Name: synthesis, cellular delivery and in vivo application of dendrimer-based ph sensors
Uploaded: 2013-09-10T10:00:00
Description: Watch this Scientific Journal Video about Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors at JoVE.com

与Isja德Feijter和马特·贝克有益的讨论表示诚挚的谢意。

1。该传感器的合成<ol><li>在以下部分中，我们提供了一个程序的pH值指标，PAMAM树枝状聚合物的缀合。相同的协议，可以以最小的修改被应用到另一种胺-轴承树枝状聚合物。5,17,13,14市售的树枝状聚合物和染料可以不经进一步纯化使用。 </li><li>溶解树枝状聚合物在无水DMSO（50μM终浓度）。在无水DMSO中制备10mM的储备溶液的荧光素-NHS和四甲基罗丹明-NHS（TMR）的。 </li><li>添加到树枝状聚合?...

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对于成功的pH成像与树枝状聚合物基传感器的关键步骤是：在正确的树突状支架ⅰ）选择和缀合到它指示的数目和传感器递送协议ⅱ）在细胞内或体内的最优化。 该合成过程是相当容易的，并且可以几乎应用于每个胺息的超支化聚合物。该传感器可以由一个单步市售的树枝状聚合物与NHS-活化的染料来获得。我们认为，这种模块化和简单的过程将有利于为各种生?...

利用荧光分子标记特定的生物相关分子已经完全改变了我们研究生物系统的方式。广角并允许生物过程的实时高分辨率的可视化和时下激光共聚焦显微镜是最流行 ​​的技术，研究在体外生物事件，在细胞和体内 。1一个相应的改进是由荧光指标的发展代表即染料的荧光是依赖于特定的分子实体的浓度。尤其是pH值和钙指标对细胞生理学研究产生了巨大影响，由于H +和Ca 2 +的离子在生物的巨大意义。2,3 然而，大多数目前的传感染料的几个固有的局限性与他们的小分子的行为，如：i）于亚细胞targeti困难纳克;在水中，因而差的生物相容性ⅱ）溶解性差;以及iii）细胞渗漏，因而缺乏长时间推移成像能力4此外，许多探针的信号不能被校正，以染料浓度的依赖性（非比例成像），因此，在细胞中或在体内的绝对测量是不可能的。 最近，我们描述一个简单而有效的方法来克服这些限制，基于传感染料的树枝状聚合物骨架共轭。5树枝状化合物是单分散性的超支化聚合物具有非常吸引人的特性对于生物学应用。6特别一些树突状结构已被开发并用于药物7和基因传递。8只最近几个小组开始研究这些分子作为支架材料用于传感设备的潜力。9,10,11 我们以前描述了基于NHS活化酯对不同聚酰胺-胺的官能化的容易合成路线（PAMAM）支架12的结合物可以在单一步骤中通过透析的方法，因为只有纯化而获得。有趣的是这种方法可以很容易地应用到各种各样的树突或聚合物骨架。13,14 实现成像比例分别为树枝状双标有两套染料：ⅰ）一个pH指示剂（ 即荧光素）和ii）的pH非依赖性的荧光部分（ 例如若丹明）。这使我们能够进行精确的pH值成像的荧光素和罗丹明之间的比率为仅依赖于pH值，没有更多的探针的浓度。另一个有趣的方法这个问题是通过使用生命周期为基础的探针。15表示为终身不依赖于探针浓度这些测量不需要比例修正。然而，LIFETIME测量需要一个更复杂的仪器的设置和它们的时间分辨率是次优的用于快速的生理过程，从而限制了它们的应用潜力。 为了进行细胞内成像，探针需要跨质膜被传递到胞质溶胶中。作为树枝状聚合物不会由于它们的尺寸和亲水性膜渗透性，细胞内递送可通过电穿孔来实现。通过这种技术，广泛应用于生物学转染的方式，标记大分子可以有效地传递到细胞内进行高品质的影像。此外，与电穿​​孔可避免与树枝状聚合物的内吞作用的复杂的大分子被直接递送到细胞质中。有趣的是电穿孔后不同树枝状显示单元，即使在没有任何具体的靶向序列内不同的本地化。5本PASSIVË目标，只是由于树枝状聚合物的物理化学性质，可以被利用来实现细胞器特定的pH成像。 比例成像可以使用共焦显微镜来进行。荧光素和若丹明，共价偶联到树枝状骨架，分别成像，并创建一个像素由像素比例的地图。据报道几个程序由离子载体的手段来控制细胞内pH值在活细胞。离子载体是小的疏水性分子能够穿过细胞膜的运输离子;离子载体为H +离子，如尼日利亚菌素，都是有效的，可用于校准树枝状聚合物为基础的传感器16，这些测量结果表明，以pH值线性响应类似于所观察到的。 体外 。在校准细胞内pH值的基础上可以正确地测定。这些测量结果表明，树枝状聚合物为基础的传感器可以在研究H +的homeost一个有价值的工具ASIS在活细胞和病理过程，涉及pH值调节故障。 我们最近表明，树枝状聚合物为基础的pH传感器也能在体内被应用，在麻醉小鼠的大脑进行成像pH值17由于生物组织体内感应一个高品质在技术上具有挑战性的复杂环境。这里，我们表明所述的实验步骤在体内 pH值成像的寻址到在大脑中进行准确的pH值成像的关键问题加重的详细描述。双光子显微镜已受聘主要有两个原因：一）使用红外光能够克服缺乏标准的共聚焦显微镜组织渗透的;二）荧光素和罗丹明的广泛双光子吸收让他们同时激发回避并发症相关的使用的两个波长进行激发。在小鼠大脑的pH测量是顺利进行;传感器容易缺氧诱导响应pH值在脑细胞外空间的变化。这些测量结果表明，树枝状聚合物为基础的指标，可以成功地用来突出pH值的生理和病理变化在体内在动物模型。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
		 <td>Company</td>
		 <td>Catalogue number</td>
		 <td>Comments (optional)</td>
 </tr>
	 <tr>
	 <td>PAMAM G4</td>
		 <td>Sigma-Aldrich</td>
		 <td>412449</td>
		 <td></td>
	 </tr>
	 <tr>
	 <td>Carboxyfluorescein NHS ester</td>
		 <td>Life technologies</td>
		 <td>C-1311</td>
		 <td></td>
	 </tr>
	 <tr>
	 <td>TMR NHS ester</td>
		 <td>Life technologies</td>
		 <td>C-1171</td>
		 <td></td>
	 </tr>
	 <tr>
	 <td>DMSO</td>
		 <td>Sigma-Aldrich</td>
		 <td>D8418</td>
		 <td></td>
	 </tr>
	 <tr>
	 <td>Dyalsis bags</td>
		 <td>Spectrum Labs</td>
		 <td>132117</td>
		 <td></td>
	 </tr>
	 <tr>
	 <td>WillCo Dishes</td>
		 <td>WillCo Wells</td>
		 <td>GWSt-3512</td>
		 <td></td>
	 </tr>
	 <tr>
	 <td>Urethane</td>
		 <td>Sigma-Aldrich</td>
		 <td>U2500</td>
		 <td></td>
	 </tr>
 </tbody>
</table>

