Name: dna nanotubes as a versatile tool to study semiflexible polymers
Uploaded: 2017-10-25T14:00:00.000+00:00
Duration: 8 min
Description: Watch this Scientific Journal Video about DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers at JoVE.com

我们承认 DFG (1116/17-1) 和莱比锡自然科学学校 "BuildMoNa" (GSC 185) 的资助。这项工作得到了通过弗劳恩霍夫吸引项目 601 683。骅承认欧洲社会基金 (ESF-100077106) 提供的资金。

1. 准备 n 高温超导 注: 在这里, n 表示在某一大小的螺旋管的形成中所涉及的不同单 DNA 链的数量。对于 n = 8, 八不同的单个 DNA 链组成一个单位环. 
<ol> <li> 从合适的 dna 合成服务中购买 dna 序列 (HPLC 纯度级或更高), 或执行高质量的合成和纯化 (在 表 1 中给出的模范序列). </li>
	<li> 在纯化水中重冻干寡核苷酸, 并按照该公司相应手册中的进一步悬浮步骤进行操作。调整水量以获得200和 #181 的最终浓度; m. </li>
	<li> 确定单个 DNA 链的浓度以验证分销商给出的值 (例如, 通过 260 nm 的紫外-可见光谱), 因为精确的化学计量对装配过程至关重要。计算的浓度的寡核苷酸, 考虑到特定的摩尔重量和消光系数的每单 DNA 寡核苷酸. 
	注意: 消光系数值在不同的序列中固有的变化, 并在商业购买的 DNA 文档中注明。它们也可以根据最近的邻居模型, 使用一些免费的在线工具来计算。为了符合光谱仪的线性范围, 考虑稀释用于浓度测定的样品. </li>
	<li> 使用微, 混合 DNA 链-每个在同一 concentration-in 一个容器适合 thermocycler ( 例如, 200 和 #181; L PCR 管) 形成所需的 n 高温超导 (最后的缓冲条件: 1xTE (10 毫米三和1毫米 EDTA, pH 8) 和12.5 mM 氯化镁 2 ). 
	注: 最终的 DNA n HT 样本包括 n -1 股, u 1 和 #8722; u n 和 #8722; 1 和一个 T n 链。每股的浓度可以从 sub-micromolar 到超过10和 #181; m, 根据用户的需要 ( 例如, 和 #60; 1 和 #181; m 每绞线为以后稀释观察单丝或更高的集中为考试密集的网络力学). </li>
	<li> 杂交 n thermocycler 中的高温超导 (在90和 #176 中变性为10分钟; c, 随后快速下降到65和 #176; c; 缓慢的温度从65和 #176 下降; c 到55和 #176; c, 在20温度步长为-0.5 K 的互补基配对每个;从55和 #176 的快速下降; c 到20和 #176; c);请参见 图 2A . 
	注: 缓慢下降步骤从65和 #176; C 可以延长, 以增加平均管长;然而, 随后的快速下降到20和 #176; C 是至关重要的, 以避免聚合的聚合管. </li>
	<li> 存储杂交 n 高温超导3周, 在4和 #176; C 无可检测的降解. 
	注: 对于荧光显微镜, 标记的 n 高温超导是由 (部分) 取代 U1 与修改后的 U1-Cy3 ( 表 1 ) 杂交的。对于较长的观察时间, 建议添加已建立的 anti-bleaching 剂. </li>
	<li> 仔细地将样本稀释到所需的最终浓度 ( 例如, 4 和 #181; M 用于网络或 20 nM 的 single-filament 观测) (如有必要) 使用示例缓冲区。为了尽量减少可能的误差源, 在所需的最终浓度下装配样品. </li>
</ol> 2。剪切流变学 <ol> <li> 为动态剪切流变仪选择合适的几何 (板-板或锥板)。对于小容量 ( 即, 低于200和 #181; L), 使用锥板几何. </li>
	<li> 在动态剪切流变仪上加载示例. </li>
	<li> 使用以下步骤钝化采样和环境的气水界面, 以防止潜在的蒸发效果. 
	<ol> <li> 将样品与2.5 毫升的样品缓冲浴包围. </li>
		<li> 添加表面活性剂略低于胶束浓度. </li>
		<li> 使用哈密尔顿注射器将样品与油脂包围, 以避免样品与空气直接接触. </li>
	</ol> </li> <li> 用带有湿海绵的帽盖密封样品室, 如果可能的话, 用一个额外的水浴 (不与样品接触) 来进一步抑制蒸发. </li>
