我们承认从美国国立卫生研究院（NHLBI合约编号HHSN268201000044C到RW，上海和SYS）的资助。

1。 GST和纳米粒子（NP）结合物的制备<ol><li> GST-TCO准备： <ol><li>添加8微升的TCO-NHS溶液（50毫米DMSO）100μL商品及服务税（1毫克/毫升的PBS）和摇动混合物在室温下1小时。 </li><li>使用Zeba旋转脱盐柱除去过量的试剂。存储包含GST-TCO共轭回收的滤液在4℃后才能使用。 </li></ol></li><li> GST-TZ准备： <ol><li>加入6微升TZ-NHS溶液（25mM的在DMF中），以75微升的GST（1毫克/毫升在PBS中），并摇动混合物在室温下1小时。 </li><li>用25微升PBS稀释反应混合物和纯化使用旋脱盐柱。存储包含GST-TZ共轭滤液在4℃后才能使用。 </li></ol> 注意：质谱分析（MALDI-TOF）表明，平均10〜TZ或TCO分子被缀合至GST。 </li><li> NP-TCO准备： <ol><li>加入100μl的TCO-NHS溶液（50毫米DMSO）为加入150μl胺化的纳米颗粒（NP-NH 2，8.7毫克铁/毫升的PBS中，3nm的铁心〜8000的Fe / NP），并摇动混合物在室温下1小时。 </li><li>通过使用NAP-10柱纯化，用PBS缓冲液凝胶过滤除去过量的试剂。收集含NP-TCO产品的色带。 </li><li>浓缩滤液，用离心过滤装置（100 K MWCO）〜150微升的最终体积。 </li><li>加50μl的琥珀酸酐，（在DMSO 0.1M）的NP-TCO溶液并摇动在RT 1小时（酐反应与任何剩余的胺，以形成终端的羧酸。这防止非特异性相互作用的葡聚糖表面）。 </li><li>使用NAP-10柱，用PBS洗脱纯化的NP-TCO产品。存储NP-TCO在4℃至使用前的解决方案。 </li></ol></li></ol> 2。表面处理所有的表面等离子共振测定法是在在Biacore T100仪器（GE Healthcare）上进行25℃下使用CM5传感器芯片和PBS-P作为运行缓冲液，除非另有说明。的Biacore控制和评价软件随仪器提供被用于建立实验和分析数据。两种操作模式，应用程序向导和手动运行将用于表面处理和监控芯片上捕获环。该方法构建器模式将被用于建立反向方向，结合实验和测量环加成反应速率。数据是双减去参考和动力学分析是使用1:1 Langmuir结合模式进行。 <ol><li>使用固定化胺模板向导，然后选择固定的流动细胞1（参考）和2（检测）。修改胺的方法通过注射的0.4M的EDC的1:1溶液，激活表面的羧基基团：0.1M NHS为480秒，以10微升/分钟的流速。将注射乙醇胺解渴剩余活性酯，以420秒的接触时间的10微升/分钟的流速。 </li><li>输入名称配体，抗-GST（​​在醋酸盐缓冲液，pH 5.0 18微克/毫升），并设置为以10微升/分钟的流速注入为420秒的接触时间。 </li><li>将所需的解决方案在小瓶试剂架2，确保以配合内容列表中的位置。保存并开始固定。 注：以这种方式导致的〜14,000至17,000 RU的范围内抗GST固定化水平运行的固定化向导。 </li><li>编辑固定化向导420秒，5微升/分钟以上参考流通池1只注入GST配体（20微克/毫升）的解决方案。 </li></ol> 3。监控片上捕获环官能分子<ol><li>监控芯片上捕获环加成实时（ 图2）。 <ol><li>放置GST-TCO（20微克/毫升），TZ-BnNH 2（10μM）和再生（10 1mM甘氨酸，pH值= 2.0）在试剂架2溶液的小瓶中。选择男人UAL run方法，设置流量5微升/分钟和流动路径流动池2。 </li><li>注入GST-TCO的解决方案，为420秒。 </li><li>注入TZ-BnNH 2溶液600秒。 </li><li>再生的表面有两个30秒的注射再生解决方案。 </li></ol></li><li>监测多组分结构的实时（ 图3）的片上组件： <ol><li>放置GST-TZ（20微克/毫升），NP-TCO（100微克的Fe /毫升），TZ-BnNH 2（10μM）和再生（10 1mM甘氨酸，pH值= 2.0）在试剂架2溶液的小瓶中。选择仪器手动运行方法，设置流量5微升/分钟和流路径流动池2。 </li><li>注入GST-TZ的60秒的解决方案。 </li><li>注入的NP-TCO的60秒的解决方案。 </li><li>注入TZ-BnNH 2溶液60秒。 </li><li>再生的表面有两个30秒的注射再生解决方案。 </li></ol></li></ol> 4。 Monitoring环加成率的固定化密度和测定<ol><li>小分子环加成反应速率（ 图4）表征。 <ol><li>选择新的方法和一般的输入参数。在试验的步骤面板创建一个新的台阶，并命名为样本。设置目的和连接基设置到样品。 </li><li>在周期类型面板创建一个新的循环步骤，并插入下面的命令，采集，样品，再生1和再生2。 </li><li>选择捕捉命令，输入名称的配体（GST-TCO，20微克/毫升），接触时间120秒，5微升/分钟，并设置流动路径为：二。选择采样命令;输入180秒的接触时间，60秒的解离时间，加入30μl/分钟的流速，并设置流路来：两个。选择再生1命令，输入再生解决方案的名称（10 mM的甘氨酸 - 盐酸pH值2.0），30秒，30微升/分钟，并设置流动路径，接触时间：二。对于再生2命令重复相同的。&lt;/ LI&gt; <li>选择安装运行，并设置流动路径为：2-1。选择下一个，并填写抽样单与分析解决方案（TZ-BnNH 2）和浓度系列（0.3-10微米，1:2稀释）。选择下一个机架位置面板。在试剂架2和系列稀释液在96孔板处溶液的小瓶，确保以匹配的内容列表的位置。保存方法模板，并开始结合分析。结合数据和动力学分析示于图4。 </li></ol></li><li>的NP环加成反应速率（ 图4）表征。 <ol><li>从上面打开保存的方法模板并更改以下参数。选择捕捉面板，改变配体名称与GST-TZ，20微克/毫升。选择样本面板;改变接触的时间为120秒的接触时间和离解时间为120秒。 </li><li>选择示例清单;改变分析物的NP-TCO和浓度系列（7〜224纳米，1:2稀释）。在试剂架到位的解决方案小瓶2和稀释系列在96孔板中，确保以匹配的内容列表的位置。保存方法模板，并开始结合分析。结合数据和动力学分析示于图4。 </li></ol></li></ol> 5。测量FKBP12的结合固定AP1497 逆方向的结合研究使用FKBP12作为分析物和化合物AP1497作为固定的配体（ 图5）。这个实验的一般方法是设置使用的方法构建工具如下： <ol><li>选择新的方法和一般的输入参数。在试验的步骤面板创建和命名捕获，采样和再生步骤。选择相应的目的和连接的基础设置。 </li><li>选择周期类型面板并创建3个循环步骤：采集，采样和再生。 </li><li>将捕捉命令两次下捕捉循环步骤。选择捕捉1面板并选择采集解决方案为variablE，设置接触时间为300秒，5微升/分钟，并设置流路的流量：二。选择捕捉2面板并选择捕获溶液作为变量，设置接触时间，以250秒为5微升/分钟，并设置流路的流速：第二。 </li><li>将样品指挥下采样周期的一步。选择的样品板;组接触时间为60秒，解离时间为200秒以30微升/分钟的流速，并设置流路：两者。 </li><li>将再生命令两次下的再生循环步骤。选择再生1面板;输入再生解决方案的名称（10 mM的甘氨酸 - 盐酸pH值2.0），30秒，30微升/分钟，并设置流动路径，接触时间：二。对于再生2面板重复相同的。 </li><li>选择安装运行，并设置流动路径为：2-1。选择一个和输入捕捉解决方案的名称（GST-TCO和AP1497-TZ），样品名称（FKBP12），浓度系列（0.020〜5微克/毫升，1:2稀释）和MW（13,000）。在r发布的解决方案小瓶在96孔板eagent机架2和稀释系列并确保匹配的内容列表的位置。保存方法模板，并开始结合分析。动力学分析数据示于图5。 </li></ol> 6。测量FKBP12的结合，以AP1497附在纳米粒子固定化对于纳米粒子固定化，小分子衍生化FKBP12结合分析的一般方法是设置使用该方法构建工具如下： <ol><li>从上面打开保存的方法模板和修改。插入一个额外的捕获指挥下捕获周期的一步。选择捕捉1面板;取消选择捕获解决方案，因为变数和姓名采集解决方案1（GST-TZ）。一套接触时间为60秒，5微升/分钟的流速，并设置流路到二。选择捕捉2面板;取消选择捕获解决方案，因为变数和姓名采集解决方案2（NP-TCO）。一套接触时间90秒，5微升/分钟的流速和设置流路第二。选择捕捉3面板;取消选择捕获解决方案，因为变数和姓名采集解决方案3（AP1497-TZ）。一套接触时间180秒以5微升/分钟的流速，并设置流路到二。 </li><li>选择安装运行，并设置流动路径为：2-1。选择下一个两次。在试剂架2和系列稀释液在96孔板处溶液的小瓶，确保以匹配的内容列表的位置。保存方法模板，并开始结合分析。动力学分析数据示于图5。 </li></ol>

