作者感谢罗文大学支持本研究。 XH也感谢大卫L卡普兰博士塔夫茨大学和美国国立卫生研究院P41组织工程资源中心（TERC）在以前的技 ​​术培训。

1，预测蛋白质相互作用<ol><li>蛋白质分子的生物信息学分析<ol><li>访问国家生物技术信息中心网站（www.ncbi.nlm.nih.gov/protein/），并搜索将要使用的合金研究的蛋白质的名称。注意：在这个例子中，两种蛋白质分别采用：蛋白A，它是野生柞蚕丝素蛋白，以及蛋白B，这是国内桑蚕丝心蛋白。对于A蛋白的氨基酸序列中可以找到“素[柞蚕] GenBank登录号：AAC32606.1”（ 柞蚕是中国（橡木）柞蚕蛾）。对于蛋白B，所述氨基酸序列中可以找到“心蛋白重链的前体[家蚕] NCBI参考序列：NP_001106733.1”和“心蛋白轻链的前体[家蚕] NCBI参考序列：NP_001037488.1”在一起（ 家蚕家蚕是桑树的家蚕）。 </li><li>选择和SA已经将蛋白A和蛋白B从数据库中的氨基酸序列。 </li><li>访问ExPASy网站（SIB生物信息学资源门户）（www.expasy.org），或使用其他的商业软件基于其序列包括计算基本生物信息学的蛋白质的数据，但不限于，每分子的总电荷，疏水性指数在分子中，分子在不同pH值滴定曲线等，这信息将被用作用于蛋白质相互作用的计算机模拟的基本元件，并且将有助于理解这两种蛋白质是否具有强的相互作用。 [注：这一步是不是准确预测像那些小肽和功能蛋白的科学使用的蛋白质相互作用的每一个细节。这部分的目的仅仅是为了避免产生蛋白质混合物具有明显的宏观相分离，这不能被称为一个“合金”的材料。因此，估计可能是近似的，但日E蛋白合金系统可以通过使用精确的热分析]在步骤3.1中描述的实验方法进行验证。 </li></ol></li><li>蛋白质合金系统的计算机模拟 下面描述的过程来模拟蛋白质的合金系统。模拟程序写入，可以在单处理器或多处理器计算机系统中使用C语言编程。格子弹簧-质量（LSM）模型被用来模拟在合金蛋白36-39。在LSM模型给出的净力上一团一简单的描述，当连接到一个弹簧，一个可以解决力方程，了解每一个质量的议案。一个简单的程序算法，利用LSM模型为建模这种蛋白质合金系统给出： <ol><li>代表一个单一的蛋白质作为粗粒度的粒子具有质量为m。 </li><li>使用虎克或新虎克弹簧来表示债券38,39。通过互连颗粒的弹簧数量有限，可以米AKE表示合金蛋白的稳定积木子合金域。来表示不同类型的内联键，使用不同的弹簧常数/刚度。 </li><li>模型中的蛋白质合金系统的迟钝交联亚合金构成的材料。在这里再次不同刚度被用来代表该子合金的相互联系的不同的键。 </li><li>由贝尔型号40,41，通过弱键允许进行改革模式键断裂和改革进程，但强债不能改变一旦被打破。当系统被充分强调（无论是在内部suballoy和跨suballoy债券），债券可以分解和改性。 </li><li>为了研究在合金蛋白变形效果的时候都强调，申请外力到系统。求解力方程（牛顿定律），当平均分配这些部队每个粒子。 </li><li>为了模拟互动的溶液（例如水分子）和蛋白质之间，施加附加的阻力或摩擦力，以每个粒子。 </li><li>求出每一个粒子与每个力（从键，外力和摩擦力的弹簧力）的动作的力方程。 </li><li>计算并提取蛋白粒子的位置作为时间的函数。 </li><li>计算出的物理量是从粒子的位置表征了合金的蛋白质。 </li><li>在节目中，了解不同的蛋白质之间的相互作用改变债券的刚度。 （平均粘结刚度，从杨氏模量的蛋白质材料的计算杨氏模量不同的纤维蛋白物质可以通过万能拉伸试验18中 ，或者直接从以前的文献2-4,18获取）。 </li></ol></li></ol> 2，生产蛋白质合金解决方案野生柞蚕丝（蛋白A）和国内桑蚕丝（蛋白B）在这里选择蛋白质合金系统的例子。该协议首先介绍如何获取野生柞蚕丝（蛋白A）的解决方案。 <ol><li>切生的野生柞丝茧或纤维在3克重。 </li><li>测量将3克钠二碳酸酯或碳酸钠（注：如果使用碳酸钠，蛋白链的分子量将在沸腾过程42减少）。 </li><li>装满蒸馏水2升玻璃烧杯（H 2 O）。然后，放置在加热阶段的玻璃烧杯中，盖上铝箔，并加热至沸腾。 </li><li>取下铝箔盖，慢慢加入被测碳酸钠放入开水，使其完全溶解。 （注：钠二碳酸酯的作用是“肥皂”清理掉附着野蚕丝纤维的表面上的可溶性的丝胶蛋白和其它杂质如使用超视距。呃性蛋白质纤维，请根据文献中选择相应的化学试剂）。 </li><li>添加粗蛋白纤维（野蚕丝纤维）入沸水，让煮2-3小时（注：煎煮时间有重要影响蛋白质链26,43的分子量每个人都应该根据选择适当的时机文献或通过进行对照实验26,43。沸点温度也可以被调整，以影响该蛋白链26,43,44）的分子量。 </li><li>煮沸后，小心地除去蛋白质纤维与从溶液刮刀和挤压它们以除去过量的水。 （注意：该纤维是非常热！） </li><li>接着，浸渍于2L烧杯用冷蒸馏水中的纤维，并两次洗涤纤维为每30分钟，以从纤维表面完全除去不纯残基。干在通风橱中的纤维，至少12小时。 </li><li>熔体45.784克钙尼特的率（的Ca（NO 3）2）在玻璃烧杯中以形成液体，在65℃下溶解该野生蚕丝蛋白纤维。 （注意：如果使用其他的天然蛋白质纤维，选择相应的溶剂溶解的蛋白质在这里，您还可以使用9.3 M LiSCN的或溴化锂溶液，或溶解不同的丝纤维的85％磷酸溶液。） </li><li>结合纤维和溶剂以1克纤维在10ml溶剂中的比率。使纤维在95℃下溶解于5至12小时。 （注：溶解时间取决于26,43-45的蛋白质的分子量） </li><li>使用注射器，注射野生蚕丝溶液进12毫升透析盒（最大1000兆瓦的截止大小）或密封的渗析管（最大1000兆瓦的截止大小）和透析对2升的蒸馏水中。 （注：注射是如果保持溶液在35℃下更有效，否则的蛋白质溶液的粘度在室温下显着地增加）。频繁更换蒸馏水除去的Ca（NO 3）2的溶剂中的溶液（30分钟，2小时，6小时，并在此之后每12小时3天，总计将有大约8换水）。 </li><li> 3天后，收集蛋白溶液从透析盒或管并放入13000 rpm额定管。 </li><li>离心1小时，将溶液在3,500 rpm转速于4℃下3次，除去沉淀物。每个离心机运行后，快速拉取上清液到新的试管中。存储解决方案，最终在4℃冰箱中。 </li><li>倾溶于5ml蛋白溶液涂布到聚二甲基硅氧烷（PDMS）基片或其他平坦疏水基底，并允许它完全干燥（此过程通常需要超过12小时）。称量剩余的固体蛋白质膜和按重量百分比（重量/体积％）=测得的重量（毫克）÷5（单位为ml）÷10计算最终溶液浓度。 </li><li>收集其他选择天然蛋白质纤维（在此CASE，家养桑蚕丝被用作蛋白B），并重复上述过程用适当的“肥皂”和溶解的溶剂，直到得到与测得的浓度的最终蛋白质水溶液。 [注意：如果该蛋白材料是粉末状的，可以使用适当的多孔管或膜过程中的“皂洗”过程来保存样品。如果蛋白质已纯化，直接进入到步骤2.8以溶解粉末。如果蛋白已经纯化并是水溶性的，使得其具有所需浓度的水溶液，然后再转到下面的步骤2.15进行混合蛋白质溶液。] </li><li>慢慢稀释该蛋白在蒸馏水中的溶液（这里是野蚕丝溶液），在4℃以形成1.0重量％的A蛋白水溶液。