支持加州大学实验室费用研究发展计划奖12-LR-237197，O.库里图鲁斯D. Dimlioglu。 P. Daggumati是支持大学的加州Davis Research的投资在科学与工程（RISE）奖。支持CA查普曼系国家需要奖学金​​研究生教育援助领域。这项工作是支持由UC实验室费用研究发展计划，加州大学戴维斯分校的上升，和加州大学戴维斯分校工程的启动资金。

1。制备纳米多孔金<ol><li> Piranha溶液清洁基板<ol><li> 25毫升的过氧化氢（30％）加入到100毫升的硫酸（96％）的结晶皿和加热混合物至65℃的加热板上对。注意:液体是腐蚀性极强，必须小心处理。将使用了的溶液不应该被存储在一个密封的容器，因为它可能会引起爆炸。 </li><li>放置1英寸×3英寸的显微镜载物片到该混合物中，用耐酸钳10分钟，并清除它们。 ，使用瓷免疫船小批量清洗盖玻片。治疗小盖玻片与空气等离子浸入到溶液中之前，在10瓦特，30秒。 </li><li>下运行3分钟，去离子水（DI）冲洗清洁样品结晶菜肴。用氮气枪吹干样品超过皮棉免费毛巾。 </li></ol></li><li>准备模板掩模（方法1:使用创建毫米级的模式） <ol><li>冲床250微米厚的硅橡胶片活检拳和/或切割用手术刀超过半硬塑料表面的区域。清洁处理后的弹性体片材，在70％的异丙醇和干燥的，用氮气枪。 </li><li>将打孔纸不起毛的洁净毛巾上，并对齐食人鱼清洗盖玻片与样品表面面临的模版模板。 </li></ol></li><li>图案剥离光致抗蚀剂（方法2:使用创建亚毫米级图案） <ol><li>纺纱夹头将食人鱼清洁显微镜幻灯片，并用氮气枪吹掉微粒。分液1.5ml的粘合促进剂（六甲基二硅氮烷）到载玻片上，用塑料吸管。差价的启动子，通过依次以500rpm旋转滑动，持续5秒和每分钟1500转，30秒。烤7分钟，在115℃的烤盘上的幻灯片，并让它冷却5分钟。 </li><li>输注4毫升到玻璃载片上的光致抗蚀剂（3英寸bY 1英寸）。传播的光致抗蚀剂，通过纺丝相同的协议作为粘合促进剂中的滑动。在烤盘上烤的光刻胶为1.5分钟，在115℃10分钟，让它冷却。 </li><li>用UV光（强度:22毫瓦/厘米2）的光致抗蚀剂涂层的滑动暴露通过透明罩，持续15秒。烘烤光致抗蚀剂在加热板上在115℃下1.5分钟，等待45分钟。曝光的光致抗蚀剂溶解在开发商至少3.5分钟。去离子水彻底冲洗。型态在光学显微镜下检查。 </li></ol></li><li>存款前体金属产生NP-金薄膜<ol><li>将样品放入溅射机可以独立存金，银，铬。溅射清洁的样品在50 W 90秒25毫托氩处理气氛下开始之前金属沉积。 </li><li> 10分钟，在300瓦下10毫托氩溅镀铬。溅射黄金90秒400 W UNDER 10毫氩。合作溅射黄金和白银与白银在10分钟200 W和Au功率在100瓦黄金溅射源银关闭前约10秒，关闭溅射源。 </li></ol></li><li>获得前体金属微细<ol><li>超声〜180毫升和10个循环20秒的超声处理2分钟的暂停周期之间的光致抗蚀剂剥离剂的光致抗蚀剂涂层的样品。用DI水冲洗样品，并用氮气枪干燥。在显微镜下检查金属图案。 </li><li>剥离非光刻胶涂层样品用两个镊子揭示熔敷金属的弹性模板。 </li></ol></li><li> Dealloy前体通过热处理纳米金属和修改<ol><li>填充的200毫升的玻璃烧杯中，用170毫升硝酸（70％），并在加热板上，保持溶液的温度在55℃。注意:硝酸是腐蚀性极强，应处理适当的防护设备。 </lI&gt; <li>对待小盖玻片空气等离子在10 W 30秒前浸入硝酸。名次1英寸×3英寸的显微镜载玻片放入烧杯中使用耐酸钳dealloy 15分钟。