholter, E. J., Zuilhof, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailor-made+functionalization+of+silicon+nitride+surfaces.">Tailor-made functionalization of silicon nitride surfaces.</a> J. Am. Chem. Soc. 126, 8600-8601 (2004).</li><li>Wanunu, M., Meller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+modified+solid-state+nanopores.">Chemically modified solid-state nanopores.</a> Nano. Lett. 7, 1580-1585 (2007).</li><li>Movileanu, L., Cheley, S., Bayley, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partitioning+of+individual+flexible+polymers+into+a+nanoscopic+protein+pore.">Partitioning of individual flexible polymers into a nanoscopic protein pore.</a> Biophys. J. 85, 897-910 (2003).</li><li>Aksimentiev, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+ionic+current+signatures+of+DNA+transport+through+a+nanopore.">Deciphering ionic current signatures of DNA transport through a nanopore.</a> Nanoscale. 2, 468-483 (2010).</li><li>Timp, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanopore+Sequencing:+Electrical+Measurements+of+the+Code+of+Life.">Nanopore Sequencing: Electrical Measurements of the Code of Life.</a> IEEE Trans. Nanotechnol. 9, 281-294 (2010).</li><li>Roach, P., Farrar, D., Perry, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+tailoring+for+controlled+protein+adsorption:+effect+of+topography+at+the+nanometer+scale+and+chemistry.">Surface tailoring for controlled protein adsorption: effect of topography at the nanometer scale and chemistry.</a> J. Am. Chem. Soc. 128, 3939-3945 (2006).</li><li>Roach, P., Farrar, D., Perry, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpretation+of+protein+adsorption:+surface-induced+conformational+changes.">Interpretation of protein adsorption: surface-induced conformational changes.</a> J. Am. Chem. Soc. 127, 8168-8173 (2005).</li><li>Brewer, S. H., Glomm, W. R., Johnson, M. C., Knag, M. K., Franzen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+BSA+binding+to+citrate-coated+gold+nanoparticles+and+surfaces.">Probing BSA binding to citrate-coated gold nanoparticles and surfaces.</a> Langmuir. 21, 9303-9307 (2005).</li><li>Schon, P., Gorlich, M., Coenen, M. J., Heus, H. A., Speller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonspecific+protein+adsorption+at+the+single+molecule+level+studied+by+atomic+force+microscopy.">Nonspecific protein adsorption at the single molecule level studied by atomic force microscopy.</a> Langmuir. 23, 9921-9923 (2007).</li><li>Rabe, M., Verdes, D., Zimmermann, J., Seeger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+organization+and+cooperativity+during+nonspecific+protein+adsorption+events.">Surface organization and cooperativity during nonspecific protein adsorption events.</a> J. Phys. Chem. B. 112, 13971-13980 (2008).</li><li>Rabe, M., Verdes, D., Rankl, M., Artus, G. R., Seeger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+study+of+concepts+and+phenomena+of+the+nonspecific+adsorption+of+beta-lactoglobulin.">A comprehensive study of concepts and phenomena of the nonspecific adsorption of beta-lactoglobulin.</a> Chemphyschem. 8, 862-872 (2007).</li><li>Micic, M., Chen, A., Leblanc, R. M., Moy, V. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+Electron+Microscopy+Studies+of+Protein-Functionalized+Atomic+Force+Microscopy+Cantilever+Tips.">Scanning Electron Microscopy Studies of Protein-Functionalized Atomic Force Microscopy Cantilever Tips.</a> Scanning. 21, 394-397 (1999).</li><li>Grant, A. W., Hu, Q. H., Kasemo, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+electron+microscopy+'windows'+for+nanofabricated+structures.">Transmission electron microscopy 'windows' for nanofabricated structures.</a> Nanotechnology. 15, 1175-1181 (2004).</li><li>Giannoulis, C. S., Desai, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+proteins+and+fibroblasts+on+thin+inorganic+films.">Characterization of proteins and fibroblasts on thin inorganic films.</a> J. Mater. Sci: Mater. Med. 13, 75-80 (2002).</li><li>Gustavsson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+modifications+of+silicon+nitride+for+cellular+biosensors+applications.">Surface modifications of silicon nitride for cellular biosensors applications.</a> J. Mater. Sci: Mater. Med. 19, 1839-1850 (2008).</li><li>Shirshov, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+response+of+planar+polarization+interferometer+to+molecular+layer+formation:+fibrinogen+adsorption+on+silicon+nitride+surface.">Analysis of the response of planar polarization interferometer to molecular layer formation: fibrinogen adsorption on silicon nitride surface.</a> Biosens. Bioelectron. 16, 381-390 (2001).</li><li>Chen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+layer+deposition+to+fine-tune+the+surface+properties+and+diamters+of+fabricated+nanopores.">Atomic layer deposition to fine-tune the surface properties and diamters of fabricated nanopores.</a> Nano. Lett. 4, 1333-1337 (2004).</li><li>Siwy, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+biosensors+based+on+biofunctionalized+conical+gold+nanotubes.">Protein biosensors based on biofunctionalized conical gold nanotubes.</a> J. Am. Chem. Soc. 127, 5000-5001 (2005).</li><li>Ding, S., Gao, C., Gu, L. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capturing+Single+Molecules+of+Immunoglobulin+and+Ricin+with+an+Aptamer-Encoded+Glass+Nanopore.">Capturing Single Molecules of Immunoglobulin and Ricin with an Aptamer-Encoded Glass Nanopore.</a> Anal. Chem. , (2009).</li><li>Kim, M. -. J., Wanunu, M., Bell, C. D., Meller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Fabrication+of+Uniform+Size+Nanopores+and+Nanopore+Arrays+for+Parallel+DNA+Analysis.">Rapid Fabrication of Uniform Size Nanopores and Nanopore Arrays for Parallel DNA Analysis.</a> Adv. Mater. 18, 3149-3153 (2006).</li><li>Bezrukov, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+channels+as+molecular+Coulter+counters+to+probe+metabolite+transport.">Ion channels as molecular Coulter counters to probe metabolite transport.</a> J. Membr. Biol. 174, 1-13 (2000).</li></ol>

