这项工作是由赠款，以资助自置居所从美国国立卫生研究院（R01 GM092993）的支持，美国国家科学基金会（NSF CAREER奖和DBI 0964216），海军研究办公室（ONR）青年研究者计划和明尼苏达合作奖的生物技术和医学基因组学。在明尼苏达大学的纳米加工中心（NFC）技术，它从美国国家科学基金会通过了国家纳米技术基础设施网络接收支持进行设备制造。这项工作也是由补助到MR卫生（NS048357，R21 NS073684），国家多发性硬化症协会（CA1060A11）时，鲍姆，希尔顿，彼得森和桑福德基础和MCNEILUS家庭的国家机构的支持。作者要感谢Hyungsoon林与插图和Shailabh库马尔与扫描电子显微镜的协助援助。

1，微细加工的微孔阵列基板<ol><li>从一个4英寸的硅晶片上热生长氧化物为100nm。 </li><li>旋SPR-955 0.7光刻胶的晶片上以4000rpm进行30秒。 </li><li>烘烤的加热板上，在115℃下进行90秒。 </li><li>曝光光刻胶。 <ol><li>使用会产生1微米的孔排列成六边形阵列具有3微米的周期，其中该阵列包括有2毫米×2毫米区域的掩模。 </li><li>使用6毫米的步长和200毫焦耳/厘米2的曝光在一个i-线步进曝光晶片。 </li></ol></li><li>烘烤的加热板上，在115℃下进行90秒。 </li><li>开发利用旋开发商沉积在晶圆上2水坑的CD-26的90秒的总开发时间。 </li><li>用超纯水去离子水和干与N 2彻底冲洗。 </li><li>在反应性离子蚀刻机蚀刻氧化物为6分钟，使用50 sccm的氩，CF 4 25 sccm的，和50sccm的CHF的3 FLO机翼在75毫托的压力。 </li><li>在ICP深沟槽反应性离子蚀刻机2分钟，以63 sccm的或C 4 F 8，27 sccm的SF 6气体，40sccm的氩气和10标准立方厘米的为14毫托的压力下O 2的流动蚀刻硅。 </li><li>漂洗在丙酮，甲醇和异丙醇，以除去抗蚀剂。 </li><li>这里的H 2 SO 4浴：H 2 O 2 1:1 10分钟来清洁表面。 </li><li>用超纯水去离子水和干与N 2彻底冲洗。 </li><li>沉积的Al 2 1,080 A O 3通过原子层沉积在250°C。 </li></ol> 2，制备聚（二甲基硅氧烷）刮刀<ol><li>如果可能的话，工作在洁净室。在可能的最清洁的环境，否则工作。 </li><li>得到的塑料培养皿（或其它清洁的一次性容器）与≥13毫米两侧。 </li><li>在那道菜，倒出来5克PDMS固化剂，接着加入50克的PDMS浅ë剂（或用1:10的比例，将大部分填补了培养皿中的任何东西）。用一次性塑料吸管或棒调匀。 </li><li>通过将混合的PDMS中的腔与真空管线，施加真空，直至气泡出来的混合物（约30分钟）除去气泡。 </li><li>如果不是已经在培养皿，倒入PDMS在培养皿中，避免产生气泡。 </li><li>一夜之间治愈的电炉在&gt; 50°C（用于更短的时间高热量也适用）。 </li><li>随着新/干净的刀片，小心地切一块长方形约2.5×2.5厘米，保持顶面尽可能的干净。剥出用干净的镊子。通过按下刀片中的一个运动，以使上边缘尽可能尖锐（这将是在所述刮墨刀的过程中使用的边缘）修剪过的一个边缘（下降到1.5厘米）。 </li><li>干净，用丙酮，甲醇，异丙醇，和超纯去离子水，然后吹干用干净的氮气。存放在一个干净的培养皿中。 </li></ol>囊泡3。准备<ol><li>收集的25毫克/毫升的蛋磷脂酰胆碱（PC）的脂质储备溶液和1 mg / ml的1,2 -二棕榈酰-sn-甘油基-3 -磷酸乙醇胺-N - （丽丝胺罗丹明B磺酰基）铵盐（ρ-DPPE）的氯仿以及0.5毫克/毫升的库存单唾液酸神经节苷脂GM1的氯仿/甲醇溶液（2:1体积/体积）。 </li><li>在一个小的玻璃瓶中作出，结果在97％（摩尔）的PC，2％（摩尔）神经节苷脂GM1和1％（摩尔）的Rho-DPPE，加入18.9微升的25毫克/毫升的PC，39.6微升的0.5毫克/毫升GM1和7.9微升的混合物1毫克/毫升的Rho-DPPE使用玻璃注射器。 </li><li>注：为Nair 等人 25的补充材料包含一个极好的可定制的Excel电子表格计算脂质创建任何所需组成的囊泡所需的金额。 </li><li>放置小瓶在真空干燥器中，并除去溶剂，留下在真空下6小时的小瓶中。 </li><li>使0.1的水溶液M氯化钠。加入0.5毫升的0.1M NaCl溶液到含有干燥的脂质膜的小瓶。 </li><li>允许水性脂质混合物休息过夜。 </li><li>简要地涡旋混合，以创建一个小泡悬浮液中，然后超声处理20分钟，在浴中超声破碎仪在室温下。 </li><li>通过聚碳酸酯膜具有100nm的孔径挤出囊悬浮液。通过过滤器通过17倍的囊泡停牌。 </li><li>储存在玻璃小瓶中挤出的膜泡在4℃下</li></ol>球面支持的脂质双层膜4。形成<ol><li>收集700纳米直径的SiO 2的珠子。珠粒被提供在蒸馏水中以原料浓度为1.4×10 11珠/毫升。 </li><li>在1.5ml Eppendorf管中，加入107微升的珠子储备溶液以893微升0.1M的氯化钠制备1毫升珠粒悬浮液以1.5×10 10珠/ ml的浓度。 </li><li> VORTEX暂停，以确保它是很好英里固定的。 </li><li>离心该悬浮液在1700×g离心20分钟，然后弃上清。重悬珠在1ml的0.1M NaCl洗涤。重复此步骤两次彻底清洗珠。 </li><li>以形成SSLBs，在1.5ml的Eppendorf管中加入25微升的SiO 2珠悬浮于200微升的1毫克/毫升的小泡悬浮液中。涡旋混合，静置1小时，在室温下进行。在这个阶段SSLB浓度应为1.7×10 9 SSLBs /毫升。 </li><li>离心机在1700×g离心20分钟以沉淀SSLBs。粒料应出现粉红色由于对珠的Rho-DPPE囊泡的破裂。 </li><li>丢弃在225微升磷酸盐缓冲盐水（PBS），pH值= 7.4的上清，重悬。重复步骤4.6-4.7两次从SSLB悬挂删除任何未破裂的小泡​​。 </li></ol> SSLB阵列5。大会<ol><li>导致矩形件4-6麦克风微孔阵列的顺劈片每件罗威尔阵列。 </li><li>旋涡混合SSLB暂停，然后将10微升SSLB悬挂的每个微孔阵列上。免得休息1小时，以允许SSLBs沉淀到表面上。 </li><li>用PBS轻轻从洗瓶清洗微孔阵列芯片上，然后浸入在浅盘制备的PBS浴。 </li><li>而淹没，轻轻将PDMS刮刀冲洗微孔阵列芯片上，并轻轻沿表面滑动5倍，除去未微孔中固定SSLBs。 </li><li>在PBS浴握微孔阵列芯片镊子，轻轻摇晃，以消除任何多余的SSLBs。 </li><li>从吸水扒浴快速去除芯片，并放置在一个新鲜的PBS浴备用。确保芯片的顶表面保持湿润以保留SSLBs。 </li></ol>注意：上述的组装方法，可用于创建天然膜颗粒的阵列，如髓鞘颗粒从小鼠脑，或坡分离spholipid无泡在SiO 2珠的支持。详细维滕贝格等 21这一工作描述和代表性的结果如下所示。 6。霍乱毒素结合分析<ol><li>准备一个2毫克/毫升的溶液的牛血清白蛋白（BSA）的PBS中。 </li><li>准备与Alexa 488缀合的霍乱毒素B亚单位的在PBS中的2毫克/毫升牛血清白蛋白的所需浓度的溶液。 </li><li>从PBS浴中取出SSLB阵列芯片和灯芯大多数的PBS溶液相差使用实验室擦拭。留在芯片上刚好够的PBS，以保持SSLB阵列的水分。 </li><li>为了防止非特异性结合，加入200μl的2毫克/毫升BSA溶液的SSLB阵列芯片。让我们休息1小时的加湿盒。 </li><li>用微量的SSLB阵列芯片的顶部取出200微升的解决方案。 </li><li>加入200μl的霍乱毒素解决SSLB阵列芯片，并让其余的1小时在加湿盒。 </li><li>用PBS轻轻从洗瓶清洗SSLB阵列芯片以除去任何未结合的霍乱毒素。 </li><li>威克用实验室走多余的PBS擦拭。前盖成像芯片装有24mm×40mm的盖玻片。 </li><li>使用过滤器一个堂堂正正的显微镜图像阵列设置适合感兴趣的荧光。 </li><li>分析使用ImageJ软件的自动颗粒分析函数数组中的各个SSLBs的荧光强度。 </li><li>编译个别SSLBs平均荧光强度成汇总SSLBs的一个给定的数组中找到的强度直方图。 </li></ol>

