Name: encapsulating cytochrome c in silica aerogel nanoarchitectures without metal nanoparticles while retaining gas-phase bioactivity
Uploaded: 2016-03-01T15:00:00.000+00:00
Duration: 11 min 6 s
Description: Watch this Scientific Journal Video about Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity at JoVE.com

这项工作和/或出版支持由艺术费尔菲尔德大学学院的科学研究所和科学，费尔菲尔德大学的教授研究基金，一科特雷尔高校科学奖从研究公司科学进步，艺术与科学费尔菲尔德大学学院和提供费尔菲尔德大学化学与生物化学系。我们非常感谢让·玛丽·华莱士在关​​于这个一般的研究领域很多有益的见解和建议。此外，我们延长一个非常特别感谢所有过去，现在和哈珀 - 莱特曼研究实验室未来研究人员本科。

安全眼镜或护目镜，实验室大衣和实验室手套在所有时间过程中被磨损。不要在没有安全眼镜或护目镜操作临界点干燥设备。含四甲氧基硅烷，甲醇，乙醇，丙酮和氨水所有解决方案应在通风橱内进行处理。 1.缓冲和细胞色素。Ç解决方案<ol><li>让〜750毫升pH为7，40 mM磷酸钾缓冲液中，首先通过称量出2.72克磷酸二氢钾并使用500ml的容量瓶中溶解在水中制成500毫升之0.04M的磷酸二氢钾。 </li><li>通过称出3.48克磷酸氢二钾的并使用500ml的容量瓶中溶解在水中制成500毫升之0.04M的磷酸氢二钾的。 </li><li>倾二元盐溶液放入大烧杯有搅拌棒，并开始搅拌在搅拌盘的溶液。 </li><li>慢慢加入的一元盐S部分olution到二元盐溶液中，同时监测pH用pH电极和计直到pH为7.00。约250-300毫升的单碱式盐溶液将被使用。 </li><li>称取玻璃闪烁瓶约0.023克CYT。c和地点。添加使用微量制备的磷酸钾缓冲液2,000微升，然后轻轻摇动该溶液混合，直到所有的固体，红细胞色素的。C具有溶解到溶液中，并没有颗粒物质存在。 </li><li>取20微升制备的细胞色素。C溶液并加入到1-cm光程塑料杯中。加入3 ml准备好的缓冲。 </li><li>在基准单元用缓冲采取紫外可见光谱，从300-700纳米。以106100 M -1 -1 409纳米，消光系数31（ε）使用细胞色素。C吸收（A）中，反应杯路径长度（L），和比尔-朗伯定律来确定浓度（C）该解决方案的（A =εlc）。 </li><li>回到计算原始制备的溶液的浓度。该混合制得细胞色素。C溶液典型地在浓度0.7毫之间至0.9。 </li><li>吹打117微升0.72毫准备CYT。C溶液变成闪烁瓶中稀释原准备CYT，C溶液800微升0.105毫米的。然后加入制备缓冲的800微升（683微升在这种情况下）的平衡。涡流混合。确切的量将根据原始制备细胞色素的确切浓度c到吸移管被作为（800微升* 0.105毫摩尔）计算而改变。C溶液作为细胞色素的体积。/（原始细胞色素。在4.0mm的C浓度）。 </li><li>在冰箱在2-8℃下保存原来的准备和稀释细胞色素。ç解决方案，直到准备使用长达两个星期。 </li></ol> 2.合成二氧化硅（SiO 2）的溶胶<ol><li>标签一次性50毫升聚丙烯烧杯"要阿克尔A&#39;。放置在分析天平上的摇动烧杯中，并用玻璃巴氏吸管1.88克四甲加入到烧杯中。零余额，然后吸取2.88克甲醇为"烧杯"。 </li><li>封面"烧杯"封口膜。 </li><li>标签一次性50毫升聚丙烯烧杯"烧杯B&#39;。添加磁性搅拌棒和地点在分析天平上的锅。使用玻璃吸管来添加0.75克水和3.00克甲醇。 </li><li>封面"烧杯B&#39;封口膜。 </li><li>开始搅拌在通风柜内部的搅拌板"烧杯B&#39;中的内容，然后用注射器插入5微升的28.0-30.0％的氢氧化铵溶液通过封口膜覆盖到混合物中，同时进行搅拌。 </li><li>只要步骤2.5完成后，"烧杯A"的内容添加到&#39;烧杯B&#39;搅拌该混合物20分钟，同时覆盖封口膜。 </li></ol> 3.准备凝胶模具注意:是时间，而该硅溶胶混合物在步骤2.6搅拌以制备凝胶的模具。 <ol><li>获得8-9聚丙烯闪烁瓶（16毫米×57毫米音量大小6.5毫升，与底部切掉）和相应的上限。放保鲜膜在小瓶的帽端以创建一个平坦表面的凝胶，以形成上和放置在盖在它确保保鲜膜撑帽内不变。 </li><li>线向上与帽式小瓶倒在工作台上，打开底部朝上。 </li></ol> 4.准备色素。ç -二氧化硅溶胶-凝胶<ol><li>经溶胶混合（步骤2.6）完成后，加入3毫升溶胶混合到一个干净的一次性50毫升聚丙烯烧杯中。 </li><li>使用玻璃巴斯德吸管缓慢下降500微升的0.105毫米稀释细胞色素。