做同样的过程蛋白B（这里驯养丝）。 </li><li>慢慢地，在4 1％（重量）的蛋白质混合使用蛋白B液A液 利用℃，吸管，避免p在混合过程中rotein聚集。 （注1：不要使用仪器旋涡混合蛋白质，因为有些蛋白质（ 如丝绸）将振动46,47过程中形成的水凝胶注2：如果可能的话，使用额外的设备来控制搅拌速度和混合尺寸制作请务必将它们混合，尽量避免聚集慢。混合过程中不迅速吸取的溶液）。 </li><li>最终混合溶液应具有一个指定的质量比或蛋白A的摩尔比为：蛋白质B.通常情况下，将它们混合以90:10的质量比，75:25，50:50，25:75，10:90，得到合金解决方案广泛。用纯蛋白A和蛋白B的解决方案作为对照。对于用蛋白A的摩尔比混合解决方案：蛋白B = R：（100-R）。计算混合比（基于相同重量1％水溶液）人：A卷：卷B = R·（一兆瓦）：（100-R）·（B兆瓦）。 </li><li>立即施放最后的解决方案上的PDMS基板形成薄膜或其他DESIgned材料。 （注意：不要将高浓度的蛋白质合金的解决方案很长一段时间，由于蛋白质 - 蛋白质相互作用在水中聚集更多的可能形态。）。如果需要的话，稀释共混物溶液与离子的蒸馏水，并保存在4℃的冰箱中，以避免在溶液中的其他蛋白的聚集。 </li></ol> 3，制造蛋白质变合金材料<ol><li>确认预测合金热分析3,9,31-35 <ol><li>准备PDMS基板和浸泡在蒸馏水中清洗。 </li><li>铸蛋白共混物溶液具有不同混合比例上的PDMS衬底。 </li><li>在化学罩与空气流干燥该溶液至少12小时，直到膜被形成（注：使用相同体积的不同的解决方案，使得膜的厚度可以是固定的）。 </li><li>从PDMS基板取下蛋白合金膜，并将其放置到干净的盘子。 </li><li>权衡许多差示扫描量热法（DSC）的铝盘和盖子的DSC研究。匹配锅和盖子对拥有相等的总重量（重量盖锅加重量等于恒重）。例如，盖在这里，总重量和平移22.50毫克，使用，和8套盖和盘的组合与该总重量进行制备。 </li><li>封装6毫克每一种干燥的蛋白质融合到DSC铝盘和密封他们与他们匹配的盖子过程3.1.5。密封一个空锅和盖子对要与样品作为基准用来使样品本身将热分析（注过程中被记录的唯一的热容量的DSC将比较的基准锅+盖子与该热容量样品+锅+盒盖。由于相等的权重，从盘和盖子的背景热容量会占试样的放置在秤盘唯一的热容量）。 </li><li>把密封的参考和样品盘成DSC仪器，用吹扫的50ml /分钟的干燥氮气气体流量，并装配有冷冻冷却系统。之前的样品测量时，DSC仪器应首先进行校准蓝宝石和铟为分别热流和温度。 </li><li>预运行DSC以2 K / min的加热速率至150℃，然后保持在该温度下15分钟以除去任何剩余的水分子的样品（通常为约3-10％的总重量的）中。 （10K /分钟）迅速冷却至25℃。 </li><li>再次运行在DSC以2 K / min的升温速率为300 ℃，或直至蛋白共混物的降解峰出现34。在此过程中记录在不同温度下的蛋白样品的热容量。降温的DSC和老样本更改为新的样本有不同的混合比例。 </li><li>计算并绘制热容量随温度的变化曲线，使用DSC软件31-35每个蛋白质混合样品。 </li><li>判断米蛋白掺合物通过以下方法iscibility（参见图4的热和图5），并且如果两种蛋白质是完全混溶的，它们可以被称为“蛋白的合金”。