我们什么都没有透露。

自发到固态表面27-29上的蛋白质吸附在一些领域，如生物芯片的应用和设计一个新的功能混合生物材料类，是从根本上重要的。以往的研究表明，吸附到固体表面的蛋白质不显示横向调动或显著解吸率，因此蛋白质的吸附通常被认为是一个不可逆转的和非特异性的过程30-32。到固态表面的蛋白质吸附被认为是由于多种因素， 包括 13在固-液界面的蛋白质和活性基团的侧链之?...

<table border="1"><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> Tecnai F20的S /透射电子显微镜</td><td>费</td><td></td><td> S /透射电子显微镜需要加速电压≥200KV和场发射源。 </td></tr><tr><td> 20纳米厚的氮化硅膜窗口用于TEM </td><td> SPI </td><td> 4163SN - BA </td><td></td></tr><tr><td>轴突Axopatch 200B中膜片钳放大器</td><td>分子器件</td><td></td><td></td></tr><tr><td>轴突Digidata 1440A </td><td>分子器件</td><td></td><td></td></tr><tr><td> pCLAMP 10软件</td><td>分子器件</td><td></td><td>电数据采集和分析软件</td></tr><tr><td>硫酸</td><td> Fisher科学</td><td> A300 </td><td></td></tr><tr><td>过氧化氢</td><td> Fisher Scientific则</td><td> H325 </td><td></td></tr><tr><td>矽O型环</td><td>麦克马斯特 - 卡尔</td><td> 003 S70 </td><td>或者使用的PDMS </td></tr><tr><td>银线</td><td> Sigma - Aldrich公司</td><td> 348759 </td><td>对于电极</td></tr><tr><td> SPC技术，D SUB接触，针</td><td>纽瓦克</td><td> 9K4978 </td><td>对于电极</td></tr><tr><td>氯化钾</td><td>西格玛</td><td> P9541 </td><td></td></tr><tr><td>磷酸钾二元</td><td>西格玛</td><td> P2222 </td><td></td></tr><tr><td>磷酸二氢钾</td><td>西格玛</td><td> P5379 </td><td></td></tr><tr><td>硅橡胶</td><td>道康宁</td><td></td><td> Sylgar 184 Ëlastomer设置。制作室。 </td></tr><tr><td>佳柏，演员密封胶</td><td>世界精密仪器</td><td> KWIK - CAST </td><td>快速代理硅酮密封胶</td></tr><tr><td>热板</td><td> Fisher Scientific则</td><td></td><td></td></tr><tr><td>法拉第笼</td><td></td><td></td><td></td></tr></table>