<ol>
	<li>Drews, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+discovery:+A+Historical+Perspective.">Drug discovery: A Historical Perspective.</a> Science. 287 (5460), 1960-1964 (1126).</li><li>Cooper, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+Membrane+Receptor+Screening+and+Analysis.">Advances in Membrane Receptor Screening and Analysis.</a> J. Mol. Recognit. 17 (4), 286-315 (2004).</li><li>Voskuhl, J., Ravoo, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Recognition+of+Bilayer+Besicles.">Molecular Recognition of Bilayer Besicles.</a> Chem. Soc. Rev. 38 (2), 495-505 (2009).</li><li>Walde, P., Cosentino, K., Engel, H., Stano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+Vesicles:+Preparations+and+Applications.">Giant Vesicles: Preparations and Applications.</a> Chembiochem. 11 (7), 848-865 (1002).</li><li>Castellana, E. T., Cremer, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid+Supported+Lipid+Bilayers:+From+Biophysical+Studies+to+Sensor+Design.">Solid Supported Lipid Bilayers: From Biophysical Studies to Sensor Design.</a> Surf. Sci. Rep. 61 (10), 429-444 (2006).</li><li>Johnson, J. M., Ha, T., Chu, S., Boxer, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Steps+of+Supported+Bilayer+Formation+Probed+by+Single+Vesicle+Fluorescence+Assays.">Early Steps of Supported Bilayer Formation Probed by Single Vesicle Fluorescence Assays.</a> Biophys. J. 83 (6), 3371-3379 (2002).</li><li>Krysinski, P., Zebrowska, A., Michota, A., Bukowska, J., Becucci, L., Moncelli, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tethered+Mono-+and+Bilayer+Lipid+Membranes+on+Au+and+Hg.">Tethered Mono- and Bilayer Lipid Membranes on Au and Hg.</a> Langmuir. 17 (13), 3852-3857 (2001).</li><li>Li, L., Wang, H. F., Cheng, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Coherent+anti-Stokes+Raman+Scattering+Imaging+of+Lipid+Distribution+in+Coexisting+Domains.">Quantitative Coherent anti-Stokes Raman Scattering Imaging of Lipid Distribution in Coexisting Domains.</a> Biophys. J. 89 (5), 3480-3490 (2005).</li><li>Richter, R. P., Brisson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Following+the+Formation+of+Supported+Lipid+Bilayers+on+Mica:+A+Study+Combining+AFM,+QCM-D,+and+Ellipsometry.">Following the Formation of Supported Lipid Bilayers on Mica: A Study Combining AFM, QCM-D, and Ellipsometry.</a> Biophys. J. 88 (5), 3422-3433 (2005).</li><li>Dahlin, A., Z&#228;ch, M., Rindzevicius, T., K&#228;ll, M., Sutherland, D. S., H&#246;&#246;k, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+Surface+Plasmon+Resonance+Sensing+of+Lipid-Membrane-Mediated+Biorecognition+Events.">Localized Surface Plasmon Resonance Sensing of Lipid-Membrane-Mediated Biorecognition Events.</a> J. Am. Chem. Soc. 127 (14), 5043-5048 (2005).</li><li>Kraft, M. L., Weber, P. K., Longo, M. L., Hutcheon, I. D., Boxer, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+Separation+of+Lipid+Membranes+Analyzed+with+High-Resolution+Secondary+Ion+Mass+Spectrometry.">Phase Separation of Lipid Membranes Analyzed with High-Resolution Secondary Ion Mass Spectrometry.</a> Science. 313 (5795), 1948-1951 (2006).</li><li>Groves, J. T., Boxer, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropattern+Formation+in+Supported+Lipid+Membranes.">Micropattern Formation in Supported Lipid Membranes.</a> Acc. Chem. Res. 35 (3), 149-157 (2002).</li><li>Bally, M., Bailey, K., Sugihara, K., Grieshaber, D., V&#246;r&#246;s, J., Stadler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome+and+Lipid+Bilayer+Arrays+Towards+Biosensing+Applications.">Liposome and Lipid Bilayer Arrays Towards Biosensing Applications.</a> Small. 6 (22), 2481-2497 (2010).