否则，所谓“蛋白质合成”的，根据聚合物描述性理论48,49）将是一个合适的名称： <ol><li>个别蛋白A和B应该有单独的单玻璃化转变温度T G（A）和T G（B）（参见图5中的绿色和蓝色曲线）3,48; </li><li>这个单一的玻璃化转变温度是那些在两个单独的蛋白组分的 Tg（A）和T 克 （B）的中间，通常中间（参见图5）3,48; </li><li>不混溶相分离共混得到，如果两者的 Tg（A）和T G（B）出现在原来的位置（ 图5），并与每个的 Tg一步高度成比例的组合物中，这两种蛋白是完全不混溶的3,48。 </li><li>半混溶的复合材料共混物的类型将有一个很宽的玻璃化转变，或者可以仍然有两个玻璃化转变，但每个已迁移彼此靠近相对于纯蛋白组分的 Tg（A）和T 克 （B）（参见图5）。在这种情况下，可能有两个蛋白组分之间所形成的微相异质结构，并且该组合物可以从一个位置变化到位置。 </li></ol></li><li>如果（3.1.11.1）是在DSC中所示的情况下，它可以被证实，该蛋白AB是一种合金系统中，然后转移到制造蛋白质的合金材料。 </li></ol></li><li>光学材料由蛋白质合金制造<ol><li>产生（在制造实验室）或购买设计地形表面铸造。在这个具体的例子中，使用的玻璃具有四个衍射图（ 图4光）。 </li><li>将玻璃与衍射图案成菜，并确保图案的表面朝上。 </li><li>传播的PDMS溶液均匀地涂在玻璃表面，并完全覆盖所述表面型态（：根据用户指令23,44 1混合比的PDMS溶液通过灌封而在9催化剂溶液制备）。 </li><li>把铸造菜到65℃的烘箱中至少2小时，同时在一个平面上。的PDMS溶液应干燥到在此过程中的固体衬底。 </li><li>从玻璃去除PDMS衬底上。的衍射图案，现在应该转移到PDMS表面。 </li><li>切出的硅橡胶模具使用合适的打孔衍射图谱。 </li><li>滴在PDMS表面与衍射图案蛋白合金溶液，并使其干燥至少12小时以获得具有衍射图案的薄膜。 </li><li>为了获得不溶性蛋白质合金材料，将整组博士Ý膜，包括PDMS模具成60℃真空烘箱中（25千帕）与水盘上的腔室的底部。泵出的空气在炉中，并让水蒸汽退火样品进行至少2小时。 （此过程称为温度控制的水蒸汽退火45，与广泛使用的甲醇的方法相比，它可以产生在丝材料45相似的β-折叠含量）。释放真空并利用镊子的PDMS衬底剥离的水不溶性膜。在这个例子中，野蚕丝，蚕丝驯化合金使用。 </li><li>通过将它们与玻璃上的原始图案的比较测试的X射线衍射图案上的薄膜的质量（ 例如 ，收集的SEM图像的微观尺度的细节;收集激光衍射型态的一般模式质量）。 </li></ol></li><li>电路的蛋白质合金材料的制备<ol><li>制作在玻璃基板上的电路图案，第一CLEA使用一些脱脂呐载玻片溶剂如Alconox清洁剂在超声波清洗5分钟，然后用5分钟在丙酮中，然后用5分钟在甲醇中。在使用甲醇最后，因为它的蒸发速度低于丙酮所以可以吹出衬底而不是干燥和留下的残余物。 </li><li>使用通过汽化从180升液氮杜瓦产生干燥的氮气吹载玻片干燥。 </li><li>基板材料引入到沉积室中。 （这些准则的溅射系统，但其他的沉积技术也可以使用。）如果该室被设计成具有负载锁，在沉积室中的真空度不显著影响。疏散负载锁到30毫托的压力。 </li><li>打开负载锁和主沉积腔室之间的门阀，引入基板进入腔室。 </li><li>打开Ar气及压力调节器和控制压力至所需沉积物等Ñ​​压力。较高的压力给低能量溅射的金属原子和更均匀的薄膜，而较低的压力下产生更好的附着速度快淀积膜。