由国防部W81XWH - 1023年10月1日（张家港），从加州大学旧金山分校中心的系统与合成生物学（张家港）批P50 GM081879，美国国立卫生研究院5R21EB015088-02（YJ）和美国国立卫生研究院1R21EB018044（张家港与YJ）提供资金。 DS是由人类前沿科学计划跨学科的博士后研究奖学金支持。

1，生产单价量子点<ol><li>量子点从有机相转移到水相<ol><li>稀释200微升的有机相量子点与400μL的氯仿5 mL玻璃小瓶1微米的解决方案。 </li><li>混合400微升的0.3M的四丁基溴化铵（TBAB）氯仿溶液用36微升整齐的mPEG硫醇（CH 3 O（CH 2 CH 2 O）6 C 2 H 5 SH）和摇O 2 / N。 </li><li>添加800μL的0.2 M氢氧化钠水溶液振摇30秒。几分钟后，由着色颗粒的上面的较密的有机相转移到水相中指示内的相转移发生。 </li><li>如果粒子凝聚在水相和有机相之间的第三阶段，增加孵育时间用的mPEG硫醇。如果将水相保持清晰，量子点都没有相转移（参见图3A）。交替地，增加concentmPEG的巯基在步骤2中的口粮，以减轻穷人的相转移。 </li><li>回收的（有颜色的）水相，然后浓缩所收集的量子点具有的Centricon离心柱（30 kDa的分子量截止）至1ml。 </li><li>添加的浓缩QD溶液成的Sephadex NAP10柱预平衡10含有30 mM氯化钠（pH 8.0）中50mM Tris缓冲液。洗脱量子点用1.5ml的洗脱缓冲液通过重力流动。 </li><li>测量量子点与吸收光谱的浓度在350纳米。 </li></ol></li><li> MQDS的制备<ol><li>购买（或合成）ptDNA。该协议使用的序列为5&#39;-A S 50（CT）10（ACTG）5 3&#39;（ 见表1）。 </li></ol></li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> DNA </td><td> 序 </td></tr><tr><td> 5&#39;-A S 50（CT），10（ACTG）5 </sub&gt; 3&#39; </td><td> A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S A S CTCTCTCTCTCTCTCTCTCTACTGACTGACTGACTGACTG </td></tr><tr><td> 5&#39;-NH 2 - （CT）10（CAGT）5 -3&#39; </td><td> NH 2 - / C6spacer / CTCTCTCTCTCTCTCTCTCTCAGTCAGTCAGTCAGTCAGT </td></tr><tr><td> BG-DNA </td><td> BG-/ C6spacer / CTCTCTCTCTCTCTCTCTCTCAGTCAGTCAGTCAGTCAGT </td></tr></tbody></table> 表1中的DNA序列用于生产和靶MQDS。 <ol start="2" style="margin-left: 40px;"><li>制备将1ml 100nM的QD溶液在10含有30 mM氯化钠（pH 8）中50mM Tris缓冲液。 </li><li>添加逐滴加入500μl的100nM的ptDNA溶液至水量子点，持续1分钟，同时剧烈搅拌。搅拌或放在摇床额外9小时。 注：此理论上应该产生1：2的比例ptDNA的：QD。然而，实际的化学计量比是决不完美 - 通常的结果是越接近1：1.5。为了产生一个1:1的比例ptDNA的：QD，凝胶电泳后密度是必要的，以量化偶联给出上面所用的已知体积的准确率。 </li><li>除去〜10微升的QD混合物的对一个分析的琼脂糖凝胶电泳。另外在去除水相（从步骤1.2.1）未结合量子点的浓度相近。 <ol><li>添加〜2μ;升的6倍的Ficoll上样缓冲液（以增加溶液的密度），并在0.8％（重量）/ v琼脂糖凝胶中的硼酸钠缓冲液中15分钟，在150V。在未缀合的控制车道上运行这两个样品一起的单一带迁移接近井，并与量子点结合的车道两个频段都看到（ 见图2B凝胶）。 </li></ol></li><li>计算使用两个谱带的相对强度的未结合的QD级分（参见图2B中的公式）。然后使用该部分来计算需要匹配量子点的数量100纳米ptDNA解决方案的额外容量。 </li><li>重复步骤1.2.3至1.2.5再一次用这个计算量，或直至结合的量子点坍塌成一个单一的条带在凝胶上显示的所有量子点的完整结合。 </li><li>加入100μl的10毫米（CO 2 H）CH 2 O（CH 2 CH 2 O）6 C 11 H 23 SH（羧基PEG6烷烃硫醇）在10毫米的Tris缓冲合作ntaining 30 mM氯化钠（pH 8）中，以上述制得的共轭MQDS，并振摇10分钟。 注意：这些PEG配体钝化量子点表面。较长的PEG会更好钝化量子点，但是，它也将增加它们的大小。疏水烷烃硫醇功能对量子点更高的亲和力，合共不下了疏水性的PEG-巯用于相位转移。