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>添加到树枝状聚合物溶液的荧光素和TMR的所需量。在混合物中的摩尔比将反映染料上的树枝状聚合物装入的量。通常为1毫升的离心管G4 PAMAM树枝状聚合物溶液与8当量的荧光素（40微升）和8当量的TMR（40微升）。搅拌该溶液在室温下搅拌12小时。 </li><li>稀释1:10去离子水，并加载该反应混合物透析袋（截留分子量= 10 kDa的）。透析24小时，用去离子水经常更换水在储。 </li><li>将该溶液转移至小瓶中并冷冻干燥24小时。紫色粉末应获得。重量所得到的固体，并在10μM的最终浓度溶解在milliQ水中。分装，在-20℃下的溶液，并存储</li></ol> 2， 体外 pH测量<ol><li>用于体外校准制备树枝状聚合物的溶液中500nm处的PBS（2 mM磷酸盐）的石英比色杯中。建议使用非常稀的PBS缓冲液（2毫摩尔），以避免pH值的滴定过程中的突然变化。 </li><li>测量荧光素（EXC 488 nm）和TMR（EXC 550 nm）的发射光谱和优化荧光计的光学设置，以达到良好的信号噪声比。 </li><li>通过添加少量的氢氧化钠0.1N的盐酸和0.1 N。进行pH滴定后每增加摇试管进行搅拌，等待1分钟进行平衡，并通过pH微电极的方法测量pH值。荧光素和TMR的发射光谱应记录没有光学设置的任何改变每一步。 </li><li>绘制荧光强度与pH值的滴定。若丹明信号应该由pH值（&lt;10％）受到影响。荧光素信号应该是一个S形曲线，并应配有与PK = 6.4单绑定模型。 </li></ol> 3。细胞培养与电穿孔<ol><li>培养在补充有10％胎牛血清和100 U / ml青霉素和100毫克/毫升链霉素（Invitrogen）的Dulbecco改良的Eagle培养基（DMEM）的HeLa细胞。保持细胞培养物在37℃下在湿润的5％CO 2的气氛。 </li><li>对于树枝状电，当细胞汇合，取出介质，并使用DPBS（Dulbecco氏磷酸盐缓冲液）洗涤细胞。除去DPBS，并添加胰蛋白酶-EDTA。中和胰通过添加含血清培养基但不犯罪的抗生素。离心机在900-1,200 rpm下在室温下2分钟。取出介质，并用DPBS冲洗颗​​粒。 </li><li>计数细胞，并采取4×10 6个细胞。离心机在1,200-1,500 rpm下在室温下2分钟。 </li><li>重悬细胞沉淀中加入200μl微穿孔缓冲液（由microporator厂家提供）和细胞转移到1.5 ml离心管。 </li><li>加树枝状聚合物水溶液为悬浮细胞。每个样品所需的树枝状聚合物的量取决于PAMAM型（通常为250纳米的阳离子和2微米中性树枝状）。 </li><li>加电穿孔缓冲液（由microporator厂家提供）在微穿孔管。吸取与100μl的体积尺寸的电穿孔尖端的细胞和树枝状聚合物的混合物。将microporator吸管插入吸管站。设置为微穿孔脉象：脉冲电压为1,005 V;妇女参与发展的脉搏TH = 35毫秒，脉冲数= 2。 </li><li>后的脉冲传递细胞到1.5ml微量离心管中，离心细胞5分钟，在1,200 rpm下以除去培养基中的过量的树枝状聚合物。板10微升电穿孔细胞到35 mm玻璃底菜（Willco公司 - 菜GWSt-3522）用新鲜培养基W / O型抗生素。 </li></ol> 4。生活在HeLa细胞的pH传感<ol><li>以电穿孔后共聚焦显微镜12小时图象的细胞。 </li><li>可用于荧光素和罗丹明标准滤波器组。若可调谐滤波器可安排从500绿色通道 - 550nm和红色通道从580纳米到650纳米。激发波长为488nm处是最佳的荧光素罗丹明时可成像要么与543nm或561激光线。 </li><li>聚焦在试样上，并调整激光功率和检测器增益最大化的信号 - 噪声比。如果电穿孔是成功的细胞应该是在两个通道的明亮的荧光。该本地化取决于所使用的树枝状聚合物的大小和电荷。往往是一些溶酶体定位（核周小囊泡）是由于内吞作用或条块存在。