压力的范围一般为3毫乇和60毫托之间，用20毫乇工作良好。 </li><li>金属，然后投射到光闸，以保护从利用RF功率100 W的指示的射频功率，以在金属靶的调谐电路是必需的涂覆基底。直流电源可以用来代替RF对金属靶。为了从目标移除氧化层和污染物，预溅射数分钟。 </li><li>打开快门和溅射的金属在基板上。沉积速率为所述的结构是每分钟约10nm。这个速率将取决于工作距离，压力，磁场强度在磁控管阴极，目标厚度和溅射的金属。调节沉积时间，以获得所需的厚度。 </li><li>除去从涂覆的玻璃载片上室。 </li><li>使用旋转器，旋转光刻胶涂布到膜的表面。许多抗蚀剂都可以使用。对于这种情况下，正性光刻胶使用。 </li><li>后的抗蚀剂被旋涂在薄膜，软烘烤在90℃下进行5分钟以干燥抗蚀剂。 </li><li>放置一个接触掩模与器件的紧贴抗蚀剂图像。紫外线光源被用于曝光光致抗蚀剂。曝光10秒，但是根据使用的光源的强度和抗蚀剂使用。 </li><li>将膜在光致抗蚀剂显影剂直到投影的图像将出现。显影剂洗去抗蚀该暴露于UV光而引起聚合物键的断裂。紧随形象出现后，沾去离子水的薄膜，从工作的未曝光光刻胶停止开发。 </li><li>吹干用干氮气的膜。 </li><li>将膜放入烘箱，在120℃下进行15分钟至“硬烤”的照片抗拒。 </li><li>后的膜冷却，将它们放置在蚀刻溶液中，直至没有被光刻胶保护的金属升空。浸在水中来停止蚀刻。 </li><li>用丙酮冲洗以除去硬化的光致抗蚀剂。 </li><li>冲洗用甲醇和吹干用干燥的氮气。 </li><li>一旦被涂覆眼镜准备，滴不同蛋白合金溶液到玻璃的表面，并使其干燥至少12小时，以获得对眼镜蛋白合金膜。 （建议先集中在合金溶液至5％（重量），得到粗蛋白合金膜。） </li><li>由于疏水-亲水相互作用，在金属薄膜会从玻璃表面被转移到连接蛋白合金膜面51。剥去蛋白合金膜与从使用镊子将玻璃基板上的薄金属图案。 </li><li>以获得不溶性蛋白的合金材料，将干燥膜成60℃的真空烘箱中（25千帕）与AW亚特盘上的腔室的底部。泵出的空气在炉中，并让水蒸汽退火样品进行至少2小时。释放真空并利用镊子基板剥离的水不溶性膜。 </li><li>检验对蛋白合金膜的金属图案的电特性，例如电阻，并将它们与玻璃上的原始图案。 </li></ol></li><li>医药材料由蛋白质合金制造<ol><li>为了制造一种蛋白质合金薄膜的药物化合物，如步骤3.2中所述先准备PDMS基板。清洗所形成的PDMS衬底由蒸馏水。 </li><li>溶解或分散药物化合物到水溶液中。使用超声或涡流，以均匀混合的药物化合物与水。如果该化合物是不溶于水，分散的粉末具有在无离子蒸馏水中均匀分布。 </li><li>计算所需的质量比的化合物溶液化合物的解x重量百分比体积：化合物对蛋白质的合金由蛋白质合金液×体重的化合物溶液的体积百分比（这里1％（重量）的合金液使用）。选择一个比例，以获得与蛋白合金膜所需的化合物密度的薄膜。 </li><li>慢慢地混合使用下列第2的过程2.16相同的指令蛋白质合金液的化合物溶液。 （注：为避免凝胶化，不要超声波降解或涡流混合过程中的溶液）。 </li><li>倒入混合到PDMS基板上的计算量和晾干至少12小时在化学罩含有药物化合物的设计比例获得蛋白质的合金膜。 </li><li>物理交联下面的第3.2节中的过程3.2.8相同的指令电影。合金膜与不溶性模型药物的低密度（LD）或高（HD）的密度的一个例子可以在图4中可以看出</strong&gt;化工。 </li></ol></li></ol>

