Name: engineering molecular recognition with bio-mimetic polymers on single walled carbon nanotubes
Uploaded: 2017-01-10T14:00:00.000+00:00
Duration: 9 min 28 s
Description: Watch this Scientific Journal Video about Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes at JoVE.com

This work was supported by Burroughs Wellcome Fund Career Award at the Scientific Interface (CASI), a Simons Foundation grant, and a Brain and Behavior Research foundation young investigator grant.

注意:使用前请咨询所有相关的材料安全数据表（SDS）。相比，他们的散料对口纳米材料可能有额外的危害。利用一切适当的安全实践，包括工程控制（通风橱，隔音罩）和个人防护设备（安全眼镜，护目镜，实验室外套，全长裤，封闭趾鞋）。 1.缓冲液，表面活性剂和聚合物溶液的制备<ol><li> 的100mM NaCl的溶液制备 <ol><li>溶解584毫克的NaCl在80毫升去离子水。加去离子水使总体积为100毫升。 </li></ol></li><li> 3％十二烷基硫酸钠的制备（SDS）溶液 <ol><li>溶解3克的SDS在80毫升去离子水。加去离子水使总体积为100毫升。 </li></ol></li><li> 2％的胆酸钠的制备（SC）溶液 <ol><li>溶解2克草皮鎓胆水合物在80毫升去离子水。加去离子水使总体积为100毫升。 </li></ol></li><li> 成像缓冲器的制备（1×的Tris:20毫摩尔Tris，100mM的NaCl）中 <ol><li>溶解22.23克Tris碱并使用磁力搅拌棒和板在500毫升去离子水58.44克氯化钠。 </li><li>小心加入浓HCl直到达到pH为8.1。 </li><li>加入去离子水，达到1升的最终体积</li></ol></li><li> RITC-PEG-RITC聚合物的合成 <ol><li>二氯甲烷和二甲基甲酰胺（DMF）的混合物:在1毫升1:1的溶解胺双官能聚乙二醇（PEG）（5 100kDa或20kDa的，0.1摩尔/升）和若丹明异硫氰酸酯（RITC，0.2摩尔/升）。 </li><li>添加0.2摩尔/升的N，N-二异丙基乙胺（DIEA）。 </li><li> 3小时后，沉淀用10倍体积的乙醚，随后通过真空过滤的。 </li><li>重新溶解在DMF和重复乙醚沉淀弗洛通过真空过滤结婚。 注意:其他异硫氰酸酯改性分子（ 如，异硫氰酸荧光素，FITC）可以连接到PEG或其它胺改性的聚合物使用类似的方法。 </li></ol></li><li> PEG化-DNA的制备（PEG-DNA）的 <ol><li>结合100三（2-羧乙基）膦盐酸盐（TCEP）溶液（0.5M，pH 7.0）中以44.9微升微升5&#39;-巯基 - 修饰的DNA（1毫克/10μL在0.1M NaCl）中，并加入到4.855毫升去离子水。 </li><li>搅拌1小时。 </li><li>溶解500毫克甲氧基聚乙二醇马来酰亚胺的5ml磷酸盐缓冲盐水。 </li><li>结合DNA和PEG溶液（10毫升总），搅拌24小时。 </li></ol></li></ol> 2.单壁碳纳米管（单壁碳纳米管）悬浮液的制备<ol><li> 单壁碳纳米管的清洗以除去催化剂和杂质。 <ol><li>添加200-300毫克没有洗过的单壁碳纳米管的成塑料离心管中CONTA进不去45毫升去离子水。 </li><li>涡旋2分钟，超声处理用水浴超声5分钟的溶液中。需要注意的是超声器设置仪器之间的不同，所以检查的SWNT的解决方案增加了光学密度（ 即变黑），以确保单壁碳纳米管分散。 </li><li>离心20分钟将溶液在16100 xg离心并丢弃上清液。 </li><li>加入约45毫升的新鲜去离子水。 </li><li>重复步骤2.1.2-2.1.4高达8倍。 </li><li>删除尽可能多的水尽量小心，不要去打扰丸状的单壁碳纳米管，并允许单壁碳纳米管颗粒在空气中干燥。 </li></ol></li><li> 单壁碳纳米管的核酸悬浮液 <ol><li>溶解在0.1M NaCl的核酸（NA）为100毫克/毫升的浓度。 </li><li>使用抗静电枪从一次性锅铲，微量离心管和单壁碳纳米管的库存除去静电。在通风橱中，加入Na溶液的20微升至0 980微升.1 M氯化钠，然后加入1毫克的单壁碳纳米管。 </li><li>使用超声发生器3毫米直径的尖端，超声处理10分钟，在40％的幅度，在冰浴中的溶液中。 </li><li>两次离心DNA的单壁碳纳米管溶液中90分钟，16100×g的。如果使用的PEG-DNA，纯化出多余的和未反应的DNA和PEG使用100kDa的自旋滤波器。加入足够的PBS，以填补自旋过滤器和旋转9300 XG为1.5分钟，重复此洗涤步骤3次。 </li><li>收集和保存上清液，小心不要去打扰含碳纳米管束和聚集沉淀。丢弃在根据适当的纳米材料的机构危险废物过程中沉淀。 </li><li>测量在632纳米用紫外/可见分光光度计，以确定使用ε= 0.036升/厘米·毫克按照朗伯 - 比尔定律和适当的稀释因子悬浮SWNT的近似浓度的溶液的吸光度。 </li></ol></li><li> 两亲聚合物悬浮单壁纳米碳管<ol><li>从一次性锅铲，微型离心管和单壁碳纳米管股票去除静电。在通风橱中，加入5毫克的SWNT至5ml的2％悬浮剂溶液（可选地，SDS溶液可以使用）。 </li><li>使用超声发生器6毫米直径的尖端，超声处理1小时，在40％的幅度，在冰浴中的溶液中。 </li><li>使用超速离心机和珍藏上清15万XG 4小时离心机样品。 </li><li>溶解两亲性聚合物（ 例如，RITC-PEG-RITC）的SC-SWNT溶液的1重量％。 </li><li>透析用3.5 kDa的透析膜的高分子的SC-SWNT溶液对1升的去离子水或缓冲液5天。改变出的水或小时2和小时4.较大透析膜可用于后缓冲，只要它允许移除所选择的表面活性剂，但单壁碳纳米管和两亲聚合物二者的保留。 </li><li>用紫外/可见分光光度计，以确定测量在632 nm处的溶液的吸光度用ε= 0.036 L /厘米*毫克按照朗伯 - 比尔定律及相应的稀释倍数暂停单壁碳纳米管的大致浓度。 </li></ol></li></ol> 3.表面固定化单壁碳纳米管传感器的制备<ol><li> BSA的生物素和的NeutrAvidin储备溶液的制备 <ol><li>溶解10毫克冻干的BSA - 生物素在1mL去离子水中，制成10毫克/毫升储液，并储存于4℃。 </li><li>在2mL去离子水中溶解10毫克的NeutrAvidin蛋白（NAV，去糖基化抗生物素蛋白的蛋白质），以使一个5毫克/毫升贮备液。商店等分于-20℃。解冻等分试样可以在4℃保存数天。 </li></ol></li><li> 准备BSA生物素涂显微镜载玻片 <ol><li>清洁的显微镜载玻片和0.17毫米遮玻璃（或以适合的显微镜物镜）用去离子水，随后用甲醇，丙酮和去离子水的最后冲洗。 </li><li&gt;通过在干净的显微镜载玻片放置双面胶带的几件相距约5mm创建信道。通过采取长玻璃盖玻片并按下它到双面胶带的顶部密封的通道。可以肯定的是集中在显微镜载玻片使盖玻片和幻灯片的边缘不冲水。 </li><li>加入100微升BSA - 生物素储液至900 1×μL的Tris缓冲液以1毫克/毫升的最终浓度。 </li><li>由溶液吹打到一个端部和使用组织芯吸走溶液在另一流的BSA的生物素溶液进入通道的50微升。孵育5分钟，随后由3-5刷新用0.1M NaCl的50微升。 </li><li>稀释的960微升1×Tris缓冲液5毫克/毫升的NAV库存40微升至0.2毫克/毫升的最终浓度。 </li><li>由溶液吹打到一个端部和使用组织芯吸走溶液在另一流的NAV溶液进入通道的50微升。孵育2-5分钟接着3-5刷新用0.1M NaCl的50微升。 </li><li>在成像缓冲器稀悬浮SWNT的储备溶液以1-10毫克/升的浓度和流量将溶液的50微升到通道并孵育5分钟。 </li><li>轻轻洗去使用成像缓冲器的50微升多余的单壁碳纳米管。 </li></ol></li><li> APTES硅烷化显微镜载玻片的制备 注:APTES硅烷化载玻片允许的方式固定带负电荷的DNA包裹的单壁碳纳米管的玻璃基板的表面上。 <ol><li>制备在乙醇（3-氨基丙基）三乙氧基硅烷（APTES）的10％溶液。 </li><li>通过使用3.2.2所述的步骤做渠道，流入100 1X的微升Tris缓冲液。 </li><li>冲洗与APTES溶液的信道和孵育5分钟。用1X Tris缓冲液清洗。 </li><li>稀释在成像缓冲器悬浮DNA的单壁碳纳米管的储备溶液以1-10毫克/土地流的浓度的溶液进入通道和incub 50微升吃了5分钟。 </li><li>轻轻洗去使用成像缓冲器的50微升多余的单壁碳纳米管。 </li></ol></li></ol> 4.荧光光谱法和单壁碳纳米管传感器的显微镜<ol><li> 近红外荧光显微镜 <ol><li>执行使用激光激发并用的InGaAs传感器阵列用于成像配备倒置显微镜的表面固定化的单壁碳纳米管的成像。 </li><li>引导激光束进入上使用可调节的可调安装座反射镜和一对设置到端口高度后安装虹膜倒置显微镜的背照明端口。确保梁是水平和直线通过确认放置后续镜之间，并在进入照明端口之前当其穿过两个虹膜。如果有必要，使用潜望镜装配调整光束的高度。删除会减弱光束照明端口上的任何短通滤波器的热量。 </li><li>将适当的过滤器立方体进入显微镜反映激发光到目标，并收集发射光。这通常包括用激发波长高于截止，（ 例如 ，750 LP）与发射长通滤波器（ 例如，850的LP），以撞击所述传感器进一步减少散射的激发光的分色长通滤波器。此外，发射滤波器可以被选择为选择性对特定手性的单壁碳纳米管的排放。 </li><li>通过插入合适的对准立方体用含有1mM针孔两个偏置，磨砂盘更换物镜进行微调整的光束对准。对准束两者的上下对准盘片的中心。 </li><li>附加使用适当的适配器和0.5X透镜在必要时，以容纳传感器尺寸的2D的InGaAs传感器阵列到显微镜的侧成像端口。 </li><li>使用100X油浸物镜（1.4 NA），适用无荧光浸油，并将immobilized SWNT样品到显微镜阶段。 </li><li>提高物镜直到油触点护罩玻璃的底部（＃1.5，170微米厚）。使如果必要的调整，以客观衣领成像条件， 例如，温度，玻璃厚度等 </li><li>慢慢提升与激励源的目标和监视的InGaAs相机的荧光信号。荧光强度应逐渐增加，因为焦平面接近表面和固定传感器成为焦点。 </li></ol></li><li> 近红外荧光光谱仪 可以使用相同的显微镜装置进行，但通过将所收集的光出显微镜主体并进入光谱仪和InGaAs线阵探测器荧光光谱:注。 <ol><li>使用运动坐骑和镜子额定NIR，直接朝着光谱仪的入射狭缝的光路。聚焦光线降低到POI核苷酸上的入口用的聚焦透镜狭缝（ 例如 ，平凸中，f = 150毫米）。这种对准是由衰减激光功率为&lt;1毫瓦，并用1"镜更换显微镜物镜并使用50/50光束分离滤波器立方体完成的，激发光随后将通过出口离开显微镜主体和可用来调节反射镜和透镜将光束聚焦到入口狭缝。 </li><li>更换显微镜物镜和长通滤波器后，放置一个孔板上舞台，并记录使用分光计和的InGaAs阵列的焦点对准样品的光谱。 </li></ol></li><li> 测量的（GT）15 DNA的单壁碳纳米管对多巴胺可逆反应 <ol><li>安装传感器被覆通道到显微镜台上，用100X油物镜和的InGaAs相机带入焦点。加在磷酸盐缓冲盐水（PBS）100μM的多巴胺溶液50μL到流动通道和记录在荧光强度的变化。 </li><li>用磷酸盐缓冲盐水洗出多巴胺溶液，并观察在各个单壁碳纳米管传感器的荧光的变化。 </li></ol></li><li> 孔板筛查单壁碳纳米管传感器的响应分析 注:可使用使用机动阶段控制的透明孔板进行不同的分析物的筛选。一种理想的孔板是透明的可见光和红外和具有黑色侧壁以最小化孔之间的串扰。 <ol><li>悬浮SWNT传感器（ 例如 ，5毫克/升浓度）到每个孔，足以覆盖阱均匀，典型地&gt; 100微升的用于96孔板和384孔板的底部&gt; 30微升的移液管等体积。 </li><li>移液器分析（ 例如 ，2μL，100μM最终体积）从库中筛选到孔板。准备一式三份各分析考虑到潜在的富裕以及变异，fluctuat激发强度或温度的离子。 </li><li>提高目标（ 例如 ，20X achroplan，0.45 NA），同时监测利用分光计和砷化铟镓阵列发射光谱。物镜的最佳位置将投入焦平面大致在孔中的样品体积，这应该对应于在测得的强度最大的中间。 </li><li>对于每个样品孔，录制1-10敞口收集光谱。 </li><li>比较的发光光谱为每孔含有无需额外的分析物只是单壁碳纳米管来量化荧光响应的控制。 </li></ol></li></ol>