synthesis, cellular delivery and in vivo application of dendrimer-based ph sensors

荧光指标的发展为代表的生命科学的一场革命。基因编码的，并与感测能力的合成的荧光团可生物相关物质的高空间和时间分辨率的可视化。合成染料是特别感兴趣得益于其高可调性和广泛的可测量的分析物。然而，这些分子蒙受小分子行为的若干限制（溶解性差，在定位困难，常无比例成像允许的）。在此工作中，我们介绍的树枝状聚合物基传感器的开发和在体外呈现程序用于pH测量，在活细胞和活体 。我们选择树枝状为我们的传感器理想的平台，他们的许多理想性能（单分散性，可调性，多价），使他们广泛使用的脚手架几个生物医学设备。荧光pH值的共轭指标，树枝状聚合物骨架导致其感测性能的增强。特别是树枝状展览减少细胞渗出，改善细胞内定位，并允许比例测量。这些新颖的传感器被成功地用于测量pH值在活的HeLa细胞和体内在小鼠脑。

图1示出的传感染料以不同的树枝状骨架的缀合的示意图。所得到的指标，可以从市售品1容易合成的步骤来获得。胺息树枝状化合物与NHS-活化的染料在DMSO中，并通过渗析提纯。这是一般的程序已经被成功地用于多种树枝状标签：我）PAMAM树形分子的产生2，4和6; 12聚乙二醇化树形分子17和PEG-树突状杂种18。作为不同的树枝状聚合物显示出与细胞和组织（本地化?...

Watch this Scientific Journal Video about Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors at JoVE.com

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

荧光传感器是在生命科学的强大工具。这里我们描述的方法来合成，并使用树枝状聚合物为基础的荧光传感器来测量pH值在活细胞和活体 。树突状支架增强的共轭荧光染料从而提高传感特性的属性。

合成，细胞和交货<em&gt;在体内</em&gt;的树状为基础的pH值传感器应用

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Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

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Lanthanide-doped upconversion nanocrystals (UCNs) have attracted much attention in recent years based on their promising and controllable optical ...

A Rapid Synthesis Method for Au, Pd, and Pt Aerogels Via Direct Solution-Based Reduction

直接溶液还原法快速合成金、钯、铂气凝胶的研究

Here, a method to synthesize gold, palladium, and platinum aerogels via a rapid, direct solution-based reduction is presented. The combination of various ...

Synthesis of Soft Polysiloxane-urea Elastomers for Intraocular Lens Application

软聚聚硅氧烷-尿素弹性体在人工晶体中的应用

This study discusses a synthesis route for soft polysiloxane-based urea (PSU) elastomers for their applications as accommodating intraocular lenses ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

硫醇反应试剂的合成与生物共轭作用--用于原位选择性修饰免疫共轭的生成

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...