没有利益冲突的声明。

一种生产“合金”蛋白系统中最关键的步骤是，以验证混合蛋白质的混溶性。否则，它是唯一的一个不混溶的蛋白混合物或蛋白质没有稳定和调谐性能的复合系统。实验热分析法可用于该目的，并确认其合金的特性。蛋白质-蛋白质相互作用可以根据弗洛里-哈金斯的晶格模型48为“溶剂”（主要的蛋白成分）之间的相互作用和“溶质”（次要蛋白成分）来查看。基于该模型中，“溶剂”和?...

大自然创造战略，以利用产生结构蛋白数量有限可调，多功能的生物基质。例如，弹性蛋白和胶原蛋白总是一起使用的体内 ，以提供所需的特定组织1,2的可调节的优点和功能。这里的关键策略是混合。共混包括混合蛋白质与特定的比例，是一个技术的方法来生成具有可调和不同性质3-5简单的材料系统。用合成的工程化策略6,7相比，共混还可以提高材料的均匀性和对材料的过程中，由于操作方便8-16的能力。因此，多功能的设计，生物相容性蛋白质合金材料是医学研究的一个新兴领域。此技术也将提供的天然蛋白基质的影响系统的知识上都在维生素细胞和组织的功能RO和体内 10,17。通过优化不同蛋白质之间的分子的接口，以蛋白质为基础的合金材料可以包括一系列的生理功能，例如在不同温度下的热稳定性，弹性，以支持不同的组织中，电灵敏度的可变机构，和光学性能用于角膜组织再生3， 18-27。这些研究成果将提供一个新的蛋白质材料平台在生物医学科学领域有直接关系的调谐组织修复和疾病治疗，并进一步导致他们在那里新的治疗和诊断功能，可以预见3可生物降解的植入装置。 许多天然结构蛋白具有关键物理和生物活性性质，可以利用作为候选生物材料矩阵。不同虫种丝绸，角蛋白来自不同组织的毛发和羊毛，弹性蛋白和胶原蛋白，并各种植物蛋白是一些用于设计变量蛋白基材料（ 图1）18-27中最常见的结构蛋白。在一般情况下，这些蛋白可形成不同的分子的二级结构（ 例如 ，β片层为丝绸，或者用于角蛋白螺旋线圈）由于其独特的重复性的一级氨基酸序列，3,28-35。这些功能推动形成自组装的宏观结构与独特的功能，在生物界面提示它们作为生物高分子材料的珍贵资源。在这里，两种结构蛋白被用于（野生柞蚕丝和家养桑蚕丝，例如，蛋白B蛋白A），以证明生产各种蛋白质合金生物材料的通用协议。演示的协议包括第1部分：蛋白质相互作用的预测和模拟，第2部分：生产蛋白质合金的解决方案，第3部分：蛋白质合金制造系统以及用于光，电，和制药应用。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/50891/50891fig1highres.jpg" width="500" /> 图是在我们的实验室中设计的基于蛋白质的材料，包括来自不同虫物种的丝绸常用的各种结构蛋白1，原料，角蛋白从头发和羊毛，从不同组织中弹性蛋白和各种植物的蛋白质。

<table border="0" cellpadding="0" cellspacing="0" width="1050">
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Q100 Differential Scanning Calorimeters (DSC)</td>
			<td style="width:143px;">TA Instruments, New Castle, DE, USA 
			&#160;</td>
			<td style="width:169px;">N/A</td>
			<td style="width:523px;">You can use any type of DSC with a software to calculate the heat capacity</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">SS30T Vacuum Sputtering System&#160;</td>
			<td style="width:143px;">T-M Vacuum Products, Inc., Cinnaminson, NJ, USA</td>
			<td style="width:169px;">N/A</td>
			<td>With custom built parts; You can use any type of sputtering system to coat</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">VWR 1415M Vacuum Oven&#160;</td>
			<td style="width:143px;">VWR International, Bridgeport, NJ, USA</td>
			<td style="width:169px;">N/A</td>
			<td>You can use any type of vacuum oven to physically crosslink the samples</td>
		</tr>
	</tbody>
</table>

designing silk-silk protein alloy materials for biomedical applications

纤维蛋白显示已用于在生物医学领域的各种应用，例如生物传感器，纳米医学，组织再生，和药物递送不同的序列和结构。设计基于这些蛋白之间的分子级材料相互作用将有助于产生新的多功能蛋白生物材料的合金具有可调性。这种合金材料系统也由于材料的生物降解性，生物相容性，并在体内可维持提供相比于传统的合成聚合物的优点。本文所用的野生柞蚕丝（ 柞蚕 ）和国内桑蚕丝（ 家蚕 ）的蛋白质混合物，例如，提供有关这些主题，包括如何计算方法，如何产生蛋白质合金预测蛋白质-蛋白质相互作用有用的协议解决方案，如何通过热分析验证合金系统，以及如何制造可变合金材料包括光学材料，具有衍射光栅，电子材料用的电路的涂层，以及用于药物释放和递送的药物的材料。这些方法可以提供重要信息，根据不同的蛋白质的合金设计新一代多功能生物材料。

典型的蛋白质-蛋白质相互作用（ 例如 ，蛋白质A和蛋白质B之间）可能含有电荷-电荷（静电）景点，氢键形成，疏水-亲水相互作用，以及偶极子，溶剂，抗衡离子和特定的熵效应这两种蛋白质的结构域（ 图2）3。因此，从根本上，我们可以预测这些互动通过计算机模拟的效果。 <img alt="图2" fo:content-width="5in" src="/files/ftp_...