	<li> 在室温下使用流变仪特定的软件开始测量; 或者, 在不同温度下执行测量, 如37和 #176; C (如果需要生理条件). 
	注: 冷却试样通常伴随着水蒸气从周围空气中凝结而来, 而加热则会导致蒸发的增强。这两种效应都可能无法改变样品的浓度, 所以系列中的所有测量都应在恒定温度下进行. </li>
	<li> 允许样品在室温下平衡2小时。时间的演变可以监测时间扫描 (每分钟一个数据点; 应变 和 #947; = 5%; 频率 f = 1 Hz). </li>
	<li> 使用所需的测试协议测量机械属性。从一系列频率扫描 ( f 扫描) 开始, 因为它们保持原样, 并允许更多的测量. 
	注意: 此外, 短的 f -扫描应在较长的测量之前和之后进行 (如长的 f --用更高的分辨率进行扫描), 以验证整个测量协议中的样本保持不变。
	<ol> <li> 执行短 f 扫描 ( 如 和 #947; = 5%; f = 0.01 赫兹到30赫兹;每十年5数据点). </li>
		<li> 执行 long f 扫描 (, 如 和 #947; = 5%; f = 0.001 赫兹到30赫兹;每十年21数据点);低频的制度可以省略, 如果没有必要减少测量时间. </li>
		<li> 执行短 f 扫描. </li>
		<li> 执行和 #947;-扫描 ( 例如, f = 1 Hz; 和 #947; = 0.0125% 到 100%;每十年20数据点). 
		注意: 首先使用短 f 扫描, 因为它通常与以前的 f 不一致-扫, 因为网络通常在 #947 时破裂;-清除或从板上分离。或者, 使用和 #947;-斜坡 ( 例如, = 0.025 s -1 , t = 六十年代);然而, 坡道通常需要改变电机模式, 所以随后的短 f 扫将是伪造的. </li>
	</ol> </li> <li> Bin 和/或使用高斯内核平滑原始数据. </li> </ol>
 3. 荧光辅助分析 DNA 退火 <ol> <li> 准备8等分12和 #181; L 为每个样本使用1-4 和 #181; 每个链的 M DNA, 12.5 mM 氯化镁 2 在 1x TE 缓冲区, 和1x 核酸凝胶染色从10,000x 亚砜股票.做一个没有 DNA 的负控制, 但包括核酸凝胶染色. </li>
	<li> 将等分转移到 real-time PCR 板上, 并用粘合剂膜密封 ( 例如, 96-井反应板). 
	注: 热退火协议中使用的高温将蒸发样品。因此, 确保水井是紧密密封的胶膜, 以防止水的蒸发和浓度增加, 特别是在长期的实验. </li>
	<li> 在室温下离心 100 x g 1 分钟以除去气泡; 如果气泡膨胀, 则无法分析油井. </li>
	<li> 将密封板装入 real-time 定量 PCR 系统. </li>
	<li> 运行退火程序。变性90和 #176 的 DNA; c 为10分钟, 将温度快速降到72和 #176; c。然后, 缓慢地将温度降低0.5 和 #176; C 每30分钟。信号衰减后, 加速温度斜坡. 
	注:72 和 #176; C 是足够以上的实际杂交温度;因此, 信号是记录在一个广泛的温度范围, 适合确定必要的温度范围. </li>
	<li> 确保循环的盖子加热到至少100和 #176; C 防止在粘合剂膜上蒸发样品的凝结. </li>
	<li> 在组装过程中, 用氩离子激光在 488 nm 激发样品, 在使用核酸凝胶染色 (或光谱类似) 时检测 550 nm 的荧光。对于其他染料, 选择合适的励磁/排放组合。使用内置摄像头, 尽可能多地测量荧光信号以提高整体信号质量. 
	注: 在这里, 测量每9秒采取. </li>
	<li> 如果应用预设的熔化和退火程序, 请使用所提供的软件对数据进行分析。如果不是, 则从每个温度步长的平均值中减去先前的平均值, 以获得荧光信号的相对变化. </li>
</ol> 4。AFM 成像 <ol> <li> 劈云母 (, 如, 云母和 #34; V1 和 #34;) 通过附加商业可用的胶带和翻录它; 云母的最高层应该被去除。
	<li> 使用微, 存入预先 n HT 在 Mg 2 + 包含在新劈开的云母的折叠缓冲区, 让它解决10分钟 </li>
	<li> 将样品以短3秒的间隔在 2000 x g 的小表离心机上旋转以除去剩余的液体。几个 n 高温超导仍应连接到云母表面. </li>
	<li> 使用具有高刚度的悬臂尖端 ( 例如, 54 Nm -1 ), 并用内部 AFM 软件确定其谐振频率. </li>
	<li> 在低扫描速率 ( 例如, 1 线/秒) 的情况下, 用 AFM 在空气中记录高度剖面以避免损坏或取代 n 高温超导. </li>
</ol>