C溶液（步骤1.9 ​​制造）超过约1分钟的过程3ml的溶胶的混合物。确保轻轻地旋转混合物，同时加入细胞色素c。要避免形成大红色的团块。假设卷是添加剂，稀释500微升0.105毫细胞色素。C溶液的至3500微升，现在在溶胶是细胞色素。C的浓度，在理论上，15微米。 </li><li>吸管将0.5ml所得的细胞色素。Ç硅溶胶到每个准备好的模具。还吸取0.5毫升其余&#39;纯&#39;硅溶胶成一个或两个模具在超临界干燥过程作为对照样品来使用。 </li><li>覆盖用Parafilm模具的面朝上的开口，并把在冰箱（〜2-8℃）过夜或至少12小时，以产生溶胶 - 凝胶。 </li><li>就拿出来模具冰箱。从包含CYT一个模具的顶部取下封口膜Ç溶胶-凝胶。还请从下方盖和塑料包装。 </li><li>从洗瓶加入一些乙醇倒入模具后，用注射器活塞的圆盘结束小心地将凝胶从模具中，并倒入干净加入20毫升玻璃闪烁瓶续癌宁大约5毫升乙醇。 </li><li>重复此凝胶去除过程（步骤4.5和4.6），直到所有的细胞色素的。Ç凝胶加入到小瓶中，并全部硅胶加入到单独的管形瓶中。如果CYT。ç凝胶多个浓度已经做出，一定要分开的小瓶中存放在一起像凝胶。然后填写小瓶与2-8°C之间乙醇，帽子和存储的顶部。 </li><li>每四小时全天，从冰箱取出凝胶，倒出乙醇落凝胶并用新鲜乙醇替换。 </li><li>期间另外处理三天，浸没在丙酮湿溶胶凝胶，倾析并加入新鲜的丙酮，一天三次。 </li></ol> 5.超临界干细胞色素。ç -二氧化硅溶胶-凝胶<ol><li>由连接的环行器的温度设定至8℃冷却的临界点干燥装置（ 见图1），以10℃。 </li><li>一旦该装置具有r个eached 10℃，通过填充转印船用丙酮和密封它的装置内执行对装置的泄漏测试。 <ol><li>打开装置的填充阀并加入二氧化碳直至该装置是半满。 </li><li>关闭进水阀，并认真听取了在门和阀门，其中O型圈密封，或可能会恶化嘶嘶。 </li><li>如果泄漏被发现更换所有O型圈或者盖章。 </li></ol></li><li>在完成泄漏测试之后，打开排水阀以释放丙酮和二氧化碳转化为一个通风橱的漏极。然后，从装置中取出传送舟。 </li><li>确保设备无泄漏后，小心地倒在闪烁瓶湿凝胶，与大多数丙酮，到转会船的三大段长沿（样本篮子或纱布盖是不是必要的）。微妙推并与钳舟内移动的凝胶，以确保所有的凝胶完全浸没我ñ丙酮。如果需要，密封船上的设备内部之前添加更多丙酮。 </li><li>打开装置的充水阀以添加二氧化碳，然后打开排水阀，只要丙酮与二氧化碳混合，观察到通过设备窗口被下沉到该装置的底部释放丙酮五分钟。会发生丙酮此渗滤到前下方的装置完全充满二氧化碳，所以填充阀应该保持开放到排水的过程中所需的程度，使得该装置将继续填充即使在漏极是开放的。 </li><li>关闭排水阀。保持裂了开来稍微进水阀。 </li><li>五分钟后，打开排水阀五分钟一次调整填充阀以使该装置保持在整个排水时间充分要足够打开。关闭排水阀，保持进水阀裂了开来，然后重复此排水一步一个更多时间的五分钟后。 </li><li>以下这些前三个排水步骤中，打开5分钟的漏极在时间大约每40分钟以上的至少6小时的跨度，以确保由液体二氧化碳在凝胶内完全替换丙酮中。总是调整填充阀各排水过程中有足够的开放，使得在装置中的液面排出期间从不低于船的顶部。 </li><li>一旦排水步骤后，关闭进水阀，以及使电平通过设备窗户看只是船上的插脚上面仍然可见排出液态二氧化碳。 </li><li>该装置连接的环行器的温度设定为40℃，以确保在液体二氧化碳升高高于其临界温度和压力（T C = 31°C，P C = 7.4兆帕）。 </li><li>后约15分钟，通过设备窗口作为液体menisc观察从液体到超临界流体的过渡我们在船的叉以上消失。允许的平衡时间至少15分钟，然后打开放空阀少量开始释放超临界流体。 </li><li>在大约45分钟的过程中，继续逐步打开排气阀越走越宽，使释放流体的稳定，但非常低的嘶嘶声可闻及观察压力表缓慢降低至零。 </li><li>该装置的压力已经到零后，打开装置的门，取出舟，并使用镊子来放置新干燥气凝胶在干净的玻璃闪烁瓶中。 </li></ol> 6.检定色素。ç -二氧化硅气凝胶，用紫外-可见和圆二色（CD）光谱<ol><li>准备一个纸板平台保持气凝胶整料在紫外可见分光光度计或CD分光计的光束路径。 <ol><li>切出2.5cm的×2.5cm的片轻质纸板（如从LABO一盒纸板ratory组织），对折，切半山腰上折，再对折了两片，通过削减创建的，回来了。 </li><li>切出5厘米×5厘米长的轻量级纸板，在中间1.5×1.5cm的方孔。然后使用黑色电胶带到保持的尺寸减小至0.5cm×0.5厘米</li><li>胶带使小弯曲表面为气凝胶整料创建针对5厘米×5厘米长纸板的折叠和切割纸板的折翼以直接在0.5厘米×0.5厘米的洞的前面坐（参见图2） 。然后用胶带的紫外可见分光光度计背面一块纸板所以孔是在与束路径线。 </li></ol></li><li>测量凝胶，这将被用于路径长度，用千分尺的厚度。 </li><li>放置纸板平台上的一个凝胶和从在紫外可见分光光度计的基准室与空气300-800毫微米测量的光谱。 </li><li>适合多项式，A = A </青霉&gt;λn，用来对波长（λ）区，其中所述吸光度（A），主要是由于背景散射，〜700-800纳米。的一个系数是通常适合之间的数〜1×10 8 1×10 6和n个系数是通常配合到2号和3之间〜。 </li><li>计算散射在其它波长，采用的系数，A和n，从拟合获得。 </li><li>减去从原始光谱这个计算散射背景吸收获得散射光谱校正。 </li><li>适合用在370的区域中的高斯曲线散射减去频谱使用适当的软件（GRAMS / AI 8.0），以确定峰高，峰中心，和气凝胶的索瑞峰的峰宽490纳米。 </li><li>使用凝胶的测得的厚度为路径长度（L），的106100 M中的消光系数31（ε）应用比尔-朗伯定律-1 -1 </SUP&gt;和索瑞峰高（A）来计算气凝胶（A =εlc）CYT，C的浓度（C）。 </li><li> C的浓度比较计算出的细胞色素。向浓缩理论上在凝胶（15微米），以确定该气凝胶内细胞色素。C的生存能力。典型百分之存活率接近100％，但应该指出的是，这些存活率只是估计，因为计算是基于在溶液中31细胞色素。Ç这被推定为比细胞色素的消光系数略有不同的消光系数。 c。在不知道的气凝胶。 </li><li>在CD仪器至少5分钟运行氮气接通灯之前。 </li><li>用胶带将纸板支架到CD分光计所以孔是在与束路径线。 </li><li>在100纳米/分测量与纸板支架没有从350-500纳米连续波长空白频谱，取三次扫描的平均。 </li><li>放置纸板平台上的一个凝胶（先前在步骤6.2测得的厚度），并在100纳米/分钟测量从350-500纳米光谱，服用的三次扫描的平均值。 </li><li>重复紫外可见和CD测量所有感兴趣的气凝胶单块。 </li></ol> 7.检测一的存在氧化氮（NO）气体与细胞色素。Ç -二氧化硅气凝胶注意:由于没有工作是危险的，根本没有气体应在通风橱处理或排放到通风橱。持续暴露于否是有毒的组织作为剧毒二氧化氮和/或四氧化二氮时否开始与空气接触将形成。当否开始与水接触时的热和腐蚀性烟雾也生产。 <ol><li>放置一个8升的一氧化氮气缸（10％的一氧化氮，90％的氮）在通风良好的通风橱并调节压力至4磅。 </li><li>连接管连接到两个氧化氮汽缸和一个氮气缸（压力设置为6 ps的i）和连接起来的管道，以T型阀的端部（参见图3a）。 </li><li>选择用于实验的气凝胶整料和测量的厚度（或路径长度）用千分尺。 </li><li>放置在与塑料盖的一次性比色皿气凝胶（〜3mm厚），并把反应杯放入分光光度计。切气凝胶略微如果需要，以适应在反应杯中。 </li><li>插入两个注射器的针插入反应杯的塑料帽，一个连接到T型阀的输出，和一个连接到一个管子用作排气进入通风柜（参见图3b）。用封口膜向针密封到管和盖的试管中。 </li><li>放置在参考单元的空的一次性杯中。 </li><li>调整气凝胶反应杯的位置以确保该气凝胶开始实验之前在于光束路径。 </li><li>取的初始光谱从800至300纳米。 </li><li>监测在4吸光度之间的​​差14纳米，并在408处的吸光度而转动T型阀以设定的时间间隔，确保在任何时候是氮或氧化氮/氮气混合物如此之高，流量的氮和一氧化氮/氮气混合物之间进行切换气凝胶中的反应杯移到周围。 </li><li>取的最终光谱从800至300纳米，一旦曝光周期结束。 </li><li>重复该过程具有三个至4个整料以获得平均检测响应。 </li></ol>

<ol>
	<li>Pettigrew, G. W., Moore, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cytochromes c. Biological Aspects. , (1987).</li><li>Moore, G. R., Pettigrew, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cytochromes c. Evolutionary, Structural, and Physicochemical Aspects. , (1990).</li><li>Scott, R. A., Mauk, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cytochrome c: A Multidisciplinary Approach. , (1996).