monitoring protein adsorption with solid-state nanopores

固态纳米孔已被用来执行在单分子水平测量，以考察当地的结构和灵活性 1-6核酸， 蛋白质 7开展，8种不同的配体结合的亲和力。通过这些孔洞耦合9-12电阻脉冲技术，这样的测量，可以做各种各样的条件下，没有标签 3 。电阻脉冲技术，离子的盐溶液中引入纳米孔的两侧。因此，离子驱动从一个会议厅一侧的其他应用的跨膜电位，造成在一个稳定的电流。分析物的纳米孔的分割，导致在当前的定义良好的偏转，它可以分析提取单分子信息。使用这种技术，纳米孔壁的单个蛋白质的吸附作用，可以广泛的监视下条件13。蛋白质吸附的重要性越来越大，因为微流体器件尺寸的缩小，这些系统与单一蛋白质的相互作用成​​为一个令人关注的问题。这一协议描述为氮化物薄膜，它可以很容易地扩展到其他薄膜进行纳米孔钻，或功能化氮化物表面蛋白结合的快速检测。下一个广泛的解决方案和变性条件下，可以探索多种蛋白质。此外，该协议可用于探索使用纳米孔光谱的基本问题。

Watch this Scientific Journal Video about Monitoring Protein Adsorption with Solid-state Nanopores at JoVE.com

Monitoring Protein Adsorption with Solid-state Nanopores

一个方法是使用固态纳米孔监测到无机表面蛋白质的非特异性吸附描述。该方法采用电阻脉冲的原则，允许实时和单分子水平探讨吸附。因为单一的蛋白质吸附过程是远离平衡，我们建议平行合成纳米孔阵列的就业，使表观一级反应速率常数蛋白质吸附定量测定以及和Langmuir吸附常数。

监测与固态纳米孔蛋白质吸附

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ...

monitoring-protein-adsorption-with-solid-state-nanopores

Research

JoVE Journal

Bioengineering

13.5K 次观看.  Syracuse University. 一个方法是使用固态纳米孔监测到无机表面蛋白质的非特异性吸附描述。该方法采用电阻脉冲的原则，允许实时和单分子水平探讨吸附。因为单一的蛋白质吸附过程是远离平衡，我们建议平行合成纳米孔阵列的就业，使表观一级反应速率常数蛋白质吸附定量测定以及和Langmuir吸附常数。

视频: Monitoring Protein Adsorption with Solid-state Nanopores

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

注册生物成像，纳米材料可用于诊断和治疗监测

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

科研

生物工程

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

荧光检测方法，为微流体液滴平台

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

在一个容器的支架部署在监测的墙力学

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

质量流式细胞术:协议，每日调整和执行细胞的样品上一个CyTOF的质量流式细胞仪

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Multi-analyte Biochip (MAB) Based on All-solid-state Ion-selective Electrodes (ASSISE) for Physiological Research