<ol>
	<li>Devaraj, N. K., Upadhyay, R., Haun, J. B., Hilderbrand, S. A., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+sensitive+pretargeted+labeling+of+cancer+cells+through+a+tetrazine/trans-cyclooctene+cycloaddition.">Fast and sensitive pretargeted labeling of cancer cells through a tetrazine/trans-cyclooctene cycloaddition.</a> Angew Chem Int Ed Engl. 48, 7013-7016 (2009).</li><li>Seitchik, J. L., 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=Genetically+encoded+tetrazine+amino+acid+directs+rapid+site-specific+in+vivo+bioorthogonal+ligation+with+trans-cyclooctenes.">Genetically encoded tetrazine amino acid directs rapid site-specific in vivo bioorthogonal ligation with trans-cyclooctenes.</a> J Am Chem Soc. 134, 2898-2901 (2012).</li><li>Haun, J. B., Devaraj, N. K., Marinelli, B. S., Lee, H., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+intracellular+biomarkers+and+mediators+of+cell+activation+using+nanosensors+and+bioorthogonal+chemistry.">Probing intracellular biomarkers and mediators of cell activation using nanosensors and bioorthogonal chemistry.</a> ACS Nano. 5, 3204-3213 (2011).</li><li>Budin, G., Yang, K. S., Reiner, T., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioorthogonal+probes+for+polo-like+kinase+1+imaging+and+quantification.">Bioorthogonal probes for polo-like kinase 1 imaging and quantification.</a> Angew Chem Int Ed Engl. 50, 9378-9381 (2011).</li><li>Liu, D. S., Tangpeerachaikul, A., Selvaraj, R., Taylor, M. T., Fox, J. M., Ting, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diels-Alder+cycloaddition+for+fluorophore+targeting+to+specific+proteins+inside+living+cells.">Diels-Alder cycloaddition for fluorophore targeting to specific proteins inside living cells.</a> J Am Chem Soc. 134, 792-795 (2012).</li><li>Haun, J. B., Devaraj, N. K., Hilderbrand, S. A., Lee, H., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioorthogonal+chemistry+amplifies+nanoparticle+binding+and+enhances+the+sensitivity+of+cell+detection.">Bioorthogonal chemistry amplifies nanoparticle binding and enhances the sensitivity of cell detection.</a> Nat Nanotechnol. 5, 660-665 (2010).</li><li>Liong, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+pathogen+detection+using+bioorthogonal+chemistry+and+diagnostic+magnetic+resonance.">Specific pathogen detection using bioorthogonal chemistry and diagnostic magnetic resonance.</a> Bioconjug Chem. 22, 2390-2394 (2011).</li><li>Tassa, C., 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=On-chip+bioorthogonal+chemistry+enables+immobilization+of+in+situ+modified+nanoparticles+and+small+molecules+for+label-free+monitoring+of+protein+binding+and+reaction+kinetics.">On-chip bioorthogonal chemistry enables immobilization of in situ modified nanoparticles and small molecules for label-free monitoring of protein binding and reaction kinetics.</a> Lab Chip. 12, 3103-3110 (2012).</li><li>Pol, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+correct+protein+concentration+for+kinetics+and+affinity+determination+in+structure-function+analysis.">The importance of correct protein concentration for kinetics and affinity determination in structure-function analysis.</a> J Vis Exp. (37), e1746 (2010).</li><li>MacBeath, G., Schreiber, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Printing+proteins+as+microarrays+for+high-throughput+function+determination.">Printing proteins as microarrays for high-throughput function determination.</a> Science. 289, 1760-1763 (2000).</li><li>Ong, S. E., 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=Identifying+the+proteins+to+which+small-molecule+probes+and+drugs+bind+in+cells.">Identifying the proteins to which small-molecule probes and drugs bind in cells.</a> Proc Natl Acad Sci U S A. 106, 4617-4622 (2009).</li><li>Tassa, C., 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=Binding+affinity+and+kinetic+analysis+of+targeted+small+molecule-modified+nanoparticles.">Binding affinity and kinetic analysis of targeted small molecule-modified nanoparticles.</a> Bioconjug Chem. 21, 14-19 (2010).</li><li>Weissleder, R., Kelly, K., Sun, E. Y., Shtatland, T., Josephson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+targeting+of+nanoparticles+by+multivalent+attachment+of+small+molecules.">Cell-specific targeting of nanoparticles by multivalent attachment of small molecules.</a> Nat Biotechnol. 23, 1418-1423 (2005).</li><li>Yuan, J., Oliver, R., Aguilar, M. I., Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+plasmon+resonance+assay+for+chloramphenicol.">Surface plasmon resonance assay for chloramphenicol.</a> Anal Chem. 80, 8329-8333 (2008).</li><li>Myung, J. H., Gajjar, K. A., Saric, J., Eddington, D. T., Hong, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendrimer-mediated+multivalent+binding+for+the+enhanced+capture+of+tumor+cells.">Dendrimer-mediated multivalent binding for the enhanced capture of tumor cells.</a> Angew Chem Int Ed Engl. 50, 11769-11772 (2011).</li><li>Keelan, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanotoxicology:+nanoparticles+versus+the+placenta.">Nanotoxicology: nanoparticles versus the placenta.</a> Nat Nanotechnol. 6, 263-264 (2011).</li><li>Lundqvist, M., Stigler, J., Elia, G., Lynch, I., Cedervall, T., Dawson, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+size+and+surface+properties+determine+the+protein+corona+with+possible+implications+for+biological+impacts.">Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts.</a> Proc Natl Acad Sci U S A. 105, 14265-14270 (2008).</li><li>Lundqvist, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+the+protein+corona+around+nanoparticles:+a+test+study.">The evolution of the protein corona around nanoparticles: a test study.</a> ACS Nano. 5, 7503-7509 (2011).</li><li>Mammen, M., Choi, S. -. K., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyvalent+interactions+in+biological+systems:+implications+for+design+and+use+of+multivalent+ligands+and+inhibitors.">Polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors.</a> Angew Chem Int Ed Engl. 37, 2754-2899 (1998).</li><li>Kausaite-Minkstimiene, A., Ramanaviciene, A., Kirlyte, J., Ramanavicius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+random+and+oriented+antibody+immobilization+techniques+on+the+binding+capacity+of+immunosensor.">Comparative study of random and oriented antibody immobilization techniques on the binding capacity of immunosensor.</a> Anal Chem. 82, 6401-6408 (2010).</li><li>Arkin, M. R., Wells, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+inhibitors+of+protein-protein+interactions:+progressing+towards+the+dream.">Small-molecule inhibitors of protein-protein interactions: progressing towards the dream.</a> Nat Rev Drug Discov. 3, 301-317 (2004).</li><li>Giannetti, A. M., Koch, B. D., Browner, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+plasmon+resonance+based+assay+for+the+detection+and+characterization+of+promiscuous+inhibitors.">Surface plasmon resonance based assay for the detection and characterization of promiscuous inhibitors.</a> J Med Chem. 51, 574-580 (2008).</li><li>Kimple, A. J., Willard, F. S., Giguere, P. M., Johnston, C. A., Mocanu, V., Siderovski, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RGS+protein+inhibitor+CCG-4986+is+a+covalent+modifier+of+the+RGS4+Galpha-interaction+face.">The RGS protein inhibitor CCG-4986 is a covalent modifier of the RGS4 Galpha-interaction face.</a> Biochim Biophys Acta. 1774, 1213-1220 (2007).</li><li>GE Healthcare. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biacore Sensor Surface Handbook BR-1005-71. , (2005).</li></ol>

