这项工作由中国国家自然科学基金 (21674018)、中国国家重点研究开发项目 (2016YFA0201702/2016YFA0201700) 和上海教育发展支持的 "曙光计划" 赞助。基金会和上海市教育委员会 (15SG30), 东华大学杰出青年教授项目 (A201302), 为中央大学的基础研究基金, 和111项目 (no. 111-2-04)。

警告: 请在使用前查阅所有相关的材料安全数据表。用于制备成型的几种化学药剂具有剧毒。请使用个人防护用品 (安全眼镜, 手套, 实验室大衣, 全长长裤, 和脚趾鞋). 
 1. 微流体纺丝 RSF 水溶液 <ol> <li> RSF 水纺涂料的制备 4 , 15 , 16 <ol> <li> 蚕茧脱胶 <ol> <li> 脱胶在 Na 2 CO 3 水溶液 (水中 0.5%) 中, 分别为100和 #176; C 为30分钟, 然后用去离子水洗涤丝绸去除丝胶. </li>
		</ol> </li> <li> 脱胶蚕茧的溶解 <ol> <li> 在空气中干燥脱胶茧丝绸; 然后在 9.0 M 溴化锂水溶液中溶解脱胶茧丝, 比例为 1:10 (w/v), 在40和 #176; C 为 2 h。例如, 每1克丝添加10毫升的溴化锂 ( 图 1a ). </li>
		</ol> </li> <li> 离心和筛选 <ol> <li> 通过去离子水稀释 RSF 溶液1.5 次。离心机和过滤器去除杂质。离心 RSF 溶液在250毫升瓶在4和 #176; C 为10分钟在 1234.8 x g. 使用20和 #181; m 过滤器和真空泵过滤 RSF 溶液。考虑试验效果, 双滤纸床更可取. </li>
		</ol> </li> <li> 透析 <ol> <li> 透析5和 #176 的去离子水中的 RSF 解决方案; C 使用纤维素透膜 (分子: 1.4万和 #177 2000) 的3天。RSF 溶液总容积约1升, 载入4透析袋。把这些透析袋放在水桶里, 里面装满了10升反渗透 (RO) 的去离子水. 
			注: 去离子水的 pH 值应高于 6, 以避免在浓缩过程中发生凝胶。去离子水的 pH 值不需要对此协议进行调整. </li>
		</ol> </li> <li> 集中 <ol> <li> 将 RSF 水溶液以5和 #176 的强制气流浓缩到 20%; c. 将3米 CaCl 2 水溶液添加到 RSF 溶液中至1.0 摩尔/克 Ca 2 + 最终浓度; 然后用强迫空气集中流到 38-47%. </li>
			<li> 将一滴 RSF 溶液放在玻片上, 然后在 105 #176 的烤箱中将其烘干2小时; C. 
			注: 与干燥前的重量相比, 剩余固体的重量百分比是蛋白质和 CaCl 的总浓度 2 。RSF 浓度是在扣除质量的 CaCl 2 后获得的。至少进行了四次重复测量。我们以前的研究表明, 钙的浓度 2 + 极大地影响了 RSF 水溶液的流变性能和可纺性。同时, Ca 2 + 的添加导致了有限的 #946;-页和 RSF 17 的聚合。在本机纺丝涂料中, CaCl 2 被认为在纺纱涂料贮存过程中起着重要作用, 避免了纺纱前的胶凝。 18 </li> </ol> </li> </ol> </li> <li> 微流控芯片的准备 8 19 <ol> <li> 准备掩 <ol> <li> 设计微型通道CAD 程序。打印 CAD 文件以生成高分辨率透明度 19 . </li>
		</ol> </li> <li> 准备模具 <ol> <li> 在化学罩中清洗玻璃幻灯片 <ol> <li>, 将浓硫酸和 30% 过氧化氢溶液 (10:1) 的混合溶液中的玻片煮沸, 在热板上20分钟. 
				注意: 硫酸和过氧化氢蒸气是非常有毒的. </li>
			</ol> </li> <li> 清洗玻璃幻灯片 <ol> <li> 使用去离子水冲洗玻璃玻片, 用高纯度氮气吹干。
			</ol> </li> <li> 涂膜 <ol> <li> 将 SU-8 的光刻胶涂在玻璃滑轨上, custom-built 涂层设备的间隙为100和 #181; m 在涂层条的下表面和玻璃的上表面之间. </li>
			</ol> </li> <li> 自旋涂层 <ol> <li> 在玻片上传播光刻胶, 在三十年代使用 40.3 x g 的旋转涂布机形成一个均匀的胶片。均匀膜的厚度约为85和 #181; m. </li>