Lab-on-a-chip (LOC) applications in environmental, biomedical, agricultural, biological, and spaceflight research require an ion-selective electrode (ISE) that can withstand prolonged storage in complex biological media 1-4. An all-solid-state ion-selective-electrode (ASSISE) is especially attractive for the aforementioned applications. The electrode should have the following favorable characteristics: easy construction, low maintenance, and (potential for) miniaturization, allowing for batch processing. A microfabricated ASSISE intended for quantifying H+, Ca2+, and CO32- ions was constructed. It consists of a noble-metal electrode layer (i.e. Pt), a transduction layer, and an ion-selective membrane (ISM) layer. The transduction layer functions to transduce the concentration-dependent chemical potential of the ion-selective membrane into a measurable electrical signal.
The lifetime of an ASSISE is found to depend on maintaining the potential at the conductive layer/membrane interface 5-7. To extend the ASSISE working lifetime and thereby maintain stable potentials at the interfacial layers, we utilized the conductive polymer (CP) poly(3,4-ethylenedioxythiophene) (PEDOT) 7-9 in place of silver/silver chloride (Ag/AgCl) as the transducer layer. We constructed the ASSISE in a lab-on-a-chip format, which we called the multi-analyte biochip (MAB) (Figure 1).
Calibrations in test solutions demonstrated that the MAB can monitor pH (operational range pH 4-9), CO32- (measured range 0.01 mM - 1 mM), and Ca2+ (log-linear range 0.01 mM to 1 mM). The MAB for pH provides a near-Nernstian slope response after almost one month storage in algal medium. The carbonate biochips show a potentiometric profile similar to that of a conventional ion-selective electrode. Physiological measurements were employed to monitor biological activity of the model system, the microalga Chlorella vulgaris.
The MAB conveys an advantage in size, versatility, and multiplexed analyte sensing capability, making it applicable to many confined monitoring situations, on Earth or in space.
Biochip Design and Experimental Methods
The biochip is 10 x 11 mm in dimension and has 9 ASSISEs designated as working electrodes (WEs) and 5 Ag/AgCl reference electrodes (REs). Each working electrode (WE) is 240 &mu;m in diameter and is equally spaced at 1.4 mm from the REs, which are 480 &mu;m in diameter. These electrodes are connected to electrical contact pads with a dimension of 0.5 mm x 0.5 mm. The schematic is shown in Figure 2.
Cyclic voltammetry (CV) and galvanostatic deposition methods are used to electropolymerize the PEDOT films using a Bioanalytical Systems Inc. (BASI) C3 cell stand (Figure 3). The counter-ion for the PEDOT film is tailored to suit the analyte ion of interest. A PEDOT with poly(styrenesulfonate) counter ion (PEDOT/PSS) is utilized for H+ and CO32-, while one with sulphate (added to the solution as CaSO4) is utilized for Ca2+. The electrochemical properties of the PEDOT-coated WE is analyzed using CVs in redox-active solution (i.e. 2 mM potassium ferricyanide (K3Fe(CN)6)). Based on the CV profile, Randles-Sevcik analysis was used to determine the effective surface area 10. Spin-coating at 1,500 rpm is used to cast ~2 &mu;m thick ion-selective membranes (ISMs) on the MAB working electrodes (WEs).
The MAB is contained in a microfluidic flow-cell chamber filled with a 150 &mu;l volume of algal medium; the contact pads are electrically connected to the BASI system (Figure 4). The photosynthetic activity of Chlorella vulgaris is monitored in ambient light and dark conditions.

Multi-analyte Biochip MAB Based on All-solid-state Ion-selective Electrodes ASSISE for Physiological Research

All-solid-state ion-selective electrodes (ASSISEs) constructed from a conductive polymer (CP) transducer provide several months of functional lifetime in liquid media. Here, we describe the fabrication and calibration process of ASSISEs in a lab-on-a-chip format. The ASSISE is demonstrated to have maintained a near-Nernstian slope profile after prolonged storage in complex biological media.

多分析物生物芯片（MAB），基于全固态离子选择性电极（ASSISE）的生理研究

Lab-on-a-chip (LOC) applications in environmental, biomedical, agricultural, biological, and spaceflight research require an ion-selective electrode (ISE) ...

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

Metallic implants, especially titanium implants, are widely used in clinical applications. Tissue in-growth and integration to these implants in the tissues are important parameters for successful clinical outcomes. In order to improve tissue integration, porous metallic implants have being developed. Open porosity of metallic foams is very advantageous, since the pore areas can be functionalized without compromising the mechanical properties of the whole structure. Here we describe such modifications using porous titanium implants based on titanium microbeads. By using inherent physical properties such as hydrophobicity of titanium, it is possible to obtain hydrophobic pore gradients within microbead based metallic implants and at the same time to have a basement membrane mimic based on hydrophilic, natural polymers. 3D pore gradients are formed by synthetic polymers such as Poly-L-lactic acid (PLLA) by freeze-extraction method. 2D nanofibrillar surfaces are formed by using collagen/alginate followed by a crosslinking step with a natural crosslinker (genipin). This nanofibrillar film was built up by layer by layer (LbL) deposition method of the two oppositely charged molecules, collagen and alginate. Finally, an implant where different areas can accommodate different cell types, as this is necessary for many multicellular tissues, can be obtained. By, this way cellular movement in different directions by different cell types can be controlled. Such a system is described for the specific case of trachea regeneration, but it can be modified for other target organs. Analysis of cell migration and the possible methods for creating different pore gradients are elaborated. The next step in the analysis of such implants is their characterization after implantation. However, histological analysis of metallic implants is a long and cumbersome process, thus for monitoring host reaction to metallic implants in vivo an alternative method based on monitoring CGA and different blood proteins is also described. These methods can be used for developing in vitro custom-made migration and colonization tests and also be used for analysis of functionalized metallic implants in vivo without histology.