作者宣称，他们有没有竞争的财务权益。

这里所描述的捕获环加成方法使改性纳米粒子和小分子无标记芯片为基础的互动和动力学研究的快速，可逆固定。固定化的协议可在要求&lt;10μM浓度的小分子配体分钟来进行。通过调节配体浓度和接触时间的固定化密度可以严格控制。我们的数据显示，芯片上的生物正交反应保持原位功能化的纳米颗粒或固定化小分子与他们的目标交互的能力。我们还常表现为两种小分子，（总拥有成本和T...

高效的偶联反应是用于将生物活性分子到表面上用于各种生物技术应用有价值的工具。最近，非常快的生物正交反式 -环辛烯（TCO）之间[4 +2]环加成反应和1,2,4,5 -四嗪（TZ）已被用于标记细胞的表面，亚细胞结构，抗体和纳米粒子1 - 7在这里，我们结合使用的[4 +2]环加成反应与抗原/抗体捕获（GST /抗-GST）为可逆的片上合成的表面等离子体共振（SPR）的相互作用分析多组分结构及监察过程中的实时（ 图1）。8,9值得注意的是，使用一个既定的协议捕获环加成策略使表面再生。8作为稳定的传感器表面有超过配体的方向和密度控制各种新的实验的结果，组装格式现在是可能的。运用这一战略，我们展示的TCO / TZ-衍生的小分子的固定化和在各种缓冲液条件表征环加成率。我们选择了FKBP12和分子AP1497之间的公知的相互作用是FKBP12 10-12结合，例如，以验证该捕集环加成策略保留了小分子与其靶相互作用的能力时，无论是直接连接到固定的GST抗原或与固定化的纳米颗粒（纳米）。 该方法提供了几个优点。首先，小分子上的传感器芯片中的可逆固定化成为可能。第二，小分子的TCO / TZ的固定化也使无标记相互作用的研究，反向规范化SPR研究的取向，并且可以提供一种结合相互作用的互补视图。第三，这种方法可以使微流体合成靶向纳米粒子，以及它们的结合蛋白的直接评定克属性。这有望改善的评价或筛选靶向纳米粒子的效率，也减少了所需的纳米颗粒的量。13-15第四，这种方法可以在连续流动测量实时的生物正交环加成反应的反应动力学。最后，在TCO / TZ固定化化学是健壮的血清中的存在。综上所述，我们预计，这种多功能的方法将广泛地施工方便稳定的传感器表面的各种与微流控相关研究，以在体外和体内细胞的应用。