作者没有什么可透露的。

要获得适当形成的网络, 组装 DNA 纳米管是一个关键的步骤。合成过程中的误差对管质量产生负面影响;因此, 建议采用 HPLC 法或更严格的工艺来纯化寡核苷酸。由于离散而非聚合的 DNA 纳米管的形成, 以及它们的长度分布, 取决于集合中的n组成的寡核苷酸的摩尔化学计量, 因此必须测量浓度被购买的子线, 因为被给的价值可能从实际集中极大地变化。例如, 可以通过紫外可见光谱在 260 nm 的吸收...

由于其独特的力学性能使复杂的行为, 半聚合物是基本的生活物质的基石。与柔性聚合物相比, 半聚合物采用了一种伸展的结构, 由于其非的骨架刚度, 同时仍受强热波动的影响1。因此, 纯粹的随机模型不能应用于他们的行为, 就像极端的完全灵活或刚性的聚合物。so-called 蠕虫样链模型2,3,4被开发为通过lp来量化此刚度, 它是沿灯丝4的切线相关的衰减常数。如果lp与灯丝的轮廓长度 (lc) 相媲美, 则该聚合物被视为半1。类似于帐篷的两极, 他们在网络或捆绑的安排稳定整个集体系统在低体积分数, 导致异常粘弹性属性5,6,7, 8,9。这些结构在大型网格大小10中提供了高弹性, 保持了机械完整性, 同时还促进了扩散和活动的传输过程。该特性特别适用于生物系统, 如骨架或胞外基质, 但它也广泛应用于食品工程1,11,12。
为了能有发展仿生材料或新型水凝胶的工具, 对这些结构的物理性质进行全面的检验, 对其意义深远。在半聚合物方面, 这意味着系统地确定由 single-filament 属性 (如lp和描述性理论框架的开发) 产生的网络的集体属性。在开创性研究中, 细胞生物肌动蛋白被建立为半聚合物的模型系统, 并仍被广泛认为是金标准5,13,14,15,16,17. 然而, 这一系统的详尽研究是有限的, 因为它们与这种蛋白质的固有特性息息相关。各种理论方法的目的是建立一个描述的非平凡的机械行为在 single-filament 水平, 并导致明显不同的比例预测的依赖线性弹性高原剪切模量, G 0 (即网络的 "弹性"), 就集中度 (c) 和lp6,7,13,14, 15,18,19,20,21,22,23。虽然在 actin-based 或其他模型系统的实验中可以方便地进行浓度缩放, 而理论预测已得到严格验证13,16,24,25, 相对于lp的缩放在实验上仍然不可访问。但是, 这是一个主要的限制, 因为lp也是一个独立变量, 它是半聚合物的定义数量。
这种中央, 自然的限制强加由固定的lp的肌动蛋白或其他生物衍生聚合物, 如胶原蛋白, 最近已经解决了使用型 DNA 管, 这是可调谐的机械性能9,26,27,28. 管结构的细微变化 (例如,在单位环内的不同数量的组成 DNA 链) 产生独特的值为lp, 可以通过荧光显微镜进行评估,通过分析一个波动管或评估几个粘附管的弯曲配置, 如前面所描述的9,28。这些分析显示, 不同管种群的lp值在多个数量级上变化, 并且不同的评估技术产生一致的结果9,28。
令人惊讶的是, 线性弹性高原切变模量的整体比例G0关于浓度和lp已报告与所有以前的理论方法不一致9, 特别是在lp上演示了比预测依赖强得多的依赖性。这些发现强调了一个新的模型系统的价值, 研究半聚合物的中心性质。使用n-螺旋 DNA 管极大地拓宽了这些调查的范围。lp不仅可以在不改变基本材料的情况下自由变化, 而且 DNA 的固有可编程性可以使系统地检查其他元素, 如通道或动态切换过程。此外, 这些管子可溶于水, 与大多数蛋白质相比, 在适当的 pH 值和离子条件下稳定数周, 没有可检测的降解9。
为了组装这些管子, 我们使用了一组离散的 DNA 寡核苷酸, 每个都包含两个域, 共享互补的碱基序列到两个相邻的链 (由于特定的序列, 单链不能形成结构, 如发夹)。互补序列杂交的循环方式, 形成封闭的, half-overlapping 环的n互联的双段 (图 1A和 B)。这些环形成在一个离散的直径 (图 1C), 其 half-overlapping 配置暴露了轴向粘性两端互补的另一环的粘性两端。这种选择性添加匹配的寡核苷酸触发了环的堆叠, 导致有效聚合的丝状 DNA 螺旋管的大小n (nHT)。他们的外形长度通常测量几微米长度, 并且他们的长度分布是可比较的肌动蛋白花丝9,26,27,28。它已经证明了类似的 DNA 纳米管, 他们确实展示聚合动力学类似的肌动蛋白丝和微管内<su></su>p 类 = "xref" > 29。根据组成基本环结构的单个 DNA 链的数字n , nHT 体系结构以及其周长和直径可控变化。使用更多的 DNA 链增加环/管的周长, 相应的结构变化将机械性能转移到更高的lp值 (图 1C), 对应于更高的刚性。在介观尺度上, 这些较大的lp值转换为更高的刚度 (图 1D和E) 的弯曲构象。