			</ol> </li> <li> 固化 <ol> <li> 使用温度控制程序在烤箱中固化光刻胶。将室温温度提高到65和 #176; c 在2和 #176; c/分钟, 并保持在65和 #176; c 为2分钟, 继续升温从65和 #176; c 到95和 #176; c 和保持在95和 #176; c 为 15 min. 关闭烤箱, 自然降温到房间烤箱里的温度. </li>
			</ol> </li> <li> 紫外线曝光 <ol> <li> 将玻璃玻片的侧面与光刻胶一起暴露在十二年代使用透明胶片作为掩的光刻 19 。
				注意: 紫外光的波为 365 nm, 曝光能量为273.6 兆焦耳/cm 2 . 
				注意: 在使用 UV 灯和烤箱时, 采取适当的安全措施. </li>
			</ol> </li> <li> 按步骤1.2.2.5 中所述固化光刻胶. </li>
			<li> 开发 <ol> <li> 在三十年代开发人员解决方案中超声波清洗光刻胶. 用异丙醇和显影液冲洗玻片, 交替两个, 直到玻璃滑梯上没有沉淀. </li>
			</ol> </li> <li> 使用温度控制程序在烤箱中固化光刻胶。将室温温度提高到170和 #176; c 在2和 #176; c/分钟, 保持在170和 #176; c 为30分钟. 关闭烤箱, 自然冷却到烤箱室温. </li>
		</ol> </li> <li> 软光刻 <ol> <li> 将8.8 克液体烷 (硅橡胶) 预在模具和固化中, 在65和 #176 上30分钟; c, 和15分钟, 在80和 #176; c. 液态预由硅橡胶和固化剂组成 (通常在 10:1 (w/w)). </li>
		</ol> </li> <li> 冲孔 <ol> <li> 通过钻孔在通道的开始处通过该复制副本来打孔。钻头直径为 1.2 mm. </li>
		</ol> </li> <li> 密封 <ol> <li> 通过在两个硅橡胶层表面上进行氧等离子体处理, 将具有该通道的硅橡胶复制件与无图案的平坦的硅橡胶层密封. 
			注: 芯片的整体制备过程约需72小时. </li>
		</ol> </li> </ol> </li> <li> 制作 RSF 光纤 <ol> <li> 旋喷剂 <ol> <li> 将 RSF 旋涂料注入2和 #181 的微通道中; 由注射器泵送至/分钟. </li>
		</ol> </li> <li> 环境调节 <ol> <li> 将相对湿度调整为40和 #177; 5 RH% 或50和 #177; 5 RH% 使用加湿器。在40和 #177; 5 RH%, 作为纺纤维固化速度超过50和 #177; 5 RH%. </li>
		</ol> </li> <li> 生产 RSF 光纤 <ol> <li> 通过在微流体通道的出口处触摸 RSF 的下落, 将 RSF 纤维拉入空气中, 然后通过10厘米的空气间隙将其卷到滚筒上, 速度为3厘米/秒 ( 图 1b ).</li>
		</ol> </li> <li> 将 RSF 光纤存储在密封的干燥中24小时. </li>
		<li> 光纤后处理 <ol> <li> 在 80% ethano 中绘制4次在 0.9 mm -1 上的旋转纤维l custom-built 机解决方案, 然后保持所绘制的纤维固定, 浸泡在溶液中的纤维1小时。由于这种处理, 纤维的长度从原来的15毫米改为60毫米. </li>
		</ol> </li> <li> 准备用于特征描述的示例 <ol> <li> 将 post-treated 光纤固定在一个长度为 10 mm 的纸框架上。至少有20光纤需要测量, 包括在拉伸试验, SEM, FTIR, 和 WAXS。post-drawn 纤维的直径范围从5到10和 #181; m. 图 1 显示了纤维生产和 WAXD 特性的示意图。RSF 纤维的力学性能由 (25、#177、2)、#176、C、(45、#177; 5) 相对湿度为% 的材料测试系统检测。延伸率和测量长度分别为2毫米/分和1厘米. </li>
		</ol> </li> </ol> </li> </ol> 2。RSF 光纤晶体结构的同步辐射表征 <ol> <li> 同步辐射特性 4 13 17 <ol> <li> 调整光束 <ol> <li> 将 X 射线和光斑大小的波长调整为 0.07746 nm 和 3 x 2 和 #181; m 2 分别. 