因此，不允许该步骤继​​续进行过长或ptDNA将由烷-PEG-硫醇代替。一〜30分钟孵育后，DNA的显著部分将被迫离开量子点（ 图3B）。 </li><li>以除去过量的烷烃的PEG-硫醇，加入0.5毫升QD溶液的Sephadex NAP5柱预平衡用洗脱缓冲液（10含有30 mM的NaCl的50mM Tris缓冲液）。收集1.0毫升的洗脱缓冲液通过重力流的MQDS。 </li><li>集中收集到的量子点具有的Centricon离心柱（30 kDa的）。存储这些MQDS在4℃下几个月。请勿酒店frEEZE他们。 注意：如果在试管底部的着色粒料可以看到的，这些点都聚集，并有可能不再可用。 </li></ol> 2，生产定位（Benzylguanine-）的DNA <ol><li>生产或购买胺封端的DNA。该协议使用的序列为5&#39;-NH 2 - （CT）10 - （CAGT）5 3&#39;（ 见表1）。聚-CT接头增加无障碍功能在多糖包被深埋，但可能并不需要对所有的应用程序。如果存在进入DNA合成仪，用适当的5&#39;氨基修饰剂（5&#39;氨基修饰剂C6）生产的氨基改性的DNA。 </li><li>溶解BG-N-羟基琥珀酰亚胺（BG-NHS）的无水二甲亚砜（DMSO）中，以10毫克/毫升的浓度。 注意：BG-NHS会吸收空气中的水分而导致的反应性NHS-酯的水解，特别是在吸湿溶剂，如DMSO和二甲基甲酰胺（DMF）中。 BG-NHS是最好的店d于-80°C作为辅助含干燥剂的容器内的粉末在自己的小瓶。暖打开小瓶之前至室温。交替地，立即分装供单人使用的BG-NHS酯成干极性溶剂并冷冻在-80℃下，或在步骤2.2中的胺的DNA反应完全。 </li><li>通过从步骤2.1混合的BG-NHS在DMSO中与胺封端的DNA在HEPES（4-（2 - 羟乙基）-1 - 哌嗪乙磺酸）缓冲液（pH8.5）水反应。在一个典型的反应，20微升10毫克/毫升的BG-NHS在DMSO中与2微升2mM的NH 2 - （CT）10 - （CAGT）缓冲用20微升的0.5M HEPES pH 8.5的5在58微升的去离子水。开展1.5 ml离心管中的反应。混合快地反应迅速进行，并且必须与NHS-酯水解竞争。 </li><li>超声处理5〜10分钟，以保证充分混合。孵育约1小时，在室温。 BG的化学计量比的DNA可以被改变，以驱动反应完成。 </li><lI&gt;使用标准NAP5列到100毫米醋酸三乙胺（TEAA，pH值7），脱盐反应。 </li><li>净化与未反应的胺-DNA通过反相HPLC对BG-DNA上运行的100mM的TEAA：8％至95％乙腈在30分钟内乙腈（ACN）梯度。未反应胺的DNA将在BG-DNA的前洗脱。收集在锥形管的BG-DNA片段。 <ol><li>使用C 18 Zorbax柱子上的标准寡核苷酸纯化及以下梯度： 0→15分钟：8→30％乙腈; 15→21分钟：30〜90％乙腈; 21→25分钟：90→8％乙腈; 25→30分：8％乙腈; </li></ol></li><li>冷冻在液氮中，并冷冻干燥所收集的级分。重新悬浮在去离子水中的DNA。要格外小心，冻干两次以上，以去除残留的TEAA。残余TEAA可在较高浓度下是有毒的细胞。 </li><li>悬浮冻干BG-DNA的水，确保洗号的两侧ê管，并稀释至约25微米的股票。分装和储存在4℃。样品保存〜9个月没有明显下降。 </li></ol> 3，标记活细胞与单价量子点<ol><li>任选地，使用的试剂，如酪蛋白（0.5％）或牛血清白蛋白（BSA，1％-3％）钝化MQDS刚刚之前的成像试验。 </li><li>板细胞中表达上的全内反射荧光（TIRF） - 质量玻璃的SNAP-标签的蛋白质。在这个实验中，我们使用表达SNAP标记的Notch1受体和高品质的玻璃表面的U2OS细胞系。 </li><li>后的细胞是否已附着在玻璃（〜24小时），除去生长培养基，用PBS洗涤，并孵育细胞10-30分钟，在RT在〜100-150微升PBS或含〜1μM细胞培养基BG- DNA从上面步骤2.6。 </li><li>孵化与BG后，小心地用PBS或细胞培养基洗细胞。培养细胞为〜5-10分钟，日在步骤1.2.9生产ËMQDS（也可以钝化步骤3.1）。 </li><li> （可选）清洗未结合MQDS并返回细胞缓冲/媒体同时适用于成像和文化。对于细胞是在全内反射荧光模式进行成像，最后的清洗步骤往往是不必要的，因为在溶液中游离MQDS扩散得如此之快与约束MQDS因为在分析过程中是无法检测比较。 </li></ol> 4，显微镜及分析<ol><li>图片用全内反射荧光显微镜细胞。 注：适用范围必须有可分离的激发和发射器，或自定义QD过滤器立方体。量子点是一个低波长激光器（405或488 nm）的最佳兴奋。不同直径的量子点具有特征发射波长，通常有较大的斯托克的转变有关。 </li><li>因为MQDS的亮度，收集在高帧速率的图像中的TIRF而成像的活细胞的基底侧。 注意：可替换地，单分子动力学可以是follo结婚时用旋转盘共聚焦显微镜等焦平面。自动化的单粒子跟踪软件是准确跟踪明亮，耐光MQDS总体上是成功。 </li></ol>