<table><tbody><tr><td>AFM cantilever ACTA</td><td>AppNano</td><td></td><td></td></tr><tr><td>AFM - NanoWizard 3</td><td>JPK Instruments</td><td></td><td></td></tr><tr><td>CCD camera</td><td>Andor</td><td>iXon DV887</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>DNA oligonucleotides</td><td>Biomers.net</td><td></td><td>For sequences see Table 1</td></tr><tr><td>DNA Cy3-labeled oligonucleotides</td><td>Biomers.net</td><td></td><td>For sequence see Table 1</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E-9884</td><td></td></tr><tr><td>Epi-fluorescence micro-scope</td><td>Leica</td><td>DM-IRB</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M-8266</td><td></td></tr><tr><td>Mica &quot;V1&quot;, 12 mm round</td><td>Plano GmbH</td><td>50-12</td><td></td></tr><tr><td>MicroAmp&amp;reg; Fast Optical 96-Well Reaction Plate</td><td>Thermo Fisher Scientific Inc.</td><td>4346907</td><td></td></tr><tr><td>MicroAmp&amp;reg; Optical Adhesive Film</td><td>Thermo Fisher Scientific Inc.</td><td>4306311</td><td></td></tr><tr><td>NanoDrop 1000 Spectrophotometer</td><td>Thermo Fisher Scientific Inc.</td><td></td><td></td></tr><tr><td>100x objective</td><td>Leica5</td><td>506168</td><td></td></tr><tr><td>Purified water</td><td>Merk Millipore - Milli-Q &amp;amp; Elix</td><td></td><td></td></tr><tr><td>Sapphire PCR tubes</td><td>Greiner Bio-One</td><td>683271</td><td></td></tr><tr><td>TProfessional Standard PCR Thermocycler</td><td>Core Life Sciences Inc.</td><td>070- Standard</td><td></td></tr><tr><td>7900HT Fast Real-Time PCR System</td><td>Applied Biosystems</td><td>4351405</td><td></td></tr><tr><td>Rheometer</td><td>TA Instruments</td><td>ARES</td><td></td></tr><tr><td>SYBR&amp;reg; Green I nucleic acid gel stain</td><td>Thermo Fisher Scientific Inc.</td><td>S7567</td><td></td></tr><tr><td>Tris</td><td>Sigma-Aldrich</td><td>T4661</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich Co.</td><td>X-100</td><td>Suppresses evaporation of sample at air-water interface</td></tr></tbody></table>