</li><li>Wallace, J. M., Rice, J. K., Pietron, J. J., Stroud, R. M., Long, J. W., Rolison, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silica+nanoarchitectures+incorporating+self-organized+protein+superstructures+with+gas-phase+bioactivity.">Silica nanoarchitectures incorporating self-organized protein superstructures with gas-phase bioactivity.</a> Nano Lett. 3 (10), 1463-1467 (2003).</li><li>Wallace, J. M., Dening, B. M., Eden, K. B., Stroud, R. M., Long, J. W., Rolison, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver-colloid-nucleated+cytochrome+c.+superstructures+encapsulated+in+silica+nanoarchitectures.">Silver-colloid-nucleated cytochrome c. superstructures encapsulated in silica nanoarchitectures.</a> Langmuir. 20 (21), 9276-9281 (2004).</li><li>Wallace, J. M., Stroud, R. M., Pietron, J. J., Long, J. W., Rolison, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+particle+size+and+protein+content+on+nanoparticle-gold-nucleated+cytochrome+c.+superstructures+encapsulated+in+silica+nanoarchitectures.">The effect of particle size and protein content on nanoparticle-gold-nucleated cytochrome c. superstructures encapsulated in silica nanoarchitectures.</a> J.Non-Cryst. Solids. 350, 31-38 (2004).</li><li>Rolison, D. R., Wallace, J. M., Pietron, J. J., Rice, J. K., Stroud, R. M. U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> US Patent. , (2007).</li><li>Harper-Leatherman, A. S., Wallace, J. M., Rolison, D. R., Minteer, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochrome+c.+stabilization+and+immobilization+in+aerogels.">Cytochrome c. stabilization and immobilization in aerogels.</a> Enzyme Stabilization and Immobilization: Methods and Protocols. 679, 193-205 (2011).</li><li>Harper-Leatherman, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+procedure+for+encapsulating+cytochrome+c.+in+silica+aerogel+nanoarchitectures+while+retaining+gas-phase+bioactivity.">Simplified procedure for encapsulating cytochrome c. in silica aerogel nanoarchitectures while retaining gas-phase bioactivity.</a> Langmuir. 28 (41), 14756-14765 (2012).</li><li>Hitihami-Mudiyanselage, A., Senevirathne, K., Brock, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+phosphide+nanocrystals+into+porous+networks:+Formation+of+InP+gels+and+aerogels.">Assembly of phosphide nanocrystals into porous networks: Formation of InP gels and aerogels.