我们承认金融支持的美国国家科学基金会奖CMMI - 1000724。

1A。硅主要成膜<ol><li>硅片切成1厘米2基板，灰尘和过滤水和乙醇冲洗。 </li><li>取出浸在玻璃含有纳米地带的菜，在75 º C的硅衬底上的有机污染物15分钟后，每个基板与去离子水，过滤水冲洗。 </li><li>每个基板放置在5％HF溶液（警告：HF是一个极其危险的物质）去除原生氧化层。 5分钟后用氮气干燥无氧化硅</li><li>为了产生氯化基板，立即淹没在闪烁小瓶含2毫升饱和PCL 5，在氯苯每个氧化物硅片。这个解决方案应该被过滤到0.2微米。 </li><li>组建一个小瓶每一瓶上冷凝器和heatblock设置为112℃一小时。 </li><li>反应完成后，让小瓶冷却和冲洗每个surfa行政长官氯苯和干燥过滤氮下。 </li><li>要形成一个丙烯终止基板，将每个氯化硅表面压力含有4毫升丙烯基氯化镁的小瓶。 heatblock在每个压力小瓶放置在130 ° C下24小时。 </li><li>取出的heatblock每个压力瓶，放凉。 </li><li>每个表面迅速与DCM和乙醇​​冲洗和干燥过滤氮下。 </li></ol> 1B。锗主要成膜<ol><li>切成1平方厘米基板锗晶圆，灰尘和冲洗过滤水和乙醇。 </li><li>取出，在一个玻璃皿含丙酮20分钟，浸表面的有机污染物</li><li>将每个面在10％盐酸15分钟的解决方案。这个过程同时去除原生氧化层和chlorinates表面。 5分钟后用氮气干燥基板。 </li><li>形成辛基终止基板，解放军CE每个氯化锗表面压力小瓶含4毫升辛基氯化镁（2毫米）。放置在130 ° C下48小时的压力heatblock在每个小瓶。 </li><li>取出的heatblock每个压力瓶，让冷却到室温。 </li><li>每个表面迅速与DCM和乙醇​​冲洗和干燥过滤氮下。 </li></ol> 2。 NHS的硅和锗基板官能<ol><li>准备在四氯化碳中筛选的0.1 M的NHS - diazirine解决方案。警告：将光线照射到最低限度。 </li><li>吸取到解决方案几滴甲基终止的表面。允许解决方案，遍布整个表面。 </li><li>下放置紫外灯（☐= 254纳米，在0.74英寸4400/cm2）的表面。允许的表面，在紫外光下反应30分钟，然后加入NHS - diazirine表面，让一个额外的30分钟进行反应。 </li><li>冲洗NHS修改小号urfaces与DCM和乙醇​​和干燥过滤氮下。 </li></ol> 3。小分子功能化<ol><li>阵营NHS的修改基板在20毫米叔丁基氨基甲酰（叔丁氧羰基）乙二胺在室温两小时的二氯甲烷（DCM）的解决方案。 </li><li>反应结束后，DCM和乙醇​​冲洗中银改性的衬底。 </li><li> Deprotect使用在室温下1小时25％三氟乙酸（TFA）在DCM中银修改基板。 </li><li>冲洗造成表面与DCM，乙醇在水和干燥过滤氮下10％（W / V）碳酸氢钾。 </li><li>所有表面的XPS分析，以确定元素组成。 </li></ol> 4。酸性的聚氨酯丙烯酸酯邮票（PUA）的制备<ol><li>稀释丙烯酸一个由30％的乙氧基化三羟甲基丙烷三丙烯酸酯B以降低粘度。添加光引发剂C和D反应混合物（ 图图6）。 </li><li>钠2 mercaptoethanesulfonate（0.2克，1.22毫摩尔）添加到4N盐酸二氧杂环己烷（10毫升）中的解决方案，并在室温下搅拌2分钟。 </li><li>过滤器通过一个精美的玻璃制品过滤器，然后通过一个0.2μ米PTFE膜注射器过滤负担二恶烷的一个明确的解决办法2 mercaptoethanesulfonic酸氯化钠第一。 </li><li>减压下蒸发的二恶烷</li><li> 2毫升prepolymeric混合物在室温下的聚氨酯 - 丙烯酸酯，然后在真空状态下发生反应产生磺酸在50 ° C。一定要彻底摆脱气泡的混合物。 </li><li>冷却至室温最终的解决方案和聚合之间的两片玻璃显微镜幻灯片或玻片上，暴露在紫外灯在室温下2小时主。 </li><li>聚合后，小心剥离了主的邮票和邮票，用乙醇和水，干燥过滤nitrogeN. </li></ol> 5。催化打印和扫描电镜/原子力显微镜分析<ol><li> NHS的修改基板上放置相应的聚氨酯 - 丙烯酸酯邮票一分钟，在室温下没有外部负载在一起。 </li><li>反应结束后，单独的邮票和基​​板。 </li><li>与乙醇，水，然后用干燥过滤氮和乙醇冲洗基板。 </li><li>与乙醇，水，然后用干燥过滤氮和乙醇冲洗邮票。 </li><li>下一个应用程序前，应保持室温的邮票。 </li><li>分析产生的模式，采用接触模式横向原子力microsopy（AFM）和扫描电子显微镜（SEM） </li></ol> 6。蛋白质的图案和荧光显微镜<ol><li>淹没NHS图案双功能基板赖氨酸N，N -二乙酸（20毫米），并在DMF 等3 N（100毫米）：H20（1:1）在室温下1小时，然后冲洗水和乙醇。 </li><li>底物孵育50毫米硫酸镍在室温下5分钟的解决方案。 </li><li>冲洗过度与水和结合缓冲液（20毫米10毫米，250毫米氯化钠，咪唑，pH值7.5 NAP）和淹没在筛选的绿色荧光蛋白溶液（〜40μM）的螯合基板为1小时0 ° C 。 </li><li>立即漱口结合缓冲液PBS（pH值7.4）的基板。 </li><li>保持在PBS基板水合在0 ° C，直到他们准备好荧光显微镜分析。 </li></ol> 7。蛋白质的图案和荧光显微镜<ol><li>淹没NHS图案双功能基板赖氨酸N，N -二乙酸（20毫米）等3 N（100毫米）在DMF中的H 2 0（1:1）在室温下1小时，然后用清水冲洗和乙醇。 </li><li>底物孵育50毫米NISO 4在室温下5分钟的解决方案。 </li><li>冲洗过度瓦特的螯合基板第i个水和结合缓冲液（20毫米10毫米，250毫米氯化钠，咪唑，pH值7.5 NAP）和淹没在过滤的GFP解决方案（〜40微米）为1小时，在0 ° C。 </li><li>立即漱口结合缓冲液PBS（pH值7.4）的基板。 </li><li>保持在PBS基板水合在0 ° C，直到他们准备好荧光显微镜分析。 </li></ol> 8。代表性的成果： 软平版催化纳米图案的一个例子是在图 7所示。方法创建无氧化硅和锗，可与不同的化学和生物基团功能化正交的化学选择性的模式。 NHS的functioanlized基板和催化图案的邮票之间的反应导致NHS的基团水解形接触的领域，产生一个图案衬底的双功能关系的NHS的地区激活和自由羧酸。由于该diffus离子自由的性质，我们的方法，我们实现光刻分辨率接近。例如， 图7显示了125纳米的特点，整个硅衬底表面均匀转载。值得注意的是，催化邮票可以重复使用多次，而不会失去效率。 图案半导体与生物分子的化学选择性功能化开辟了传统的电子材料相结合的检测，诊断和分析领域的研究，应用高选择性的生物基质的前景。这种功能化的一个例子是在图 8所示，其中NHS图案的硅是有选择性的蛋白质分子官能。通过利用激活和自由羧酸差反应性，我们首先到NHS的官能地区贴终止氮川三乙酸（NTA）的异型连接器，然后用产生的国家旅游局图案作为模板，为六组氨酸标签的绿色荧光蛋白。图8b的选择性附件表面清楚地表明GFP修饰及水解羧酸地区的荧光强度的差别。双方NHS图案的表面（ 图8a）和GFP修饰的表面（ 图8b）的大小和形状的复制功能，确认碳钝化表面的显着的稳定性和冲压方法的选择性。该协议不仅限于他的标签蛋白，并可以使用模式，包括DNA和抗体等生物分子。 <img src="/files/ftp_upload/3478/3478fig1.jpg" alt="图1"/> 图1计划，催化微接触印刷 。 <img src="/files/ftp_upload/3478/3478fig2.jpg" alt="图2"/> 双向分层米结构图2。Ge和Si olecular系统。主烷基单层形式与基板稳定的Ge - C或Si - C键，并提供一个化学惰性和关闭包装系统退化保护下垫面（二）中学重迭层形成稳定的CC键与主保护层，并提供终端功能团体<img src="/files/ftp_upload/3478/3478fig3.jpg" alt="图3"/> 图3。反应计划在Si（A）和GE的主要保护单分子膜的形成（二） <img src="/files/ftp_upload/3478/3478fig4.jpg" alt="图4"/> 图4化学官能主保护单层与异型卡宾捐助。 <img src="/files/ftp_upload/3478/3478fig5.jpg" alt="图5"/> 图5。反应计划展示NHS的官能子的小分子修饰strates和相应的XPS谱<img src="/files/ftp_upload/3478/3478fig6.jpg" alt="图6"/> 图6。组成催化预聚合物的混合物，聚合条件，磺酸修饰图案邮票SEM图像和相应的PMMA硅主<img src="/files/ftp_upload/3478/3478fig7.jpg" alt="图7"/> 图7。SEM和原子力显微镜的硅和锗的图案地空导弹摩擦与酸性邮票的图像<img src="/files/ftp_upload/3478/3478fig8.jpg" alt="图8"/> 图8软平版印刷图案和功能化的有机和生物分子的钝化芯片 ：。SEM照片图案NHS的修改基板B：GFP修饰底物荧光显微镜。

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft-Lithographic+Approach+to+Functionalization+and+Nanopatterning+Oxide-Free+Silicon.">Soft-Lithographic Approach to Functionalization and Nanopatterning Oxide-Free Silicon.</a> Langmuir. 27 (10), 6478-6485 (2011).</li></ol>

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所提出的协议是inkless微接触印刷的形式或可普遍适用于任何能够支持简单有序的单分子膜的基板。在此方法中，邮票固定的催化剂转移到表面附有相应的功能组别的格局。由于这一过程从邮票上的油墨转移不依靠表面的扩散决议的限制，传统和反应μCP，是省却，允许常规制造纳米级物体。注册成立的一所小学的高度有序的分子系统提供从底层的半导体氧化损伤的完整保护。同时，该方法支持固定笨重的活性基团，利用一个二次反应重迭层;在一起，该系统实现了保护与功能。 该技术开始为化学惰性PRIMAR允许形成稳定的碳表面债券Ÿ单层氧化物的形成提供一个有效的屏障。一所中学的反应重迭层的形成提供终端NHS的各种化学和生物基团的附着点的功能组别。使用我们的催化μCP方法，这种稳定的双层分子系统随后图案。在这项研究中提出的方法提供了广泛的有机和生物材料为图案的半导体基板的一般方法。能够创建图案的有机半导体界面，无需昂贵的，复杂的仪器，如电子，纳米技术，生物化学和生物物理学等领域的众多机会。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司/型号</th></tr><tr><td> XPS光谱仪</td><td>奎托斯轴超</td></tr><tr><td>原子力显微镜</td><td> Veeco的D3100 </td></tr><tr><td> SEM - FEG显微镜</td><td>费XL30 </td></tr><tr><td>荧光显微镜</td><td>蔡司Axio上成像仪</td></tr><tr><td> Heatblock </td><td> VWR </td></tr><tr><td>真空泵</td><td> BOC爱德华</td></tr><tr><td>水净化系统</td><td> Millipore公司</td></tr><tr><td> TESP硅探针</td><td> Veeco公司</td></tr><tr><td>硅</td><td></td></tr><tr><td>压力样品瓶</td><td> Chemglass </td></tr><tr><td>真空歧管</td><td> Chemglass </td></tr><tr><td>紫外灯</td><td> UVP </td></tr><tr><td>邮票材料</td><td>见参考文献20和18 </td></tr><tr><td> PFTE注射器过滤器</td><td> VWR </td></tr><tr><td>纳米地带</td><td> Cyantek </td></tr><tr><td>盐酸</td><td>西格玛</td></tr><tr><td>乙醇</td><td>西格玛</td></tr><tr><td>丙酮</td><td>西格玛</td></tr><tr><td>高频</td><td>西格玛</td></tr><tr><td>氯苯</td><td>西格玛</td></tr><tr><td> PCL5 </td><td>西格玛</td></tr><tr><td>氯化烯丙基镁</td><td>西格玛</td></tr><tr><td>氯化辛基镁</td><td>西格玛</td></tr><tr><td>四氯化碳</td><td>西格玛</td></tr><tr><td>中银保护乙二胺</td><td>西格玛</td></tr><tr><td>反式脂肪酸</td><td>西格玛</td></tr><tr><td>钠2 - mercaptoethanesulfonate </td><td>西格玛</td></tr><tr><td> 4N盐酸溶液中二恶烷</td><td>西格玛</td></tr><tr><td>赖氨酸N，N -二乙酸</td><td>西格玛</td></tr><tr><td>等3 N </td><td>西格玛</td></tr><tr><td>二甲基甲酰胺</td><td>西格玛</td></tr><tr><td> NISO 4 </td><td>西格玛</td></tr><tr><td> NAP </td><td>西格玛</td></tr><tr><td>氯化钠</td><td>西格玛</td></tr><tr><td>咪唑</td><td>西格玛</td></tr><tr><td> PBS </TD&gt; <td>西格玛</td></tr></tbody></table>

soft lithographic functionalization and patterning oxide-free silicon and germanium

混合型电子设备的发展在很大程度上依赖（生物）通过一个稳定的接口，允许高效率的电子传输和保护底层基板氧化降解的有机材料和无机半导体的集成。作为有机和水溶液的防渗障碍法简单烷基链组成的高度有序的自组装单层膜（SAMS），第四组的半导体可以得到有效的保护。然而，简单的烷基地空导弹惰性和不服从传统图案的技术。固定有机半导体分子系统的动机是传授新的功能，可以提供的光学，电子，和机械的功能，以及化学和生物活性的表面。 微接触印刷（μCP）是无数表面上的图案地空导弹的软光刻技术。尽管其简体1-9licity和多功能性，该方法已在很大程度上限制以高尚的金属表面，并没有得到很好的开发模式转移到技术上重要的基板，如无氧化硅和锗。此外，由于这项技术依赖油墨扩散传输模式从弹性基板上，这种传统印刷的分辨率本质上是有限的近1μ 米 10-16 在传统印刷相比，inklessμCP的图案依赖于一个特定的反应之间的表面固定基板和邮票结合的催化剂。由于该技术不依赖于扩散SAM的形成，它显著扩大patternable表面的多样性。此外，inkless技术省却了分子扩散的特征尺寸限制，促进复制非常小（&lt;200 nm的） 的功能。17-23然而，直至现在 ， inklessμ彗星P已经被主要用于图案相对无序的分子系统，它不保护退化下垫面。 在这里，我们报告一个简单，可靠的钝化与活性有机单层硅和锗的图案的高通量的方法和表现与小分子和蛋白质的图案基板选择性官能。该技术利用一个预制NHS的反应无氧化硅和锗的双层系统。 NHS的基团水解，在一个特定的方式，与一个磺酸改性丙烯酸酯邮票NHS活化和自由羧酸的化学性质不同的模式的模式。一个显著的限制，以解决许多 μCP技术是硅橡胶材料的使用缺乏高保真传输所需的机械强度。为了缓解这一限制，我们使用的聚氨酯丙烯酸酯聚合物，相对刚性的材料，可轻松地与不同的有机基团功能化。我们的图案的做法完全保护化学氧化硅和锗，在形状和大小的图案特征提供精确的控制，并随时获得化学歧视可以进一步与有机和生物分子功能化的模式。该方法是一般和适用于其他技术相关的表面。

Watch this Scientific Journal Video about Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium at JoVE.com

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

在这里，我们描述了一个简单的方法，图案无氧化硅和锗活性有机单层和展示与小分子和蛋白质的图案基板官能。这种方法完全保护表面化学氧化，提供以上功能形态的精确控制，并提供随时获得化学歧视模式。

软平版功能化和图案无氧化硅和锗

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

soft-lithographic-functionalization-patterning-oxide-free-silicon

Research

JoVE Journal

Bioengineering

14.6K 次观看.  Duke University. 在这里，我们描述了一个简单的方法，图案无氧化硅和锗活性有机单层和展示与小分子和蛋白质的图案基板官能。这种方法完全保护表面化学氧化，提供以上功能形态的精确控制，并提供随时获得化学歧视模式。

视频: Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

等离子体表面图案光刻技术的蜂窝网络创作

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

科研

生物工程

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

碳水化合物和聚酸酐纳米粒子功能化的高通量合成

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

皮层组织和动态可视化的微生物，利用全内反射荧光显微镜

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

图案蛋白质和细胞的通用的方法

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

Soft Lithographic Procedure for Producing Plastic Microfluidic Devices with View-ports Transparent to Visible and Infrared Light

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable microfluidic devices. Here, a protocol for the fabrication of plastic microfluidic devices is demonstrated, where soft lithographic techniques are used to embed transparent Calcium Fluoride (CaF2) view-ports in connection with observation chamber(s). The method is based on a replica casting approach, where a polydimethylsiloxane (PDMS) mold is produced through standard lithographic procedures and then used as the template to produce a plastic device. The plastic device features ultraviolet/visible/infrared (UV/Vis/IR) -transparent windows made of CaF2 to allow for direct observation with visible and IR light. The advantages of the proposed method include: a reduced need for accessing a clean room micro-fabrication facility, multiple view-ports, an easy and versatile connection to an external pumping system through the plastic body, flexibility of the design, e.g., open/closed channels configuration, and the possibility to add sophisticated features such as nanoporous membranes.

A protocol for the fabrication of plastic microfluidic devices with transparent view-ports for visible and infrared light imaging is described.

生产塑料微流控装置与查看端口对可见光和红外透明软光刻过程

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable ...

Designing Porous Silicon Films as Carriers of Nerve Growth Factor

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Here, we present a protocol to design and fabricate nanostructured porous silicon (PSi) films as degradable carriers for the nerve growth factor (NGF). Neuronal differentiation and outgrowth of PC12 cells and mice dorsal root ganglion (DRG) neurons are characterized upon treatment with the NGF-loaded PSi carriers.

多孔硅薄膜作为神经生长因子载体的设计

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the ...

Bioinspired Soft Robot with Incorporated Microelectrodes

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics paradigm, especially in biomedical research. Recreating artificial life-like actuation dynamics is crucial for a soft-robotic system. However, the precise control and tuning of actuation behavior still represents one of the main challenges of modern soft robotic systems. This method describes a low-cost, highly scalable, and easy-to-use procedure to fabricate an electrically controllable soft robot with life-like movements that is activated and controlled by the contraction of cardiac muscle tissue on a micropatterned sting ray-like hydrogel scaffold. The use of soft photolithography methods makes it possible to successfully integrate multiple components in the soft robotic system, including micropatterned hydrogel-based scaffolds with carbon nanotubes (CNTs) embedded gelatin methacryloyl (CNT-GelMA), poly(ethylene glycol) diacrylate (PEGDA), flexible gold (Au) microelectrodes, and cardiac muscle tissue. In particular, the hydrogels alignment and micropattern are designed to mimic the muscle and cartilage structure of the sting ray. The electrically conductive CNT-GelMA hydrogel acts as a cell scaffold that improves the maturation and contraction behavior of cardiomyocytes, while the mechanically robust PEGDA hydrogel provides structural cartilage-like support to the whole soft robot. To overcome the hard and brittle nature of metal-based microelectrodes, we designed a serpentine pattern that has high flexibility and can avoid hampering the beating dynamics of cardiomyocytes. The incorporated flexible Au microelectrodes provide electrical stimulation across the soft robot, making it easier to control the contraction behavior of cardiac tissue.

A bioinspired scaffold is fabricated by a soft photolithography technique using mechanically robust and electrically conductive hydrogels. The micropatterned hydrogels provide directional cardiomyocyte cell alignment, resulting in a tailored direction of actuation. Flexible microelectrodes are also integrated into the scaffold to bring electrical controllability for a self-actuating cardiac tissue.

生物灵感软机器人与合并微电极

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics ...

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.

硅纳米线与光学刺激研究细胞内和细胞间电耦合

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These ...

Simple, Affordable, and Modular Patterning of Cells using DNA

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells of the same or different type, micropatterning techniques have proved useful. DNA Programmed Assembly of Cells (DPAC) is a micropatterning technique that targets the adhesion of cells to a substrate or other cells using DNA hybridization. The most basic operations in DPAC begin with decorating cell membranes with lipid-modified oligonucleotides, then flowing them over a substrate that has been patterned with complementary DNA sequences. Cells adhere selectively to the substrate only where they find a complementary DNA sequence. Non-adherent cells are washed away, revealing a pattern of adherent cells. Additional operations include further rounds of cell-substrate or cell-cell adhesion, as well as transferring the patterns formed by DPAC to an embedding hydrogel for long-term culture. Previously, methods for patterning oligonucleotides on surfaces and decorating cells with DNA sequences required specialized equipment and custom DNA synthesis, respectively. We report an updated version of the protocol, utilizing an inexpensive benchtop photolithography setup and commercially available cholesterol modified oligonucleotides (CMOs) deployed using a modular format. CMO-labeled cells adhere with high efficiency to DNA-patterned substrates. This approach can be used to pattern multiple cell types at once with high precision and to create arrays of microtissues embedded within an extracellular matrix. Advantages of this method include its high resolution, ability to embed cells into a three-dimensional microenvironment without disrupting the micropattern, and flexibility in patterning any cell type.

Here we present a protocol to micropattern cells at single-cell resolution using DNA-programmed adhesion. This protocol uses a benchtop photolithography platform to create patterns of DNA oligonucleotides on a glass slide and then labels cell membranes with commercially available complementary oligonucleotides. Hybridization of the oligos results in programmed cell adhesion.

使用DNA对细胞进行简单、经济、模块化的图案化

The relative positioning of cells is a key feature of the microenvironment that organizes cell-cell interactions. To study the interactions between cells ...