dna nanotubes as a versatile tool to study semiflexible polymers

复杂的, 聚合物的软物质的机械性能, 如细胞或生物网络, 可以理解在既不经典框架的柔性聚合物也不是刚性棒。由于其非的主干刚度 (通过持久长度 (lp) 进行量化), 底层细丝仍会伸展, 但它们也会受到强烈的热波动的影响。它们的有限弯曲刚度导致了独特的、非平凡的体积网络集体力学, 使得在低体积分数下形成稳定的支架, 同时提供大的网格尺寸。这一基本原理在自然界中很普遍 (例如,在细胞或组织中的), 使高分子含量最小化, 从而促进扩散或主动运输。由于其生物学意义和潜在的技术应用在生物相容的水凝胶, 半聚合物已受到相当多的研究。然而, 可理解的调查仍然具有挑战性, 因为他们依赖天然的聚合物, 如肌动蛋白丝, 这是不能自由调谐。尽管有这些限制, 由于缺乏合成, 机械可调谐, 和半聚合物, 肌动蛋白花丝被建立作为共同的模型系统。一个主要的限制是, 不能自由调整中心数量lp来研究其对宏观体积结构的影响。通过采用结构化可编程的 DNA 纳米管, 实现了对灯丝刚度的控制改变, 从而解决了这一缺陷。它们是通过型的设计形成的, 在这里, 一组离散的部分互补的链杂交在一个具有离散圆周的环形结构中。这些环的特点是粘性的两端, 使有效的聚合成丝数微米的长度, 并显示类似的聚合动力学作为天然的生物聚合物。由于他们的可编程的力学, 这些管是多才多艺的, 新颖的工具, 研究的影响, lp对分子和散装规模。与肌动蛋白丝相比, 它们在几周内保持稳定, 没有明显的变性, 它们的处理也比较简单。

通过温度斜坡 (图 2) 进行 DNA 纳米管的组装是形成这些人造半聚合物的一种非常可靠的方法。这些聚合物与自然发生的对应物有相似的特征, 例如肌动蛋白丝, 但提供一个更加宽广的实验框架, 因为他们的机械物产可以被控改变9,27.与生物聚合物一样, 它们可以在网络中进行排列 (图 3), ?...

Watch this Scientific Journal Video about DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers at JoVE.com

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

半聚合物显示独特的机械性能, 广泛应用于生活系统。然而, 由于聚合物刚性等性质无法进入, 对生物聚合物的系统研究是有限的。这篇手稿描述了如何利用可编程的 DNA 纳米管规避这一限制, 从而对纤维刚性的影响进行实验研究。

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 ...

dna-nanotubes-as-a-versatile-tool-to-study-semiflexible-polymers

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

6.8K 次观看.  Fraunhofer Institute for Cell Therapy and Immunology. 半聚合物显示独特的机械性能, 广泛应用于生活系统。然而, 由于聚合物刚性等性质无法进入, 对生物聚合物的系统研究是有限的。这篇手稿描述了如何利用可编程的 DNA 纳米管规避这一限制, 从而对纤维刚性的影响进行实验研究。

视频: DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Scalable Nanohelices for Predictive Studies and Enhanced 3D Visualization