			注: 该协议是使用 BL15U1 光束在上海同步辐射设施. </li>
		</ol> </li> <li> x 射线点的位置 <ol> <li> 查找 x 射线点的位置. 
			注: x 光点的位置由同步辐射装置的实验室技术员进行调整. </li>
		</ol> </li> <li> 标准样品测试: 二氧化铈 (ceo 2 ) <ol> <li> 测试标准样品 ceo 2 粉末。CeO 2 粉末的特征是计算圆中心和从样品到检测器的距离. </li>
		</ol> </li> <li> 示例准备 <ol> <li> 在纸框架上以 10 mm 的长度与对方并行修复 RSF 丝。将纸张框架粘附在测试阶段. 
			注意: 保持纤维水平. </li>
		</ol> </li> <li> SR WAXD 测试 <ol> <li> 打开快门 <ol> <li> 关闭光束站的门, 确保房间内没有人。打开 X 射线光源的快门. </li>
			</ol> </li> <li> 聚焦 <ol> <li> 将纤程稍稍移至焦点。通过软件 ( 图 1c ) 远程调整光纤沿 x、y、z 方向的位置. </li>
			</ol> </li> <li> 示例曝光 <ol> <li> 通过软件远程上下移动光纤, 直到它位于 x 射线点上。按软件上的 "开始" 按钮将光纤暴露给二十年代的 X 射线光束 ( 图 1c ). </li>
			</ol> </li> <li> 背景衍射 <ol> <li> 以二十年代的曝光时间来测试空气背景的衍射. 通过软件将光纤从 X 射线点远程移开。按下 #34; 启动和 #34; 软件上的按钮, 以暴露 X 射线光束到 #34; 空气介质和 #34; 二十年代. </li>
			</ol> </li> </ol> </li> </ol> </li> <li> WAXD 数据处理 13 <ol> <li> 软件校准 <ol> <li> 使用 FIT2D (V12.077) 处理 WAXD 数据。使用 CeO 2 粉末的衍射数据校准圆中心和 sample-to-detector 的距离. </li>
		</ol> </li> <li> 2 维衍射图案 <ol> <li> 使用 FIT2D (V12.077) 从光纤衍射图案中减去空气背景. </li>
		</ol> </li> <li> 结晶度和微晶尺寸 <ol> <li> 将衍射强度集成为散射角2和 #952 的函数;。通过自动峰值分离软件 (版本 4.12) 12 来执行强度积分的反褶积。结晶度, x c , 从关系估计: <img alt="Equation" src="/files/ftp_upload/56271/56271eq1.jpg"/> , 其中 i c 是晶体峰的综合强度和 i 的总和 是非晶晕的综合强度 4 , 11 。WAXD 模式的集成显示了 [200]、[020]、[002] 反射的峰值宽度。这些峰值的 FWHM 使用谢瑞和 #39 的公式 4 来确定沿 a、b 和 c 轴的微晶大小. </li>
		</ol> </li> <li> 确定微晶方向 <ol> <li> 根据 (020) 和 (210) 峰值 11 的方位积分计算微晶和无定形的取向。根据赫尔曼和 #39 的取向, 可以定量地计算晶体的取向。在这里, (020) 和 (210) 峰都装有两个高斯函数。较窄的一个是晶体方向和其他更广泛的是面向非晶态材料 13 。在这里, 对 RSF 纤维的微晶取向进行了比较, 利用了方位积分 (002) 峰值的半最大 (FWHM) 的全宽度. </li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
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P., Vollrath, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+crystals+and+flow+elongation+in+a+spider's+silk+production+line.">Liquid crystals and flow elongation in a spider's silk production line.</a> P Roy Soc B-Biol Sci. 266 (1418), 519-523 (1999).</li></ol>