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The authors have nothing to disclose.

在MQD设计的模块化使得实验的灵活性程度增加。例如，各种MQDS可以快速的在独特的颜色，允许多个目标的同时成像制备。单链DNA靶序列可以直接MQDS蛋白质，糖类12，脂类和表面13。是许多酶标记可用正交反应性，允许多个目标，同时以差异有针对性的MQDS成像。除了使用SNAP标签定位，靶蛋白与使用CLIP标签，哈洛标签，MQDS和生物素蛋白标记也是成功的。此协议表明活细胞上与这些MQDS表面受体的特异性标记，但该协议可以很容易地适应于许多不同的场合。 生产MQDS与定义的化合价的显著方法论的观点是〜50硫代联碱是必需的，以包住量子点（并因此防止两个分子从同时绑定）。多聚A S 50序列可重复和稳定结合自Life Technologies 605纳米的量子点。虽然这款产品已经停产，空间排阻策略推广到由具有不同尺寸，形状，光谱特性等厂商的同类产品。 ptDNA包裹量子点的效率和稳定性主要取决于量子点的表面化学性质和结构。因此，协议的成功将取决于量子点的商业来源和化学结构。对于这个协议的目的，量子点的各种商业渠道之间存在差异的三大要点：在必要的相转移条件的差异;在最初的PEG-硫醇配体结合，这可能阻碍位移由ptDNA的强度差;以及在暴露的CdSe核心的量的差异，从而可以勒广告到QD的由MPEG硫醇相转移条件下淬火。 至于出版，量子点与生产MQDS的最佳结构是4-10自Life Technologies在545，585，605和625纳米（ 图2A）购买了发射光谱波长的CdSe / ZnS核/壳量子点。基于&#39;鲜艳&#39;制剂（545，605 等 ） 的量子点的淬灭后，另外的mPEG硫醇，并且不适合于这种应用。自Aldrich和海洋纳米量子点的工作很好，但需要更长的相转移步骤和预​​处理与三辛基氧化。该协议进行了优化，从生命技术的量子点。 用来包裹量子点聚硫代磷酸酯序列终止与含（ACTG）5到靶向股可杂交序列的原生20个碱基的DNA尾巴。这个序列是方便的，因为它具有很少或几乎没有二级结构，并且将保持hybridiz版在37℃的PBS中。如果存在进入DNA合成仪中，ptDNA可以使用C 8柱通过合成，然后通过反相高效液相色谱法（HPLC）纯化。该ptDNA将在高效液相色谱比同等的寡核苷酸与本地骨干后来洗脱。我们通常会留下5&#39;DMT保护基团对我们的硫代磷酸酯寡核苷酸纯化后。 通常需要为了提高量子点的胶体稳定性和降低背景的大多数实验应用结合ptDNA包裹的量子点的钝化。该协议采用的PEG层钝化量子点。羧基的PEG链烷硫醇与额外的PEG单元（（CO 2 H）CH 2 O（CH 2 CH 2 O）12 C 11 H 23 SH基，羧基-PEG12烷硫醇）提供显著降低背景，虽然更长的PEG都较大，和通常更昂贵。 MQDS涂上羧基聚乙二醇ALKANE硫醇配位体是在生理缓冲液非常稳定，例如磷酸盐缓冲生理盐水，并培养介质。 MQDS在4℃下长期储存（&gt; 8个月）没有表现出显著聚集或ptDNA支队11。根据该实验时，在量子点的PEG钝化本身并不总是充分地降低非特异性的MQDS的结合。在含有3％BSA的基本上在使用前20分钟的磷酸盐缓冲盐水（PBS）中温育两细胞和MQDS减少非特异性结合到细胞中，尽管它由〜50％增加的MQDS的表观流体动力学半径。钝化，用0.5％酪蛋白降低了非特异性结合，甚至进一步，但它增加了表观尺寸比BSA更大的程度。 DNA链互补的MQDS的5&#39;末端可以进行修改，以使多个不同的生物分子的靶向。有一批建立的技术可用于共价修饰的蛋白，升ipids与糖的单链DNA（ssDNA）。只要单链DNA在细胞外出现，它就可以到达易溶MQDS。 MQDS与上述序列将迅速与细胞培养条件下与其互补的DNA链杂交。可能需要的ptDNA和靶向序列之间的10-20x聚（CT）间隔为有效的靶向，从而提高了结合序列的厚和带负电荷的多糖包被的细胞之上。对于这个协议，我们选择了以生产BG-DNA与（CAGT）5的互补序列，将二者杂交的MQDS和共价本身链接到SNAP-标签蛋白快速，特异标记。类似的协议功能以及用于连接其它的NHS-酯，以氨基修饰的寡核苷酸。 为单分子成像，通常需要标记的低密度解决单个分子。对〜0.5纳米的PBS最后MQD浓度钝化剂是一个很好的目标。然而，这些高稀释度有时导致下标记的细胞。如果出现这种情况，附加MQD可以添加到标签的最佳密度是观察。在SNAP-标签的人类Notch1基因的情况下，&gt; 10nM的MQD浓度产生致密的标记细胞，同时为0.5nM MQD导致的附着〜20 MQDS在靶向细胞的基底表面（参见图4B）。与MQDS细胞标记高度依赖镀细胞的汇合。过度融合细胞不标注在自己的基础面。 总之，一个简单的方法来生成一价和模块化量子点被描述。这些MQDS找到实用的多种活细胞成像的应用，主要表现在成像活U2OS细胞的Notch受体。 MQDS的适用性并不局限于这个特定的情况下，但也可以潜在地延长了其它细胞靶标，如其它蛋白质，核酸和酶。