</li><li>Groves, J. T., Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supported+Planar+Bilayers+in+Studies+on+Immune+Cell+Adhesion+and+Communication.">Supported Planar Bilayers in Studies on Immune Cell Adhesion and Communication.</a> J. Immunol. Methods. 278 (1-2), 19-32 .</li><li>Yang, T. L., Jung, S. Y., Mao, H. B., Cremer, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+Phospholipid+Bilayer-Coated+Microchannels+for+On-Chip+Immunoassays.">Fabrication of Phospholipid Bilayer-Coated Microchannels for On-Chip Immunoassays.</a> Anal. Chem. 73 (2), 165-169 (2001).</li><li>Groves, J. T., Ulman, N., Boxer, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micropatterning+Fluid+Lipid+Bilayers+on+Solid+Supports.">Micropatterning Fluid Lipid Bilayers on Solid Supports.</a> Science. 275 (5300), 651-653 (1997).</li><li>Hovis, J. S., Boxer, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+and+Composition+Arrays+of+Supported+Lipid+Bilayers+by+Microcontact+Printing.">Patterning and Composition Arrays of Supported Lipid Bilayers by Microcontact Printing.</a> Langmuir. 17 (11), 3400-3405 (2001).</li><li>Yee, C. K., Amweg, M. L., Parikh, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Photochemical+Patterning+and+Refunctionalization+of+Supported+Phospholipid+Bilayers.">Direct Photochemical Patterning and Refunctionalization of Supported Phospholipid Bilayers.</a> J. Am. Chem. Soc. 126 (43), 13962-13972 (2004).</li><li>Kelly, C. V., Craighead, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanofabrication+for+the+Analysis+and+Manipulation+of+Membranes.">Nanofabrication for the Analysis and Manipulation of Membranes.</a> Ann. Biomed. Eng. 40 (6), 1356-1366 (2012).</li><li>Wittenberg, N. J., Johnson, T. W., Oh, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Density+Arrays+of+Submicron+Spherical+Supported+Lipid+Bilayers.">High-Density Arrays of Submicron Spherical Supported Lipid Bilayers.</a> Anal. Chem. 84 (19), 8207-8213 (2012).</li><li>Wittenberg, N. J., 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=Facile+Assembly+of+Micro-+and+Nanoarrays+for+Sensing+with+Natural+Cell+Membranes.">Facile Assembly of Micro- and Nanoarrays for Sensing with Natural Cell Membranes.</a> ACS Nano. 5 (9), 7555-7564 (2011).</li><li>Stamou, D., Duschl, C., Delamarche, E., Vogel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-Assembled+Microarrays+of+Attoliter+Molecular+Vessels.">Self-Assembled Microarrays of Attoliter Molecular Vessels.</a> Angew. Chem. Int. Ed. 42 (45), 5580-5583 (2003).</li><li>Kalyankar, N. 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=Arraying+of+Intact+Liposomes+into+Chemically+Functionalized+Microwells.">Arraying of Intact Liposomes into Chemically Functionalized Microwells.</a> Langmuir. 22 (12), 5403-5411 (2006).</li><li>Dahlin, A. B., Jonsson, M. P., H&#246;&#246;k, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+Self-Assembly+of+Single+Lipid+Vesicles+in+Nanoplasmonic+Apertures+in+Gold.">Specific Self-Assembly of Single Lipid Vesicles in Nanoplasmonic Apertures in Gold.</a> Adv. Mater. 20 (8), 1436-1442 (2008).</li><li>Nair, P. M., Salaita, K., Petit, R. S., Groves, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Patterned+Supported+Lipid+Membranes+to+Investigate+the+Role+of+Receptor+Organization+in+Intercellular+Signaling.">Using Patterned Supported Lipid Membranes to Investigate the Role of Receptor Organization in Intercellular Signaling.</a> Nat. Protoc. 6 (4), 523-539 (2011).</li><li>Kuziemko, G. M., Stroh, M., Stevens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholera+Toxin+Binding+Affinity+and+Specificity+for+Gangliosides+Determined+by+Surface+Plasmon+Resonance.">Cholera Toxin Binding Affinity and Specificity for Gangliosides Determined by Surface Plasmon Resonance.