作者没有什么可透露的。

在 RSF 溶液的透析过程中, pH 值对以下浓度过程至关重要。如果去离子水的 pH 值小于 6, RSF 溶液在浓缩过程中会更容易凝胶。为了避免凝胶, CaCl2被添加到 RSF 解决方案中。CaCl2的浓度是每重量 RSF 的1摩尔。
我们以前的工作证明了微流控 RSF 水溶液干纺的可能性8。微流控通道的几何形状是一个简化的单级指数函数。对于蜘蛛和蚕, 纺纱兴奋剂是?...

在环境温度和压力下, 蜘蛛和蚕可以从水蛋白溶液中生产出优异的蚕丝纤维。剪切和伸展流动可诱导丝腺中的液晶纹理的形成1。近年来, 为了生产高强度的人造纤维, 模拟蜘蛛的纺丝工艺有着很大的兴趣。然而, 大量的蜘蛛丝蛋白不能生产效率和经济的养殖蜘蛛由于食人。大量的蚕丝绸可以通过耕作容易获得。否则, 蚕和蜘蛛有类似的纺纱工艺和氨基酸组成。因此, 蚕丝丝素被许多研究者选为人工动物丝的替代品。
蜘蛛和蚕通过他们的旋转导管挤出蛋白质溶液到空气中的纤维。沿着纺丝管道产生的高应力力很可能将丝素分子拉伸到更长的构象2。采用传统的湿纺和干纺工艺纺制了人造蚕丝纤维,3,4, 不考虑纺丝管产生的流体力。
首先, 采用微流控方法对蚕丝蛋白的组装进行了研究5,6。然后, 通过对剪切和伸展力的建模, 研究了 RSF 的微流体制备7,8。RSF 纤维的杨氏模量和直径可通过微流控湿纺纱进行调谐, 但拉伸纤维的抗拉强度小于100兆帕斯卡7。最后, 用微流控干纺法成功地制备了高强度 RSF 纤维, 但其直径仅2µm8。近年来, 微流控湿纺在高强度重组蜘蛛丝纤维生产中得到了成功的应用。空气中的 post-spinning 图改善了人造纤维的表面和内部缺陷9。
本文介绍了改进的微流控纺丝工艺在 RSF 光纤中的应用。它的目的是模仿蚕丝的纺纱过程, 包括纺纱涂料, 剪切力, 和干纺工艺。这种纺纱方法不仅可以生产高强度的人造蚕丝纤维, 而且可以调节纤维的直径。首先, 在具有二阶指数衰减的仿生通道中, RSF 纺涂料被剪切和拉长。其次, 研究了微流控干法纺丝过程中相对湿度 (RH) 对纤维形貌和性能的影响10。与传统的纺丝喷相比, 我们的微流控系统具有高度的仿生性, 可以用干湿纺法在室温下从溶液中产生高强度纤维。
由于同步辐射微 X 射线的高分辨率、亮度和高能性, 可以用来表征单个纤维的显微结构, 其直径为数微米4,11,12,13,14. 用 SR WAXD 技术计算了 RSF 纤维的结晶度、晶粒尺寸和晶体取向。