单个分子上活细胞的动力学有利于它们的生物学功能。单分子荧光成像技术是研究细胞表面上的1,2,3单分子动力学的常用方法。然而，在这些研究中最常用的成像探头具有几个重要的缺点。例如，传统的有机染料和荧光蛋白质提供中等亮度，大约10 5 -10 6 M -1 -1，但是光化学不稳定的，大约10 5 -10 6的光子典型的活细胞成像的条件下的发光后，漂4,5。与此相反，半导体纳米颗粒，常称为量子点（QDs），是显著更明亮，更稳定，具有消光系数为10 6 -10 7 M -1 -1的范围和超过photobl前10 7 -10 8发射的光子eaching 5。量子点在有机荧光团的改进的亮度和光使单个分子的显著更快的帧速率的观察和对更长的轨迹6。 尽管他们的优势和商业可用性，连带责任仍是这些强大的显像剂。第一，他们定义不清靶向价，这可能导致在目标生物分子6的交联。第二，它们通常具有较大的流体动力学大小（&gt; 20纳米），限制访问某些拥挤的蜂窝环境7。第三，它们具有有限的定位模块7。几种策略试图解决这些问题8,9,10，但一般需要专门的知识和试剂来实现。 为了解决这些问题，我们最近报道了“空间排阻”战略制备单价，小，模块化的量子点11。该量子点包裹着一个长硫代磷酸酯的DNA（ptDNA）聚合物。该ptDNA结合通过表面暴露的锌原子和ptDNA聚合物的磷酸酯基团之间的多个锌-S相互作用的量子点表面。一个单一的结合聚合物的空间位和静电不包括聚合物的额外当量的结合而不显著增加粒子的总尺寸（约2nm）。所有试剂均为市售产品，形成以高收率，并且该过程仅需要脱盐工序进行精制。一旦标记，量子点包裹着一个ptDNA（MQDS）结合轴承的目标领域（ 如 ，苄基鸟嘌呤（BG），苄基胞嘧啶，或烷基卤化物）互补DNA链。 这些功能针对MQDS具体酶标记，如SNAP，CLIP＆HALO被基因融合到感兴趣的蛋白质。这是一个协议，用于合成，目标，并通过空间排阻生产MQDS的活细胞成像。