如果溶酶体定位是主要的， 即大部分的荧光是局部内囊泡，差信号在胞液中被观测到，这意味着毒性和测量应该被丢弃。顺序地获取两个通道，如果需要获取多个图像和平均化图像，以提高图像质量。 </li><li>使用缓冲液校准夹具细胞的pH与离子载体在不同的pH和如上所述获得每个pH值至少为20的细胞。对于该过程的详细描述和缓冲器的组合物，请参阅Bizzarri的同事和16中，我们建议至少测量5个点在pH = 5.5至pH = 7.5。 pH值低于6是有毒的细胞，但容忍的时间较短，我们建议获得尽可能快的图像。如果细胞证明细胞凋亡的迹象，丢弃电池，然后重新启动。 </li><li>使用ImageJ的或用于数据分析的类似软件。导入的绿色和红色通道的图像，减去背景和由像素比成像与工具“图像计算器”创建一个像素。 </li><li>绘制感兴趣区（ROI）选择所需的单元格的区域，并测量细胞内的绿色到红色的比例。分析所有获取的图像，然后绘制比与pH值。在从5.5到7.5的范围内的趋势应该是线性的。所得到的点的线性拟合会给将用于绿色到红色比转换至pH的校准曲线。 </li><li>作为进一步的控制收购一些未经处理的细胞（无离子载体），并尝试计算pH值与所获得的校准曲线。应获得7.2和7.4之间的值。 </li></ol> 5， 体内样品制备<ol><li>实验产后d所对C57BL/6J（男性和女性）进行AY 28和70。麻醉的小鼠腹膜内注射乌拉坦（ 即乙基氨基甲酸酯）（20％w / v的在生理盐水中，20毫克/公斤的聚氨酯）。实验与聚氨酯的后跟一个心内注射相同的麻醉剂过量后处死动物。 </li><li>进行注射地塞米松磷酸钠（2毫克/千克体重）肌肉内，以减少外科手术过程中的皮质应激反应和脑水肿。 </li><li>剃除动物的头部和应用2.5％利多卡因凝胶在头皮上。 </li><li>用剪刀剪下的皮肤覆盖南北半球的颅骨皮瓣</li><li>用生理盐水冲洗骨外露，轻轻地用镊子取出骨膜。这将提供胶水和牙科用粘固剂附着有更好的基础。 </li><li>套用一个特制的钢头后与中央成像室，并与氰基丙烯酸酯在一个平面上胶水约与头骨i的皮层区域平行nterest并修复到位，白色的牙齿水泥（Paladur）。 </li><li>修正了鼠标的头部，以执行2-3毫米直径钻孔在该地区的利益开颅手术。 </li><li>尝试手术，硬脑膜撕裂，或出血期间尽量减少皮质加热。 </li><li>保持灌流用无菌ACSF（126 mM氯化钠，3 mM的氯化钾，1.2mM的KH 2 PO 4，1.3 mM的MgSO 4干燥，26毫米的NaHCO 3，2.4毫米氯化钙2皮层，15mM的葡萄糖，1.2毫摩尔HEPES中蒸馏H 2 O的，pH值= 7.4）。 </li></ol> 6。在小鼠脑成像pH值<ol><li>在实验过程中帮助动物的呼吸提供氧气的富氧空气。氧被浓缩至高达80％。 O 2分压和流量经常调整，以获得正确的呼吸辅助。保持体温恒定在37℃与反馈控制的加热毯。 </li><li>通过以下的两个照片的目标的钢后的修复动物Ñ​​成像设置。 </li><li>为了将传感器在大脑皮质注入装入含有氯化银电极（尖端直径为4毫米），树枝状聚合物溶液（1μM）的玻璃移液管。电极将允许录制外场电位。 </li><li>用显微注射法设置插入吸移管在皮层，大约150微米的深度。注射1-2分钟在0.5 psi的压力。 </li><li>优化的光学装置的成像。激光功率应调整，以减少光漂白和光损伤。采用通常的激光功率大约为20毫瓦，而光电倍增管的增益保持恒定在667因为V先前校正表明，该电压提供了最好的S / N比。 </li><li>对于成像激励传感器在820 nm和通过标准FITC和TRITC过滤器同时检测荧光素和罗丹明荧光。 </li><li>时间分辨测量采集时间的推移扳成2赫兹。 </li><li>背景校正获得一个黑暗的框架落色ř快门关闭来衡量，通常由电子添加的光电倍增管和底座所产生的平均热噪声。 </li><li>数据分析报告遵循在第4相同的步骤。 </li></ol>