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The authors have nothing to disclose.

单壁碳纳米管可以容易地悬浮在通过直接超声水溶液用SDS或ssDNA的，由所得到的单壁碳纳米管的聚合物混合的胶态分散体提供的增加光密度所指示的。 SDS和单链DNA分散并通过疏水性或π-π相互作用吸附在SWNT表面溶解的单壁碳纳米管束。此外，其他的聚合物，例如基因组DNA，两亲聚合物，共轭聚合物和脂质，可以通过使用SC或SDS悬浮样品的透析吸附到单壁碳纳米管的表面上。亲水性聚合物，如PEG，可以与疏水性"锚"，如RITC或FITC末端改性，以使嵌段共聚物的表面吸附。对于易受在暴露于探头尖端超声处理的高功率剪切或降解，或对于具有低结合亲和力SWNT聚合物的聚合物，透析，以产生一个稳定的SWNT聚合物悬浮液的最佳方法。继聚合物encapsulation，离心除去大的SWNT束，无定形碳，残留的金属催化剂，以及其他不溶的污染物离开一个均匀分散的样品。分散的单壁碳纳米管的典型终浓度后10-100毫克/升之间离心范围。 超声功率和持续时间可以调节和用于分散剂的特定选择优化。这是在用于涂层的单壁碳纳米管的方法的关键步骤，因为太少或弱超声处理可导致分散性差，而过多或过强大超声处理可导致荧光差。通常情况下，较低的权力是必要时使用容易剪切，如DNA聚合物时减少损失。再超声处理持续时间或更高的强度可导致减少单壁碳纳米管，其中所述〜100nm的激子复合长度以下的单壁碳纳米管成为非荧光的长度。超声波仪探头尖端的大小也应符合样本量，以避免飞溅和泡沫最佳结果（通常由厂家提供）。避免接触探头尖端的容器的侧面，并放置该溶液用冰块上，以尽量减少溶液的加热。一旦分散，单壁碳纳米管的溶液是在室温下无限期地稳定。 分散产生的单壁碳纳米管的溶液的吸光度和发射光谱包含多个峰，表明不同手性的分散的单壁碳纳米管的混合物中的存在。的单壁碳纳米管的产生或纯化方法的替代方法可以改变手性分布，从而导致不同的峰值激发和发射荧光光谱。此外，不同的合成方法可以产生不同的手性人口分布的单壁碳纳米管样品。例如:CoMoCAT（钴 - 钼催化剂）生长单壁碳纳米管富含（6,5）手性，而的Hipco（与羰基铁催化剂高压）生长单壁碳纳米管富含（7,6）手性，导致在吸收差异和辐透oluminescence光谱。 某些吸附聚合物通过在管表面改变局部环境调节的SWNT的荧光发射使分析物的特异性检测。这种方法提供了明显的优势的由不会永久破坏的SWNT晶格，这可以减少荧光发射强度结合部分共价连接。 6,32 - 34此外，CoPhMoRe方法具有开发无抗体的传感器在有可能不存在一个已知的结合部分指标的潜力。 28具体而言，（GT）15 -DNA已经显示出选择性地增强多巴胺的存在单壁碳纳米管的荧光发射，使得其用作多巴胺传感器。散装荧光的测量结果表明在某些SWNT手性峰值发射增加多达80％。固定化（GT）上的载玻片15 -DNA的SWNT使荧光RESP个体（GT）15 -DNA官能单壁碳纳米管的ONSE测量，显示出荧光可以超过3倍于多巴胺的存在单一的SWNT传感器增加，而没有任何明显的漂白，在连续的激光照射。多巴胺和SWNT的传感器之间的相互作用是可逆的，因为显然由原始荧光信号用缓冲液洗涤多巴胺出微流体腔室的后的恢复。此外，单分子测量可以用于量化聚合物吸附（ 图4）或结合事件的动力学的强大表征技术。此外，比率传感可以通过化学分离奇异SWNT手性与唯一激发发光峰，与（GT）15 -DNA官能它，并分离出第二单壁碳纳米管的手性是不敏感的多巴胺来实现。同时监测SWNT的手性提供了一个可以相比米稳定控制荧光通道<html> odulating的（GT）15-DNA单壁碳纳米管的荧光。结合不同的聚合物（ 例如，聚乙二醇和（GT）15 -DNA）可以添加额外的功能如修改扩散或细胞摄取的特性， 在体内实验进行时是关键的性质。 目前，CoPhMoRe方法的限制传感器的发展包括聚合物图书馆的发展。因为结合部分是未知的先验 ，开发用于一个特定对象的传感器可以是时间密集的，并且需要大量的化学变化的聚合物的用于构造所述传感器文库进行筛选。此外， 在体内环境中的稳定性和传感器的兼容性在可以从传感器变化到传感器。然而，一旦一个候选传感器已被确定，进一步的修改策略可以用于优化性能所必需的体内应用。 ve_content"&gt;这里，我们已经证明用于分散在适用于各种各样的分散剂的水溶液的SWNT的方法，这种方法可用于创建分散的单壁碳纳米管的库新颖新NIR传感器的发现为小分子和生物标记物。特别感兴趣的是用于检测神经递质，它可以使实时，在复杂的生物环境中的这些分子的空间精确检测传感器。