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Sensor Chip CM5</td>
			<td>GE Healthcare</td>
			<td>BR-1005-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Amine coupling kit</td>
			<td>GE Healthcare</td>
			<td>BR-1000-50</td>
			<td></td>
		</tr>
		<tr>
			<td>GST capture kit</td>
			<td>GE Healthcare</td>
			<td>BR-1002-23</td>
			<td></td>
		</tr>
		<tr>
			<td>NAP-10 Columns</td>
			<td>GE Healthcare</td>
			<td>17-0854-01</td>
			<td></td>
		</tr>
		<tr>
			<td>GST, lyophilized in 1X PBS</td>
			<td>Genscript</td>
			<td>Z02039</td>
			<td>1 mg/ml</td>
		</tr>
		<tr>
			<td>rhFKBP12</td>
			<td>R&#38;D Systems</td>
			<td>3777-FK</td>
			<td></td>
		</tr>
		<tr>
			<td>Surfactant P-20</td>
			<td>GE Healthcare</td>
			<td>BR-1000-54</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine 2.0</td>
			<td>GE Healthcare</td>
			<td>BR-1003-55</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeba spin desalting column</td>
			<td>Thermo</td>
			<td>89882</td>
			<td>7 K MWCO</td>
		</tr>
		<tr>
			<td>Amicon Ultra 4</td>
			<td>Fisher</td>
			<td>UFC810096</td>
			<td>100 K centrifugal filter</td>
		</tr>
		<tr>
			<td>TCO-OH</td>
			<td>Ref. 8</td>
			<td></td>
			<td>Synthesized in-house</td>
		</tr>
		<tr>
			<td>TCO-NHS</td>
			<td>Ref. 8</td>
			<td></td>
			<td>Synthesized in-house, *Commercially available from Click Chemistry Tools # 1016-25</td>
		</tr>
		<tr>
			<td>Tz-BnNH2</td>
			<td>Ref. 8</td>
			<td></td>
			<td>Synthesized in-house</td>
		</tr>
		<tr>
			<td>Tz-NHS</td>
			<td>Ref. 8</td>
			<td>764701</td>
			<td>Synthesized in-house, *Commercially available from Sigma Aldrich # 764701</td>
		</tr>
		<tr>
			<td>NP-NH2 = CLIO-NH2</td>
			<td>Ref. 8</td>
			<td></td>
			<td>Synthesized in-house</td>
		</tr>
		<tr>
			<td>AP1497, AP1497-Tz</td>
			<td>Ref. 8</td>
			<td></td>
			<td>Synthesized in-house</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>SPR Biosensor</td>
			<td>GE Healthcare</td>
			<td>Biacore T100</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidic on-chip capture-cycloaddition reaction to reversibly immobilize small molecules or multi-component structures for biosensor applications

方法有超过取向和固定化密度控制生物活性小分子的快速表面固定有用于生物传感器和微阵列应用非常理想。在这项研究中，我们使用了高效共价生物正交反式 -环辛烯（TCO）的[4 +2]环加成反应和1,2,4,5 -四嗪（TZ），使TCO / TZ衍生分子的固定化微流控。我们监测使用表面等离子体共振（SPR）在连续流动条件下的过程中的实时性。以使可逆固定化，并延长了实验范围传感器表面的，我们结合一个非共价的抗原 - 抗体捕获组分与环加成反应。通过交替呈现的TCO或TZ部分到传感器表面，多个捕获环的过程，现在可能在一个传感器表面的各种多组分结构的芯片组装和相互作用的研究。我们我一种小分子，AP1497结合FK506结合蛋白12（FKBP12），与上一个生物传感器芯片2的不同的固定化实验llustrate这种方法和相同的小分子作为固定化和原位功能化的纳米颗粒的一部分。

数据和数字已经改编自参考8。 生物活性小分子与在方向和密度控制高效的可逆固定在新的生物传感器应用的发展起着关键的作用。使用TCO与Tz之间的快速生物正交反应中，我们描述了一种用于逐步组装和配位体的表面与保持生物活性的再生。 图2显示的TZ-BnNH 2固定化的实时监测。 GST-TCO的溶液注射到产生〜400 RU上升响应预先固定化的抗-GST表面。第二次注?...

Watch this Scientific Journal Video about Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications at JoVE.

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

我们提出了一种小分子的快速，可逆的固定化和功能化的纳米粒子组合件表面等离子体共振（SPR）的研究中，使用连续的片上生物正交环化学和抗体 - 抗原捕获。

微流控芯片上捕获环加成反应可逆地固定小分子或多组分结构的生物传感器应用

Methods  for rapid surface immobilization of bioactive small molecules with control over  orientation and immobilization density are highly desirable for ...

microfluidic-on-chip-capture-cycloaddition-reaction-to-reversibly

Research

JoVE Journal

Chemistry

10.7K 次观看.  Massachusetts General Hospital. 我们提出了一种小分子的快速，可逆的固定化和功能化的纳米粒子组合件表面等离子体共振（SPR）的研究中，使用连续的片上生物正交环化学和抗体 - 抗原捕获。 

