这项工作是由美国农业部国家研究所的粮食和农业，国家竞争力的资助计划，国家研究计划项目71.1 2007年度部分资金作为批准号:2007-35503-18392和美国国立卫生研究院，研究院过敏和传染病，R33AI94514-03。

1，纺丝原液的制备<ol><li>准备进行调查一系列生物聚合物的浓度（0.1％〜30％，W / V），并一定要考虑生物​​聚合物粉末在这些计算中的水分含量。对于每个浓度，称量该生物聚合物（淀粉或支链淀粉）的粉末置于50ml试管中。添加水二甲基亚砜（DMSO）溶液和搅拌棒。 </li><li>将管子放入沸水与磁力搅拌器加热板不断搅拌。 </li><li>经过约1小时，熄火并允许该分散冷却至室温。分散体，然后准备进行流变试验和静电。 </li></ol> 2，稳定剪切流变<ol><li>热身流变仪和设置阶段的温度在20℃。校准探头（用于25毫米锥形）和级（板）之间的间隙。 </li><li>该生物聚合物分散体的负载0.41毫升到台上的中心和降低探头，设置positi在（0.053毫米差距25毫米锥）。确保分散均匀敷于间隙内。 </li><li>用下面的实验参数进行流变学试验:扫描模式:日志，初始速度:100秒-1，最终速率:0.1秒-1，每十点:10，延迟测量结果之前:5秒，测量时间:10秒，而每小节的方向:2（顺时针和逆时针）。 </li><li>分析流变数据来估算适当的分散浓度静电试验。 <ol><li>绘制表观剪切粘度对剪切速率的聚合物浓度的函数。针对每一个流的曲线，近似零剪切粘度，η0，由实际或外推值（ 例如 ，在低浓度时，其中的低剪切速率下的数据是不可靠的）为表观粘度在0.1秒- 1。 </li><li>计算比粘度:ηSP =（η0 - ηS）/ηs，其中ηs是溶剂的粘度。 </li><li>绘制特定粘度随浓度的函数。确定亚浓非缠结和纠​​缠制度。在亚浓非缠结政权从低浓度结尾加上一个小山坡开始，和亚浓纠缠制度具有更大的斜率以下非缠结制度。飞度幂回归模型中的两种制度。功率值是在一个log-log图亚浓非缠结和纠​​缠制度的斜坡（浓度依赖性）。两个拟合线的交点就是纠缠浓度， 居权证。 </li></ol></li></ol> 3，静电参数变化<ol><li>组装静电设置为如图1中所示。装入注射器用的适当组合物的分散性， 例如 ，15％（重量/体积），淀粉或支链淀粉在100％DMSO中，到注射器泵。夹在高压线（POSITIVE）到针。通过浸渍的接地线（负）进入浴缸连接含有纯乙醇对地面的凝固浴中。用一个实验室千斤顶调整注射器针头和凝固浴之间的距离。沉浸金属网在洗澡后，静电收集纤维毡。 </li><li>旋转生物聚合物在下列参数范围:进料速率为0.1至0.4毫升/小时，从5到10厘米纺丝距离，并且电压从0到15千伏。 <ol><li>开始用5厘米的纺丝距离。用于第一进给速率（0.1毫升/小时），缓慢地从0V。讲究斜坡电压上升至挤出在针尖分散体的形状，并注意滴分散体被加速，然后拉长。 </li><li>请注意，在这一个微小的飞机从下拉表面引发，这说明解决方案的electrospinnability的电压。记录在其中的连续喷射启动，如果任何电压。 </li><li>检查全系列为三个paramete的RS和记成功的纺丝条件。收集的纤维时，才会有从前端连续喷射。 </li></ol></li><li>收集了几分钟后，冲洗纤维毡用纯乙醇。将纤维毡为含真空除湿干燥器。 </li><li>重复每个生物聚合物浓度为完整的特征。 </li></ol><img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51933/51933fig1highres.jpg" width="500" /> 电湿式纺丝机的图1示意图。生物聚合物分散体是由注射泵挤出。高压直流电源提供高电压的钝针和地面凝固浴。从针尖的聚合物射流穿过直通道，然后开发了快速搅打路径（又名搅打稳定性）。 4，形态特征<ol><li>剪下一块干燥的纤维毡​​，并使用碳胶带固定其上的SEM​​存根。 </li><li>将样品存根放入扫描电镜仪器获取图像进行分析。 </li></ol>