单壁碳纳米管（SWNT）的原子的碳原子薄层轧成表现出独特的电子和光学性质长而细的圆柱体。 1这些性质包括一个带隙产生近红外（NIR）荧光经由激子的重组也就是它的本地环境高度敏感发射。 SWNT的NIR发射落在近红外窗口，在其中的光的穿透深度为最大的生物组织内。 2,3此外，单壁碳纳米管表现出相对于有机荧光非典型几个独特的功能:SWNT呈现大的斯托克斯位移，不要光漂白，不闪烁。 4最近，利用这些特性已导致新颖分子传感器的与应用到生物学各种各样的发展。 5,6-未修改然而，单壁碳纳米管是不溶于水，并且获得各个单壁碳纳米管的悬浮液可能是一个挑战。 7,8 BundliNg和在溶液中的单壁碳纳米管的聚集可以混淆他们的带隙的荧光，使2它们不适合用于传感应用。 在水溶液中分散个别碳纳米管要求修改其表面以防止疏水驱动聚集。 9虽然共价修饰可以呈现的单壁碳纳米管的水溶性，10以及赋予特异性结合化学，在单壁碳纳米管的晶格缺陷部位减少或减轻它们的荧光发射。通过疏水和π-π堆积相互作用吸附到纳米管表面13 -相反，单壁碳纳米管的官能化可以通过使用表面活性剂，脂质，聚合物和DNA 9,11来完成。周围表面官能化的单壁碳纳米管的产生的化学环境被称为其电晕相。扰动电晕相位可对激子的纳米管表面上行驶的很大的影响，引起调制到SWNT氟escence排放。它是电晕相和可利用通过将特异性结合模式到单壁碳纳米管的大的表面积，以开发新的分子传感器的SWNT荧光之间的这种敏感的关系。扰动在结合分析物的单壁碳纳米管的电晕相位可以导致在局部电介质环境的变化，电荷转移，或引入晶格缺陷，所有这些都可以调节SWNT的荧光发射以作为信号转导机制。 14这种方法是在新型荧光传感器的发展用于检测许多不同类型的分子中的包括DNA，15,16葡萄糖17和小分子如ATP，18活性氧19和一氧化氮。 20,21然而，这些方法被限制的，因为它们依赖于对靶分析物的已知结合形态的存在。 近日，一个更通用的应用程序蟑螂对设计荧光传感器用单壁碳纳米管与两亲杂，磷脂和多核酸非共价地官能化的发展。这些分子吸附到碳纳米管表面，以产生单个的单壁碳纳米管22的高度稳定的悬浮液- 25具有独特的电晕阶段可以特异性结合蛋白26,27或小分子包括神经递质多巴胺。 28 - 30工程电晕相位分散的单壁碳纳米管和特异性结合目标分析物被称为电晕相分子识别（CoPhMoRe）。 28小尺寸，低毒性，高稳定性和CoPhMoRe单壁碳纳米管传感器unbleaching NIR荧光使他们为延长时间分辨测量体内检测的最佳候选。 6最近的工作表明它们在植物组织应用活性氮和氧的光学检测。 31为CoPhMoRe SWNT传感器特别激动人心的应用是用于神经递质如在体内 ，多巴胺标签免费检测的电位在其他技术，如电化学感测或免疫组织化学，从缺乏空间分辨率，时间分辨率和特异性的痛苦。 设计和发现CoPhMoRe SWNT传感器迄今被分散库的大小和化学多样性限制，限制找到一个传感器为特定的目标的可能性。到目前为止，研究人员已经只是触及可结合，共同块，生物，可作为功能活跃分散剂SWNT仿生传感器的聚合物的表面。在这里，我们提出了两个单壁碳纳米管的分散和表征他们的高通量筛选和单一的单壁碳纳米管传感器分析荧光不同的方法。具体来说，我们概述了与多核苷酸oligom涂层单壁碳纳米管的方法器使用直接超声处理以及如何通过透析通过表面活性剂交换官能与两亲聚合物的SWNT。我们使用若丹明异硫氰酸酯（RITC-PEG-RITC）官能化（GT）15 -DNA和聚乙二醇作为例子。我们证明了使用的（GT）15-DNA单壁碳纳米管作为用于检测多巴胺CoPhMoRe传感器。最后，我们概述用于执行单分子传感器测量，其可用于表征或单分子检测程序。