<table border="0" cellpadding="0" cellspacing="0" width="497">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:91px;">Comments/Description</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">mQD Production:</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Phosphorothioate DNA:</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">(A*)x50-(ACTG)x5</td>
			<td style="width:71px;">IDT</td>
			<td style="width:119px;">N/A</td>
			<td style="width:91px;">Most DNA Synthesis companies</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Quantum Dots:</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:216px;">QDots 525, 585, 605 &#38; 625</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">Q21791MP (545), Q21711MP (585), Q21701MP (605)</td>
			<td style="width:91px;">Custom QD synthesis for QD625.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">QD610</td>
			<td style="width:71px;">Ocean Nanotech</td>
			<td style="width:119px;">QSP-610-10&#160;</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">QD 610 lamda</td>
			<td style="width:71px;">Aldrich</td>
			<td style="width:119px;">731854</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Chloroform, 99.8%</td>
			<td style="width:71px;">ACROS</td>
			<td style="width:119px;">67-66-3</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Tetrabutylammonium bromide, 98.0%</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">426288</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">mPEG thiol [2,5,8,11,14,17,20-Heptaoxadocosane-22-thiol], MW 354.5, 95%</td>
			<td style="width:71px;">Polypure</td>
			<td style="width:119px;">11156-0695</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:216px;">HSC11EG6CO2H&#160; [HS-(CH2)11-(OCH2CH2)6-OCH2CO2H]</td>
			<td style="width:71px;">ProChimia</td>
			<td style="width:119px;">TH 003-m11.n6-0.1</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Boric Acid, 99.5%</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">B0394</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Sodium Hydroxide, 99.0%</td>
			<td style="width:71px;">ACROS</td>
			<td style="width:119px;">S/4845</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Sodium Chloride, 98%</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">310166</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Agarose LE</td>
			<td style="width:71px;">U.S. Biotech Sources</td>
			<td style="width:119px;">G02PD-125</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Ethanol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">459828</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">BG-DNA Production:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Amine DNA:</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">(CAGT)5-NH2</td>
			<td style="width:71px;">IDT</td>
			<td style="width:119px;">N/A</td>
			<td style="width:91px;">Most DNA Synthesis companies</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">(CAGT)5(T)40-NH2</td>
			<td style="width:71px;">IDT</td>
			<td style="width:119px;">N/A</td>
			<td style="width:91px;">Most DNA Synthesis companies</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">NHS-GLA-Benzylguanine</td>
			<td style="width:71px;">New England Biosciences</td>
			<td style="width:119px;">S9151</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">DMSO</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8428</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">HEPES Buffer</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">83264</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">NAP5 Column</td>
			<td style="width:71px;">GE Healthcare</td>
			<td style="width:119px;">17-0853-01</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">C18 Column</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Acetonitrile</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">Mammalian Cell Culture &#38; Imaging:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Cell line expressing SNAP-tagged protein</td>
			<td style="width:71px;">New England Biosciences</td>
			<td style="width:119px;">E9100S</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">McCoys 5A</td>
			<td style="width:71px;">UCSF Cell Culture Facility (?)</td>
			<td style="width:119px;"></td>
			<td style="width:91px;">Specific to U2OS culture</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:71px;">UCSF Cell Culture Facility (?)</td>
			<td style="width:119px;"></td>
			<td style="width:91px;">Specific to U2OS culture</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">PBS</td>
			<td style="width:71px;">UCSF Cell Culture Facility (?)</td>
			<td style="width:119px;"></td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Nunc Lab-tek II Chambered Coverglass</td>
			<td style="width:71px;">Thermo-Fischer Scientific</td>
			<td style="width:119px;">155409</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Matriplate 96-well plate</td>
			<td style="width:71px;">Brooks Life Science Systems</td>
			<td style="width:119px;">MGB096-1-2-LG-L</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">BSA</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">5% Alkali-soluble Casein</td>
			<td style="width:71px;">EMD Millipore</td>
			<td style="width:119px;">70955</td>
			<td style="width:91px;">Not all caseins are the same</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">(Optional) DNA Synthesis Reagents:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">5&#8217; Amine modifier C6</td>
			<td style="width:71px;">Glen Research</td>
			<td style="width:119px;">Oct-06</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">dA-Thiophosphoramidite</td>
			<td style="width:71px;">Glen Research</td>
			<td style="width:119px;">10-700</td>
			<td style="width:91px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Analysis Software:</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">FIJI</td>
			<td colspan="3">http://valelab.ucsf.edu/~schindelin/</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">RyTrack.pro</td>
			<td colspan="3">http://sun.iwu.edu/~gspaldin/rytrack.html</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Tracker</td>
			<td colspan="3">http://www.gartnerlab.ucsf.edu/more/software</td>
		</tr>
	</tbody>
</table>

production and targeting of monovalent quantum dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