视频: Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

生物分子有序结构由短肽的自组装形成

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

科研

化学

Preparation of Hydrophobic Metal-Organic Frameworks via Plasma Enhanced Chemical Vapor Deposition of Perfluoroalkanes for the Removal of Ammonia

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high surface area microporous materials, such as metal-organic frameworks (MOFs), unique challenges present themselves for PECVD treatments. Herein the protocol for development of a MOF that was previously unstable to humid conditions is presented. The protocol describes the synthesis of Cu-BTC (also known as HKUST-1), the treatment of Cu-BTC with PECVD of perfluoroalkanes, the aging of materials under humid conditions, and the subsequent ammonia microbreakthrough experiments on milligram quantities of microporous materials. Cu-BTC has an extremely high surface area (~1,800 m2/g) when compared to most materials or surfaces that have been previously treated by PECVD methods. Parameters such as chamber pressure and treatment time are extremely important to ensure the perfluoroalkane plasma penetrates to and reacts with the inner MOF surfaces. Furthermore, the protocol for ammonia microbreakthrough experiments set forth here can be utilized for a variety of test gases and microporous materials.

Herein the procedures for plasma enhanced chemical vapor deposition of perfluoroalkanes on microporous materials such as metal-organic frameworks to enhance their stability and hydrophobicity are described. Furthermore, breakthrough testing of milligram quantities of samples is described in detail.

增强型氟烷烃的化学气相沉积等离子通过疏水金属 - 有机骨架的制备氨的去除

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high ...

Activating Molecules, Ions, and Solid Particles with Acoustic Cavitation

The chemical and physical effects of ultrasound arise not from a direct interaction of molecules with sound waves, but rather from the acoustic cavitation: the nucleation, growth, and implosive collapse of microbubbles in liquids submitted to power ultrasound. The violent implosion of bubbles leads to the formation of chemically reactive species and to the emission of light, named sonoluminescence. In this manuscript, we describe the techniques allowing study of extreme intrabubble conditions and chemical reactivity of acoustic cavitation in solutions. The analysis of sonoluminescence spectra of water sparged with noble gases provides evidence for nonequilibrium plasma formation. The photons and the "hot" particles generated by cavitation bubbles enable to excite the non-volatile species in solutions increasing their chemical reactivity. For example the mechanism of ultrabright sonoluminescence of uranyl ions in acidic solutions varies with uranium concentration: sonophotoluminescence dominates in diluted solutions, and collisional excitation contributes at higher uranium concentration. Secondary sonochemical products may arise from chemically active species that are formed inside the bubble, but then diffuse into the liquid phase and react with solution precursors to form a variety of products. For instance, the sonochemical reduction of Pt(IV) in pure water provides an innovative synthetic route for monodispersed nanoparticles of metallic platinum without any templates or capping agents. Many studies reveal the advantages of ultrasound to activate the divided solids. In general, the mechanical effects of ultrasound strongly contribute in heterogeneous systems in addition to chemical effects. In particular, the sonolysis of PuO2 powder in pure water yields stable colloids of plutonium due to both effects.

Acoustic cavitation in liquids submitted to power ultrasound creates transient extreme conditions inside the collapsing bubbles, which are the origin of unusual chemical reactivity and light emission, known as sonoluminescence. In the presence of noble gases, nonequilibrium plasma is formed. The "hot" particles and the photons generated by collapsing bubbles are able to excite species in solution.

活化分子，离子和固体颗粒与声空化

The chemical and physical effects of ultrasound arise not from a direct interaction of molecules with sound waves, but rather from the acoustic ...

Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for Biomedical Applications

Fluorescent nanocrystals, specifically quantum dots, have been a useful tool for many biomedical applications. For successful use in biological systems, quantum dots should be highly fluorescent and small/monodisperse in size. While commonly used cadmium-based quantum dots possess these qualities, they are potentially toxic due to the possible release of Cd2+ ions through nanoparticle degradation. Indium-based quantum dots, specifically InP/ZnS, have recently been explored as a viable alternative to cadmium-based quantum dots due to their relatively similar fluorescence characteristics and size. The synthesis presented here uses standard hot-injection techniques for effective nanoparticle growth; however, nanoparticle properties such as size, emission wavelength, and emission intensity can drastically change due to small changes in the reaction conditions. Therefore, reaction conditions such temperature, reaction duration, and precursor concentration should be maintained precisely to yield reproducible products. Because quantum dots are not inherently soluble in aqueous solutions, they must also undergo surface modification to impart solubility in water. In this protocol, an amphiphilic polymer is used to interact with both hydrophobic ligands on the quantum dot surface and bulk solvent water molecules. Here, a detailed protocol is provided for the synthesis of highly fluorescent InP/ZnS quantum dots that are suitable for use in biomedical applications.

In this protocol, the synthesis of Cd-free InP/ZnS quantum dots (QDs) is detailed. InP-based QDs are gaining popularity due to the toxicity of Cd2+ ions that may be released through nanoparticle degradation. After synthesis, QDs are solubilized in water using an amphiphilic polymer for use in biomedical applications.