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默认：作者什么都没有透露。

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

The development of  fluorescent indicators represented a revolution for life sciences. Genetically  encoded and synthetic fluorophores with sensing ...

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

Unit 1

Acid–Base Titration

Acid-base and Solubility Equilibria

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11.6K 次观看.  Eindhoven University of Technology & NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR. 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。 

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

Synthesis of Hypervalent Iodonium Alkynyl Triflates for the Application of Generating Cyanocarbenes

The procedures described in this article involve the synthesis and isolation of hypervalent iodonium alkynyl triflates (HIATs) and their subsequent reactions with azides to form cyanocarbene intermediates. The synthesis of hypervalent iodonium alkynyl triflates can be facile, but difficulties stem from their isolation and reactivity. In particular, the necessity to use filtration under inert atmosphere at -45 &deg;C for some HIATs requires special care and equipment. Once isolated, the compounds can be stored and used in reactions with azides to form cyanocarbene intermediates. 
The evidence for cyanocarbene generation is shown by visible extrusion of dinitrogen as well as the characterization of products that occur from O-H insertion, sulfoxide complexation, and cyclopropanation. A side reaction of the cyanocarbene formation is the generation of a vinylidene-carbene and the conditions to control this process are discussed. There is also potential to form a hypervalent iodonium alkenyl triflate and the means of isolation and control of its generation are provided. The O-H insertion reaction involves using a HIAT, sodium azide or tetrabutylammonium azide, and methanol as solvent/substrate. The sulfoxide complexation reaction uses a HIAT, sodium azide or tetrabutylammonium azide, and dimethyl sulfoxide as solvent. The cyclopropanations can be performed with or without the use of solvent. The azide source must be tetrabutylammonium azide and the substrate shown is styrene.

Herein is described the synthesis, isolation, and reactions of hypervalent iodonium alkynyl triflates (HIATs) with azides to generate cyanocarbenes. The procedures will involve air-sensitive and cryogen techniques, including cold filtration under an inert atmosphere. Handling and safety of the synthesized compounds will also be discussed.

高价碘鎓炔基三氟甲磺酸酯的生成Cyanocarbenes的合成应用

The procedures described in this article involve the synthesis and isolation of hypervalent iodonium alkynyl triflates (HIATs) and their subsequent ...

科研

化学

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended solvent window, low background currents, corrosion resistance, etc., arise from the catalytically inert nature of the surface. However, if during the growth process, non-diamond-carbon (NDC) becomes incorporated into the electrode matrix, the electrochemical properties will change as the surface becomes more catalytically active. As such it is important that the electrochemist is aware of the quality and resulting key electrochemical properties of the BDD electrode prior to use. This paper describes a series of characterization steps, including Raman microscopy, capacitance, solvent window and redox electrochemistry, to ascertain whether the BDD electrode contains negligible NDC i.e. negligible sp2 carbon. One application is highlighted which takes advantage of the catalytically inert and corrosion resistant nature of an NDC-free surface i.e. stable and quantifiable local proton and hydroxide production due to water electrolysis at a BDD electrode. An approach to measuring the local pH change induced by water electrolysis using iridium oxide coated BDD electrodes is also described in detail.

Assessment of Boron Doped Diamond Electrode Quality and Application to In Situ Modification of Local pH by Water Electrolysis

A protocol is described for the characterization of the key electrochemical parameters of a boron doped diamond (BDD) electrode and subsequent application for in situ pH generation experiments.

掺硼金刚石电极质量和应用，以评估<em&gt;原位</em&gt;本地pH值通过水电改造

Boron doped diamond (BDD) electrodes have shown considerable promise as an electrode material where many of their reported properties such as extended ...