<table><tbody><tr><td>sodium chloride</td><td>Fisher Scientific</td><td>S271-1</td><td></td></tr><tr><td>sodium dodecyl sulfate</td><td>Sigma Aldrich</td><td>L6026</td><td></td></tr><tr><td>sodium cholate hydrate</td><td>Sigma Aldrich</td><td>C6445</td><td></td></tr><tr><td>tris base (Trizma base)</td><td>Sigma Aldrich</td><td>93362</td><td></td></tr><tr><td>hydrochloric acid</td><td>Fisher Scientific</td><td>A144-212</td><td></td></tr><tr><td>Amine-PEG-amine,NH&lt;sub&gt;2&lt;/sub&gt;-PEG-NH&lt;sub&gt;2&lt;/sub&gt;</td><td>Nanocs Inc</td><td>PG2-AM-5k</td><td></td></tr><tr><td>rhodamine B isothiocyanate</td><td>Sigma Aldrich</td><td>283924</td><td></td></tr><tr><td>fluorescein isothiocyanate</td><td>Sigma Aldrich</td><td>F7250</td><td></td></tr><tr><td>dichloromethane</td><td>Sigma Aldrich</td><td>676853</td><td></td></tr><tr><td>dimethylformamide</td><td>Sigma Aldrich</td><td>D4551</td><td></td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-diisopropylethylamine</td><td>Sigma Aldrich</td><td>D125806</td><td></td></tr><tr><td>diethyl ether</td><td>Sigma Aldrich</td><td>673811</td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride</td><td>Sigma Aldrich</td><td>C4706&amp;nbsp;</td><td></td></tr><tr><td>5&amp;rsquo;-thiol-modified DNA&amp;nbsp;</td><td>Integrated DNA Technologies</td><td></td><td></td></tr><tr><td>methoxypolyethylene glycol maleimide</td><td>Sigma Aldrich</td><td>63187</td><td></td></tr><tr><td>100 kDa spin filters</td><td>Millipore</td><td></td><td></td></tr><tr><td>HiPCO Super purified single walled carbon nanotubes</td><td>Integris</td><td>HiPco SuperPurified</td><td></td></tr><tr><td>phosphate buffered saline</td><td>Sigma Aldrich</td><td>P5493</td><td></td></tr><tr><td>anti static gun</td><td>Milty</td><td>Milty Zerostat 3</td><td></td></tr><tr><td>centrifuge</td><td>Eppendorf</td><td>5415 D</td><td></td></tr><tr><td>ultra sonicator</td><td>Cole Parmer</td><td>CV18</td><td></td></tr><tr><td>dialysis cassettes</td><td>Thermo scientific</td><td>Slide-A-Lyzer G2 87722</td><td></td></tr><tr><td>BSA-biotin</td><td>Thermo scientific</td><td>29130</td><td></td></tr><tr><td>Neutravidin protein</td><td>Thermo scientific</td><td>31000</td><td></td></tr><tr><td>(3-Aminopropyl)triethoxysilane (APTES)</td><td>Sigma Aldrich</td><td>440140</td><td></td></tr><tr><td>inverted microscope</td><td>Zeiss</td><td>Axio Observer.Z1</td><td></td></tr><tr><td>kinematic mirrors</td><td>ThorLabs</td><td>KM200-E03</td><td></td></tr><tr><td>periscope</td><td>ThorLabs</td><td>RS99</td><td></td></tr><tr><td>immersion oil</td><td>Zeiss</td><td>Immersol 518f</td><td></td></tr><tr><td>100X objective</td><td>Zeiss</td><td>Plan-apochromat 100X oil, 1.4NA, PH3, 420791-9911-000</td><td></td></tr><tr><td>20X objective</td><td>Zeiss</td><td>N-Achroplan 0.45 NA, 420953-9901-000&amp;nbsp;</td><td></td></tr><tr><td>cover glass</td><td>Healthrow Scientific</td><td>HS159879H</td><td></td></tr><tr><td>dopamine hydrochloride</td><td>Sigma Aldrich</td><td>H8502&amp;nbsp;</td><td></td></tr><tr><td>infrared 2D array camera</td><td>Princeton Instruments</td><td>NIRvana</td><td></td></tr><tr><td>infrared 1D sensor array</td><td>Princeton Instruments</td><td>PyLoN IR</td><td></td></tr><tr><td>nIR spectrograph</td><td>Princeton Instruments</td><td>SCT-320</td><td></td></tr><tr><td>planoconvex lens</td><td>ThorLabs</td><td>LA1384</td><td></td></tr><tr><td>well plates (glass bottom)</td><td>Corning</td><td>4580</td><td></td></tr></tbody></table>

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.

单壁碳纳米管悬浮在同时使用表面活性剂和通过直接超声处理和通过透析交换两亲聚合物水溶液。 图1显示了单壁碳纳米管，采用羰基铁催化法（的Hipco）增长，使用SC，RITC-PEF20-RITC暂停，（GT）15 -DNA。用SDS（或聚合物）一个单壁碳纳米管的光密度在超声处理后显着增加，并在除去聚集物和污染物通过纯化通过离心（ 图1）减小。在632吸光度测量纳米定量悬浮SWNT的浓度。 28 单壁碳纳米管悬浮液的光物理性质使用吸收光谱和荧光光谱表征。 图2示出了使用（GT）15 -DNA和RITC-PEG20-RITC悬浮的单壁碳纳米管的吸光度和荧光发射光谱。钍È吸收光谱是单个吸收峰为样品中存在的纳米管的每个不同手性的叠加。类似地，每个手性显示出其特有的荧光发射峰。相对发光峰强度的差异是使用721纳米的激光在手性的人口分布，以及在激发效率差的差异的结果。 的（GT）15 DNA的单壁碳纳米管的荧光响应（其中I 0是初始的SWNT荧光强度和I是多巴胺添加后的单壁碳纳米管的强度）在不同浓度的多巴胺的存在是通过使用监视的样品的荧光测定分光光度计的InGaAs线性阵列（ 图3）。 （GT）15-DNA，单壁碳纳米管与多巴胺浓度的增加而增加（ 图3a）的总荧光。荧光řesponse是发射峰的函数（ 图3b），表明该响应可以是手性的特定。在1044纳米的多巴胺浓度接近2微米波长峰的强度增加2倍的荧光和1078。 图3e示出的PEG-（GT）15 DNA的单壁碳纳米管的增加整个发射光谱的响应于加法多巴胺的强度。 使用替代的DNA序列涂覆单个的单壁碳纳米管，C 26 -DNA，拴显微镜载玻片的表面上（用在（GT）15相同的方法制备）被激光照射下使用的InGaAs相机和100X油浸物镜测量（ 图4）。监测拴到表面单个发射器可以用来验证由远用缓冲溶液洗涤靶分子传感器响应的可逆性。全内反射荧光（TIRF）百万分之一拷贝还可用于吸附在单壁碳纳米管的量化通过光漂白实验附加到每个管的DNA分子的数量影像染料缀合的DNA。 图4显示了Cy3标记的DNA的3个不同的白化事件从一个单一的发射器的荧光强度曲线的量化步骤的推断。这些结果表明，三种DNA分子附着到单壁碳纳米管。 <img alt="图1" src="/files/ftp_upload/55030/55030fig1.jpg" /> 图1: 聚合物，表面活性暂停单壁碳纳米管。 （ 一 ）RITC-PEG20-RITC单壁碳纳米管的照片用2％SC编写的各个点暂停。左:单壁碳纳米管直接超声之前加入到SC的解决方案。中心:浴超声处理10分钟，然后用10分钟探针针尖超声处理在90％振幅随后离心后。该溶液的光密度增大为单壁碳纳米管束是分散（约100毫克/升的SWNT浓度）。右:经过透析RITC-PEG20-RITC聚合物，RITC-PEG-RITC的最终浓度悬浮的单壁碳纳米管是〜20毫克/升。 （ 二 ）的SWNT悬浮产量可根据用于悬浮SWNT在聚合物上会有所不同。该溶液的光密度提供溶液相SWNT浓度的良好估计。图为不同浓度（GT）15在不同浓度悬浮-DNA管。从左至右:100毫克/升，10毫克/升，1毫克/升，0毫克/升。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55030/55030fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55030/55030fig2.jpg" /> 图2: 表面活性剂和聚合物的吸收光谱和荧光发射光谱暂停单壁碳纳米管。 （ 一 ）代表吸光度藻使用（GT）15-DNA直接超声悬浮的单壁碳纳米管的ectra。 SWNT的浓度为10毫克/升。插图:吸收光谱的UV区域示出了在260nm处的特性的DNA吸收峰。 （ 二 ）RITC-PEG20-RITC的吸收光谱通过透析交换SC后的单壁碳纳米管。插图:RITC-PEG-RITC单壁碳纳米管的10倍稀释样品吸光度显示罗丹明的特征吸收。 （c）使用（GT）15直接超声（785 nm激发）-DNA暂停单壁碳纳米管的代表近红外发射光谱。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55030/55030fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55030/55030fig3.jpg" /> 图3:使用多巴胺荧光检测（GT）15-DNA 包裹的单壁碳纳米管。 （ 一 </<html>强&gt;）荧光反应（GT）15-DNA包裹的单壁碳纳米管的加入多巴胺。以5mg / L的浓度的传感器的样品激发用500毫瓦，721纳米CW激光器。传感器发射的从900-1,350集成荧光纳米添加了多巴胺浓度为1μM增加至250微米。 （ 二 ）PEG-的荧光发射光谱（GT）15-DNA单壁碳纳米管前后除了多巴胺后包装。传感器的浓度为10毫克/毫升到加入100μM的终浓度多巴胺。该样品用500毫瓦，721纳米CW激光器激发。两个最高强度峰强度增加多巴胺后约一倍。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55030/55030fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <html> G"/&gt; 图4:单固定于表面的单壁碳纳米管的荧光成像。 （ 一 ）固定到使用APTES硅烷化过程的二氧化硅盖玻片（＃1.5）个人C 26 [-DNA-SWNT（红色箭头）的荧光发射和使用2D的InGaAs传感器阵列成像，倒置显微镜以100X油浸物镜（计划复消色差透镜，1.4的NA），以及一个500毫瓦，721纳米CW激光器。 （ 二 ）C 26 [-Cy3 DNA的单壁碳纳米管荧光漂白实验用APTES拴在表面上。 DNA链的3&#39;用Cy3管悬挂前末端标记。跟踪单个传感器的增量逐步漂白（红色拟合曲线）来确定吸附到单壁碳纳米管的表面的DNA分子的数量。图像用在TIRF模式倒置显微镜以100X油浸物镜（计划复消色差透镜，1.4 NA）和561纳米的激光激发获得的。比例尺:10微米。https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55030/55030fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes at JoVE.com

