作者想感谢Byoung哲李，菲利普财和塞缪尔何宝贵援助。在美国能源部劳伦斯伯克利国家实验室，这是科学办公室基础能源科学办公室支持这项工作是在分子铸造，根据合同号DE - AC02 - 05CH11231和国防威胁降低机构根据合约编号：IACRO - B0845281。

1。固相Polypeptoids Submonomer合成固相合成（SPS）是一种常见的技术，用于直接合成特定序列的生物大分子的一步，明智的惰性固体支持，如高分子树脂珠。高耦合的产量和缓解过剩的反应物去除的卫生和植物检疫措施的主要优点。耦合反应的树脂后，干脆倒掉多余的试剂和珠洗净，准备下一个反应步骤。最终合成反应后，全长低聚物树脂切割，可进一步研究和解决方案相材料。在这里，我们适应的卫生和植物检疫程序生成序列特异性peptoid聚合物。 <ol><li>设置：手​​动peptoid合成的所有步骤，就可以进行一次性，聚丙烯（PP）烧结墨盒或多孔玻璃反应釜配有3路阀。在通风橱中执行的所有操作。对于在G孵化姑娘的船只或塑料盒，连接一只手臂轻轻泡沫适当的混合解决方案的一个氮气供应。另外，对于反应孵化一次性墨盒，墨盒盖帽和旋转摇床两端密封。排反应混合物或清洗，连接真空房子通过废物陷阱。船只应与粗熔块烧结。硅化（siliconize）玻璃反应釜，以避免粘到玻璃容器壁珠。准备5％的二甲基二氯硅烷二氯乙烷（V / V）溶液。一个清洁，干燥的反应釜填充硅化解决方案，让我们坐了30分钟，然后沥干。在DCE洗一次船，然后一次用甲醇。硅化解决方案，可重复使用，因此它应该被保存。无论是空气干燥或摆脱任何多余的解决方案和烘烤干，直到取出后，活塞的玻璃器皿。酷之前加入树脂反应釜。 </li><li>溜冰场酰胺重新加入100毫克（0.06毫摩尔）罪，以一个多孔的反应容器。加入2毫升二甲基甲酰胺（DMF）的膨胀的树脂。搅拌摇晃或10分钟冒泡。漏隔离膨胀的树脂真空解决方案。 </li><li>加入1毫升20％的四甲基在DMF（V / V），以deprotect Fmoc基团。鼓动2分钟，漏。重复12分钟孵化。 </li><li>加入2毫升的DMF，搅拌15秒，排水冲洗树脂。重复3倍。 </li><li> Bromoacetylation：加入1 ml 0.6 M溴乙酸（0.6毫摩尔）在DMF和86μL的N，N&#39; - diisopropylcarbodiimide（相当于0.93，0.56毫摩尔）。孵育30分钟的温和的冒泡，然后与二甲基甲酰胺2毫升（重复4X）排水和冲洗。 </li><li>排量：加入1毫升1-2米胺的N - methylpyrrolidinone。孵育30-120分钟冒泡，然后与DMF（4X 2毫升）排水和冲洗。 </li><li>继续增长，重复submonomer周期，步骤1.5（bromoacetylation）和1.6（displacemen peptoid链T）。 </li></ol><ol start="8"><li>后做最后的位移，二甲基甲酰胺2毫升（重复4X）冲洗，然后2 mL二氯甲烷（重复3倍）。第和存储，直到裂解反应釜。 </li><li>暂停合成（可选）：要在一个peptoid合成暂停，完成置换反应，并继续加强1.8。为了继续保持增长peptoid链，重新启动重新膨胀的干燥树脂（步骤1.2）和重复submonomer周期（1.5和1.6步）的合成。树脂可烘干和储存以外的任何第二位移的位移后因为树脂peptoid共轭可能形成一个循环diketopiperazine端产品。 </li><li>对于多个同步合成，固相萃取真空多方面的建议，以最大限度地提高效率。 Peptoid合成也可以通过适当的编程方法，如在Aapptec APEX 396，CEM自由微波合成器和蛋白质TECHNO市售肽合成器自动logies公司前奏曲的集大成者。 </li></ol> 2。乳沟和侧链脱保护<ol><li>干树脂转移到20毫升闪烁玻璃小瓶。 </li><li>引擎盖内的工作，并使用适当的个人防护设备，添加4毫升三氟乙酸（TFA），裂解鸡尾酒（如95％AQ。TFA的，见讨论）闪烁的玻璃小瓶和第紧密。摇10分钟到2小时在室温下（见讨论）。 </li><li>收集TFA的乳沟解决方案，通过一个一次性的，聚丙烯烧结滤芯过滤到一个新的，预先称重20毫升闪烁玻璃小瓶树脂。一次性PP吸管是方便转移裂解鸡尾酒解决方案。 </li><li>加入1毫升的新鲜裂解鸡尾酒冲洗树脂和收集任何剩余peptoid。重复2倍。 </li><li>吹温柔的氮流或通过使用一个Biotage V10蒸发器蒸发，反式脂肪酸。 </li><li>在6毫升溶解原油乙腈/水1:1（V / V）为色谱。冷冻和冻干。重复。 </li><li>记录的原油产品的重量。存储作为一个在-20 ° C干粉</li><li>可以执行的测试乳沟（可选）：0.5％的树脂的测试裂解合成peptoid的纯度和质量迅速确定是否选择正确的裂解条件。测试分裂是特别有用监察合成的进展。 </li></ol> 3。表征及净化Polypeptoid <ol><li>通过结合分析法，电的LC - MS和/或MALDI - TOF，确定的原油产品的纯度和所需的分子量是否存在。 </li><li>准备一个〜5-10毫克/毫升的水peptoid粉干的解决方案，以最小的乙腈需要溶解度。筛选明确的解决办法，用0.45μm注射器过滤器去除灰尘和颗粒peptoid生产原油。 </li><li>分析HPLC和电的LC - MS：准备好了〜20微克/毫升原油peptoid解决方案。过滤200μL0.45μm的过滤器，并注入20μL。 </li><li> MALDI：混合1〜20毫克/毫升与1μL的矩阵peptoidμL。 SPOT 1μL的MALDI板和风干。矩阵和采集模式是依赖于样品（图5）。 </li><li>与反相制备高效液相色谱法净化原油peptoid混合。选择梯度和列（C4或C18）的基础上，polypeptoid疏水。结合纯化的组分，冻结，冻干，蓬松的白色粉末。记录最终产品的重量。 </li><li>形成的盐酸盐（可选）：溶解在100毫米盐酸（aq.）冻干粉，用最少的乙腈。转移到预先称重的玻璃小瓶。冻结和再冻干。重复2倍。 Reweigh确定peptoid粉的质量。 </li></ol> 4。 Peptoid薄片形成本节DEScribes协议形式从单链序列特异性，双亲的36 - mer peptoid（图1）张。后peptoid链合成，纯化，冻干如上所述，由此产生的白色粉末，溶于DMSO，使原液2毫米。 <ol><li>准备500μL20μM的peptoid表形成缓冲的解决方案（10毫米的Tris - HCl，100 mM氯化钠，在水中的pH值8.0）1 DRAM的玻璃小瓶。首先，添加445μL的Milli - Q水，50μL10X表形成缓冲，涡旋混合。然后，添加5μL2毫米peptoid原液，轻轻漩涡的解决方案。第玻璃小瓶。 </li><li>轻轻摇动的淡化水peptoid解决方案所形成的表。慢慢地倾斜的玻璃小瓶从水平位置，在张的直立位置结果。轻轻摇动也产生张;但是，张倾向于体积更小，用较少的直边。一个更透彻的分析表形成机制是reporteð分开。 </li><li>对于许多高品质的纸张，约横轴旋转的玻璃小瓶缓慢（&lt;1 RPM）为一至三天。适当的技术资源RKVSD Rotamix管肩或一个定制的摇杆可以执行这个不断。 </li><li>透析纳米片（可选）：在某些应用中，它可能是必要的，以消除任何免费peptoid链或缓冲区/盐。浸泡一个Float - A -仪100 kD的膜在15分钟的缓冲区。装入样品室500μLpeptoid表解决方案。浸泡在500毫升所需的缓冲区，磁力搅拌棒在60 RPM搅拌。让张进行4小时的透析。每隔一小时，交流与缓冲溶液中的新鲜的股票。 </li></ol> 5。荧光显微镜的纳米片<ol><li>尼罗红，环境敏感的染料，其荧光强度增加，当它在hydr本地化的薄片的荧光图像成像ophobic环境（图2）。 </li><li> 100μL的纳米片的解决方案，获得了尼罗河红1微米的终浓度添加1μL到100微米的尼罗红。 </li><li>在热水中1％的琼脂糖溶液倒入一个塑料培养皿。确保琼脂糖溶液约1 / 8英寸厚，并让溶液冷却在一个平面上不受干扰。琼脂糖集后，用抹刀削减和转移1厘米× 1厘米正方形载玻片。 </li><li>要收集在同一平面上，当场负债表上的一块琼脂糖溶液1μL的表。允许琼脂糖吸收2分钟的缓冲，留在纸张表面。在15分钟内的形象，否则琼脂糖将开始变形，由于脱水。 </li><li>在溶液中的形象表，负载15μL，20毫米直径0.12毫米载玻片上的垫片内。盖上盖玻片。如果纸张根本没有垫片，载玻片和盖玻片之间夹着无线许多张将剪切和看似微小的蒸发将导致表经常移动。 </li><li> epifluorescence照明下的图像表（如奥林巴斯IX81倒置显微镜下安装与安道尔iXonEM +与得克萨斯州的红色过滤器的EMCCD光谱）。 </li></ol> 6。扫描电子显微镜（SEM）的纳米片<ol><li>等离子体刻蚀硅衬底（可选）：硅芯片等离子蚀刻，以帮助在张的吸附。放置在真空室的等离子清洁（如Harrick等离子清洗机/灭菌器PDC - 32G）中的硅芯片。泵下降到200毫托和18W（PDC - 32G的高设置）设置射频线圈。 2分钟蚀刻。 </li><li>下降20μL一个等离子体处理的硅衬底上peptoid表解决方案。允许坐下来3分钟。删除的提示金正日多余的溶液擦拭。吸取20μL的水在表面上，并去除多余的解决方案，再次删除缓冲区和盐。重复4倍。 </li><li>或者，请拨打YZE peptoid表对水的解决方案，以消除缓冲区和盐。下降等离子体处理硅衬底上20μL透析表的解决方案。空气干燥样品。 </li><li>与扫描电镜（如蔡司双子座超55分析扫描电子显微镜）镜头探测器和1千伏及5千伏（图3）之间的束能量图片张。 </li></ol> 7。安全注意事项： <ol><li>二甲基甲酰胺，二氯甲烷有理由怀疑致癌物。 </li><li> N，N - Diisopropylcarbodiimide，4 methylpiperdine和溴乙酸对皮肤，眼睛和呼吸道造成危害。他们应该用在引擎盖与护理。它可能是有毒的，如果吸入或经皮肤吸收，接触可能会导致在敏。空容器中保留产品残留物（液体/蒸气）从罩中取出之前，应彻底清洗。 </li><li> TFA是一种强酸，具有极大的破坏性，上呼吸道，眼睛和皮肤。 TFA是挥发性保持在任何时候都罩的TFA的集中解决方案，以避免呼吸系统损害。使用适当的个人防护装备，并谨慎处理的TFA的解决方案时。及时更换手套，如果他们来与TFA的接触，并立即清理任何泄漏。 </li></ol> 8。代表性的成果： 本节介绍，序列特异的36 - mer peptoid链的合成，表征和净化，进入一个高度有序的纳米片（图1）3倍。 peptoid的H - [NAE - NPE] 9 - [NCE - NPE] 9 - NH 2块负责100毫克Rink酰胺树脂合成。 2米胺溶液被用于所有的位移反应，其中残留1-18为60分钟和120分钟残留19-36。叔丁基β-丙氨酸盐酸转化为游离碱（见讨论），而苯乙胺和银乙二胺均使用directlY.树脂切割与TFA的95％，2.5％triispropylsilane，2.5％的水2小时。 TFA的蒸发和由此产生的稠油（〜180毫克）重新溶解于6 mL乙腈：水1:1（V / V）。产品纯度（图4）和产品质量的存在被证实由反相高效液相色谱分析（30-80％乙腈水梯度，都含有1毫升/分钟超过30分钟的0.1％（V / V）TFA的，在60 ° c用的C18，5微米，50 x 2毫米列）和MALDI（图5）。 在Vydac C18柱（10微米，22毫米× 250毫米）反相高效液相色谱法分离纯化过程中，使用0.1％TFA超过60分钟，10毫升/分钟的30-60％乙腈水梯度。列装有60毫克粗品为每个色谱运行。纯化的组分结合的基础上从反相高效液相色谱法分析（图4）的纯度和冻干产生蓬松的白色粉末〜80毫克。 净化块负责peptoid分子重量也证实了电离。 1μL100μM纯化乙腈peptoid：水1:1（V / V）混合1μL矩阵（5 mg / mL的α-氰基-4 - 羟基乙腈酸：水1:1 V / V和0.1％TFA）和1μL发现电离板。样品风干后，它被放置在应用生物系统/ MDS SCIEX 4800 MALDI TOF / TOF分析仪。采集和处理模式是线性的低质量。在有针对性的大规模进入自动调整延迟时间计算重量。激光的强度设置为3400。所观察到的质量，4981.2，与密切合作，以4981.74计算质量。 纯化的冻干粉溶于DMSO，使2毫米原液，它可以存储在4 ° C。上述协议表和荧光光学显微镜和扫描电镜（图2和3）成像。各种形状与功能可达300微米大小的观察，并notabLY，直边突出。 <img alt="图1" src="/files/ftp_upload/3373/3373fig1.jpg" /> 图1序列的块负责peptoid的H - [NAE - NPE] 9 - [NCE - NPE] 9 - NH 2。单链块负责，双亲polypeptoid的36 - mer自组装成高度有序的二维薄片 3 。计算分子量为4981.74。 <img alt="图2" src="/files/ftp_upload/3373/3373fig2.jpg" /> 图2。peptoid薄片的荧光显微镜图像。形成了从20微米peptoid解决方案在10毫米，100毫米氯化钠，pH 8.0三表。琼脂糖1μM尼罗红的床单上成像。比例尺是100微米。 <img alt="图3" src="/files/ftp_upload/3373/3373fig3.jpg" /> 图3扫描电子显微镜图像。peptoid薄片。形成了从20微米peptoid解决方案在10毫米，100毫米氯化钠，pH 8.0三表。比例尺是5微米。 <img alt="图4" src="/files/ftp_upload/3373/3373fig4.jpg" /> 图4分析反相高效液相色谱法跟踪的H - [NAE - NPE] 9 - [NCE - NPE] 9 - NH 2。原油和纯化分析HPLC跟踪（30-80％的坡度超过30分钟1毫升/分钟，60 °的C18，5微米，50 x 2毫米列C）的原油和纯化块负责peptoid的H - [ NAE - NPE] 9 - [NCE - NPE] 9 - NH 2所示。 <img alt="图5" src="/files/ftp_upload/3373/3373fig5.jpg" /> 图5的MALDI - TOF质谱跟踪的H - [NAE - NPE] 9 - [NCE - NPE] 9 - NH 2。所观察到的质量，4981.2，密切协议，以计算出的质量，4981.74。