Synthesis and Exfoliation of Discotic Zirconium Phosphates to Obtain Colloidal Liquid Crystals

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and engineers. Growth of such anisotropic nanocrystals through a simple chemical method is a challenging task. In this study, we fabricate discotic nanodisks of zirconium phosphate [Zr(HPO4)2&#183;H2O] as a model material using hydrothermal, reflux and microwave-assisted methods. Growth of crystals is controlled by duration time, temperature, and concentration of reacting species. The novelty of the adopted methods is that discotic crystals of size ranging from hundred nanometers to few micrometers can be obtained while keeping the polydispersity well within control. The layered discotic crystals are converted to monolayers by exfoliation with tetra-(n)-butyl ammonium hydroxide [(C4H9)4NOH, TBAOH]. Exfoliated disks show isotropic and nematic liquid crystal phases. Size and polydispersity of disk suspensions is highly important in deciding their phase behavior.

A two dimensional model material of discotic zirconium phosphate was developed. The inorganic crystal with lamellar structure was synthesized by hydrothermal, reflux, and microwave-assisted methods. On exfoliation with organic molecules, layered crystals can be converted to monolayers, and nematic liquid crystal phase was formed at sufficient concentration of monolayers.

合成与盘状磷酸盐锆剥落获取胶体液晶

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

放射性标记和磷酸肌醇的细胞水平的定量高效液相色谱耦合流动闪烁

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

蛋白质生物缀合物的合成<em&gt;通过</em&gt;半胱氨酸马来酰亚胺化学

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance imaging (MRI). Their purpose is to enhance the relaxation rate of water protons in tissue, thus increasing the MR image contrast and the specificity of the MRI measurements. Current clinically approved contrast agents are low molecular weight molecules that are rapidly cleared from the body. The use of dendrimers as carriers of paramagnetic chelators can play an important role in the future development of more efficient MRI contrast agents. Specifically, the increase in local concentration of the paramagnetic species results in a higher signal contrast. Furthermore, this CA provides a longer tissue retention time due to its high molecular weight and size. Here, we demonstrate a convenient procedure for the preparation of macromolecular MRI contrast agents based on poly(amidoamine) (PAMAM) dendrimers with monomacrocyclic DOTA-type chelators (DOTA &#8211; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate). The chelating unit was appended by exploiting the reactivity of the isothiocyanate (NCS) group towards the amine surface groups of the PAMAM dendrimer to form thiourea bridges. Dendrimeric products were purified and analyzed by means of nuclear magnetic resonance spectroscopy, mass spectrometry, and elemental analysis. Finally, high resolution MR images were recorded and the signal contrasts obtained from the prepared dendrimeric and the commercially available monomeric agents were compared.

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

This protocol describes the preparation and characterization of a dendrimeric magnetic resonance imaging (MRI) contrast agent that carries cyclen-based macrocyclic chelates coordinating paramagnetic gadolinium ions. In a series of MRI experiments in vitro, this agent produced an amplified MRI signal when compared to the commercially available monomeric analogue.

制备及<em&gt;体外</em&gt;磁共振成像基于树状大分子造影剂的表征

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance ...

Synthesis of Core-shell Lanthanide-doped Upconversion Nanocrystals for Cellular Applications

Lanthanide-doped upconversion nanocrystals (UCNs) have attracted much attention in recent years based on their promising and controllable optical properties, which allow for the absorption of near-infrared (NIR) light and can subsequently convert it into multiplexed emissions that span over a broad range of regions from the UV to the visible to the NIR. This article presents detailed experimental procedures for high-temperature co-precipitation synthesis of core-shell UCNs that incorporate different lanthanide ions into nanocrystals for efficiently converting deep-tissue penetrable NIR excitation (808 nm) into a strong blue emission at 480 nm. By controlling the surface modification with biocompatible polymer (polyacrylic acid, PAA), the as-prepared UCNs acquires great solubility in buffer solutions. The hydrophilic nanocrystals are further functionalized with specific ligands (dibenzyl cyclooctyne, DBCO) for localization on the cell membrane. Upon NIR light (808 nm) irradiation, the upconverted blue emission can effectively activate the light-gated channel protein on the cell membrane and specifically regulate the cation (e.g., Ca2+) influx in the cytoplasm. This protocol provides a feasible methodology for the synthesis of core-shell lanthanide-doped UCNs and subsequent biocompatible surface modification for further cellular applications.