Spring-like materials are ubiquitous in nature and of interest in nanotechnology for energy harvesting, hydrogen storage, and biological sensing applications.&#160; For predictive simulations, it has become increasingly important to be able to model the structure of nanohelices accurately.&#160; To study the effect of local structure on the properties of these complex geometries one must develop realistic models.&#160; To date, software packages are rather limited in creating atomistic helical models.&#160; This work focuses on producing atomistic models of silica glass (SiO2) nanoribbons and nanosprings for molecular dynamics (MD) simulations. Using an MD model of &#8220;bulk&#8221; silica glass, two computational procedures to precisely create the shape of nanoribbons and nanosprings are presented.&#160; The first method employs the AWK programming language and open-source software to effectively carve various shapes of silica nanoribbons from the initial bulk model, using desired dimensions and parametric equations to define a helix.&#160; With this method, accurate atomistic silica nanoribbons can be generated for a range of pitch values and dimensions.&#160; The second method involves a more robust code which allows flexibility in modeling nanohelical structures.&#160; This approach utilizes a C++ code particularly written to implement pre-screening methods as well as the mathematical equations for a helix, resulting in greater precision and efficiency when creating nanospring models.&#160; Using these codes, well-defined and scalable nanoribbons and nanosprings suited for atomistic simulations can be effectively created.&#160; An added value in both open-source codes is that they can be adapted to reproduce different helical structures, independent of material.&#160; In addition, a MATLAB graphical user interface (GUI) is used to enhance learning through visualization and interaction for a general user with the atomistic helical structures.&#160; One application of these methods is the recent study of nanohelices via MD simulations for mechanical energy harvesting purposes.

Accurate modeling of nanohelical structures is important for predictive simulation studies leading to novel nanotechnology applications.&#160; Currently, software packages and codes are limited in creating atomistic helical models.&#160; We present two procedures designed to create atomistic nanohelical models for simulations, and a graphical interface to enhance research through visualization.

可扩展的Nanohelices的预测研究和增强的3D可视化

Spring-like materials are ubiquitous in nature and of interest in nanotechnology for energy harvesting, hydrogen storage, and biological sensing ...

科研

工程学

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

内部自组装脂质颗粒的简易制备碳纳米管稳定

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

化学

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 ...

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past two decades1. Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules2-4, and even long microtubules have measured persistence lengths which vary by an order of magnitude5-9.
	Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay10. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores attached to the microtubule, the gliding trajectories of single microtubules are tracked with nanometer-level precision. The persistence length of the trajectories is the same as the persistence length of the microtubule under the conditions used11. An automated tracking routine is used to create microtubule trajectories from fluorophores attached to individual microtubules, and the persistence length of this trajectory is calculated using routines written in IDL.
	This technique is rapidly implementable, and capable of measuring the persistence length of 100 microtubules in one day of experimentation. The method can be extended to measure persistence length under a variety of conditions, including persistence length as a function of length along microtubules. Moreover, the analysis routines used can be extended to myosin-based acting gliding assays, to measure the persistence length of actin filaments as well.

A method to measure the persistence length or flexural rigidity of biopolymers is described. The method uses a kinesin-driven microtubule gliding assay to experimentally determine the persistence length of individual microtubules and is adaptable to actin-based gliding assays.

使用滑翔分析生物大分子的抗弯刚度测量

Microtubules are cytoskeletal polymers which  play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires ...

生物工程

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 ...

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

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

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

由小圆形 DNA 分子构建的稳定 DNA 分子、一维和二维纳米结构

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

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 ...

Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor individual reactions live in well-controlled conditions. To do so, live single-filament experiments have emerged over the past 20 years, mostly using total internal reflection fluorescence (TIRF) microscopy, and have provided a trove of key results. In 2011, in order to further expand the possibilities of these experiments and to avoid recurring problematic artifacts, we introduced simple microfluidics in these assays. This study details our basic protocol, where individual actin filaments are anchored by one end to the passivated coverslip surface, align with the flow, and can be successively exposed to different protein solutions. We also present the protocols for specific applications and explain how controlled mechanical forces can be applied, thanks to the viscous drag of the flowing solution. We highlight the technical caveats of these experiments and briefly present possible developments based on this technique. These protocols and explanations, along with today&#39;s availability of easy-to-use microfluidics equipment, should allow non-specialists to implement this assay in their labs.