<table><tbody><tr><td>&lt;em&gt;B. mori&lt;/em&gt; Cocoons</td><td>Farmer in Tongxiang, Zhejiang Province, China</td><td></td><td></td></tr><tr><td>Sodium carbonate, anhydrous, 99.8%</td><td>Shanghai Lingfeng Chemical Reagent Co., Ltd., China</td><td></td><td>Analytically Pure</td></tr><tr><td>Lithium bromide, 99.1%</td><td>Shanghai China Lithium Industrial Co., Ltd., China</td><td></td><td>Analytically Pure</td></tr><tr><td>Calcium chloride, anhydrous, 96.0%</td><td>Shanghai Lingfeng Chemical Reagent Co., Ltd., China</td><td></td><td>Analytically Pure</td></tr><tr><td>Ethanol, anhydrous, 99.7%</td><td>Sinopharm Group Chemical Reagent Co.,Ltd., China</td><td>10009218</td><td>Analytically Pure</td></tr><tr><td>SU-8 photoresist</td><td>MicroChem Corp., USA</td><td></td><td></td></tr><tr><td>Developing solution</td><td>MicroChem Corp., USA</td><td></td><td></td></tr><tr><td>Sylgard 184</td><td>Dow Corning, USA</td><td></td><td></td></tr><tr><td>Isopropanol</td><td>Shanghai Lingfeng Chemical Reagent Co., Ltd., China</td><td></td><td>Analytically Pure</td></tr><tr><td>Concentrated sulfuric acid</td><td>Pinghu Chemical Reagent Factory, China</td><td></td><td>Analytically Pure</td></tr><tr><td>30 vol% hydrogen peroxide</td><td>Shanghai Jinlu Chemical reagent Co., Ltd., China</td><td></td><td>Analytically Pure</td></tr><tr><td>Acetone</td><td>Shanghai Zhengxing Chemical Reagent Factory, China</td><td></td><td>Analytically Pure</td></tr><tr><td>Oxygen plasma treatment</td><td>DT-01, Suzhou Omega Machinery Electronic Technology Co., Ltd., China</td><td></td><td></td></tr><tr><td>Syringe pump</td><td>&amp;nbsp;KD Scientific, USA</td><td></td><td>KDS 200P</td></tr><tr><td>Humidifier</td><td>SEN electric</td><td></td><td></td></tr><tr><td>Driller</td><td>Hangzhou Bo Yang Machinery Co., Ltd., China</td><td></td><td>bench drilling machine Z406c</td></tr><tr><td>Material testing system</td><td>Instron, USA</td><td></td><td>Model: 5565</td></tr><tr><td>PeakFit</td><td>Systat Software, Inc., USA</td><td></td><td>Version 4.12</td></tr></tbody></table>

microfluidic dry-spinning and characterization of regenerated silk fibroin fibers

该协议展示了一种仿蚕纺纱过程的方法。在本机纺纱过程中, 收缩纺丝管使蚕丝蛋白具有紧凑和有序的剪切和伸长力。在此, 设计了一种仿生微流控通道, 模拟了蚕纺管的特定几何形状。以高浓度掺杂的再生蚕丝丝素 (RSF) 为原料, 在室温和压力下通过微通道向干纺纤维挤出。在 post-treated 过程中, 在乙醇水溶液中提取和贮存成纱纤维。采用同步辐射 wide-angle x 射线衍射 (SR WAXD) 技术对单 RSF 纤维的显微组织进行了研究, 并将其固定在与 X 射线微的 RSF 纤维轴垂直的试样持有者身上。从 WAXD 数据计算了纤维的结晶度、晶粒尺寸和晶体取向。two-dimensional WAXD 模式赤道附近的衍射弧表明, post-treated RSF 纤维具有很高的取向度。

采用微流控纺丝方法成功地制备了高强度 RSF 纤维。在图 2中显示了拉伸 RSF 光纤 C44R40 的应力-应变曲线和 SEM 图像。在拉伸试验中测量了至少10纤维。根据纤维断裂应力和应变的平均值选择应力应变曲线。光纤的 WAXD 数据显示在图 3中。根据 WAXD 数据计算了结晶度和结晶取向。对于样品的指定, 我们用 C 和 R 分别介绍了纺丝涂料?...