</a> Biochemistry. 35 (20), 6375-6384 (1996).</li><li>Moran-Mirabal, J. M., Edel, J. B., Meyer, G. D., Throckmorton, D., Singh, A. K., Craighead, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micrometer-Sized+Supported+Lipid+Bilayer+Arrays+for+Bacterial+Toxin+Binding+Studies+Through+Total+Internal+Reflection+Fluorescence+Microscopy.">Micrometer-Sized Supported Lipid Bilayer Arrays for Bacterial Toxin Binding Studies Through Total Internal Reflection Fluorescence Microscopy.</a> Biophys. J. 89 (1), 296-305 (2005).</li><li>Shi, J. J., Yang, T. L., Kataoka, S., Zhang, Y. J., Diaz, A. J., Cremer, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GM1+Clustering+Inhibits+Cholera+Toxin+Binding+in+Supported+Phospholipid+Membranes.">GM1 Clustering Inhibits Cholera Toxin Binding in Supported Phospholipid Membranes.</a> J. Am. Chem. Soc. 129 (18), 5954-5961 (2007).</li><li>Gill, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arrangement+of+Subunits+in+Cholera+Toxin.">The Arrangement of Subunits in Cholera Toxin.</a> Biochemistry. 15 (6), 1242-1248 (1021).</li><li>Weng, K. C., Kanter, J. L., Robinson, W. H., Frank, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+Supported+Lipid+Bilayers+Containing+Monosialoganglioside+GM1:+A+QCM-D+and+FRAP+study.">Fluid Supported Lipid Bilayers Containing Monosialoganglioside GM1: A QCM-D and FRAP study.</a> Colloid Surface B. 50 (1), 76-84 (1016).</li><li>Walt, 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=Fibre+Optic+Microarrays.">Fibre Optic Microarrays.</a> Chem. Soc. Rev. 39 (1), 38-50 (2010).</li><li>Bake, K. D., Walt, 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=Multiplexed+Spectroscopic+Detections.">Multiplexed Spectroscopic Detections.</a> Annu. Rev. Anal. Chem. 1 (1), 515-547 (2008).</li><li>Kundu, J., Levin, C. S., Halas, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-Time+Monitoring+of+Lipid+Transfer+Between+Vesicles+and+Hybrid+Bilayers+on+Au+Nanoshells+Using+Surface+Enhanced+Raman+Scattering+(SERS).">Real-Time Monitoring of Lipid Transfer Between Vesicles and Hybrid Bilayers on Au Nanoshells Using Surface Enhanced Raman Scattering (SERS).</a> Nanoscale. 1 (1), 114-117 (2009).</li><li>Junesch, J., Sannomiya, T., Dahlin, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Properties+of+Nanohole+Arrays+in+Metal-Dielectric+Double+Films+Prepared+by+Mask-on-Metal+Colloidal+Lithography.">Optical Properties of Nanohole Arrays in Metal-Dielectric Double Films Prepared by Mask-on-Metal Colloidal Lithography.</a> ACS Nano. 6 (11), 10405-10415 (2012).</li><li>Wu, M., Holowka, D., Craighead, H. G., Baird, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+Plasma+Membrane+Compartmentalization+with+Patterned+Lipid+Bilayers.">Visualization of Plasma Membrane Compartmentalization with Patterned Lipid Bilayers.</a> Proc. Natl. Acad. Sci. U.S.A. 101 (38), 13798-13803 (2004).</li><li>Hatzakis, N. S., Bhatia, V. K., Larsen, J., Madsen, K. L., Bolinger, P. Y., Kunding, A. H., Castillo, J., Gether, U., Hedegard, P., Stamou, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Curved+Membranes+Recruit+Amphipathic+Helices+and+Protein+Anchoring+Motifs.">How Curved Membranes Recruit Amphipathic Helices and Protein Anchoring Motifs.</a> Nat. Chem. Biol. 5 (11), 835-841 (2009).</li><li>Roizard, S., Danelon, C., Hassaine, G., Piguett, J., Schulze, K., Hovius, R., Tampe, R., Vogel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+G-Protein-Coupled+Receptors+in+Cell-Derived+Plasma+Membranes+Supported+on+Porous+Beads.">Activation of G-Protein-Coupled Receptors in Cell-Derived Plasma Membranes Supported on Porous Beads.</a> J. Am. Chem. Soc. 133 (42), 16868-16874 (2011).</li></ol>