</a> ACS Nano. 7 (2), 1163-1170 (2013).</li><li>Fricke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Aerogels. , (1986).</li><li>H&#252;sing, N., Schubert, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aerogels-airy+materials:+chemistry,+structure,+and+properties.">Aerogels-airy materials: chemistry, structure, and properties.</a> Angew. Chem. Int. Edit. 37 (1-2), 22-45 (1998).</li><li>Aegerter, A. M., Leventis, N., Koebel, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Aerogels Handbook. , (2011).</li><li>Kazuyoshi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+aerogel+science+and+technology.">Recent progress in aerogel science and technology.</a> Adv. Porous Mater. 1 (2), 147-163 (2013).</li><li>Leventis, N., Elder, I. A., Anderson, M. L., Rolison, D. R., Merzbacher, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Durable+modification+of+silica+aerogel+monoliths+with+fluorescent+2,7-diazapyrenium+moieties.+Sensing+oxygen+near+the+speed+of+open-air+diffusion.">Durable modification of silica aerogel monoliths with fluorescent 2,7-diazapyrenium moieties. Sensing oxygen near the speed of open-air diffusion.</a> Chem. Mater. 11 (10), 2837-2845 (1999).</li><li>Plata, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aerogel-platform+optical+sensors+for+oxygen+gas.">Aerogel-platform optical sensors for oxygen gas.</a> J. Non-Cryst. Solids. 350, 326-335 (2004).</li><li>Rolison, D. R., Pietron, J. J., Long, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+sensitivity,+specificity,+and+time+signature+of+sensors+through+architectural+design+on+the+nanoscale.">Controlling the sensitivity, specificity, and time signature of sensors through architectural design on the nanoscale.</a> ECS Trans. 19 (6), 171-179 (2009).</li><li>Carroll, M. K., Anderson, A. M., Aegerter, A. M., Leventis, N., Koebel, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aerogels+as+platforms+for+chemical+sensors.">Aerogels as platforms for chemical sensors.</a> Aerogels Handbook. , 637-650 (2011).