Watch this Scientific Journal Video about Designing Silk-silk Protein Alloy Materials for Biomedical Applications at JoVE.com

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

勾兑是一种有效的方法来生成生物材料具有广泛的性能和功能相结合的。通过预测不同的天然丝蛋白之间的分子相互作用，新丝的丝蛋白合金平台具有可调的机械弹性，电响应，光学透明性，化学加工性，生物降解性，或热稳定性可以被设计。

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, ...

designing-silk-silk-protein-alloy-materials-for-biomedical

Research

JoVE Journal

Bioengineering

18.2K 次观看.  Rowan University. 勾兑是一种有效的方法来生成生物材料具有广泛的性能和功能相结合的。通过预测不同的天然丝蛋白之间的分子相互作用，新丝的丝蛋白合金平台具有可调的机械弹性，电响应，光学透明性，化学加工性，生物降解性，或热稳定性可以被设计。 

视频: Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Designing a Bio-responsive Robot from DNA Origami

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in parallel computing2,3 , recognize and bind molecular or cellular targets4,5 , catalyze chemical reactions6,7 , and generate calculated responses in a biological system8,9. Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures10-12, enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect.
Creating shapes from nucleic acids was first proposed by Seeman13, and several variations on this theme have since been realized using various techniques11,12,14,15 . However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami16. In this technique, the folding of a long (&gt;7,000 bases) single-stranded DNA 'scaffold' is directed to a desired shape by hundreds of short complementary strands termed 'staples'. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well17,18 .
 
The current paper will focus on the caDNAno 2.0 software19 developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient.
In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described20. This robot is 'robotic' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando21 to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here, we describe how DNA origami can be utilized to design a robotic robot capable of sensing biological cues and responding by shape shifting, subsequently relayed to a desired effect.

DNA折纸设计生物反应灵敏的机器人

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information1, they can be utilized in ...

科研

生物工程

Graphene Coatings for Biomedical Implants

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with nanometer thick layers of graphene for applications as a stent material. Graphene was grown on copper substrates via chemical vapor deposition and then transferred onto nitinol substrates. In order to understand how the graphene coating could change biological response, cell viability of rat aortic endothelial cells and rat aortic smooth muscle cells was investigated. Moreover, the effect of graphene-coatings on cell adhesion and morphology was examined with fluorescent confocal microscopy. Cells were stained for actin and nuclei, and there were noticeable differences between pristine nitinol samples compared to graphene-coated samples. Total actin expression from rat aortic smooth muscle cells was found using western blot. Protein adsorption characteristics, an indicator for potential thrombogenicity, were determined for serum albumin and fibrinogen with gel electrophoresis. Moreover, the transfer of charge from fibrinogen to substrate was deduced using Raman spectroscopy. It was found that graphene coating on nitinol substrates met the functional requirements for a stent material and improved the biological response compared to uncoated nitinol. Thus, graphene-coated nitinol is a viable candidate for a stent material.

Graphene offers potential as a coating material for biomedical implants. In this study we demonstrate a method for coating nitinol alloys with nanometer thick layers of graphene and determine how graphene may influence implant response.

生物医学植入物的石墨涂料

Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with ...

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

细胞外基质蛋白纳米纤维和纳米结构工程采用表面引发大会

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

制造超疏水高分子材料生物医学应用

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

一种快速定量荧光方法蛋白靶向小分子药物筛选

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

加工机械可调和生物活性金属支架的生物医学应用

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

RGD功能化水凝胶作为治疗应用的工具合成

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

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 ...

Extraction of Plant-based Capsules for Microencapsulation Applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

基于植物的提取胶囊微胶囊化应用

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

仿生纳米羟基磷灰石的快速混合制备生物医学应用

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...