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应用和意义 此协议，描述了一个peptoid合成和水自组装成薄片的peptoids简单而有效的方法。大多数实验室很容易合成，因为廉价的材料，基本知识和简单的技术是利用 4 peptoids 。同样，超薄，高度有序的纳米片自组装仅仅需要反复倾斜稀水溶液peptoid解决方案2一小瓶。 Peptoids是有前途的生物医学和纳米研究材料，因为它们是强大和综合灵活且具有序列特异性和高度可调5。 Peptoids已经证明生物活性（6,7疗法，诊断8，细胞内交付9-10）和折叠成层次纳米结构3，11-14。由于其模块化的合成，组合peptoid溴化锂白羊座15-19可以很容易地合成了一系列广泛的活动或属性筛选。尤其是纳米片作为一个三维显示棚架，膜类似物，生物传感器，蛋白质类似物和设备制造的潜在平台。几乎取之不尽，用之不竭的的可能不同的序列，peptoid研究领域迅速扩大。 在固相合成polypeptoids submonomer的变量由于选择的能力，从一个单体20的令人难以置信的庞大和多样化的的拼音，submonomer方法需要偶尔修改的情况下增加的每一个步骤的耦合效率将提高产品的整体收益率。不受保护的杂环侧链纳入需要使用氯乙酸， 溴乙酸21。扩展位移时间和更高的胺浓度通常采用长peptoid序列或较少的亲核胺后约20接头。反应容器中加热至35 ° C，水夹套反应釜使用，有助于推动反应。对于高挥发性胺，如异丙胺，必须小心，以避免蒸发。 如叔丁基β-丙氨酸盐酸胺盐酸盐的形式，需要在位移反应之前自由为基础的。溶解或悬浮在DCM（〜5G amine/25毫升DCM）胺，并与摩尔的氢氧化钠水溶液在分液漏斗解决方案中和，这样就可以实现。 DCM层是收集和洗涤水层是额外的DCM。合并DCM层硫酸钠干燥和过滤到一个预先称重的圆底烧瓶中。旋转蒸发除去溶剂油产量，并记录产品的重量。 Durin克裂解步骤，TFA的卵裂鸡尾酒和裂解时间是保护组使用的数量和品种上的依赖。裂解鸡尾酒的指导方针是类似传统的肽脱保护分裂1。一般来说，10分钟孵化所需的序列没有几个高酸不稳定保护团体（如银，三苯甲基）保护团体或序列。两个小时的孵化建议更难以保护团体（如T -丁酯，地铁，PBF）或与许多保护组序列的序列，以确保每一个链的全脱保护。原油peptoid产品一般会溶解在乙腈：水1:1（V / V），但更高的乙腈比例共同具有较高的整体疏水性侧链。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>二甲基甲酰胺</td><td>电解二氧化锰</td><td> EM - DX1726P - 1 </td><td> 99％ </td></tr><tr><td> N - methylpyrrolidinone </td><td> BDH </td><td> BDH1141 - 4LP </td><td> 99％ </td></tr><tr><td>溴乙酸</td><td> Acros Organics公司</td><td> 200000-106 </td><td> 99％ </td></tr><tr><td>四甲基</td><td>西格玛爱秩序</td><td> M73206 </td><td> 96％ </td></tr><tr><td> N，N - diisopropylcarbodiimide </td><td>化工，Impex公司</td><td> 001100 </td><td> 99.5％ </td></tr><tr><td>二氯甲烷</td><td>电解二氧化锰</td><td> EMD - DX0835 </td><td> ACS级</td></tr><tr><td>乙腈</td><td>电解二氧化锰</td><td> EM - AX0145P - 1 </td><td> 99.8％ </td></tr><tr><td>三氟乙酸</td><td>西格玛爱秩序</td><td> T6508 </td><td> 99％ </td></tr><tr><td> Triisopropylsilane </td><td>西格玛爱秩序</td><td> 233781 -10G </td><td>对于TFA的乳沟</td></tr><tr><td> 1,2 - 二氯乙烷</td><td> JT贝克</td><td> JTH076 - 