<ol>
	<li>Greiner, A., Wendorff, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+self-assembled+nanofibers+by+electrospinning.">Functional self-assembled nanofibers by electrospinning.</a> Self-aseembled nanomaterials. 1, 107-171 (2008).</li><li>Ramakrishna, S., Fujihara, K., Teo, W. E., Lim, T. C., Ma, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> An Introduction to Electrospinning and Nanofibers. , (2005).</li><li>Klossner, R. R., Queen, H. A., Coughlin, A. J., Krause, W. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+rheological+behavior+and+electrospinning+of+cationic+polyelectrolytes.">Solution rheological behavior and electrospinning of cationic polyelectrolytes.</a> Macromolecules. 39 (2), 575-583 (2006).</li><li>Kong, L., Ziegler, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+pure+starch+fibers+by+electrospinning.">Fabrication of pure starch fibers by electrospinning.</a> Food Hydrocolloids. 36, 20-25 (2014).</li><li>Singh, R. S., Saini, G. K., Kennedy, J. 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作者宣称，他们什么都没有透露。

流变学是研究聚合物，包括常规纤维纺丝和静电13的处理的一个重要工具。从稳态剪 ​​切流变学研究，聚合物的构象和它们在不同溶剂中的相互作用可以得到解决（ 图2和3）。在浓度不够高的生物聚合物分子彼此重叠，它们的浓度依赖性为1.4左右（ 图3），这是与其它聚合物的良溶剂3,4报道值相符。经过生物聚合物分子开始纠缠，具体粘度表现出的浓度要高得多的依赖。较大的N值表示一个强大的分子间相互作用。许多无规线圈多糖表现出类似的浓度依赖性，具有大约3.3 14的n值。普鲁兰多糖表现出比淀粉相互作用较强HIG中的溶剂ħDMSO含量，这可能是由于两种生物聚合物的分子性质。所使用的淀粉有一些高度支化的组分（约20％支链淀粉），而支链淀粉应该是线性的。当然，分子量，这是未知的，也有一定的影响。 缠结浓度将取决于在该分散液中的生物聚合物的构象。例如，淀粉在92.5％的缠结浓度（重量/体积）的DMSO水溶液是远低于在纯DMSO中11。这意味着，在一个更​​扩展的构象存在于92.5％的淀粉分子（重量/体积）的DMSO水溶液中以使它们占据了较大的流体动力学体积，并趋向于更容易地重叠。支链淀粉的缠绕浓度没有变化为显着的那些淀粉具有不同溶剂的质量，这可能是因为水和DMSO都是良溶剂的支链淀粉和对分子的构象的影响不大。水，这是不是一个好溶剂淀粉，制成场景复杂得多，因为不溶解的淀粉分子会影响流变响应。 纺好的纤维中，浓度必须是1.2-2.7和1.9-2.3x缠结浓度为淀粉和支链淀粉，分别（ 图4和5）。这个范围是支链淀粉窄，大概也由于在溶剂中较少的构象差异。这是非常有趣地注意到，在缠结浓度的分散体中，当聚合物开始相互缠结，没有electrospinnable。大概，涉及可能已经建立在静态和低剪切条件，从而增强和足够缠结，需要静电阻碍链重叠和长范围的聚合物相互作用的高剪切力。此外，剪切粘度也发挥了重要作用（ 图6）。该electrospinnable淀粉和普鲁兰DISPERS离子陷入了类似的一系列剪切粘度在100秒-1，用2.2帕·秒的上限。 本文所描述的方法可以对应于该设备与在其他研究中所使用的材料进行修改。聚合物的溶解是在这个协议中的第一个关键步骤，因为我们发现，部分溶解的淀粉分散体（ 例如 ，在85％（W / V）的DMSO）产生，阻止准确测定居权证不稳定的稳态剪 ​​切粘度数据。在进行稳态剪切测量，我们宁愿从最高剪切速率下启动。通过这样做，将分散液均匀地通过高剪切速率的帮助分布在间隙内。静电纺丝工序需要大量的实践。应注意，以液滴的针尖的形状变化。静电纺丝过程中的安全注意事项是不容忽视的。静电纺丝的主要危险来自于日使用高压ê过程中，虽然在当前是比较低的。电纺丝实验应在通风橱中以驱逐溶剂蒸气可能构成对健康的危害，如果一个被暴露于它很长一段时间来进行。避免接近的距离，甚至在带电针尖和凝固浴之间的联系，因为这些会导致短路和火灾的危险。 在目前的研究中所用的流变性的方法是有限制的。例如，应当注意的是，涉及在电纺丝的实际剪切速度是大于100秒-1 1高得多。除了 ​​剪切流变学研究，伸长流变，其特征分散体的拉伸沿着轨迹，也可发挥重要作用15。在本研究中使用的流变仪是不能够表征拉伸粘度的。 流变学研究可以提供有关dispe生物大分子构象有价值的信息rsions和其加工性能。该协议是在许多其他生物聚合物及其共混静电可能有用，在溶剂系统的选择，参数优化和纤维形成机制在分子水平上。