镉免费的InP /硫化锌量子点的合成适合生物医学应用

Fluorescent nanocrystals, specifically quantum dots, have been a useful tool for many biomedical applications. For successful use in biological systems, ...

Highly Stable, Functional Hairy Nanoparticles and Biopolymers from Wood Fibers: Towards Sustainable Nanotechnology

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While metal-based nanoparticles are associated with synthetic and environmental hassles, cellulose introduces a green, sustainable alternative for nanoparticle synthesis. Here, we present the chemical synthesis and separation procedures to produce new classes of hairy nanoparticles (bearing both amorphous and crystalline regions) and biopolymers based on wood fibers. Through periodate oxidation of soft wood pulp, the glucose ring of cellulose is opened at the C2-C3 bond to form 2,3-dialdehyde groups. Further heating of the partially oxidized fibers (e.g., T = 80 &#176;C) results in three products, namely fibrous oxidized cellulose, sterically stabilized nanocrystalline cellulose (SNCC), and dissolved dialdehyde modified cellulose (DAMC), which are well separated by intermittent centrifugation and co-solvent addition. The partially oxidized fibers (without heating) were used as a highly reactive intermediate to react with chlorite for converting almost all aldehyde to carboxyl groups. Co-solvent precipitation and centrifugation resulted in electrosterically stabilized nanocrystalline cellulose (ENCC) and dicarboxylated cellulose (DCC). The aldehyde content of SNCC and consequently surface charge of ENCC (carboxyl content) were precisely controlled by controlling the periodate oxidation reaction time, resulting in highly stable nanoparticles bearing more than 7 mmol functional groups per gram of nanoparticles (e.g., as compared to conventional NCC bearing &#60;&#60; 1 mmol functional group/g). Atomic force microscopy (AFM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) attested to the rod-like morphology. Conductometric titration, Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), dynamic light scattering (DLS), electrokinetic-sonic-amplitude (ESA) and acoustic attenuation spectroscopy shed light on the superior properties of these nanomaterials.

Synthesis schemes to prepare highly stable wood fiber-based hairy nanoparticles and functional cellulose-based biopolymers have been detailed.

高稳定性，功能性毛纳米粒子和木纤维生物聚合物：迈向可持续纳米技术

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While ...

Low-energy Cathodoluminescence for (Oxy)Nitride Phosphors

Nitride and oxynitride (Sialon) phosphors are good candidates for the ultraviolet and visible emission applications. High performance, good stability and flexibility of their emission properties can be achieved by controlling their composition and dopants. However, a lot of work is still required to improve their properties and to reduce the production cost. A possible approach is to correlate the luminescence properties of the Sialon particles with their local structural and chemical environment in order to optimize their growth parameters and find novel phosphors. For such a purpose, the low-voltage cathodoluminescence (CL) microscopy is a powerful technique. The use of electron as an excitation source allows detecting most of the luminescence centers, revealing their luminescence distribution spatially and in depth, directly comparing CL results with the other electron-based techniques, and investigating the stability of their luminescence properties under stress. Such advantages for phosphors characterization will be highlighted through examples of investigation on several Sialon phosphors by low-energy CL.

Low-energy Cathodoluminescence for OxyNitride Phosphors

An excellent chemical and luminescence stabilities of (oxy)nitride phosphors present it as an promising alternative to currently used sulfide and oxide phosphors. In this paper, we present the way to investigate its local luminescence properties using low-energy cathodoluminescence (CL).

低能量的阴极射线（氧）氮化物荧光粉

Nitride and oxynitride (Sialon) phosphors are good candidates for the ultraviolet and visible emission applications. High performance, good stability and ...

The Preparation and Properties of Thermo-reversibly Cross-linked Rubber Via Diels-Alder Chemistry

A method for using Diels Alder thermo-reversible chemistry as cross-linking tool for rubber products is demonstrated. In this work, a commercial ethylene-propylene rubber, grafted with maleic anhydride, is thermo-reversibly cross-linked in two steps. The pending anhydride moieties are first modified with furfurylamine to graft furan groups to the rubber backbone. These pendant furan groups are then cross-linked with a bis-maleimide via a Diels-Alder coupling reaction. Both reactions can be performed under a broad range of experimental conditions and can easily be applied on a large scale. The material properties of the resulting Diels-Alder cross-linked rubbers are similar to a peroxide-cured ethylene/propylene/diene rubber (EPDM) reference. The cross-links break at elevated temperatures (&#62; 150 &#176;C) via the retro-Diels-Alder reaction and can be reformed by thermal annealing at lower temperatures (50-70 &#176;C). Reversibility of the system was proven with infrared spectroscopy, solubility tests and mechanical properties. Recyclability of the material was also shown in a practical way, i.e., by cutting a cross-linked sample into small parts and compression molding them into new samples displaying comparable mechanical properties, which is not possible for conventionally cross-linked rubbers.

A simple two-step approach involving rubber modification and cross-linking yields fully reworkable, elastic rubber products.