33 </td><td>对于硅化玻璃反应釜</td></tr><tr><td>苯乙胺</td><td>西格玛爱秩序</td><td> 407267 - 100ML </td><td> &gt; 99.5％，疏水性侧链胺</td></tr><tr><td>叔丁氧羰基乙二胺</td><td> CNH公司技术</td><td> C - 1112 </td><td>阳离子侧链胺</td></tr><tr><td>叔丁基β-丙氨酸盐酸</td><td> Impex公司国际化</td><td> 04407 </td><td>阴离子侧链胺</td></tr><tr><td> α-氰基-4 - 羟基苯甲酸</td><td>西格玛爱秩序</td><td> C8982 - 10X10MG </td><td>用于MALDI基质</td></tr><tr><td>尼罗红</td><td>西格玛爱秩序</td><td> 19123 - 10MG </td><td>对于荧光成像</td></tr><tr><td>二甲基二氯硅烷</td><td>西格玛爱秩序</td><td> 80430 - 500G - F </td><td>对于硅化玻璃反应釜</td></tr><tr><td>一次性聚丙烯烧结车脊</td><td>应用分离</td><td> 2416 </td><td> 6 mL聚丙烯盒20毫米的聚乙烯熔块</td></tr><tr><td>一次性3路露儿适配器</td><td>科尔帕默</td><td> 31200-80 </td><td>旋塞阀一次性人工合成反应釜</td></tr><tr><td>鲁尔锁定环</td><td>科尔帕默</td><td> 45503-19 </td><td> ¼“装修为一次性使用人工合成反应釜</td></tr><tr><td>配件露儿</td><td>科尔帕默</td><td> 45500-20 </td><td> ¼“装修为一次性使用人工合成反应釜</td></tr><tr><td>一次性聚丙烯吸管</td><td> VWR </td><td> 16001-194 </td><td>对于TFA的转让</td></tr><tr><td>鲁尔锁塑料注射器</td><td>国家科学技术进步奖</td><td> S7515 - 5 </td><td> 6毫升注射器</td></tr><tr><td> 1 DRAM玻璃小瓶</td><td> VWR </td><td> 66011-041 </td><td>以酚醛树脂成型聚氯乙烯面临的纸浆班轮螺丝帽</td></tr><tr><td> 20毫升SCINtillation小瓶</td><td> VWR </td><td> 66022-060 </td><td>附有PP瓶盖和纸浆箔班轮</td></tr><tr><td>安全密封胶间隔</td><td> Invitrogen公司</td><td> S - 24736 </td><td>对于荧光成像</td></tr><tr><td>载玻片</td><td>电子显微镜科学</td><td> 63411 </td><td>对于荧光成像</td></tr><tr><td>盖玻片</td><td> VWR </td><td> 48366-067 </td><td>对于荧光成像</td></tr><tr><td> 4“硅片</td><td>泰德佩拉</td><td> 16007 </td><td>预骰子在5X7毫米芯片</td></tr><tr><td> 0.45过滤器</td><td> VWR，Acrodisc </td><td> 28143-924 </td><td>为色谱。 PTFE膜</td></tr><tr><td>琼脂糖</td><td>屋宇署</td><td> 212272 </td><td>对于荧光成像</td></tr><tr><td> SPE真空歧管</td><td>西格玛爱秩序</td><td> 57044 </td><td> SPE的真空系统的例子</td></tr><tr><td>烧结玻璃容器</td><td>王牌玻璃</td><td> 6402-12 </td><td>孔隙彗星熔块</td></tr><tr><td>等离子清洁/消毒器</td><td> Harrick等离子</td><td> PDC - 32G </td><td>等离子清洁的范例，准备扫描电镜的硅芯片</td></tr></tbody></table>