Watch this Scientific Journal Video about Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers at JoVE.com

Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers

微流控干纺及再生丝素纤维的表征

提出了一种微流体纺丝的协议, 并给出了再生丝素丝的显微组织表征。

The protocol demonstrates a method for mimicking the spinning process of silkworm. In the native spinning process, the contracting spinning duct enables ...

microfluidic-dry-spinning-characterization-regenerated-silk-fibroin

Research

JoVE Journal

Chemistry

9.7K 次观看.  Donghua University. 提出了一种微流体纺丝的协议, 并给出了再生丝素丝的显微组织表征。

视频: Microfluidic Dry-spinning and Characterization of Regenerated Silk Fibroin Fibers

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

丝绸电影文化系统在体外分析和生物材料设计

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

科研

生物工程

Synthetic Spider Silk Production on a Laboratory Scale

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks.

Despite the outstanding mechanical and biochemical properties of spider silks, this material cannot be harvested in large quantities by conventional means. Here we describe an efficient strategy to spin artificial spider silk fibers, which is an important process for investigators studying spider silk production and their use as next-generation biomaterials.

实验室规模的人工合成蜘蛛丝生产

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation ...

Air Filter Devices Including Nonwoven Meshes of Electrospun Recombinant Spider Silk Proteins

Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly repetitive protein has a molecular weight of 48kDa and is soluble in different solvents (hexafluoroisopropanol (HFIP), formic acid and aqueous buffers). eADF4(C16) provides a high potential for various technical applications when processed into morphologies such as films, capsules, particles, hydrogels, coatings, fibers and nonwoven meshes. Due to their chemical stability and controlled morphology, the latter can be used to improve filter materials. In this protocol, we present a procedure to enhance the efficiency of different air filter devices, by deposition of nonwoven meshes of electrospun recombinant spider silk proteins. Electrospinning of eADF4(C16) dissolved in HFIP results in smooth fibers. Variation of the protein concentration (5-25% w/v) results in different fiber diameters (80-1,100 nm) and thus pore sizes of the nonwoven mesh.
Post-treatment of eADF4(C16) electrospun from HFIP is necessary since the protein displays a predominantly &alpha;-helical secondary structure in freshly spun fibers, and therefore the fibers are water soluble. Subsequent treatment with ethanol vapor induces formation of water resistant, stable &beta;-sheet structures, preserving the morphology of the silk fibers and meshes. Secondary structure analysis was performed using Fourier transform infrared spectroscopy (FTIR) and subsequent Fourier self-deconvolution (FSD).
The primary goal was to improve the filter efficiency of existing filter substrates by adding silk nonwoven layers on top. To evaluate the influence of electrospinning duration and thus nonwoven layer thickness on the filter efficiency, we performed air permeability tests in combination with particle deposition measurements. The experiments were carried out according to standard protocols.

Spider silk fibers display extraordinary mechanical properties. Engineered Araneus diadematus Fibroin 4 (eADF4) can be processed into nonwoven meshes using electrospinning. Here, the eADF4 nonwoven meshes are used to improve the performance of air filtering devices.

空气过滤设备，包括非织造网格静电重组蜘蛛丝蛋白

Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly ...

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

设计丝绸蚕丝蛋白合金生物医用材料

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

Reactive Inkjet Printing and Propulsion Analysis of Silk-based Self-propelled Micro-stirrers

In this study, a protocol for using reactive inkjet printing to fabricate enzymatically propelled silk swimmers with well-defined shapes is reported. The resulting devices are an example of self-propelled objects capable of generating motion without external actuation and have potential applications in medicine and environmental sciences for a variety of purposes ranging from micro-stirring, targeted therapeutic delivery, to water remediation (e.g., cleaning oil spills). This method employs reactive inkjet printing to generate well-defined small-scale solid silk structures by converting water soluble regenerated silk fibroin (silk I) to insoluble silk fibroin (silk II). These structures are also selectively doped in specific regions with the enzyme catalase in order to produce motion via bubble generation and detachment. The number of layers printed determines the three-dimensional (3D) structure of the device, and so here the effect of this parameter on the propulsive trajectories is reported. The results demonstrate the ability to tune the motion by varying the dimensions of the printed structures.

This protocol demonstrates the ability to utilize reactive inkjet printing to print self-motile biocompatible and environmentally friendly micro-stirrers for use in biomedical and environmental applications.

基于丝绸的自行式微搅拌器的反应式喷墨打印及推进分析

In this study, a protocol for using reactive inkjet printing to fabricate enzymatically propelled silk swimmers with well-defined shapes is reported. The ...

化学

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group&#8217;s method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

Here, we present a protocol to produce water soluble recombinant spider silk protein solutions and the material forms that can be formed from those solutions.

热、压水固化重组蜘蛛丝的材料形成

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order ...

Procedure for Fabricating Biofunctional Nanofibers

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups may be utilized to study material-biomarker interactions i.e. act as biosensors with potential as single molecule detectors. We have developed an effective approach for preparing functional polymers where the functionality has the capacity of specifically binding with a model protein. In our model system, the functional group is 2,4-dinitrophenyl (DNP) and the protein is anti-DNP IgE (Immunoglobulin E). The functional polymer, &alpha;,&omega;-bi[2,4-dinitrophenyl caproic][poly(ethylene oxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP), is prepared by anionic living polymerization. The difunctional initiator utilized in the polymerization was prepared by electron transfer reaction of &alpha;-methylstyrene and potassium (mirror) metal. The 2-methoxystyrene monomer was added first to the initiator, followed by the addition of the second monomer, ethylene oxide, and finally the living polymer was terminated by methanol. The &alpha;,&omega;-dihydroxyl polymer [HO-PEO-P2MS-PEO-OH] was reacted with N-2,4-DNP-&isin;-amino caproic acid, by DCC coupling, resulting in the formation of &alpha;,&omega;-bi[2,4-dinitrophenylcaproic][poly(ethyleneoxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP). The polymers were characterized by FT-IR, 1H NMR and Gel Permeation Chromatography (GPC). The molecular weight distributions of the polymers were narrow (1.1-1.2) and polymers with molecular weights greater than 50,000 was used in this study. The polymers were yellow powders and soluble in tetrahydrofuran. A water soluble CDNP-PEO-P2MS-PEO-CDNP/ DMEG (dimethoxyethylene glycol) complex binds and achieves steady state binding with solution IgE within a few seconds. Higher molecular weight (water insoluble i.e. around 50,000) CDNP-PEO-P2MS-PEO-CDNP polymers, containing 1% single wall carbon nanotubes (SWCNT) were processed into electroactive nanofibers (100 nm to 500 nm in diameter) on silicon substrate. Fluorescence spectroscopy shows that anti-DNP IgE interacts with the nanofibers by binding with the DNP functional groups decorating the fibers. These observations suggest that appropriately functionalized nanofibers hold promise for developing biomarker detection device.

An efficient approach for preparing nanofibers decorated with functional groups capable of specifically interacting with proteins is described. The approach first requires the preparation of a polymer functionalized with the appropriate functional group. The functional polymer is fabricated into nanofibers by electrospinning. The effectiveness of the binding of the nanofibers with a protein is studied by confocal microscopy.

制作生物功能纳米纤维的过程

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups ...