作者没有竞争经济利益披露。

在这项工作中，我们表明，单分散的SiO 2珠粒涂有支持的脂质双层可排列成微孔阵列，而不需要在脂质双层或在基板表面的靶向配体，并且阵列可用于表征毒素-脂质相互作用。计算CTx/GM1结合的解离常数，我们绝不逊色，给定的值在文献悬殊，与先前的报告Winter 等 ，其中脂质包被的磁珠胶体组装用于计算的有关解离常数30纳米。由数据拟合的山，WAUD结合的模式，我们确定CTx/GM1互动?...

脂双层膜的性质是基本的结构。细胞质膜和细胞器的膜构成的，即包括许多分子，是生命所必需的脂质双层中。许多维持生命过程发生的细胞的表面上，或通过与脂质双层膜相关分子介导的。事实上，许多药品目标进程或分子被发现，或在膜1,2。因此，有必要对解析调查过程，如在膜表面发生的化学反应或非共价结合事件。因为天然的膜可能难以分离和/或接口的传感器，许多研究人员采用简化模型膜，进行分析研究。一些模型膜系统在文献中有描述，从巨囊，可以是几十到几百微米的直径，以脂质体具有纳米级尺寸的3,4。 ALTERNatively，沉积在固体载体上的平面脂双层， 即 ，支持的脂质双层（SLBs中），可以形成多种不同的表面和已被广泛应用于生物物理，生物化学，和分析应用程序5。耦合SLBs的电气或光学材料通过使用不同的分析技术使膜生物化学和生物物理学研究。荧光显微镜6，7电化学，光学光谱8，扫描探针显微镜9，表面等离子体共振10，和质谱11都被用来研究的SLBs的结构和性能。 SLB阵列提供额外的多功能性传感器的多重检测12,13的设计。其他应用程序使用SLB阵列来模拟免疫细胞14之间形成的交界处。为SLB阵列的制备方法从microflu变化IDIC接近15到那些采用SLB相邻斑块之间的物理障碍。16其他团体使用的印刷方法17，光化学图案18以及各种纳米工程学接近19创建SLB阵列。 在本文中和所附的视频为大家演示了SLB-涂SiO 2的珠子存入微孔20的有序排列形成阵列负载均衡的方法。我们指的是SLB-涂上二氧化硅珠为球形支持的脂质双层（SSLBs）。这种技术是创建从天然来源21，从中我们也显示例子的结果得出磷脂囊泡和生物膜的数组早期工作的延伸。其他方法排列生物膜颗粒或小泡都依赖于特定的靶向配体的模式上，与包含在囊泡表面的互补关联的配体表面。例子包括生物素 -抗生物素蛋白关联22,23和DNA杂交方案24。我们的方法只需要一个微孔阵列，没有必要定位或识别部分。 SSLBs的大小是由SiO 2的珠载体，它具有低的聚分散度的直径限定。通过调节微孔的直径只比SSLB直径大，只有一个单一的SSLB稳定于各微孔。聚（二甲基硅氧烷）（PDMS）刮刀然后从表面移除，但在固定微孔所有SSLBs。微孔和所得SSLB阵列具有高密度（〜10 5 SSLBs 个 / mm 2）与3微米的中心到中心的间距和六边形周期性。通过串联使用不同脂质组合物沉积SSLBs，它可以创建多组阵列与随机定位SSLBs。以证明SSLB阵列的感测能力，我们使用了一个并入SSLBs神经节苷脂（GM1）霍乱毒素（CTX）的相互作用。同天然膜颗粒，我们能够检测细胞特异性脂质中含有的膜材料从两个不同的细胞类型的多组分阵列。

<table border="0" cellpadding="0" cellspacing="0" width="615">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">4-inch silicon wafers</td>
			<td style="width:91px;">University Wafer</td>
			<td align="right" style="width:119px;">425</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Shipley MEGAPOSIT SPR955-CM 0.7 photoresist</td>
			<td style="width:91px;">MicroChem</td>
			<td style="width:119px;">SPR955-CM</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Shipley MICROPOSIT CD-26 developer</td>
			<td style="width:91px;">MicroChem</td>
			<td style="width:119px;">CD-26</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">i-line stepper</td>
			<td style="width:91px;">Canon</td>
			<td style="width:119px;">2500 i3 stepper</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Vision 320 reactive ion etcher</td>
			<td style="width:91px;">Advanced Vacuum</td>