solid-phase submonomer synthesis of peptoid polymers and their self-assembly into highly-ordered nanosheets

Peptoids是类新型的仿生，非天然，序列特定的抵抗水解heteropolymers，表现出强有力的生物活性，和高阶的纳米结构倍。结构相似的多肽，peptoids聚甘氨酸，N -取代侧链连接到α-碳，而氮比。他们易于合成和结构的多样性，允许测试的基本设计原则来驱动全新设计和新的生物活性和纳米材料的工程。 这里，一个简单的手动peptoid合成的协议，使长链polypeptoids的合成，在良好的收益（50mers）。只有基本设备，简单的技术（如液体输送，过滤），和市售试剂，使peptoids访问除了许多研究人员的工具包。 peptoid骨干生长在一个时间V单体IA的单体除了周期由两个步骤：酰化和位移submonomer方法。第一，溴乙酸激活就地与N，N&#39; - diisopropylcarbodiimide acylates树脂绑定的二级胺。二，由伯胺的亲核取代的溴化如下引入侧链。两步循环迭代，直到达到所需的链长。这两步周期的耦合效率经常超过98％，使长50残留peptoids合成。高度可调，精确和化学不同的序列与submonomer方法实现，数百个现成的伯胺，可直接纳入。 Peptoids正在成为一个多功能的仿生材料的纳米生物学的研究，因为他们合成的灵活性，健壮性，并在原子水平上的顺序。单链，双亲，信息折叠最近展示了丰富TION成高度有序的纳米片polypeptoid。这peptoid是一个36 - MER只有三个不同的市售单体组成：疏水，阳离子和阴离子。苯疏水性侧链都埋在纳米片的核心，而离子胺和羧基侧链亲水面临调整。 peptoid纳米片作为一个膜类似物，蛋白类似物，设备制造和传感器的潜在平台。 peptoid合成，表的形成，和显微成像方法进行了介绍，并提供一个简单的方法，以使未来的peptoid薄片设计。