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

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 ...

engineering-molecular-recognition-with-bio-mimetic-polymers-on-single

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

8.1K 次观看.  University of California Berkeley. 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.

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

Fabrication, Densification, and Replica Molding of 3D Carbon Nanotube Microstructures

The introduction of new materials and processes to microfabrication has, in large part, enabled many important advances in microsystems, lab-on-a-chip devices, and their applications. In particular, capabilities for cost-effective fabrication of polymer microstructures were transformed by the advent of soft lithography and other micromolding techniques 1, 2, and this led a revolution in applications of microfabrication to biomedical engineering and biology. Nevertheless, it remains challenging to fabricate microstructures with well-defined nanoscale surface textures, and to fabricate arbitrary 3D shapes at the micro-scale. Robustness of master molds and maintenance of shape integrity is especially important to achieve high fidelity replication of complex structures and preserving their nanoscale surface texture. The combination of hierarchical textures, and heterogeneous shapes, is a profound challenge to existing microfabrication methods that largely rely upon top-down etching using fixed mask templates. On the other hand, the bottom-up synthesis of nanostructures such as nanotubes and nanowires can offer new capabilities to microfabrication, in particular by taking advantage of the collective self-organization of nanostructures, and local control of their growth behavior with respect to microfabricated patterns. 
Our goal is to introduce vertically aligned carbon nanotubes (CNTs), which we refer to as CNT "forests", as a new microfabrication material. We present details of a suite of related methods recently developed by our group: fabrication of CNT forest microstructures by thermal CVD from lithographically patterned catalyst thin films; self-directed elastocapillary densification of CNT microstructures; and replica molding of polymer microstructures using CNT composite master molds. In particular, our work shows that self-directed capillary densification ("capillary forming"), which is performed by condensation of a solvent onto the substrate with CNT microstructures, significantly increases the packing density of CNTs. This process enables directed transformation of vertical CNT microstructures into straight, inclined, and twisted shapes, which have robust mechanical properties exceeding those of typical microfabrication polymers. This in turn enables formation of nanocomposite CNT master molds by capillary-driven infiltration of polymers. The replica structures exhibit the anisotropic nanoscale texture of the aligned CNTs, and can have walls with sub-micron thickness and aspect ratios exceeding 50:1. Integration of CNT microstructures in fabrication offers further opportunity to exploit the electrical and thermal properties of CNTs, and diverse capabilities for chemical and biochemical functionalization 3.

We present methods for fabrication of patterned microstructures of vertically aligned carbon nanotubes (CNTs), and their use as master molds for production of polymer microstructures with organized nanoscale surface texture. The CNT forests are densified by condensation of solvent onto the substrate, which significantly increases their packing density and enables self-directed formation of 3D shapes.

制备，致密，副本三维碳纳米管微结构成型

The introduction of new materials and processes to microfabrication has, in large part, enabled many important advances in microsystems, lab-on-a-chip ...

科研

工程学

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 ...

生物工程

Surface Enhanced Raman Spectroscopy Detection of Biomolecules Using EBL Fabricated Nanostructured Substrates

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules is reported. The entire sequence of relevant experimental steps is described, involving the fabrication of nanostructured substrates using electron beam lithography, immobilization of biomolecules on the substrates, and their characterization utilizing surface-enhanced Raman spectroscopy (SERS). Three different designs of nano-biological systems are employed, including protein A, glucose binding protein, and a dopamine binding DNA aptamer. In the latter two cases, the binding of respective ligands, D-glucose and dopamine, is also included. The three kinds of biomolecules are immobilized on nanostructured substrates by different methods, and the results of SERS imaging are reported. The capabilities of SERS to detect vibrational modes from surface-immobilized proteins, as well as to capture the protein-ligand and aptamer-ligand binding are demonstrated. The results also illustrate the influence of the surface nanostructure geometry, biomolecules immobilization strategy, Raman activity of the molecules and presence or absence of the ligand binding on the SERS spectra acquired.