</li><li>Rolison, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalytic+nanoarchitectures-The+importance+of+nothing+and+the+unimportance+of+periodicity.">Catalytic nanoarchitectures-The importance of nothing and the unimportance of periodicity.</a> Science. 299 (5613), 1698-1701 (2003).</li><li>Pietron, J. J., Stroud, R. M., Rolison, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+three+dimensions+in+catalytic+mesoporous+nanoarchitectures.">Using three dimensions in catalytic mesoporous nanoarchitectures.</a> Nano Lett. 2 (5), 545-549 (2002).</li><li>Anderson, M. L., Morris, C. A., Stroud, R. M., Merzbacher, C. I., Rolison, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colloidal+gold+aerogels:+Preparation,+properties,+and+characterization.">Colloidal gold aerogels: Preparation, properties, and characterization.</a> Langmuir. 15 (3), 674-681 (1999).</li><li>Anderson, M. L., Stroud, R. M., Rolison, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+the+activity+of+fuel-cell+reactions+by+designing+three-dimensional+nanostructured+architectures:+Catalyst-modified+carbon-silica+composite+aerogels.">Enhancing the activity of fuel-cell reactions by designing three-dimensional nanostructured architectures: Catalyst-modified carbon-silica composite aerogels.</a> Nano Lett. 3 (9), 1321 (2003).</li><li>Chervin, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+by+design:+vanadium-substituted+iron+oxide+nanoarchitectures+as+cation-insertion+hosts+for+electrochemical+charge+storage.">Defective by design: vanadium-substituted iron oxide nanoarchitectures as cation-insertion hosts for electrochemical charge storage.</a> J. Mater. Chem. A. 3 (22), 12059-12068 (2015).</li><li>Ellerby, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+of+proteins+in+transparent+porous+silicate-glasses+prepared+by+the+sol-gel+method.">Encapsulation of proteins in transparent porous silicate-glasses prepared by the sol-gel method.</a> Science. 255 (5048), 1113-1115 (1992).</li><li>Massari, A. M., Finkelstein, I. J., Fayer, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+proteins+encapsulated+in+silica+sol-gel+glasses+studied+with+IR+vibrational+echo+spectroscopy.">Dynamics of proteins encapsulated in silica sol-gel glasses studied with IR vibrational echo spectroscopy.