使用瓷免疫船批次去合金​​小盖玻片。 </li><li>通过连续地浸渍在充满了新鲜的去离子水的烧杯中三次冲洗去合的样品。店内样品在去离子水中，并更换新鲜的去离子水的水，每天至少一个星期。在无绒毛巾在使用前用氮气枪吹干样品。 </li><li>将样品加载到一个干净的硅晶片的快速热处理设备。调节温度至200℃和450℃，升温速率10℃/秒。样品暴露到规定的温度，在氮气环境下的10分钟。让腔冷却（&lt;100℃），取出样品。另外，慢慢地将样品在烤盘上热treatm耳鼻喉科。 </li></ol></li></ol> 2。细胞培养<ol><li>准备NP-坳样品细胞培养<ol><li>广场NP金样品在聚苯乙烯菜和空气等离子处理30秒10 W的样品转移到24孔细胞培养板。 </li><li>向每孔中加入500μl的完整的培养基（Dulbecco改进的Eagle培养基，用10％胎牛血清和1％青霉素/链霉素）。储存增湿培养箱中，在37℃和5％CO 2中，直到播种的细胞（&lt;1小时）。 </li></ol></li><li>维护，通道和种子细胞<ol><li>保持在T75培养瓶，培养基及通过细胞70％汇合时，感兴趣的细胞（在这种情况下，NIH 3T3成纤维细胞或鼠星形胶质细胞）。 </li><li>对于传代的细胞，从烧瓶中取出介质，清洗两次，每次用10毫升磷酸盐缓冲盐水（PBS）中，加入2毫升1个胰蛋白酶/ EDTA和孵育直到细胞分离（约5分钟）。加入3毫升新鲜的媒体和在1,200 rpm离心3分钟。吸上清液，，暂停沉淀在2毫升媒体。 </li><li>从孔和种子细胞的盖玻片上，在密度为25,000细胞/ cm 2的最终体积为1毫升，取出用过的介质。向前摇动培养板中后卫和右左，以确保细胞均匀涂布在样品上。日常检查的同时，培养的细胞，直到分析。 </li></ol></li></ol> 3。细胞与材料分析<ol><li>细胞染色可视化细胞骨架和细胞核<ol><li>从水井中取出废媒体，用PBS洗涤细胞两次。修复的细胞在4％多聚甲醛的PBS中15分钟。 </li><li>准备300纳米的Alexa Fluor 488的鬼笔环肽在PBS溶液中染色，用1％牛血清白蛋白。 </li><li>用PBS洗涤细胞两次，通透于500μl的0.1％的Triton X-100的PBS中5分钟。 </li><li>用PBS洗涤细胞两次，他们转移到清洁井。 5印迹0 - 200微升到20分钟的样品并储存在黑暗的染色溶液。 </li><li>洗涤细胞，用PBS和反污垢与3纳米的在PBS中的DAPI染色5分钟。 </li><li>用PBS洗涤细胞，并浸在去离子水中，然后安装在玻璃罩幻灯片安装媒体和密封，与透明指甲油。 </li></ol></li><li> NP-金样品和细胞培养对NP金表面获取图像<ol><li>图片NP-凹表面的50000倍放大的扫描电子显微镜（SEM），在10千伏电子能量使用二次电子探测器。 </li><li>在不同的点上样品使用倒置荧光显微镜在10倍的放大倍率，用适当的滤光块捕获复合细胞图像。 </li></ol></li><li>处理图像，以确定孔隙和细胞形态<ol><li>打开ImageJ的图像分割成单独的通道，如果适用。将图像转换为8位，减的背景下，并顺利地把它们由位数filteri的纳克。调整阈值手动或通过内置的阈值的算法，以反白显示的孔隙（空隙）和胞体/细胞核。 </li><li>使用分水岭命令合并毛孔或细胞分离。颗粒分析参数设置和执行命令提取颗粒，平均面积和覆盖率颗粒数。使用DAPI染色细胞图像量化％小区覆盖的细胞计数和鬼笔环肽染色的细胞图像。 </li><li>修改包括宏文件执行批处理多个图像分析。 </li></ol></li></ol>

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作者有没有冲突的财务权益。

我们表现​​出两种不同的技术来扩大使用这些薄膜微系统和生物研究微细NP-Au膜。溅射镀膜的金及银是一种多用途的方法来创建NP-互惠模式，溅射是与传统的微细加工工艺和合金的组成和厚度可以很容易地通过改变个别溅射枪功率控制（适用于黄金和银子目标）和兼容沉积时间。典型的NP-Au膜的厚度从200纳米到2微米的范围内。允许创建毫米尺度格局，规避光刻法需要使用活检冲头和手术刀的模?...

<p class="jove_content"&gt;材料的纳米尺度特征表明承诺在提高各种应用，包括燃料电池<sup&gt; 1</sup&gt;，传感器<sup&gt; 2,3</sup&gt;，和生物医学设备<sup&gt; 4,5</sup&gt;。一个相对较新的材料是纳米多孔金性（np-Au）的，它是由纳米尺度的自组装过程。 NP-互惠的前体是最常见的由以原子百分比计为60％至80％的银金合金。简单地说，开孔纳米结构特征是在集群中的金原子的重排的结果，银溶于强酸（<em&gt;如</em&gt;硝酸70％）的电化学电势或根据<sup&gt; 6-8</sup&gt;。 Np的金从几个理想的属性，包括大的有效表面积，高导电性，完善的表面官能化的技术，和生物相容性的好处<sup&gt; 9</sup&gt;。即使出现了快速扩张研究NP金，其中大部分集中于NP-金的机械性能<sup&gt; 10,11</sup&gt;，催化活性;<sup&gt; 12</sup&gt;，和生物分子传感性能<sup&gt; 13-15</sup&gt;。虽然理想的属性数的生物医学工具是非常有用的<sup&gt; 16,17</sup&gt;，在这方面的应​​用受到了限制。这种情况的一个可能的原因是，大多数的研究主要用于宏观尺度试样（<em&gt;如</em&gt;片材，金属箔，锭），将在小型化的系统中的NP-互惠的技术仍然不够。事实上，也有只有极少数的例子，使用传统的微细加工技术，采用NP-Au膜<sup&gt; 16-20</sup&gt;。随着小型化技术和新的生物医学工具的需要，它已成为关键的是能够集成到设备中的新材料。通常，这要求该材料可以与传统的微细加工技术，淀积和构图。此外，快速量化细胞材料的相互作用通常是要评估的一种新材料的生物相容性。本文的目的是要证明微细NP-Au膜的基本技术和量化纳米结构和细胞物质的相互作用，通过数字图像处理。</p

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Gold target</td>
 <td>Lesker</td>
 <td>EJTAUXX403A2</td>
 <td >Precursor to alloy for producing np-Au</td>
 </tr>
 <tr>
 <td>Chrome target</td>
 <td>Lesker</td>
 <td>EJTCRXX353A2</td>
 <td >Adhesive layer</td>
 </tr>
 <tr>
 <td>Silver target</td>
 <td>Lesker</td>
 <td>EJTAGXX403A2</td>
 <td >Precursor to alloy for producing np-Au</td>
 </tr>
 <tr>
 <td>Porcelain boat</td>
 <td>Thomas Scientific </td>
 <td>8542E40</td>
 <td >Used for processing small samples</td>
 </tr>
 <tr>
 <td>Nitric acid</td>
 <td>Sigma-Aldrich</td>
 <td>43873</td>
 <td >Used at 70% for dealloying</td>
 </tr>
 <tr>
 <td>Sulfuric acid</td>
 <td>J.T Baker </td>
 <td>7664-93-9</td>
 <td >Used at 96% for piranha cleaning</td>
 </tr>
 <tr>
 <td>Hydrogen peroxide</td>
 <td>J.T Baker </td>
 <td>7722-84-1</td>
 <td >Used at 30% for piranha cleaning</td>
 </tr>
 <tr>
 <td>Biopsy punches</td>
 <td>Ted Pella</td>
 <td>150xx</td>
 <td >Available in several sizes</td>
 </tr>
 <tr>
 <td>Silicone elastomer sheets</td>
 <td>Rogers Corporation </td>
 <td>HT 6240</td>
 <td >Available in several thicknesses</td>
 </tr>
 <tr>
 <td>Hexamethyldisilazane</td>
 <td>Sigma-Aldrich</td>
 <td>440191-100ML</td>
 <td >Used as adhesion promoter for positive resist</td>
 </tr>
 <tr>
 <td>Microposit MF CD26</td>
 <td>Shipley</td>
 <td>38490</td>
 <td >Positive photoresist developer</td>
 </tr>
 <tr>
 <td>PRS 3000</td>
 <td>J.T Baker</td>
 <td>JT6403-5</td>
 <td >Positive photoresist stripper</td>
 </tr>
 <tr>
 <td>Circular glass coverslips (12 mm)</td>
 <td>Ted Pella</td>
 <td>26023</td>
 <td >Used as substrate for metal patterns and cell culture</td>
 </tr>
 <tr>
 <td>Glass slides (1 x 3 inch)</td>
 <td>Ted Pella</td>
 <td>26007</td>
 <td >Used as substrate for metal patterns</td>
 </tr>
 <tr>
 <td>Kapton polyimide tape</td>
 <td>VWR</td>
 <td>82030-950</td>
 <td >Used for securing elastomer</td>
 </tr>
 <tr>
 <td>Transparency masks</td>
 <td>Output City</td>
 <td></td>
 <td>Used in photolithography <a href="http://www.outputcity.com/" target="_blank">http://www.outputcity.com/</a></td>
 </tr>
 <tr>
 <td>Plasma cleaner</td>
 <td>Harrick Plasma</td>
 <td>PDC-32G</td>
 <td >Used for activating glass surfaces</td>
 </tr>
 <tr>
 <td>Sputtering machine</td>
 <td>Kurt J. Lesker</td>
 <td>LAB18</td>
 <td >Used for depositing metals</td>
 </tr>
 </tbody>
 </table>

microfabrication of nanoporous gold patterns for cell-material interaction studies

在几十纳米特征尺寸的纳米材料，增强了多种技术，包括燃料电池，生物传感器，生物医学设备涂料，药物输送工具的性能。纳米多孔金（NP-金），纳米尺度的自组装过程中所产生的，是一种相对较新的材料，具有大的有效表面积，高导电性，催化活性。这些特性NP-坳科学界的一个有吸引力的材料。 NP-金的大部分研究采用宏观尺度的标本，并侧重于基础科学的物质和其催化和传感器应用。宏观尺度的标本限制NP-金的潜力，在小型化的系统，包括生物医学设备。为了解决这些问题，我们初步描述了两种不同的方法在刚性基板上的微细NP-金薄膜。第一种方法是采用手工制作的模板创建毫米级NP-金模式的whIL口罩e的第二种方法是使用剥离光刻图案亚毫米级图案。作为NP金薄膜溅射沉积过程中获得的，它们与传统的微细加工技术，从而适合轻便整合成微兼容。这些系统包括电寻址的生物传感器平台，受益于高的有效表面积，导电性，和的金硫醇类表面生物耦合。我们描述了细胞培养，免疫组化技术和图像处理技术到量化NP-互惠与哺乳动物细胞中，这是一个重要的性能参数对某些生物传感器的交互。我们这里演示的技术将协助整合平台，各种长度尺度和众多的应用，包括生物传感器，储能系统和催化剂的NP-金。

图1概括了主要的程序步骤，包括创建NP-金模式，培养细胞，量化的纳米结构，细胞形态特征。 图2a中所示的弹性体的模具中（顶部）用于创建下面的图像中所示的np-互惠型态， 图2b是一个照片进行批量处理样品的瓷舟。 图2c显示所沉积的金属图案的颜色变化前后去合金。银色的完成（之前去合金）由于富银合金含量。膜去合金后，获得一个明显...

Watch this Scientific Journal Video about Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies at JoVE.com

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

我们报告的微细纳米多孔金薄膜，通过模版印刷，光刻法，以及培养细胞的微细加工图案的方法的技术。此外，我们描述的图像分析方法来表征形态的物质和培养的细胞，使用扫描电子显微镜和荧光显微镜技术。

细胞物质相互作用研究微细的纳米多孔金模式

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

microfabrication-nanoporous-gold-patterns-for-cell-material

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

11.3K 次观看.  University of California, Davis. 我们报告的微细纳米多孔金薄膜，通过模版印刷，光刻法，以及培养细胞的微细加工图案的方法的技术。此外，我们描述的图像分析方法来表征形态的物质和培养的细胞，使用扫描电子显微镜和荧光显微镜技术。

视频: Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Studying Cell Rolling Trajectories on Asymmetric Receptor Patterns

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new devices for label-free separation and analysis of cells1. Such devices may use lateral displacement for continuous-flow separation, or receptor patterns that modulate adhesion to distinguish between different cell phenotypes or levels of receptor expression. Understanding the nature of cell rolling trajectories on receptor-patterned substrates is necessary for engineering of the substrates and design of such devices.
Here, we demonstrate a protocol for studying cell rolling trajectories on asymmetric receptor patterns that support cell rolling adhesion2. Well-defined, &mu;m-scale patterns of P-selectin receptors were fabricated using microcontact printing on gold-coated slides that were incorporated in a flow chamber. HL60 cells expressing the PSGL-1 ligand 3were flowed across a field of patterned lines and visualized on an inverted bright field microscope. The cells rolled and tracked along the inclined edges of the patterns, resulting in lateral deflection1. Each cell typically rolled for a certain distance along the pattern edges (defined as the edge tracking length), detached from the edge, and reattached to a downstream pattern. Although this detachment makes it difficult to track the entire trajectory of a cell from entrance to exit in the flow chamber, particle-tracking software was used to analyze and yield the rolling trajectories of the cells during the time when they were moving on a single receptor-patterned line. The trajectories were then examined to obtain distributions of cell rolling velocities and the edge tracking lengths for each cell for different patterns.
This protocol is useful for quantifying cell rolling trajectories on receptor patterns and relating these to engineering parameters such as pattern angle and shear stress. Such data will be useful for design of microfluidic devices for label-free cell separation and analysis.

We describe a protocol to observe and analyze cell rolling trajectories on asymmetric receptor-patterned substrates. The resulting data are useful for engineering of receptor-patterned substrates for label-free cell separation and analysis.

不对称受体模式研究细胞滚动轨迹

Lateral displacement of cells orthogonal to a flow stream by rolling on asymmetric receptor patterns presents an opportunity for development of new ...

科研

生物工程

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

单层的合成，装配和表征蛋白质单分子膜的电化学保护的金纳米粒子薄膜

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the first, synthesis of ultra-small copper nanoclusters is described. The second example provides their utility for investigating time resolved kinetics of chemical reactions by analyzing gold nanoparticle formation using in situ X-ray absorption spectroscopy. The final example demonstrates continuous flow catalysis of reactions inside millifluidic channel coated with nanostructured catalyst.

Millifluidic devices are utilized for controlled synthesis of nanomaterials, time-resolved analysis of reaction mechanisms and continuous flow catalysis.

Millifluidics的化学合成和时间分辨机理研究

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

一种新型三维流室装置研究生理流量条件下的趋化因子的细胞循环外渗

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.

A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination.

电子舌生成连续的识别模式的蛋白质分析

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

三明治般的微环境来驾驭细胞/材料相互作用

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

新生儿心脏支架:再生研究新矩阵

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

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 ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

多路人工细胞微环境阵列的制备

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

在 Vivo 双色 2 光子成像中,基因标记报告器细胞在皮肤中

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...