We describe the fabrication and characterization of nano-biological systems interfacing nanostructured substrates with immobilized proteins and aptamers. The relevant experimental steps involving lithographic fabrication of nanostructured substrates, bio-functionalization, and surface-enhanced Raman spectroscopy (SERS) characterization, are reported. SERS detection of surface-immobilized proteins, and probing of protein-ligand and aptamer-ligand binding is demonstrated.

表面增强拉曼光谱检测生物分子利用EBL装配纳米衬底

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules ...

Functionalization of Single-walled Carbon Nanotubes with Thermo-reversible Block Copolymers and Characterization by Small-angle Neutron Scattering

We demonstrate a protocol for single-walled carbon nanotube functionalization using thermo-sensitive PEO-PPO-PEO triblock copolymers in an aqueous solution. In a carbon nanotube/PEO105-PPO70-PEO105 (poloxamer 407) aqueous solution, the amphiphilic poloxamer 407 adsorbs onto the carbon nanotube surfaces and self-assembles into continuous layers, driven by intermolecular interactions between constituent molecules. The addition of 5-methylsalicylic acid changes the self-assembled structure from spherical-micellar to a cylindrical morphology. The fabricated poloxamer 407/carbon nanotube hybrid particles exhibit thermo-responsive structural features so that the density and thickness of poloxamer 407 layers are also reversibly controllable by varying temperature. The detailed structural properties of the poloxamer 407/carbon nanotube particles in suspension can be characterized by small-angle neutron scattering experiments and model fit analyses. The distinct curve shapes of the scattering intensities depending on temperature control or addition of aromatic additives are well described by a modified core-shell cylinder model consisting of a carbon nanotube core cylinder, a hydrophobic shell, and a hydrated polymer layer. This method can provide a simple but efficient way for the fabrication and in-situ characterization of carbon nanotube-based nano particles with a structure-tunable encapsulation.

A method for the functionalization of carbon nanotubes with structure-tunable polymeric encapsulation layers and structural characterization using small-angle neutron scattering is presented.

单壁碳纳米管与热可逆嵌段共聚物及表征功能化小角度中子散射

We demonstrate a protocol for single-walled carbon nanotube functionalization using thermo-sensitive PEO-PPO-PEO triblock copolymers in an aqueous ...

化学

Repression of Multiple Myeloma Cell Growth In Vivo by Single-wall Carbon Nanotube (SWCNT)-delivered MALAT1 Antisense Oligos

The single-wall carbon nanotube (SWCNT) is a new type of nanoparticle, which has been used to deliver multiple kinds of drugs into cells, such as proteins, oligonucleotides, and synthetic small-molecule drugs. The SWCNT has customizable dimensions, a large superficial area, and can flexibly bind with drugs through different modifications on its surface; therefore, it is an ideal system to transport drugs into cells. Long noncoding RNAs (lncRNAs) are a cluster of noncoding RNA longer than 200 nt, which cannot be translated to protein but play an important role in biological and pathophysiological processes. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a highly conserved lncRNA. It was demonstrated that higher MALAT1 levels are related to the poor prognosis of various cancers, including multiple myeloma (MM). We have revealed that MALAT1 regulates DNA repair and cell death in MM; thus, MALAT1 can be considered as a therapeutic target for MM. However, the efficient delivery of the antisense oligo to inhibit/knockdown MALAT1 in vivo is still a problem. In this study, we modify the SWCNT with PEG-2000 and conjugate an anti-MALAT1 oligo to it, test the delivery of this compound in vitro, inject it intravenously into a disseminated MM mouse model, and observe a significant inhibition of MM progression, which indicates that SWCNT is an ideal delivery shuttle for anti-MALAT1 gapmer DNA.

Repression of Multiple Myeloma Cell Growth In Vivo by Single-wall Carbon Nanotube SWCNT-delivered MALAT1 Antisense Oligos

This manuscript describes the synthesis of a single-wall carbon nanotube (SWCNT)-conjugated MALAT1 antisense gapmer DNA oligonucleotide (SWCNT-anti-MALAT1), which demonstrates the reliable delivery of the SWCNT and the potent therapeutic effect of anti-MALAT1 in vitro and in vivo. Methods used for synthesis, modification, conjugation, and injection of SWCNT-anti-MALAT1 are described.

单壁碳纳米管 (sscnt) (mallat1 反义寡 3号) 抑制多骨髓瘤细胞在体内的生长

The single-wall carbon nanotube (SWCNT) is a new type of nanoparticle, which has been used to deliver multiple kinds of drugs into cells, such as ...

癌症研究

Fabrication of Carbon Nanotube High-Frequency Nanoelectronic Biosensor for Sensing in High Ionic Strength Solutions

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them good candidates for high sensitivity biosensors. When a charged molecule binds to such a sensor surface, it alters the carrier density5 in the sensor, resulting in changes in its DC conductance. However, in an ionic solution a charged surface also attracts counter-ions from the solution, forming an electrical double layer (EDL). This EDL effectively screens off the charge, and in physiologically relevant conditions ~100 millimolar (mM), the characteristic charge screening length (Debye length) is less than a nanometer (nm). Thus, in high ionic strength solutions, charge based (DC) detection is fundamentally impeded6-8.
We overcome charge screening effects by detecting molecular dipoles rather than charges at high frequency, by operating carbon nanotube field effect transistors as high frequency mixers9-11. At high frequencies, the AC drive force can no longer overcome the solution drag and the ions in solution do not have sufficient time to form the EDL. Further, frequency mixing technique allows us to operate at frequencies high enough to overcome ionic screening, and yet detect the sensing signals at lower frequencies11-12. Also, the high transconductance of SWNT transistors provides an internal gain for the sensing signal, which obviates the need for external signal amplifier.
Here, we describe the protocol to (a) fabricate SWNT transistors, (b) functionalize biomolecules to the nanotube13, (c) design and stamp a poly-dimethylsiloxane (PDMS) micro-fluidic chamber14 onto the device, and (d) carry out high frequency sensing in different ionic strength solutions11.