该量子点从有机相转移到水相是用于生产MQDS的关键的，但既可以是条件 - 和QD特异性。如果出现转移更像小瓶3第1.1节相转移应显示为干净的， 如图3中的前两小瓶。，那么人们应该再次尝试与不同的条件。 一旦量子点都涂有带负电荷的DNA就应该​​分别迁移凝胶上从非包裹的量子点。利用展开的量子点作为对照，第二个等分试样，更快的迁移带应该出现在加入ptDNA中，如在图2B中的第一凝胶看出。完全形成MQDS被证实与不动的带的损耗，以及其坍塌成如在图2B中的最后一个凝胶看到移动带。如果您的MQD产物的等分试样迁移为单一条带，从展开的量子点可分离，那么你MQDS是一价和R<html>爱迪在进一步的步骤中使用。 细胞标记应具体到你所选择的目标，应该是依赖于蛋白表达水平和MQD浓度。通常需要两个MQD浓度和MQD钝化的经验优化对于任何给定的应用程序。 图4B展示的U2OS细胞中表达与MQDS范围中的浓度为0.5纳米至10纳米的SNAP-标签的Notch受体的标记。 <img fo:content-width="6in" width="600" alt="图1" src="/files/ftp_upload/52198/52198fig1highres.jpg" /> 图1：模块化瞄准MQDS的。MQDS杂交的单链DNA通过各种靶向分子官能化。苄基鸟嘌呤-DNA的挂钩对象MQDS到SNAP标签的融合蛋白。其他5&#39;修饰和DNA序列能MQDS为目标，以各种其他生物分子。/files/ftp_upload/52198/52198fig1highres.jpg“目标=”_ blank将“&gt;请点击这里查看该图的放大版本。 <img fo:content-width="6in" width="600" alt="图2" src="/files/ftp_upload/52198/52198fig2highres.jpg" /> 图2计划生产MQDS的 。 （A） 的有机相的量子点被转移到水相，通过用的mPEG硫醇和TBAB，包裹着ptDNA和钝化羧基PEG6烷烃硫醇。代表TEM照片是有机QD545，QD585和QD605分别。 （B）为经验性地确定量子点和ptDNA在上面步骤2之间相互作用的化学计量法。 QD＆ptDNA化学计量是通过密度经验证实，使得最终的反应的化学计量比为1：1（QD：ptDNA）。 ptDNA的摩尔初始数量由QD的比率相乘：MQD，并通过实际体积对焦调整<html>器结合，产生达到1所需的摩尔数：1共轭<a href="https://www.jove.com/files/ftp_upload/52198/52198fig2highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img fo:content-width="6in" width="600" alt="图3" src="/files/ftp_upload/52198/52198fig3highres.jpg" /> 图3实验细节进行生产MQDS的（A）的成功代表的照片和失败的相转移。量子点应该致密的有机相和较少稠密的水相之间在视觉上传送。不完全相分离的指示转移不良。 （B）ptDNA可由烷-PEG-硫醇代替。正确的凝胶数据代表的地方ptDNA的显著部分流离失所的量子点，由于过度钝化反应的。es.jpg“目标=”_ blank将“&gt;请点击这里查看该图的放大版本。 <img fo:content-width="6in" width="600" alt="图4" src="/files/ftp_upload/52198/52198fig4highres.jpg" /> 图4标签上的活细胞的SNAP-标记蛋白。（一）示意图展示了一个单元标记的受体与BG-MQD针对性的表达，附件和标签。 （ 二）细胞表达的卡扣缺口mCherry构建在不同MQD浓度的代表性标志。 MQDS钝化PEG12共定位与mCherry指示特定的标签。较低的标记密度（&lt;0.5 nm）的一般都是最好的单粒子跟踪。比例尺为10微米。 <a href="https://www.jove.com/files/ftp_upload/52198/52198fig4highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about Production and Targeting of Monovalent Quantum Dots at JoVE.com

Production and Targeting of Monovalent Quantum Dots

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

生产和目标单价量子点

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

production-and-targeting-of-monovalent-quantum-dots

Research

JoVE Journal

Bioengineering

25.4K 次观看.  University of California, San Francisco. We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications. 

视频: Production and Targeting of Monovalent Quantum Dots

Solubilization and Bio-conjugation of Quantum Dots and Bacterial Toxicity Assays by Growth Curve and Plate Count

Quantum dots (QDs) are fluorescent semiconductor nanoparticles with size-dependent emission spectra that can be excited by a broad choice of wavelengths. QDs have attracted a lot of interest for imaging, diagnostics, and therapy due to their bright, stable fluorescence1,2 3,4,5. QDs can be conjugated to a variety of bio-active molecules for binding to bacteria and mammalian cells6.
QDs are also being widely investigated as cytotoxic agents for targeted killing of bacteria. The emergence of multiply-resistant bacterial strains is rapidly becoming a public health crisis, particularly in the case of Gram negative pathogens 7. Because of the well-known antimicrobial effect of certain nanomaterials, especially Ag, there are hundreds of studies examining the toxicity of nanoparticles to bacteria 8. Bacterial studies have been performed with other types of semiconductor nanoparticles as well, especially TiO2 9,10-11, but also ZnO12 and others including CuO 13. Some comparisons of bacterial strains have been performed in these studies, usually comparing a Gram negative strain with a Gram positive. With all of these particles, mechanisms of toxicity are attributed to oxidation: either the photogeneration of reactive oxygen species (ROS) by the particles or the direct release of metal ions that can cause oxidative toxicity. Even with these materials, results of different studies vary greatly. In some studies the Gram positive test strain is reportedly more sensitive than the Gram negative 10; in others it is the opposite 14. These studies have been well reviewed 15.
In all nanoparticle studies, particle composition, size, surface chemistry, sample aging/breakdown, and wavelength, power, and duration of light exposure can all dramatically affect the results. In addition, synthesis byproducts and solvents must be considered16 17. High-throughput screening techniques are needed to be able to develop effective new nanomedicine agents.
CdTe QDs have anti-microbial effects alone18 or in combination with antibiotics. In a previous study, we showed that coupling of antibiotics to CdTe can increase toxicity to bacteria but decrease toxicity to mammalian cells, due to decreased production of reactive oxygen species from the conjugates19. Although it is unlikely that cadmium-containing compounds will be approved for use in humans, such preparations could be used for disinfection of surfaces or sterilization of water.
In this protocol, we give a straightforward approach to solubilizing CdTe QDs with mercaptopropionic acid (MPA). The QDs are ready to use within an hour. We then demonstrate coupling to an antimicrobial agent.
The second part of the protocol demonstrates a 96-well bacterial inhibition assay using the conjugated and unconjugated QDs. The optical density is read over many hours, permitting the effects of QD addition and light exposure to be evaluated immediately as well as after a recovery period. We also illustrate a colony count for quantifying bacterial survival.