A protocol is presented for the synthesis of core-shell lanthanide-doped upconversion nanocrystals (UCNs) and their cellular applications for channel protein regulation upon near-infrared (NIR) light illumination.

核壳镧系掺杂转换纳米晶的合成及其在蜂窝中的应用

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

Here, a method to synthesize gold, palladium, and platinum aerogels via a rapid, direct solution-based reduction is presented. The combination of various precursor noble metal ions with reducing agents in a 1:1 (v/v) ratio results in the formation of metal gels within seconds to minutes compared to much longer synthesis times for other techniques such as sol-gel. Conducting the reduction step in a microcentrifuge tube or small volume conical tube facilitates a proposed nucleation, growth, densification, fusion, equilibration model for gel formation, with final gel geometry smaller than the initial reaction volume. This method takes advantage of the vigorous hydrogen gas evolution as a by-product of the reduction step, and as a consequence of reagent concentrations. The solvent accessible specific surface area is determined with both electrochemical impedance spectroscopy and cyclic voltammetry. After rinsing and freeze drying, the resulting aerogel structure is examined with scanning electron microscopy, X-ray diffractometry, and nitrogen gas adsorption. The synthesis method and characterization techniques result in a close correspondence of aerogel ligament sizes. This synthesis method for noble metal aerogels demonstrates that high specific surface area monoliths may be achieved with a rapid and direct reduction approach.

A rapid, direct solution-based reduction synthesis method to obtain Au, Pd, and Pt aerogels is presented.

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

This study discusses a synthesis route for soft polysiloxane-based urea (PSU) elastomers for their applications as accommodating intraocular lenses (a-IOLs). Aminopropyl-terminated polydimethylsiloxanes (PDMS) were previously prepared via the ring-chain equilibration of the cyclic siloxane octamethylcyclotetrasiloxane (D4) and 1,3-bis(3-aminopropyl)-tetramethyldisiloxane (APTMDS). Phenyl groups were introduced into the siloxane backbone via the copolymerization of D4 and 2,4,6,8-tetramethyl-2,4,6,8-tetraphenyl-cyclotetrasiloxane (D4Me,Ph). These polydimethyl-methyl-phenyl-siloxane-block copolymers were synthesized for increasing the refractive indices of polysiloxanes. For applications as an a-IOL, the refractive index of the polysiloxanes must be equivalent to that of a young human eye lens. The polysiloxane molecular weight is controlled by the ratio of the cyclic siloxane to the endblocker APTMDS. The transparency of the PSU elastomers is examined by the transmittance measurement of films between 200 and 750 nm, using a UV-Vis spectrophotometer. Transmittance values at 750 nm (upper end of the visible spectrum) are plotted against the PDMS molecular weight, and &#62; 90% of the transmittance is observed until a molecular weight of 18,000 g&#183;mol&#8722;1. Mechanical properties of the PSU elastomers are investigated using stress-strain tests on die-cut dog-bone-shaped specimens. For evaluating mechanical stability, mechanical hysteresis is measured by repeatedly stretching (10x) the specimens to 5% and 100% elongation. Hysteresis considerably decreases with the increase in the PDMS molecular weight. In vitro cytotoxicity of some selected PSU elastomers is evaluated using an MTS cell viability assay. The methods described herein permit the synthesis of a soft, transparent, and noncytotoxic PSU elastomer with a refractive index approximately equal to that of a young human eye lens.

This study describes synthetic routes for aminopropyl-terminated polydimethylsiloxanes and polydimethyl-methyl-phenyl-siloxane-block copolymers and for soft polysiloxane-based urea (PSU) elastomers. It presents the application of PSUs as accommodating an intraocular lens. An evaluation method for in vitro cytotoxicity is also described.

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 of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.

In this protocol, doxorubicin-loaded AS1411-g-PEI-g-PEG modified gold nanoparticles are synthesized via three-step amide reactions. Then, doxorubicin is loaded and delivered to target cancer cells for cancer therapy.

合成阿普塔默-PEI-g-PEG改性金纳米粒子装载多索鲁比辛用于靶向药物输送

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the ...