We present protocols for simple actin filament microfluidic assays, in combination with fluorescence microscopy, that allow one to accurately monitor individual actin filaments in real-time while sequentially exposing them to different protein solutions.

利用微流体和荧光显微镜研究单根肌动蛋白丝和束的组装动力学

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor ...

生物化学

Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated media. In this article, we describe detailed protocols that enable the construction of nanofiber architectures in organic solvent mixtures through the self-assembly of uniquely addressable, single-stranded, gamma-modified peptide nucleic acid (&#947;PNA) tiles. Each single-stranded tile (SST) is a 12-base &#947;PNA oligomer composed of two concatenated modular domains of 6 bases each. Each domain can bind to a mutually complimentary domain present on neighboring strands using programmed complementarity to form nanofibers that can grow to microns in length. The SST motif is made of 9 total oligomers to enable the formation of 3-helix nanofibers. In contrast with analogous DNA nanostructures, which form diameter-monodisperse structures, these &#947;PNA systems form nanofibers that bundle along their widths during self-assembly in organic solvent mixtures. Self-assembly protocols described here therefore also include a conventional surfactant, Sodium Dodecyl Sulfate (SDS), to reduce bundling effects.

This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures.

将伽马改性肽核酸自组装成有机溶剂混合物中复杂的纳米结构

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated ...

Procedure for Fabricating Biofunctional Nanofibers

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups may be utilized to study material-biomarker interactions i.e. act as biosensors with potential as single molecule detectors. We have developed an effective approach for preparing functional polymers where the functionality has the capacity of specifically binding with a model protein. In our model system, the functional group is 2,4-dinitrophenyl (DNP) and the protein is anti-DNP IgE (Immunoglobulin E). The functional polymer, &alpha;,&omega;-bi[2,4-dinitrophenyl caproic][poly(ethylene oxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP), is prepared by anionic living polymerization. The difunctional initiator utilized in the polymerization was prepared by electron transfer reaction of &alpha;-methylstyrene and potassium (mirror) metal. The 2-methoxystyrene monomer was added first to the initiator, followed by the addition of the second monomer, ethylene oxide, and finally the living polymer was terminated by methanol. The &alpha;,&omega;-dihydroxyl polymer [HO-PEO-P2MS-PEO-OH] was reacted with N-2,4-DNP-&isin;-amino caproic acid, by DCC coupling, resulting in the formation of &alpha;,&omega;-bi[2,4-dinitrophenylcaproic][poly(ethyleneoxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP). The polymers were characterized by FT-IR, 1H NMR and Gel Permeation Chromatography (GPC). The molecular weight distributions of the polymers were narrow (1.1-1.2) and polymers with molecular weights greater than 50,000 was used in this study. The polymers were yellow powders and soluble in tetrahydrofuran. A water soluble CDNP-PEO-P2MS-PEO-CDNP/ DMEG (dimethoxyethylene glycol) complex binds and achieves steady state binding with solution IgE within a few seconds. Higher molecular weight (water insoluble i.e. around 50,000) CDNP-PEO-P2MS-PEO-CDNP polymers, containing 1% single wall carbon nanotubes (SWCNT) were processed into electroactive nanofibers (100 nm to 500 nm in diameter) on silicon substrate. Fluorescence spectroscopy shows that anti-DNP IgE interacts with the nanofibers by binding with the DNP functional groups decorating the fibers. These observations suggest that appropriately functionalized nanofibers hold promise for developing biomarker detection device.

An efficient approach for preparing nanofibers decorated with functional groups capable of specifically interacting with proteins is described. The approach first requires the preparation of a polymer functionalized with the appropriate functional group. The functional polymer is fabricated into nanofibers by electrospinning. The effectiveness of the binding of the nanofibers with a protein is studied by confocal microscopy.

制作生物功能纳米纤维的过程

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups ...