本工作由瑞士国家科学基金会（ - 智能材料NRP 62）的支持。也承认是B HANSELMANN，K.凯尔，U.茨和B Leuthold的支持。

1。等离子体引发聚合<ol><li>准备的单体溶液。 <ol><li>在200毫升水中，并用己烷（100毫升）的分液漏斗中洗涤3次，溶解HEMA（0.718摩尔100ml）中。饱和水相用氯化钠和提取的HEMA，用乙醚（50毫升）中。干燥有机相用MgSO 4和除去溶剂在真空中 （100毫巴，40℃）。蒸馏减压HEMA（15毫巴; 99​​℃）。 </li><li>准备在第1.1.1节中产生的无抑制剂的HEMA的0.62 2M甲醇溶液。倾30毫升溶液的成1颈烧瓶中，并通过1小时的溶液中鼓入氩气排除氧。 </li></ol></li><li>表面引发聚合。 <ol><li>位置2的聚碳酸酯膜彼此相邻到等离子体腔室（ 图2）。将膜朝向气相的光泽面。 </li><li>等离子腔连接到啊IGH真空（20毫巴），持续5分钟。关闭阀至真空，并打开另一个阀，其连接到氩气和氧气的气体和2小时15 sccm的氩气和2.5 sccm的氧气吹扫腔室与该混合物。 </li><li>启动等离子体，并减少功率到所需的功率（用于聚碳酸酯膜:12瓦）和治疗的膜为4分钟，等离子体。通过打开相应的阀连接的单体溶液与腔室。关闭血浆和疏散室。 </li><li>通过打开相应的阀连接的单体溶液与所述腔室和将溶液倾入腔室。确保该膜覆盖与单体溶液。打开连接在氩气阀门和12小时存储该反应混合物在20°C（空调房间）。 </li><li>除去单体溶液。用甲醇在超声浴中5分钟，洗膜。用水重复洗涤过程。 </li><li>干燥该膜在真空下在分子筛上2小时。 </li></ol></li></ol> 2。涂层膜的修改后的<ol><li>准备spirobenzopyran 1的溶液（ 图1）（100毫克，0.27毫摩尔），在叔 butylmetylether和二甲基氨基吡啶（DMAP）（33毫克，0.27毫摩尔），N，N-二环己基碳（DCC）（0.27毫摩尔55毫克） （TBME）（12毫升）中。 </li><li>放置一保护搅拌棒和一个保护网格成一个圆底烧瓶中。干燥瓶和洪水量瓶中，用氩气。 </li><li>将溶液倒入烧瓶，随后涂覆的隔膜。 </li><li>轻轻搅拌在室温下搅拌12小时。 </li><li>除去溶液，并用叔 butylmetylether洗膜在超声浴中5分钟。用乙醇和水重复洗涤过程。 </li><li>干燥该膜在真空中在分子筛上2小时。 </li></ol> 3。表面张力测试<ol><l我&gt;修复膜与金属O型圈标准的磁带。定位超纯水（3.3微升）的在膜表面上，这是不与O形圈接触的那一部分的下降。测量接触角（CA）在膜的5个不同的斑点。 </li><li>用于测试样品的长期稳定性，测量接触角在三个不同的膜的斑点0后，1，2，3，7，14，和21天。 </li></ol> 4。光致变色膜的透气性测试<ol><li>装满水的Franz扩散池（12毫升）的受体室。 </li><li>修复膜在Franz扩散池。确保该膜是在与受体腔室中的水接触。用含水咖啡因溶液（;3.0毫升20毫摩尔）填充供料腔室（在膜的顶部的腔室）。从供体室用白色光（ 图3）的顶部照射该膜。从受体细胞收集样品（200微升）; FO用200nm的细孔孔径r轨道边的聚碳酸酯膜，收集样品每10分钟。 </li><li>如重复步骤4.2所描述的实验。但照射整个渗透性试验过程中，用紫外灯（366纳米，80瓦/米2）的膜。 </li><li>测定采集的样本的咖啡因浓度。 <ol><li>绘制校准曲线与使用的UV / Vis光谱仪15个不同的咖啡因浓度（0.05毫克/毫升和1.5mM / L）。校准在293 nm处。 </li><li>确定每个使用校正曲线所收集的样品的浓度。 </li><li>画出所确定的浓度比 。所收集的样本的时间。做线性拟合，通过点，并确定ΔC从斜坡。 </li></ol></li></ol>

<ol>
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作者什么都没有透露。

等离子体工艺产生紫色气体，该气体是由离子化的氩气。橙色则表示不希望的氮气从泄漏的存在。等离子体处理不只是在表面上形成自由基而且蚀刻膜7,12。太多的蚀刻可以显著改变孔径，这将影响到膜的渗透性。所提出的设置的受控反应条件允许提高等离子体引发的接枝方法的再现性。然而，在等离子体腔室中的膜的确切位置仍然可以在表面上形成自由基的密度影响由于等离子体的不均匀性。边沿速率也依赖于所施加的功率和上确切的气体组合物。 这种薄涂层的表征是不平凡的，由于商用膜的同等粗糙的表面。如12,21之前所描述的，层厚度使用椭偏仪和XPS实验确定。为了分析一个平坦的表面，聚碳酸酯，通过旋涂在Si-晶圆模型聚碳酸酯表面。这些聚碳酸酯涂层则视为所述的方法在聚碳酸酯膜。另外，多光子显微镜研究揭示是一种非常宝贵的测量技术来评估，如果膜的唯一的外表面涂覆或如果涂层中的孔隙发生，以及21。 由于无规聚合用官能团的相容性高，各种各样的丙烯酸酯可以用作单体。这允许使用的单体具有的官能团。在本示例中，醇基可酯化的羧酸基团。在接枝过程的限制是，可以使用溶剂。由于聚碳酸酯膜溶解于多种有机溶剂，如乙酸乙酯，四氢呋喃，氯化物roform，或丙酮，应避免在聚合以及后来的修改后的过程中，这些溶剂。适合的溶剂是水，醇如甲醇，乙醇，丙醇，脂族和芳族溶剂，如己烷，二甲苯，和一些醚。单体溶液中的浓度不显著改变涂层的厚度。因此，不适合于厚涂层的形成这一过程。然而，薄的涂层允许使用脆和硬质聚合物（ 例如 ，聚甲基丙烯酸甲酯）作为涂覆材料，而不影响散装柔性膜的柔韧性。如前所示，该聚合物也可包含不同的单体形成共聚物17。 由于聚碳酸酯膜稍有溶胀在乙醚中，加入TBME是用在本例中为修改后的程序。用TBME作为溶剂和DCC作为偶联剂用于e变更后发生在室温下sterification与spirobenzopyran化合物1 17的羧酸基团的醇。自TBME作为非极性溶剂中不润湿孔壁，只外膜表面官能化spirobenzopyran。的修改后的方法也可用于改变表面的表面张力，或使其他功能到表面12。演示的示例修改了膜进入光敏感膜。响应于其他刺激的pH，温度，化学物质或电是supposable。 与证明方法中，光响应性膜与一个关于咖啡因的渗透率显着的反应来制备。有趣的是，当spirobenzopyran单元共聚在一个步骤中的HEMA，响应要低得多17。由于涂层是比孔径（即使当在水中溶胀）的变化非常薄孔径可以被排除作为原因，在磁导率的变化。无论如何，由于极性较大的部花青将溶胀的接枝聚合物更好在水中比在它的极性较小的螺吡喃状态下，扭转光开关随后将被预期。究其原因，在磁导率的变化是在表面张力的变化，它定义了含水体系的渗透速率如前所示12。 这种刺激响应膜可以在切换的药物输送系统或智能传感器系统中的应用。这样的智能药物递送系统可用于防止早产新生儿21的呼吸暂停。其他方面，在其中光响应性膜可使用的生物技术，微流体或用于光动力分子梭22。

材料的等离子体改性已成为许多工业领域一个重要的过程。表面和表面官能化，而 ​​不改变该材料的整体性质的清洗已在等离子体处理在表面科学1-8必不可少的过程。 聚合物的等离子处理结果在均裂键的断裂。这导致了聚合物材料的一个边和自由基丰富的表面的形成。通过采用等离子体含有氧分子，表面变得富氧从而更亲水9-11。然而，表面的亲水性并不稳定随着时间的推移12。为了提高长期稳定性，等离子体处理的表面可进行化学后或在等离子体处理13-15修改。通常通过添加反应性单体物种进入气相中的等离子体处理进行该处理;这些单体聚合，然后从聚合物表面产生自由基。如果与一个非易失性单体进行化学处理，该聚合物接枝有等离子体改性后发生。为了执行控制接枝基团在表面上形成后，等离子体的设置被描述，其允许从所述表面在溶液中在控制条件下12,16等离子体引发的表面诱导聚合。 这次报告的重点是轨道边聚合物膜12,17的修改。通过修改这些膜的表面张力，透气率可变化12。这种清洁和快速的过程可以创建非常薄的层（&lt;5 nm）的，涵盖整个膜表面不改变聚合物膜的大部分财产。由于在等离子体处理磨边，轨迹一柄膜的孔径增大略12。磨边率dependi纳克的聚合物上，并具有线性特性。 当使用具有反应性官能团的单体，接枝聚合物可进一步官能化。这表现在一个PHEMA接枝膜用羧酸功能化的螺吡喃的修改后的。这导致光致变色表面，由于螺吡喃已知变换成部花青类时用UV光照射。螺吡喃形式可以通过照射部花青形式与可见光（ 图1）18,19重新建立。由于花青形式比螺吡喃状态更极性的，该涂层的表面张力可与光20被触发。在表面张​​力的变化会影响对水溶液的膜的渗透性阻力。的设立如何执行这些光响应膜的渗透性测试将显示在阻透性的显著变化（减少我97％不阻透性）是证明。这样的膜可以被集成在一个药物递送的设置或在智能传感系统。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51680/51680fig1highres.jpg" src="/files/ftp_upload/51680/51680fig1.jpg" /> 图1。光异构化spirobenzopyran化合物1。

<table><tbody><tr><td>2-Hydroxyethyl methacrylate, 97%</td><td>Sigma-Aldrich</td><td>128635</td><td></td></tr><tr><td>Hexane 99%</td><td>Biosolve</td><td></td><td></td></tr><tr><td>Magnesium sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;, anhydrous)</td><td>Sigma-Aldrich</td><td>M7506</td><td></td></tr><tr><td>Methanol, 99%&amp;nbsp;</td><td>Sigma-Aldrich</td><td>14262</td><td>dried over molecular sieves</td></tr><tr><td>N,N-Dicylcohexylcarbodiimide, 99%</td><td>Sigma-Aldrich</td><td>D8002</td><td></td></tr><tr><td>Dimethyl aminopyridine, 99%</td><td>Sigma-Aldrich</td><td>107700</td><td></td></tr><tr><td>Tert-butylmethylether, 98%</td><td>Fluka</td><td>306975</td><td></td></tr><tr><td>Polycarbonate membrane</td><td>Whatman</td><td></td><td>Nanopore Track Etched (TE) (1.0 &amp;mu;m, 0.2 &amp;mu;m, 0.1 &amp;mu;m, 50 nm, 30 nm, and 15 nm pore diameter; 47 mm or 25 mm membrane diameter)</td></tr><tr><td>Caffeine (reagent plus)</td><td>Sigma-Aldrich</td><td>C0750</td><td></td></tr><tr><td>Franz diffusion cell (12 ml)</td><td>SES-Analysesysteme</td><td>6C010015</td><td>15 mm unjacheted Franz Cell, 12 ml Receptor volume, Flat ground, clear glass, stirbar and clamp</td></tr><tr><td>UV-Lamp</td><td></td><td></td><td>UV irradiation (366 nm, 15 W/m&lt;sup&gt;2&lt;/sup&gt;)</td></tr><tr><td>White light lamp</td><td></td><td></td><td>White light irradiation (500 W bulb)</td></tr><tr><td>UV/Vis spectrophotometer</td><td>Varian 50Bio/50MPR</td><td></td><td></td></tr><tr><td>Polyester membranes</td><td>Sterlitech</td><td>PET0225100</td><td>Polyester Membrane Filters, 0.2 &amp;mu;m pore diameter, 25 mm diameter</td></tr><tr><td>Polyvinylidene fluoride membranes</td><td>Millipore</td><td></td><td>PVDF Membranes Durapore (0.22 &amp;mu;m pore diameter; 47 mm membrane diameter)</td></tr><tr><td>Argon (99.9995%)</td><td>Alphagaz</td><td></td><td></td></tr><tr><td>Dressler Cesar RF Power Generator</td><td></td><td></td><td>Plasma chamber setup</td></tr><tr><td>MKS Multi Gas Controller 647C</td><td></td><td></td><td>Plasma chamber setup</td></tr><tr><td>MKS Mass-Flow controllers</td><td></td><td></td><td>Plasma chamber setup</td></tr><tr><td>Vacuubrand RE 2.5 rotary vane vacuum pump</td><td></td><td></td><td>Plasma chamber setup</td></tr><tr><td>Contact angle measurement device</td><td>Kr&amp;uuml;ss</td><td>G10</td><td></td></tr><tr><td>Balances</td><td>Mettler Toledo</td><td>AB204-S and Mettler ME30</td><td></td></tr></tbody></table>

preparation of light-responsive membranes by a combined surface grafting and postmodification process

为了修改商业可用的轨道边缘的聚合物膜的表面张力，表面引发聚合反应的过程被呈现。从膜表面的聚合反应是由等离子体处理的膜中，接着在膜表面具有2 - 羟乙酯（HEMA）的甲醇溶液反应诱导。特别注意的是考虑到工艺参数对等离子体处理的表面上的聚合反应之前。例如，等离子体处理对不同类型的膜（ 例如聚酯，聚碳酸酯，聚偏二氟乙烯）的影响进行了研究。另外，表面接枝膜的依赖于时间的稳定性示出由接触角测量。当接枝聚（2 - 羟乙基甲基丙烯酸酯）（PHEMA），以这种方式，表面可进一步通过与所需物质的羧酸功能的聚合物的醇部分酯化改性。这些反应，因此可用于对膜表面的官能化。例如，在膜的表面张力可以改变或可以被插入期望的功能所呈现的光响应性。这表现在与这导致了光响应性膜的羧酸官能spirobenzopyran单元反应PHEMA。溶剂的选择起着在修改后的工序的主要作用，并更详细地在本文中进行了讨论。这样的官能化膜的渗透性测量使用Franz池与外部光源进行的。通过改变光的波长从可见光到紫外光范围内，水的咖啡因溶液的渗透性的变化被观察到。

刻蚀速率可以跟随不同的时间段后称量该膜。如可以从图4中可以看出，蚀刻速率如下聚酯，聚偏二氟乙烯，和聚碳酸酯膜的线性刻蚀速率，这可从蚀刻时间与重量损失的线性相关的斜率来确定。 如图4所示 ，聚碳酸酯膜显示所有三个聚合物膜的最低蚀刻速率。该蚀刻的结果之一是在孔径的变化。孔的后等离子体处理增加了约20％12,17的直径。随后的接枝聚合物具有在另一方面上的孔的直径，这是由于为1-4纳米12的非常薄的聚合物层没有显著影响。最重要的是，整个过程中不影响膜的孔结构。 在整个包衣过程可以很容易地跟随测量期间，球员接触角。原来的聚碳酸酯膜具有约60°的低接触角，这是由于市售的聚碳酸酯膜的聚乙烯吡咯烷酮（PVP）的涂层。在等离子体处理期间所述切边破坏PVP涂层和接枝聚合物之前所得到的接触角变得更加亲水（25°），由于该含氧等离子体。不稳定的表面变得越来越疏水性随时间（80°后21天）12。随后PHEMA接枝导致涂层具有大约90°的接触角，也取决于膜的孔径大小。在图5中未涂覆的膜和PHEMA接枝膜之间的接触角的差（为0.2微米和1微米孔径）所示， 图6示出了一个PHEMA涂布聚碳酸酯膜相对于时间的另外的接触角。它是清晰可见的，所述接触角不随着时间的推移，这是一个表示为一个长期稳定的涂料不会改变。与化合物1的修改后的增加，接触角为100°。然而，spirobenzopyran可以转移到更极性花青种通过用UV光照射（ 图1），并且这种转化再次降低膜表面的接触角为90°。 该膜的渗透性是使用Franz扩散池（ 图3）进行测定。将样品从受体室中取来确定膜的渗透性阻力。该spirobenzopyran改性膜的膜通透性在UV光照射下的白色光照射的影响。如可以从图7中可以看出，磁导率变化的电阻由97％时，膜被照射的白色光而减小。这表明光responsi的存在已经膜。 另外，也可以附加额外的光源，以在Franz扩散池（ 图3）。在该装置中光纤束连接到一个白光和UV光（360 nm）的源，它允许从一个波长更快的切换到另一个。因为光纤在辐照过程中保持温度，不增温度由或白光照明或通过UV光照射观察。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51680/51680fig2highres.jpg" src="/files/ftp_upload/51680/51680fig2.jpg" /> 图2 T 他等离子体室与室内的两个定位膜和两个阀的真空和气体混合物分别。 <img alt="图3" fo:con帐篷宽度="5英寸"FO:SRC ="/ files/ftp_upload/51680/51680fig3highres.jpg"SRC ="/ files/ftp_upload/51680/51680fig3.jpg"/&gt; 图3。 。与受体室（底部）和供料腔室（顶部）之间的固定膜的Franz扩散池 ，光源固定在Franz扩散池的顶部（在这里UV光）。 <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/51680/51680fig4highres.jpg" src="/files/ftp_upload/51680/51680fig4.jpg" /> 图4。刻蚀速率在​​10膜组成不同的聚合物的W上。 <img alt="图5" fo:content-width="5in" fo:src="/files/ftp_upload/51680/51680fig5highres.jpg" src="/files/ftp_upload/51680/51680fig5.jpg" /> 图5。水滴的接触角的变化时，多孔聚碳酸酯膜（RO上W:0.2微米孔径，下排:1微米孔径）是通过等离子体诱导聚合（左侧涂PHEMA:聚合前，右侧:聚合后）。 <img alt="图6" fo:content-width="5in" fo:src="/files/ftp_upload/51680/51680fig6highres.jpg" src="/files/ftp_upload/51680/51680fig6.jpg" /> 图6。 PHEMA的接触角测定接枝膜示出了涂层的长期稳定性。 <img alt="图7" fo:content-width="5in" fo:src="/files/ftp_upload/51680/51680fig7highres.jpg" src="/files/ftp_upload/51680/51680fig7.jpg" /> 通过白色光照射薄膜，并通过UV光水性咖啡因溶液（20mM）图7。渗透性测量照射膜。

Watch this Scientific Journal Video about Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process at JoVE.com

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

一种等离子体诱导聚合反应过程被描述为在聚合物膜的表面引发聚合反应。与光致变色物质的接枝聚合物的进一步修改后的呈现与开展的光响应膜渗透性测量的协议。

通过结合表面接枝和修改后的过程光敏感膜的制备

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

preparation-light-responsive-membranes-combined-surface-grafting

Nanyang Technological University

Research

JoVE Journal

Bioengineering

11.7K 次观看.  Empa, Swiss Federal Laboratories for Materials Science and Technology. 一种等离子体诱导聚合反应过程被描述为在聚合物膜的表面引发聚合反应。与光致变色物质的接枝聚合物的进一步修改后的呈现与开展的光响应膜渗透性测量的协议。 

视频: Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

Microstereolit​​hography和静电结合，以产生膜配备有利基的角膜再生

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

科研

生物工程

Fabricating Reactive Surfaces with Brush-like and Crosslinked Films of Azlactone-Functionalized Block Co-Polymers

In this paper, fabrication methods that generate novel surfaces using the azlactone-based block co-polymer, poly (glycidyl methacrylate)-block-poly (vinyl dimethyl azlactone) (PGMA-b-PVDMA), are presented. Due to the high reactivity of azlactone groups towards amine, thiol, and hydroxyl groups, PGMA-b-PVDMA surfaces can be modified with secondary molecules to create chemically or biologically functionalized interfaces for a variety of applications. Previous reports of patterned PGMA-b-PVDMA interfaces have used traditional top-down patterning techniques that generate non-uniform films and poorly controlled background chemistries. Here, we describe customized patterning techniques that enable precise deposition of highly uniform PGMA-b-PVDMA films in backgrounds that are chemically inert or that have biomolecule-repellent properties. Importantly, these methods are designed to deposit PGMA-b-PVDMA films in a manner that completely preserves azlactone functionality through each processing step. Patterned films show well-controlled thicknesses that correspond to polymer brushes (~90 nm) or to highly crosslinked structures (~1-10 &#956;m). Brush patterns are generated using either the parylene lift-off or interface directed assembly methods described and are useful for precise modulation of overall chemical surface reactivity by adjusting either the PGMA-b-PVDMA pattern density or the length of the VDMA block. In contrast, the thick, crosslinked PGMA-b-PVDMA patterns are obtained using a customized micro-contact printing technique and offer the benefit of higher loading or capture of secondary material due to higher surface area to volume ratios. Detailed experimental steps, critical film characterizations, and trouble-shooting guides for each fabrication method are discussed.

Surface fabrication methods for patterned deposition of nanometer thick brushes or micron thick, crosslinked films of an azlactone block co-polymer are reported. Critical experimental steps, representative results, and limitations of each method are discussed. These methods are useful for creating functional interfaces with tailored physical features and tunable surface reactivity.

Azlactone 功能块共聚物的刷状和交联膜制备反应表面

In this paper, fabrication methods that generate novel surfaces using the azlactone-based block co-polymer, poly (glycidyl methacrylate)-block-poly (vinyl ...

工程学

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe a novel protocol employing the TiO2 photocatalysis to locally alter cell affinity of the substrate surface. For this experiment, a thin TiO2 film was sputter-coated on a glass coverslip, and the TiO2 surface was subsequently modified with an organosilane monolayer derived from octadecyltrichlorosilane (OTS), which inhibits cell adhesion. The sample was immersed in a cell culture medium, and focused UV light was irradiated to an octagonal region. When a neuronal cell line PC12 cells were plated on the sample, cells adhered only on the UV-irradiated area. We further show that this surface modification can also be performed in situ, i.e., even when cells are growing on the substrate. Proper modification of the surface required an extracellular matrix protein collagen to be present in the medium at the time of UV irradiation. The technique presented here can potentially be employed in patterning multiple cell types for constructing coculture systems or to arbitrarily manipulate cells under culture.

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

光图案蛋白质和细胞中的水环境中使用二氧化钛<sub&gt; 2</sub&gt;光催化

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

生物膜制作的溶剂辅助脂质双分子层（SALB）方法

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

温敏纳米结构表面的组织工程准备

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

自动化脂双层膜的形成使用聚二甲基硅氧烷薄膜

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

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 ...

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

在膜上重构隔膜组件以研究生物物理性质和功能

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component ...

生物化学

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

聚合物拥挤脂质膜上的单分子扩散和组装

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs)

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma membrane and the numerous factors that influence protein behavior in live cells. Giant unilamellar vesicles (GUVs) are a biomimetic and highly tunable in vitro model system for investigating protein-membrane interactions and probing protein behavior in a precise, stimulus-dependent manner. In this protocol, we present an inexpensive and effective method for fabricating GUVs with the human serotonin 1A receptor (5-HT1AR) stably integrated in the membrane. We fabricate GUVs using a modified hydrogel swelling method; by depositing a lipid film on top of a mixture of agarose and 5-HT1AR and then hydrating the entire system, vesicles can be formed with properly oriented and functional 5-HT1AR incorporated into the membrane. These GUVs can then be used to examine protein-membrane interactions and localization behavior via microscopy. Ultimately, this protocol can advance our understanding of the functionality of integral membrane proteins, providing profound physiological insight.

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors GPCRs

This protocol utilizes agarose swelling as a powerful and generalizable technique for incorporating integral membrane proteins (IMPs) into giant unilamellar lipid vesicles (GUVs), as described here for the reconstitution of the human 1A serotonin receptor protein (5-HT1AR), one of the classes of pharmacologically important G protein-coupled receptors.

构建含有G蛋白偶联受体（GPCR）的脂质膜模型

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma ...

通过结合表面接枝和修改后的过程光敏感膜的制备

摘要

探索更多视频

此视频中的章节

Microstereolithography和静电结合，以产生膜配备有利基的角膜再生

Azlactone 功能块共聚物的刷状和交联膜制备反应表面

光图案蛋白质和细胞中的水环境中使用二氧化钛

生物膜制作的溶剂辅助脂质双分子层（SALB）方法

温敏纳米结构表面的组织工程准备

自动化脂双层膜的形成使用聚二甲基硅氧烷薄膜

用于细胞培养应用光介导的形成和构图的水凝胶

在膜上重构隔膜组件以研究生物物理性质和功能

聚合物拥挤脂质膜上的单分子扩散和组装

构建含有G蛋白偶联受体（GPCR）的脂质膜模型

通过结合表面接枝和修改后的过程光敏感膜的制备

摘要

探索更多视频

此视频中的章节

Microstereolit​​hography和静电结合，以产生膜配备有利基的角膜再生

Azlactone 功能块共聚物的刷状和交联膜制备反应表面

光图案蛋白质和细胞中的水环境中使用二氧化钛

生物膜制作的溶剂辅助脂质双分子层（SALB）方法

温敏纳米结构表面的组织工程准备

自动化脂双层膜的形成使用聚二甲基硅氧烷薄膜

用于细胞培养应用光介导的形成和构图的水凝胶

在膜上重构隔膜组件以研究生物物理性质和功能

聚合物拥挤脂质膜上的单分子扩散和组装

构建含有G蛋白偶联受体（GPCR）的脂质膜模型

Microstereolithography和静电结合，以产生膜配备有利基的角膜再生