Nanoparticles such as semiconductor quantum dots (QDs) can be used to create photoactivatable agents for anti-microbial or anti-cancer applications. This technique shows how to water-solubilize cadmium telluride (CdTe) QDs, conjugate them to an antibiotic, and perform a bacterial inhibition assay based upon growth curves and plate count.

量子点的生长曲线和平皿计数的细菌毒性检测的增溶作用和生物共轭

Quantum dots (QDs) are fluorescent semiconductor nanoparticles with size-dependent emission spectra that can be excited by a broad choice of wavelengths. ...

科研

生物工程

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

细胞标记和超顺磁性氧化铁纳米粒子靶向

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

固体脂质纳米粒（前哨淋巴结）细胞内定位的应用

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

一种快速定量荧光方法蛋白靶向小分子药物筛选

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used in cancer treatment and imaging due to increased cellular accumulation over current ACs. During early AC in vitro development, fluorescence techniques and radioimmunoassays are sufficient for determining intracellular localization, accumulation efficiency, and target cell specificity. Currently, there is no consensus on standardized methods for preparing cells for evaluating AC intracellular accumulation and localization. The initial testing of ACs modified with virus-derived peptides is critical especially if several candidates have been constructed. Determining intracellular accumulation by fluorescence can be affected by background signal from ACs at the cell surface and complicate the interpretation of accumulation. For radioimmunoassays, typically treated cells are fractionated and the radioactivity in different cell compartments measured. However, cell lysis varies from cell to cell and often nuclear and cytoplasmic compartments are not adequately isolated. This can produce misleading data on payload delivery properties. The intravenous injection of radiolabeled virus-derived peptide-modified ACs in tumor bearing mice followed by radionuclide imaging is a powerful method for determining tumor targeting and payload delivery properties at the in vivo phase of development. However, this is a relatively recent advancement and few groups have evaluated virus-derived peptide-modified ACs in this manner. We describe the processing of treated cells to more accurately evaluate virus-derived peptide-modified AC accumulation when using confocal microscopy and radioimmunoassays. Specifically, a method for trypsinizing cells to remove cell surface bound ACs. We also provide a method for improving cellular fractionation. Lastly, this protocol provides an in vivo method using positron emission tomography (PET) for evaluating initial tumor targeting properties in tumor-bearing mice. We use the radioisotope 64Cu (t1/2 = 12.7 h) as an example payload in this protocol.

Viral-derived peptides coupled to antibody-conjugates (ACs) is an approach gaining momentum due to the potential of delivering molecular payloads with increased tumor cell accumulation. Utilizing common methods to evaluate peptide conjugation, AC and payload intracellular accumulation, and tumor targeting, this protocol helps researchers during the key initial development phases.

病毒衍生肽修饰抗体结合物对增强细胞积累和改善肿瘤靶向性的初步评价

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

20 mJ, 1 ps Yb:YAG Thin-disk Regenerative Amplifier

This is a report on a 100 W, 20 mJ, 1 ps Yb:YAG thin-disk regenerative amplifier. A homemade Yb:YAG thin-disk, Kerr-lens mode-locked oscillator with turn-key performance and microjoule-level pulse energy is used to seed the regenerative chirped-pulse amplifier. The amplifier is placed in airtight housing. It operates at room temperature and exhibits stable operation at a 5 kHz repetition rate, with a pulse-to-pulse stability less than 1%. By employing a 1.5 mm-thick beta barium borate crystal, the frequency of the laser output is doubled to 515 nm, with an average power of 70 W, which corresponds to an optical-to-optical efficiency of 70%. This superior performance makes the system an attractive pump source for optical parametric chirped-pulse amplifiers in the near-infrared and mid-infrared spectral range. Combining the turn-key performance and the superior stability of the regenerative amplifier, the system facilitates the generation of a broadband, CEP-stable seed. Providing the seed and pump of the optical parametric chirped-pulse amplification (OPCPA) from one laser source eliminates the demand of active temporal synchronization between these pulses. This work presents a detailed guide to set up and operate a Yb:YAG thin-disk regenerative amplifier, based on chirped-pulse amplification (CPA), as a pump source for an optical parametric chirped-pulse amplifier.