静电纺丝是一种技术，其能够从各种各样的材料生产连续微纳米级纤维。它已经获得了越来越多的学术和产业的利益1。虽然设置和静电的做法看似简单，预测electrospinnability和控制纤维性能的能力仍然是一个挑战。其原因可能在于这一事实有影响的电纺丝过程2和过程，特别是由光纤行进的路径，是混沌1许多因素。往往是经验的"煮和 - 看"的方法，用于筛选潜在electrospinnable材料。然而，为了更好地控制电纺丝过程和所得纤维的性能，支配electrospinnability所需的机制的一个更完整的理解。一些研究人员已经发现，在纺丝原液的聚合物的分子缠结是一个essentia升前提成功的静电3-5。 流变学是一个强大的工具来探测分子构象和互动的聚合物分散体。例如，McKee 等 。研究了线性的含有氯仿/对苯二甲酸二甲酯（7:3，V / V）的分子构象和支链聚（对苯二甲酸乙二酯 - 共 - 间苯二甲酸乙二醇酯）的共聚物在溶剂中，并确定该聚合物的浓度必须是2-2.5x纠缠浓度为成功的静电4。 有从的，因为它们的生物降解性，生物相容性和可再生相，以对比它们的合成的对应的优点的生物聚合物在纤维目前新的兴趣。然而，从业者面临来自其结构的复杂性，难以热加工和差的机械性能一般产生许多挑战。淀粉，植物组织中发现，是阿蒙G于地球上最丰富和廉价的生物聚合物。纯淀粉纤维的使用电-湿式纺丝装置最近被描述6制成。普鲁兰多糖是由某种细菌细胞外产生的线性多糖。 （1→4）和（1→6） ​​经常交替糖苷键被认为是负责支链淀粉的几个独特的性质，包括优异的纤维/薄膜形成能力7,8。从水分散体的支链淀粉的纤维的静电纺丝已经报道了许多研究者9,10。在我们先前的出版物中，两个生物聚合物，淀粉11和支链淀粉12 electrospinnability，已经讨论过。本报告侧重于展示协议利用流变学原理，这两种生物聚合物的electrospinnability的调查。

<table border="0" cellpadding="0" cellspacing="0" width="762">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:253px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:175px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gelose 80 starch</td>
			<td style="width:253px;">Ingredion</td>
			<td style="width:119px;"></td>
			<td>Used as it is</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pullulan</td>
			<td style="width:253px;">Hayashibara Co. Ltd</td>
			<td style="width:119px;"></td>
			<td>Used as it is</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethyl Sulfoxide</td>
			<td style="width:253px;">BDH Chemicals</td>
			<td style="width:119px;">BDH1115-4LP</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Ethanol</td>
			<td style="width:253px;">VWR International</td>
			<td style="width:119px;">89125-172</td>
			<td>200 proof</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:253px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rheometer</td>
			<td style="width:253px;">TA Instruments</td>
			<td style="width:119px;">ARES&#160;</td>
			<td>50 mm cone and plate geometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe (10 mL)</td>
			<td style="width:253px;">Becton, Dickinson and Company</td>
			<td style="width:119px;">309604</td>
			<td>Syringe with Luer-Lok&#174; Tip</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">High voltage generator</td>
			<td style="width:253px;">Gamma High Voltage Research, Inc.</td>
			<td style="width:119px;">ES40P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe pump</td>
			<td style="width:253px;">Hamilton Company</td>
			<td style="width:119px;">81620</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Environmental scanning electron microscope</td>
			<td style="width:253px;">FEI Company</td>
			<td style="width:119px;">Quanta 200</td>
			<td>for starch fibers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Environmental scanning electron microscope</td>
			<td style="width:253px;">Phenom-World</td>
			<td style="width:119px;">Phenom G2 Pro</td>
			<td>for pullulan fibers</td>
		</tr>
	</tbody>
</table>

molecular entanglement and electrospinnability of biopolymers

静电是一种令人着迷的技术制造微纳米级纤维的各种材料。为生物聚合物，在纺丝原液的构成聚合物的分子缠结被发现是成功的静电纺丝的基本先决条件。流变学是一个强大的工具来探测生物聚合物的分子构象和互动。在本报告中，我们证明了协议用于利用流变学来评价两种生物聚合物，淀粉和支链淀粉的electrospinnability，从他们的二甲亚砜（DMSO）/水分散液。良好的淀粉和普鲁兰多糖纤维中的亚微米到微米范围内的平均直径分别获得。 Electrospinnability通过肉眼和显微镜观察所形成的纤维进行评价。通过关联分散到其electrospinnability的流变学特性，我们证明了分子构象，分子的缠结和剪切粘度都影响选rospinning。流变不仅是有用的溶剂体系的选择和过程的优化，而且还了解的纤维形成的机制在分子水平上。