			<td style="width:119px;">Vision 320 RIE</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Deep trench reactive ion etcher</td>
			<td style="width:91px;">Plasma Therm</td>
			<td style="width:119px;">SLR-770</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Atomic layer depostion system</td>
			<td style="width:91px;">Cambridge NanoTech</td>
			<td style="width:119px;">Savannah</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Dow Corning Sylgard 184 poly(dimethylsiloxane) kit</td>
			<td style="width:91px;">Ellsworth Adhesives</td>
			<td style="width:119px;">184 SIL ELAST KIT 0.5KG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">egg phosphatidylcholine</td>
			<td style="width:91px;">Avanti Polar Lipids</td>
			<td>840051C</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:239px;">1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) ammonium salt</td>
			<td style="width:91px;">Avanti Polar Lipids</td>
			<td>810158C</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">monosialoganglioside GM1&#160;</td>
			<td style="width:91px;">Avanti Polar Lipids</td>
			<td style="width:119px;">860065P</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Silica beads</td>
			<td style="width:91px;">Bangs Laboratories</td>
			<td style="width:119px;">SS03N/4666</td>
			<td>Packaging on the bead container states the beads are 900 nm in diameter. However, after light-scattering and electron microscopy we determined the beads are roughly 700 nm in diameter.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Cholera toxin B-subunit, Alexa 488 conjugate</td>
			<td style="width:91px;">Molecular Probes</td>
			<td style="width:119px;">C-34775</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Anti-oligodentrocyte antibody IgM O4, NorthernLights 557 conjugate</td>
			<td style="width:91px;">R&#38;D Systems</td>
			<td style="width:119px;">NL1326R</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">FM1-43</td>
			<td style="width:91px;">Molecular Probes</td>
			<td style="width:119px;">T-3136</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Eppendorf MiniSpin centrifuge</td>
			<td style="width:91px;">Fisher Scientific</td>
			<td style="width:119px;">05-401-09</td>
			<td></td>
		</tr>
	</tbody>
</table>