Watch this Scientific Journal Video about Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets at JoVE.com

Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets

一个简单的和一般的手动peptoid涉及基本设备和市售试剂的合成方法概述，使peptoids可以很容易地在大多数实验室中合成。双亲peptoid 36mer的合成，纯化及表征的描述，以及其自组装成高度有序的纳米片。

固相Submonomer Peptoid聚合物及其自组装成高度有序的纳米片的合成

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and ...

solid-phase-submonomer-synthesis-peptoid-polymers-their-self-assembly

Research

JoVE Journal

Bioengineering

29.1K 次观看.  Lawrence Berkeley National Laboratory. 一个简单的和一般的手动peptoid涉及基本设备和市售试剂的合成方法概述，使peptoids可以很容易地在大多数实验室中合成。双亲peptoid 36mer的合成，纯化及表征的描述，以及其自组装成高度有序的纳米片。

视频: Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

形状记忆聚合物主动细胞培养

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

科研

生物工程

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

定向细胞自我组装制造，为生物力学分析与组织工程细胞衍生的组织环

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

由共轭聚合物活细胞电活动的光控制

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

Fabrication of a Dipole-assisted Solid Phase Extraction Microchip for Trace Metal Analysis in Water Samples

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water samples. A brief overview of the evolution of chip-based SPE techniques is provided. This is followed by an introduction to specific polymeric materials and their role in SPE. To develop an innovative dipole-assisted SPE technique, a chlorine (Cl)-containing SPE functionality was implanted into a poly(methyl methacrylate) (PMMA) microchip. Herein, diverse analytical techniques including contact angle analysis, Raman spectroscopic analysis, and laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) analysis were employed to validate the utility of the implantation protocol of the C-Cl moieties on the PMMA. The analytical results of the X-ray absorption near-edge structure (XANES) analysis also demonstrated the feasibility of the Cl-containing PMMA used as an extraction medium by virtue of the dipole-ion interactions between the highly electronegative C-Cl moieties and the positively charged metal ions.

The fabrication protocol of a dipole-assisted solid phase extraction microchip for the trace metal analysis is presented.

水样中偶极辅助固相萃取Microchip的痕量金属分析的制作

This paper describes a fabrication protocol for a dipole-assisted solid phase extraction (SPE) microchip available for trace metal analysis in water ...

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

在脉冲激光激发金纳米粒子集成光电响应脂质体及其微泡空化的测量的合成

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

医疗器械细菌高通量鉴定驱蚊聚合物

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

通过反应挤出、微流体、自组装和电纺丝制备多长尺度可降解 Thermoresponsive 水凝胶

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In conventional lab-on-a-chip-platforms, solvents and samples are passed through defined microfluidic channels with complex flow control installations. The droplet actuation platform presented here is a promising alternative. With it, it is possible to move a liquid drop (microreactor) on a planar surface of a reaction platform (lab-in-a-drop). The actuation of microreactors on the hydrophobic surface of the platform is based on the use of magnetic forces acting on the outer shell of the liquid drops which is made of a thin layer of superhydrophobic magnetite particles. The hydrophobic surface of the platform is needed to avoid any contact between the liquid core and the surface to allow a smooth movement of the microreactor. On the platform, one or more microreactors with volumes of 10 &#181;L can be positioned and moved simultaneously. The platform itself consists of a 3 x 3 matrix of electrical double coils which accommodate either neodymium or iron cores. The magnetic field gradients are automatically controlled. By variation of the magnetic field gradients, the microreactors' magnetic hydrophobic shell can be manipulated automatically to move the microreactor or open the shell reversibly. Reactions of substrates and corresponding enzymes can be initiated by merging the microreactors or bringing them into contact with surface immobilized catalysts.

Lab-in-a-drop reaction systems allow the versatile implementation of complex reactions in a microfluidic scale. An automated actuation platform consisting of a 3 x 3 matrix of electromagnetic coils was developed and successfully used to merge two 10 &#181;L microreactors and thereby initiate an enzymatic reaction in the resulting liquid marbles.

用作酶反应器的水滴及其电磁驱动

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

对细胞的超快磁化阳离子化Magnetoferritin的合成

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

工程分子识别与单壁碳纳米管仿生聚合物

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...