热可逆交联橡胶通过的Diels-Alder反应的化学制备与性能

A method for using Diels Alder thermo-reversible chemistry as cross-linking tool for rubber products is demonstrated. In this work, a commercial ...

From Molecules to Materials: Engineering New Ionic Liquid Crystals Through Halogen Bonding

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid crystals (ILCs). Taking advantages of the high specificity of XB for haloperfluorocarbons and the ability of anions to act as XB-acceptors, we obtained supramolecular complexes based on 1-alkyl-3-methylimidazolium iodides and iodoperfluorocarbons, overcoming the well-known immiscibility between hydrocarbons (HCs) and perfluorocarbons (PFCs). The high directionality of the XB combined with the fluorophobic effect, allowed us to obtain enantiotropic liquid crystals where a rigid, non-aromatic, XB supramolecular anion acts as mesogenic core.
X-ray structure analysis of the complex between 1-ethyl-3-methylimidazolium iodide and iodoperfluorooctane showed the presence of a layered structure, which is a manifestation of the well-known tendency to segregation of perfluoroalkyl chains. This is consistent with the observation of smectic mesophases. Moreover, all the reported complexes melt below 100 &#176;C, and most are mesomorphic even at room temperature, despite that the starting materials were non-mesomorphic in nature.
The supramolecular strategy reported here provides new design principles for mesogen design allowing a totally new class of functional materials.

A protocol for the synthesis of a new type of mesogens, based on the halogen-bonded supramolecular anion [CnF2n+1-I&#183;&#183;&#183;I&#183;&#183;&#183;I-CnF2n+1]&#8722;, is reported.

从分子到材料: 通过卤素键合工程新的离子液体晶体

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid ...

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the average content of fluorinated drugs in their formulated drug product forms: tablets or capsules. This method is specific to fluorinated drugs because it detects only the content of fluorine, avoiding interference from the excipients that lack fluorine. The advantages of measuring the active content of fluorinated drugs using low-field 19F TD-NMR versus high-field 19F solid-state (SS) NMR are the simplicity of the method; the low cost; and the non-destructive nature of the technique, with all samples recoverable in intact forms (e.g., powders, tablets, and capsules), making this technique affordable for any laboratory.
We have tested the method with three fluorinated drug products available on the market - cinacalcet, lansoprazole, and ciprofloxacin - with doses ranging from 15 to 500 mg. The results of the analyses, measured by low-field 19F TD-NMR, supported the reported label claims for the average drug content.
Based on the simplicity and reproducibility of the analysis, we envision this methodology being implemented in any laboratory, including manufacturing plants, as a process analytical technology (PAT) tool in the pharmaceutical industry.

Application and Methodology of the Non-destructive 19F Time-domain NMR Technique to Measure the Content in Fluorine-containing Drug Products

A simple and non-destructive technique that measures the average content of drug substances in formulated drug products containing fluorine using low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) is presented here. The technique can be applied to the development and manufacturing of drugs in the pharmaceutical industry.

应用程序和非破坏性19F 时间域核磁共振技术的方法来衡量中含氟药物产品的内容

Here, we describe a protocol developed by our group that uses low-field fluorine-19 (19F) time-domain (TD) nuclear magnetic resonance (NMR) to measure the ...

Stable DNA Motifs, 1D and 2D Nanostructures Constructed from Small Circular DNA Molecules

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D DNA nanostructures. Over decades, the rapid development of DNA nanotechnology is attributed to the use of linear DNAs as the source materials. For example, the DAO (double crossover, antiparallel, odd half-turns) tile is well-known as a building block for construction of 2D DNA lattices; the core structure of DAO is made from two linear single-stranded (ss) oligonucleotides, like two ropes making a right hand granny&#160;knot. Herein, a new type of DNA tiles called cDAO (coupled DAO) are built using a small circular ss-DNA of c64nt or c84nt (circular 64 or 84 nucleotides) as the scaffold strand and several linear ss-DNAs as the staple strands. Perfect 1D and 2D nanostructures are assembled from cDAO tiles: infinite nanowires, nanospirals, nanotubes, nanoribbons; and finite nano-rectangles. Detailed protocols are described: 1) preparation by T4 ligase and purification by denaturing PAGE (polyacrylamide gel electrophoresis) of small circular oligonucleotides, 2) annealing of stable circular tiles, followed by native PAGE analysis, 3) assembling of infinite 1D nanowires, nanorings, nanospirals, infinite 2D lattices of nanotubes and nanoribbons, and finite 2D nano-rectangles, followed by AFM (Atomic Force Microscopy) imaging. The method is simple, robust, and affordable for most labs.

This article presents a detailed protocol for T4 ligation and denaturing PAGE purification of small circular DNA molecules, annealing and native PAGE analysis of circular tiles, assembling and AFM imaging of 1D and 2D DNA nanostructures, as well as agarose gel electrophoresis and centrifugation purification of finite DNA nanostructures.

由小圆形 DNA 分子构建的稳定 DNA 分子、一维和二维纳米结构

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D ...