formation of biomembrane microarrays with a squeegee-based assembly method

脂质双层膜形成细胞的质膜和限定亚细胞器的边界。在自然界中，这些膜有许多种脂质的多相混合物，含有膜结合蛋白，并装饰有碳水化合物。在一些实验中，最好是去耦从这些天然细胞膜的脂质双层的生物物理或生物化学特性。这种情况下要求使用的模型系统如巨泡，脂质体或支撑脂质双层（SLBs中）。 SLBs中的阵列是用于感测应用和模仿细胞 - 细胞相互作用特别有吸引力的。在这里，我们描述了形成SLB阵列的新方法。亚微米直径的SiO 2小珠首先用脂质双层，以形成球形的SLBs（SSLBs）。然后使小球沉积到微制造亚微米直径的微孔阵列。制备工艺采用了“刮刀”，以清洁衬底表面，而要走背后的落户为微孔SSLBs克。这种方法需要的微孔基质的无化学修饰，也没有对SSLB任何特定的靶向配体。微孔是由单个珠子占据，因为井径调到只是比珠直径。典型地，孔的75％以上都被占用，而其余的保持为空。在缓冲SSLB阵列显示大于一个星期的长期稳定性。多种类型SSLBs可以放置在一个单独的阵列通过串行沉积和阵列可以用于检测，这表明我们通过表征霍乱毒素与神经节苷脂的相互作用。我们还表明，磷脂泡囊，而不珠载体和生物膜从蜂窝源可以排列具有相同的方法和细胞特异性膜脂质可以被识别。

当SiO 2的珠粒与囊泡磷脂，荧光脂质和其他脂类如神经节苷脂组成的溶液混合时，囊泡破裂，在SiO 2胎圈表面上以形成SSLBs，如在图1a中示意性示出。洗涤SSLBs后，一滴SSLB溶液置于一个微孔阵列上，并且小珠静置至表面。 （ 图1B1）这也可以与磷脂小泡或天然膜颗粒，其显示在图1B2中的悬浮液进行。吸附到微孔阵列基板磷脂囊泡的荧光图像显示?...

Watch this Scientific Journal Video about Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method at JoVE.com

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

支持的脂质双层和天然膜颗粒是方便的系统，可以近似细胞膜的性质和在各种分析战略中。这里，我们证明了制备微阵列支持的脂质双层包被的SiO 2的有孔玻璃珠，磷脂囊泡或天然膜颗粒组成的方法。

生物膜的微阵列用刮墨刀型组合方式形成

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

formation-biomembrane-microarrays-with-squeegee-based-assembly

Research

JoVE Journal

Bioengineering

13.6K 次观看.  University of Minnesota. 支持的脂质双层和天然膜颗粒是方便的系统，可以近似细胞膜的性质和在各种分析战略中。这里，我们证明了制备微阵列支持的脂质双层包被的SiO 2的有孔玻璃珠，磷脂囊泡或天然膜颗粒组成的方法。 

视频: Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

黄金Nanostar合成银种子介导的生长方法

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

科研

生物工程

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

聚合物芯片高通量发现的生物材料

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

协议生物膜形成的流光在微流体器件与微柱

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

生物膜荧光探针力：受体 - 配体动力学并行和定量结合诱导细胞内信号作用于单细胞

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

生物膜制作的溶剂辅助脂质双分子层（SALB）方法

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

用 3 d-印刷模具制作细胞播种和组织环自组装用的自定义琼脂糖井

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

从癌细胞培养物中收集的介质光谱分析的选定金核素的同步量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.