A protocol for the operation of a high-energy, high-power optical parametric chirped pulse amplifier pump source based on an Yb:YAG thin-disk regenerative amplifier is presented here.

20 mJ，1 ps Yb：YAG薄盘再生放大器

This is a report on a 100 W, 20 mJ, 1 ps Yb:YAG thin-disk regenerative amplifier. A homemade Yb:YAG thin-disk, Kerr-lens mode-locked oscillator with ...

Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. Atherosclerosis is also the leading cause of sudden death and myocardial infarction, instigated by unstable plaques that rupture and occlude the blood vessel without warning. Current imaging modalities cannot differentiate between stable and unstable plaques that rupture. Peptide amphiphiles micelles (PAMs) can overcome this drawback as they can be modified with a variety of targeting moieties that bind specifically to diseased tissue. Monocytes have been shown to be early markers of atherosclerosis, while large accumulation of monocytes is associated with plaques prone to rupture. Hence, nanoparticles that can target monocytes can be used to discriminate different stages of atherosclerosis. To that end, here, we describe a protocol for the preparation of monocyte-targeting PAMs (monocyte chemoattractant protein-1 (MCP-1) PAMs). MCP-1 PAMs are self-assembled through synthesis under mild conditions to form nanoparticles of 15 nm in diameter with near neutral surface charge. In vitro, PAMs were found to be biocompatible and had a high binding affinity for monocytes. The methods described herein show promise for a wide range of applications in atherosclerosis as well as other inflammatory diseases.

This paper presents a method involving the synthesis and characterization of monocyte-targeting peptide amphiphile micelles and the corresponding assays to test for biocompatibility and ability of the micelle to bind to monocytes.

单核靶向肽 Amphiphile 胶束合成动脉粥样硬化的研究

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. ...

Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting and drug delivery are some of the applications of these AuNPs, but endothelial extracellular matrices&#39; defensive properties hamper particle uptake. To address this issue, we describe a synthesis method for ultrasmall gold nanoparticles to improve vascular delivery, with customizable functional groups and polymer lengths for further adjustments. The protocol yields 2.5 nm AuNPs that are capped with tetrakis(hydroxymethyl)phosphonium chloride (THPC). The replacement of THPC with hetero-functional polyethylene glycol (PEG) on the surface of the AuNP increases the hydrodynamic radius to 10.5 nm while providing various functional groups on the surface. The last part of the protocol includes an optional addition of a fluorophore to allow the AuNPs to be visualized under fluorescence to track nanoparticle uptake. Dialysis and lyophilization were used to purify and isolate the AuNPs. These fluorescent nanoparticles can be visualized in both in vitro and in vivo experiments due to the biocompatible PEG coating and fluorescent probes. Additionally, the size range of these nanoparticles render them an ideal candidate for probing the glycocalyx without disrupting normal vasculature function, which may lead to improved delivery and therapeutics.

We describe a method of synthesizing biocompatible 10-nm gold nanoparticles, functionalized by coating poly-ethylene glycol onto the surface. These particles can be used in vitro and in vivo for delivering therapeutics to nanoscale cellular and extracellular spaces that are difficult to access with conventional nanoparticle sizes.

用于内皮靶向和药物传递的功能化10纳米高分子涂层金粒子的合成

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting ...

Targeting Neuronal Fiber Tracts for Deep Brain Stimulation Therapy Using Interactive, Patient-Specific Models

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for movement disorders and is being applied to a growing number of disorders. Computational modeling has been successfully used to predict the clinical effects of DBS; however, there is a need for novel modeling techniques to keep pace with the growing complexity of DBS devices. These models also need to generate predictions quickly and accurately. The goal of this project is to develop an image processing pipeline to incorporate structural magnetic resonance imaging (MRI) and diffusion weighted imaging (DWI) into an interactive, patient specific model to simulate the effects of DBS. A virtual DBS lead can be placed inside of the patient model, along with active contacts and stimulation settings, where changes in lead position or orientation generate a new finite element mesh and solution of the bioelectric field problem in near real-time, a timespan of approximately 10 seconds. This system also enables the simulation of multiple leads in close proximity to allow for current steering by varying anodes and cathodes on different leads. The techniques presented in this paper reduce the burden of generating and using computational models while providing meaningful feedback about the effects of electrode position, electrode design, and stimulation configurations to researchers or clinicians who may not be modeling experts.

The goal of this project is to develop an interactive, patient-specific modeling pipeline to simulate the effects of deep brain stimulation in near real-time and provide meaningful feedback as to how these devices influence neural activity in the brain.

应用交互式、患者特异性模型对神经纤维束进行深部脑刺激治疗的靶向性

Deep brain stimulation (DBS), which involves insertion of an electrode to deliver stimulation to a localized brain region, is an established therapy for ...