得到的生物聚合物的分散体作为生物聚合物的浓度和溶剂的DMSO浓度的函数的流动曲线。两种有代表性的图显示的淀粉（ 图2A）和支链淀粉（ 图2B）的流动曲线，作为其浓度在纯的DMSO溶剂的功能。特定粘度作图对生物聚合物的浓度（ 图3A为淀粉和图3B为支链淀粉）。从这些曲线，缠结浓度得到的半稀释非缠结和亚浓缠结制度的拟合线的截距。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51933/51933fig2highres.jpg" width="500" /> 图2:流量（一）琼脂糖80的淀粉和（B），普鲁兰多糖纯DMSO为functi曲线对浓度（％，重量/体积）在20℃下。在这两个图中，淀粉和低浓度的支链淀粉是低粘性的，以产生在低剪切速率足够的扭矩。这些不可靠的数据，因此并不绘制。在一般情况下，这两个生物聚合物表现出牛顿特性在低浓度时， 即表观剪切粘度是剪切速率无关。剪切稀化变得明显，它们的浓度增加，特别是超过10％（重量/体积）。然而，剪切变稀的行为是弱者。的15％和20％（重量/体积）的支链淀粉的分散体仅表现为幂律区的早期阶段时的高剪切速率，而淀粉分散体没有在粘度在剪切速率为0.1〜100秒显示显著还原- 1。从REF 12，著作权（2014）爱思唯尔授权转载自文献11，版权所有（2012）美国化学学会和许可。 <img alt="图3" fo:content-width ="5英寸"SRC ="/文件/ ftp_upload / 51933 / 51933fig3highres.jpg"WIDTH ="500"/&gt; 图3地块的具体粘度与（A）琼脂糖80淀粉和纯DMSO（二）普鲁兰多糖浓度。在亚浓非缠结（左侧）的拟合线和缠绕的半稀释的斜率（右侧）制度表示的浓度的比浓粘度的依赖，又名缩放依赖性4。普鲁兰多糖表现出比淀粉的纠缠政权强大浓度依赖性。两个拟合线的截距被命名为纠缠浓度（C E）在其生物聚合物开始在分散重叠。淀粉所需的浓度高于普鲁兰开始纠缠不清。从REF 12，著作权（2014）爱思唯尔授权转载自文献11，版权所有（2012）美国化学学会和许可。 静电纺丝试图对所有的生物聚合物的分散体，并判断在electrospinnability方面的结果， 即喷射的纤维的静电纺丝过程中形成的能力，和形态形成。好electrospinnability的色散形成了稳定和连续喷射，导致连续和光滑的纤维无水滴。分散体，这是无法静电纺丝不能形成稳定的喷射或开发鞭打不稳定。任一微小液滴或粗纤维被沉积到凝固浴中， 图4示出了代表良好，并从它们的外观进行评价差的纤维。 图5以不同的DMSO溶剂中的浓度和生物高分子中分散的淀粉和支链淀粉总结electrospinnability的评价，分别。除了 ​​纠缠浓度，剪切粘度在100秒-1作图对生物聚合物的浓度，其中electrospinnability的区域被表示为（ 图6）。 </p<html> &gt; <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51933/51933fig4highres.jpg" width="500" /> 图4:好（左）和差（右）的淀粉和支链淀粉的纤维的扫描电子显微镜照片。良好的纤维是平滑的，连续的和随机取向的，而差的纤维可以具有珠粒，休息和液滴，如图中所示（红色圆圈）。 （A）10％（重量/体积）琼脂糖80淀粉在95％（体积/体积）的DMSO，（B）8％（重量/体积）琼脂糖80淀粉在80％（体积/体积）的DMSO，（三）17 ％（重量/体积）的支链淀粉在40％（体积/体积）的DMSO，和（d）9％（重量/体积）的支链淀粉在80％（体积/体积）的DMSO。从REF 12，著作权（2014）爱思唯尔授权转载自文献11，版权所有（2012）美国化学学会和许可。 JPG"WIDTH ="500"/&gt; 图5的评价（A） 的琼脂糖80淀粉和（B）的支链淀粉分散体electrospinnability作为溶剂和生物聚合物的浓度，在分散的DMSO浓度的函数:良好electrospinnability（圆圈），electrospinnability差（菱形），以及无法静电纺丝（X的）。阴影区域大致代表electrospinnable地区。纠缠的浓度也大致标注。从REF 12，著作权（2014）爱思唯尔授权转载自文献11，版权所有（2012）美国化学学会和许可。 <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/51933/51933fig6highres.jpg" width="500" /> 图6剪切粘度（一）琼脂糖80的淀粉和（B）分散普鲁兰多糖的功能（在100秒-1）在不同的DMSO /水溶剂中的生物聚合物的浓度。阴影部分大致代表了electrospinnable区域。从REF 12，著作权（2014）爱思唯尔授权转载自文献11，版权所有（2012）美国化学学会和许可。