</a> J. Am. Chem. Soc. 128 (12), 3990-3997 (2006).</li><li>Ray, A., Feng, M., Tachikawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+electrochemistry+and+Raman+spectroscopy+of+sol-gel-encapsulated+myoglobin.">Direct electrochemistry and Raman spectroscopy of sol-gel-encapsulated myoglobin.</a> Langmuir. 21 (16), 7456-7460 (2005).</li><li>Blyth, D. J., Aylott, J. W., Richardson, D. J., Russell, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sol-gel+encapsulation+of+metalloproteins+for+the+development+of+optical+biosensors+for+nitrogen-monoxide+and+carbon-monoxide.">Sol-gel encapsulation of metalloproteins for the development of optical biosensors for nitrogen-monoxide and carbon-monoxide.</a> Analyst. 120 (11), 2725-2730 (1995).</li><li>Lan, E. H., Dave, B. C., Fukuto, J. M., Dunn, B., Zink, J. I., Valentine, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+sol-gel+encapsulated+heme+proteins+with+chemical+sensing+properties.">Synthesis of sol-gel encapsulated heme proteins with chemical sensing properties.</a> J. Mater. Chem. 9 (1), 45-53 (1999).</li><li>Miller, J. M., Dunn, B., Valentine, J. S., Zink, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+conditions+for+encapsulating+cytochrome+c.+and+catalase+in+SiO2+sol-gel+materials.">Synthesis conditions for encapsulating cytochrome c. and catalase in SiO2 sol-gel materials.</a> J. Non-Cryst. Solids. 202 (3), 279-289 (1996).</li><li>Ronda, L., Bruno, S., Faggiano, S., Bettati, S., Mozzarelli, A., Poole, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+binding+to+heme+proteins+in+solution,+encapsulated+in+silica+gels,+and+in+the+crystalline+state.">Oxygen binding to heme proteins in solution, encapsulated in silica gels, and in the crystalline state.</a> Methods in Enzymology. 437, 311-328 (2008).</li><li>Margoliash, E., Frohwirt, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrum+of+Horse-Heart+Cytochrome+c.">Spectrum of Horse-Heart Cytochrome c.</a> Biochem. J. 71 (3), 570-572 (1959).</li></ol>

作者宣称，他们没有竞争的经济利益。