We describe the device fabrication and measurement protocol for carbon nanotube based high frequency biosensors. The high frequency sensing technique mitigates the fundamental ionic (Debye) screening effect and allows nanotube biosensor to be operated in high ionic strength solutions where conventional electronic biosensors fail. Our technology provides a unique platform for point-of-care (POC) electronic biosensors operating in physiologically relevant conditions.

制备碳纳米管高频纳电子生物传感器的传感高离子强度的解决方案

The unique electronic properties and high surface-to-volume ratios of single-walled carbon nanotubes (SWNT) and semiconductor nanowires (NW) 1-4 make them ...

Self-assembling Morphologies Obtained from Helical Polycarbodiimide Copolymers and Their Triazole Derivatives

A facile method for the preparation of polycarbodiimide-based secondary structures (e.g., nano-rings, &#34;craters,&#34; fibers, looped fibers, fibrous networks, ribbons, worm-like aggregates, toroidal structures, and spherical particles) is described. These aggregates are morphologically influenced by extensive hydrophobic side chain-side chain interactions of the singular polycarbodiimide strands, as inferred by atomic force microscopy (AFM) and scanning electron microscopy (SEM) techniques. Polycarbodiimide-g-polystyrene copolymers (PS-PCDs) were prepared by a combination of synthetic methods, including coordination-insertion polymerization, copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) &#34;click&#34; chemistry, and atom transfer radical polymerization (ATRP). PS-PCDs were found to form specific toroidal architectures at low concentrations in CHCl3. To determine the influence of a more polar solvent medium (i.e., THF and THF/EtOH) on polymer aggregation behavior, a number of representative PS-PCD composites have been tested to show discrete concentration-dependent spherical particles. These fundamental studies are of practical interest to the development of experimental procedures for desirable architectures by directed self-assembly in thin film. These architectures may be exploited as drug carriers, whereas other morphological findings represent certain interest in the area of novel functional materials.

Here, we present a protocol to prepare and visualize secondary structures (e.g., fibers, toroidal architectures, and nano-spheres) derived from helical polycarbodiimides. The morphology characterized by both atomic force microscopy (AFM) and scanning electron microscopy (SEM) was shown to depend on molecular structure, concentration, and the solvent of choice.

从螺旋聚碳化二亚胺共聚物及其三唑衍生物获得自组装形貌

A facile method for the preparation of polycarbodiimide-based secondary structures (e.g., nano-rings, &#34;craters,&#34; fibers, looped fibers, fibrous ...

Grafting Multiwalled Carbon Nanotubes with Polystyrene to Enable Self-Assembly and Anisotropic Patchiness

We demonstrate a straightforward protocol to graft pristine multiwalled carbon nanotubes (MWCNTs) with polystyrene (PS) chains at the sidewalls through a free-radical polymerization strategy to enable the modulation of the nanotube surface properties and produce supramolecular self-assembly of the nanostructures. First, a selective hydroxylation of the pristine nanotubes through a biphasic catalytically mediated oxidation reaction creates superficially distributed reactive sites at the sidewalls. The latter reactive sites are subsequently modified with methacrylic moieties using a silylated methacrylic precursor to create polymerizable sites. Those polymerizable groups can address further polymerization of styrene to produce a hybrid nanomaterial containing PS chains grafted to the nanotube sidewalls. The polymer-graft content, amount of silylated methacrylic moieties introduced and hydroxylation modification of the nanotubes are identified and quantified by Thermogravimetric Analysis (TGA). The presence of reactive functional groups hydroxyl and silylated methacrylate are confirmed by Fourier Transform Infrared Spectroscopy (FT-IR). Polystyrene-grafted carbon nanotube solutions in tetrahydrofuran (THF) provide wall-to-wall collinearly self-assembled nanotubes when cast samples are analyzed by transmission electron microscopy (TEM). Those self-assemblies are not obtained when suitable blanks are similarly cast from analogous solutions containing non-grafted counterparts. Therefore, this method enables the modification of the nanotube anisotropic patchiness at the sidewalls which results into spontaneous auto-organization at the nanoscale.

A procedure for the synthesis of polystyrene-grafted multiwalled carbon nanotubes using successive chemical modification steps to selectively introduce the polymer chains to the sidewalls and their self-assembly via anisotropic patchiness is presented.

用聚苯乙烯接枝多壁纳米碳纳米管使自组装和各向异性斑块

We demonstrate a straightforward protocol to graft pristine multiwalled carbon nanotubes (MWCNTs) with polystyrene (PS) chains at the sidewalls through a ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

DNA 纳米管作为研究半聚合物的万能工具

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

<meta charset="utf8"></head><body>用于鉴别性 RNA 和 DNA 分析的精密 DNAzyme 纳米机器

DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their functions include tight analyte binding, highly selective analyte recognition, fluorescent signal amplification by multiple catalytic cleavages of a fluorogenic reporter substrate, and fluorogenic substrate attraction for an increase in sensor response. Functional units are attached to a common DNA scaffold for their cooperative action. The RNA-cleaving 10-23 DNMs feature&#160;improved sensitivity in comparison with non-catalytic hybridization probes. The stability of the DNM and the increased chances of substrate recognition are provided&#160;by a double-stranded DNA fragment,&#160;a tile. DNM can differentiate two analytes with a single nucleotide difference in a folded RNA and a double-stranded DNA and detect analytes at concentrations ~1000 times lower than other protein-free hybridization probes. This article presents the concept behind the diagnostic potential of DNA-nanomachine activity and overviews DNM design, assembly, and application&#160;in nucleic acid detection assays.

<meta charset="utf8"></head><body>作者聚焦:DNA 纳米传感器的进步 – 解决分子检测中的灵敏度和选择性挑战

The DNAzyme-based nanomachines can be used for highly selective and sensitive detection of nucleic acids. This article describes a detailed protocol for the design of DNAzyme-based nanomachines with a 10-23 core using free software and their application in the detection of an Epstein-Barr virus fragment as an example.

DNAzyme 10-23 - 用于核酸识别的基于纳米机器

DNAzyme-based nanomachines (DNM) for the detection of DNA and RNA sequences (analytes) are multifunctional structures made of oligonucleotides. Their ...