Watch this Scientific Journal Video about Molecular Entanglement and Electrospinnability of Biopolymers at JoVE.com

Molecular Entanglement and Electrospinnability of Biopolymers

静电是用来从各种材料制作的微纳米级纤维的迷人的技术。在纺丝液的构成聚合物的分子缠结是成功的电纺丝是必不可少的。我们提出了一个协议，利用流变评估两个生物聚合物，淀粉的支链淀粉的electrospinnability。

分子纠缠与生物大分子的Electrospinnability

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular ...

molecular-entanglement-and-electrospinnability-of-biopolymers

Nanyang Technological University

Research

JoVE Journal

Bioengineering

13.7K 次观看.  Pennsylvania State University. 静电是用来从各种材料制作的微纳米级纤维的迷人的技术。在纺丝液的构成聚合物的分子缠结是成功的电纺丝是必不可少的。我们提出了一个协议，利用流变评估两个生物聚合物，淀粉的支链淀粉的electrospinnability。 

视频: Molecular Entanglement and Electrospinnability of Biopolymers

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

在活细胞中的荧光寿命成像分子转子

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

科研

生物工程

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

在检测DNA损伤的体外半人工分子机器，其使用的大会

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

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 ...

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Marine snail Aplysia californica has been widely used as a neurobiology model for the studies on cellular and molecular basis of behavior. Here a methodology is described for exploring the nervous system of Aplysia for the electrophysiological and molecular analyses of single neurons of identified neural circuitry.

阿普利西亚 单一神经元电生理学和分子分析的甘利亚制备

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

微型涡辅助电穿孔的顺序传递分子

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Biofunctionalized普鲁士蓝纳米粒子多式联运分子成像应用

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

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 ...

Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and macro-structures, as seen in the cases of moving water in vascular bundles and moisturizing water in skin layers. In this study, we developed a method for assessing the effect of aqueous liquid crystalline (LC) solutions composed of biopolymers on drying. As LC biopolymers have megamolecular weight, we chose to study polysaccharides, cytoskeletal proteins, and DNA. The observation of biopolymer solutions during drying under polarized light reveals milliscale self-integration starting from the unstable air-LC interface. The dynamics of the aqueous LC biopolymer solutions can be monitored by evaporating water from a one-side-open cell. By analyzing the images taken using cross-polarized light, it is possible to recognize the spatio-temporal changes in the orientational order parameter. This method can be useful for the characterization of not only artificial materials in various fields, but also natural living tissues. We believe that it will provide an evaluation method for soft materials in the biomedical and environmental fields.

A method for the drying-induced self-integration of megamolecular biopolymers on the air-liquid crystalline interface is provided here. This methodology will be useful not only for understanding the macroscopic potentials of biopolymers, but also as an evaluation method for soft materials in biomedical and environmental fields.

对干燥空气-LC接口Megamolecular生物大分子的自我整合方法

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

人体神经细胞在体外热的化学和分子分析的生物粒子微阵列

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...

分子纠缠与生物大分子的Electrospinnability

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此视频中的章节

在活细胞中的荧光寿命成像分子转子

在检测DNA损伤的体外半人工分子机器，其使用的大会

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

使用滑翔分析生物大分子的抗弯刚度测量

阿普利西亚单一神经元电生理学和分子分析的甘利亚制备

微型涡辅助电穿孔的顺序传递分子

Biofunctionalized普鲁士蓝纳米粒子多式联运分子成像应用

工程分子识别与单壁碳纳米管仿生聚合物

对干燥空气-LC接口Megamolecular生物大分子的自我整合方法

人体神经细胞在体外热的化学和分子分析的生物粒子微阵列

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