生物学

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical methods for manipulating and monitoring the activity of neurons in vivo. These types of experiments rely on two main components: 1) implantable devices that provide optical access to the brain, and 2) light-sensitive proteins that change neuronal excitability or provide a readout of neuronal activity. There are a number of ways to express light-sensitive proteins, but stereotaxic injection of viral vectors is currently the most flexible approach because expression can be controlled with genetic, anatomical, and temporal precision. Despite the great utility of viral vectors, delivering the virus to the site of optical implants poses numerous challenges. Stereotaxic virus injections are demanding surgeries that increase surgical time, increase the cost of studies, and pose a risk to the animal's health. The surrounding tissue can be physically damaged by the injection syringe, and by immunogenic inflammation caused by the abrupt delivery of a bolus of high-titer virus. Aligning injections with optical implants is especially difficult when targeting small regions deep in the brain. To overcome these challenges, we describe a method for coating multiple types of optical implants with films composed of silk fibroin and Adeno-associated viral (AAV) vectors. Fibroin, a polymer derived from the cocoon of Bombyx mori, can encapsulate and protect biomolecules and can be processed into forms ranging from soluble films to ceramics. When implanted into the brain, silk/AAV coatings release virus at the interface between optical elements and the surrounding brain, driving expression precisely where it is needed. This method is easily implemented and promises to greatly facilitate in vivo studies of neural circuit function.

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

Here, we present a method for delivering viral expression vectors into the brain using silk fibroin films. This method allows targeted delivery of expression vectors using silk/AAV coated optical fibers, tapered optical fibers, and cranial windows.

在 vivosilk/aav 薄膜在光基因表达中的靶向表达

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical ...

神经科学

<meta charset="utf8"></head><body>FN-Silk 膜的形成和附着到细胞培养物的插入物上

Nanofibrillar Basement Membrane Mimic Made of Recombinant Functionalized Spider Silk in Custom-Made Tissue Culture Inserts

Replicating tissue barriers&#160;is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using tissue culture inserts with a plastic membrane, which generates an apical and a basal side. Besides providing support for the cells, these membranes come far from emulating their native counterpart, the basement membrane, which is a nanofibrillar, protein-based matrix. In this work, we show a simple way to considerably improve the biological relevance of the tissue culture inserts by replacing the plastic membrane with one made from a pure recombinant functionalized spider silk protein. The silk membrane forms through self-assembly and will spontaneously adhere to a membrane-free tissue culture insert, where it can provide support for cells. Custom-designed tissue culture inserts can be printed using a standard 3D printer, following the instructions provided in the protocol, or commercial ones can be purchased and used instead. This protocol shows how the culture system with silk membranes in inserts is set up and, subsequently, how the same cell culturing techniques that are used with traditional, commercially available inserts can be implemented.

<meta charset="utf8"></head><body>作者聚焦：使用重组功能化蜘蛛丝膜增强体外细胞培养模型

Tissue culture inserts with plastic membranes are the golden standard in cell culture labs as permeable supports to establish cell layers and models of barrier tissues. Herein, we present a simple method to replace the plastic membrane with a more biologically relevant membrane made from a recombinant functionalized spider silk protein.

纳米原纤维基底膜模拟物，由重组功能化蜘蛛丝制成，用于定制组织培养小室

Replicating tissue barriers&#160;is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using ...

微流控干纺及再生丝素纤维的表征

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此视频中的章节

丝绸电影文化系统在体外分析和生物材料设计

实验室规模的人工合成蜘蛛丝生产

空气过滤设备，包括非织造网格静电重组蜘蛛丝蛋白

设计丝绸蚕丝蛋白合金生物医用材料

基于丝绸的自行式微搅拌器的反应式喷墨打印及推进分析

热、压水固化重组蜘蛛丝的材料形成

制作生物功能纳米纤维的过程

在 vivosilk/aav 薄膜在光基因表达中的靶向表达

纳米原纤维基底膜模拟物，由重组功能化蜘蛛丝制成，用于定制组织培养小室

纳米原纤维基底膜模拟物，由重组功能化蜘蛛丝制成，用于定制组织培养小室