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

In this video, we will demonstrate modification techniques for porous metallic implants to improve their functionality and to control cell migration. Techniques include development of pore gradients to control cell movement in 3D and production of basement membrane mimics to control cell movement in 2-D. Also, a HPLC-based method for monitoring implant integration in-vivo via analysis of blood proteins is described.

多尺度孔梯度，聚电解质和间接监测改性金属植入物<em&gt;在体内</em

Metallic implants, especially titanium implants, are widely used in  clinical applications. Tissue in-growth and integration to these implants in  the ...

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic performance of microalgae-based biofuel production. Models that estimate microalgal growth under different conditions can help to optimize PBR design and operation. To be effective, the growth parameters used in these models must be accurately determined. Algal growth experiments are often constrained by the dynamic nature of the culture environment, and control systems are needed to accurately determine the kinetic parameters. The first step in setting up a controlled batch experiment is live data acquisition and monitoring. This protocol outlines a process for the assembly and operation of a bench-scale photosynthetic bioreactor that can be used to conduct microalgal growth experiments. This protocol describes how to size and assemble a flat-plate, bench-scale PBR from acrylic. It also details how to configure a PBR with continuous pH, light, and temperature monitoring using a data acquisition and control unit, analog sensors, and open-source data acquisition software.

This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

用于动态生长测试的温度，光照和pH监测的台式藻类光合生物反应器的构建和设置

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

在肽膜前体的维西图洛合成中,用于自主囊泡生长

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

Functional Transcranial Doppler Ultrasound for Monitoring Cerebral Blood Flow

Functional transcranial Doppler ultrasound (fTCD) is the use of transcranial Doppler ultrasound (TCD) to study neural activation occurring during stimuli such as physical movement, activation of tactile sensors in the skin, and viewing images. Neural activation is inferred from an increase in the cerebral blood flow velocity (CBFV) supplying the region of the brain involved in processing sensory input. For example, viewing bright light causes increased neural activity in the occipital lobe of the cerebral cortex, leading to increased blood flow in the posterior cerebral artery, which supplies the occipital lobe. In fTCD, changes in CBFV are used to estimate changes in cerebral blood flow (CBF).
With its high temporal resolution measurement of blood flow velocities in the major cerebral arteries, fTCD complements other established functional imaging techniques. The goal of this Methods paper is to give step-by-step instructions for using fTCD to perform a functional imaging experiment. First, the basic steps for identifying the middle cerebral artery (MCA) and optimizing the signal will be described. Next, placement of a fixation device for holding the TCD probe in place during the experiment will be described. Finally, the breath-holding experiment, which is a specific example of a functional imaging experiment using fTCD, will be demonstrated.

Functional transcranial Doppler ultrasound complements other functional imaging modalities, with its high temporal resolution measurement of stimulus-induced changes in cerebral blood flow within the basal cerebral arteries. This Methods paper gives step-by-step instructions for using functional transcranial Doppler ultrasound to perform a functional imaging experiment.&#160;

功能颅内多普勒超声监测脑血流

Functional transcranial Doppler ultrasound (fTCD) is the use of transcranial Doppler ultrasound (TCD) to study neural activation occurring during stimuli ...

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their cellular source, such as platelet lysate (PL). When used as treatment, EVs are expected to enter the target cells and effect a response from these. In this research, PL-derived EVs have been studied as a cell-free treatment for osteoarthritis (OA). Thus, a method was set up to label EVs and test their uptake on cartilage explants. PL-derived EVs are labeled with the lipophilic dye PKH26, washed twice through a column, and then tested in an in vitro inflammation-driven&#160;OA model for 5 h after particle quantification by nanoparticle tracking analysis (NTA). Hourly, cartilage explants are fixed, paraffined, cut into 6 &#181;m sections to mount on slides, and observed under a confocal microscope. This allows verification of whether EVs enter the target cells (chondrocytes) during this period and analyze their direct effect.

Here, we present a protocol to label platelet lysate-derived extracellular vesicles to monitor their migration and uptake in cartilage explants used as a model for osteoarthritis.