如上所述，这个过程一直产生可行的细胞色素。ç气凝胶中封装。细胞色素c。将气凝胶中的浓度可从5至15微米，气凝胶可从4.4至70毫摩尔磷酸而对蛋白质生存能力严重不利影响改变内包封的初始细胞色素。C溶液的缓冲液浓度而变化。然而，峰值中心和特色CYT的峰宽c。在气凝胶索瑞峰是最接近他们是什么在溶液中细胞色素。c当细胞色素c。在气凝胶被封装从40毫米缓冲9解决方案。 的细胞色素的合成。Ç-SiO 2气凝胶是由一些起始试剂的年龄的影响。甲醇，四甲氧基硅烷，和氢氧化铵溶液都是吸湿性和应每一到两个月取代。增加的水，在积聚这些试剂随着时间的推移会影响凝胶的结构特征和溶胶至凝胶转变的时间。 当进行超临界干燥，临界点干燥设备的传输船可容纳多达18厚0.5厘米，直径1厘米的凝胶。在协议部分中概述，一个特定的填充和排放过程应该遵循二氧化碳转移到溶胶 - 凝胶。要注意，在排水协议的开始是很重要的，二氧化碳和丙酮的排出混合物在该引流管冻结僵硬与湿气冷凝成冰在外面如此高的速率流动。引流出混合物含有一些水，因为丙酮是不是无水的，并且这种水可能偶尔冻结的引流管堵塞实际的程度。有必要观看这种堵塞和监听流动的停止。排水阀应关闭几分钟，以便如果检测到阻塞的阻塞会熔化。在最坏的情况下，如果排水阀没有关闭，一个堵塞会引起这么大的压力，建立了排水管强行弹出关闭设备。前几个漏极周期后，大部分的丙酮将已冲洗的装置，和湿冰块的发生将显着降低。放电会逐渐类似于干冰作为排水协议用丙酮存在下（如气味）的任何残余证据继续变得检测不到由排水过程结束。 该装置中的二氧化碳已从液体转变到超临界流体后及放出过程已经开始，有必要在至少45分钟，以释放流体以缓慢的速度如在步骤9所示。释放的较高的速率可以在气凝胶内减少细胞色素的生存能力。C（ 如图9）和气凝胶本身实际上可能掰开作为第Ë流体七嘴八舌从凝胶逃脱。在一般情况下，即使当气凝胶保持打开装置的门后完好无损，仔细并轻轻处理它们是重要的，因为它们是易碎，并且可以容易破裂。 被浇沿着细胞色素控制硅胶。Ç-SiO 2凝胶超临界干燥后使用，以确定是否将二氧化碳转移到凝胶是成功的。有时，细胞色素。Ç-SiO可能会出现2凝胶混浊，而且重要的是要确定这是否是由于不完整的溶剂转移，或者它可能做的菜园，C浓度或缓冲封装在凝胶内。如果硅胶无细胞色素。Ç出现在整个具有均匀的，半透明的外观，这可作为证据溶剂转移发生完全即使细胞色素。Ç-SiO 2凝胶具有一些混浊给他们。在硅胶内云量没有细胞色素，C干燥后表明，一些丙酮排气中保持凝胶内。 在协议中部分指出，重要的安全预防措施需要采取与一氧化氮（NO）时。来检测NO使用气凝胶，这是必要的，以密封试管非常好，用尽流过的气凝胶成通风柜气体。可替代地，整个分光光度计可移动到与NO气体筒作为附加的预防措施沿通风柜限制暴露NO气体。在与空气接触NO会立即产生剧毒二氧化氮，四氧化二氮或两者兼而有之。 NO也可以与水反应产生热量和腐蚀性烟雾。因此，持续暴露于NO可能导致直接组织的毒性。 当使用细胞色素。Ç-SiO 2气凝胶以检测一氧化氮的存在下，Soret带最初将在〜408 nm和将转向在一氧化氮的存在下〜414纳米。切换回氮气后，Soret带应转回到〜408纳米为中心。这也可能是可以使用细胞色素。Ç-SiO 2气凝胶以检测诸如一氧化碳27其它配体的存在。 不同公开的程序包括金或银纳米颗粒与细胞色素c。在溶液与溶胶混合和超临界干燥，形成气凝胶4-8之前结合附加的步骤。比较细胞色素的紫外可见光谱。Ç包封在用金属纳米粒子到细胞色素的气凝胶。Ç包封在气凝胶无金属纳米颗粒显示了这两种类型的封装技术产生气凝胶内细胞色素。类似生存能力的C（ 图5） 。然而，封装金属纳米粒子的细胞色素。c是略高于CYT更稳定。Ç封装Ð没有气凝胶9内的金属纳米粒子。这两种类型的细胞色素。Ç气凝胶的CD光谱也类似，虽然两者从细胞色素c。将在缓冲器指示部分解折叠的气凝胶内细胞色素。C的光谱（ 图7）不同。上细胞色素以前的报告。Ç包封在气凝胶表明圆二色光谱是最有可能评估蛋白质的最外层，展现在与硅胶接触，无论是金属纳米颗粒成核的多层细胞色素内。C结构或组织松散结构，其形成当没有金属纳米颗粒存在于气凝胶4,9。如由紫外-可见光谱，虽然测量的气凝胶内任一类型的自组织结构的内部的大部分细胞色素c。将保持折叠。本文描述的sans纳米颗粒的协议的优点是，昂贵的购买或金属的费时合成纳米颗粒是没有必要的。蛋白质已不经常被成功气凝胶内包封，所以此过程中，它可能导致一个更一般的方法的开发在气凝胶与未来的生物分析设备潜在意义包封其它蛋白质是重要的。

色素c（cyt的。c）为参与机体的细胞呼吸反应的关键的电子转移蛋白。它已被证明是参与细胞凋亡，细胞死亡的受控的形式，并且它可以检测这样小的毒性分子作为一氧化氮和一氧化碳1-3。一氧化氮（NO）起着多种发生在神经，心血管和免疫系统的生理过程的作用。而细胞色素。Ç通常需要缓冲至pH中性值保持结构完整和活性的含水环境中，有研究表明，细胞色素。C能够保持其结构和功能在一定的条件下4-9称为气凝胶的固体材料。 气凝胶通常是由合成的溶胶 - 凝胶金属氧化物（尽管金属氧化物气凝胶是很常见的，碳和其他类型的气凝胶的已经合成形成高度多孔的材料，其中一个例子是磷化铟AERogels）10和以这样的方式进行干燥，这些溶胶-凝胶，多孔固体基质保持不变11-14。所有的固体气凝胶的毛孔造成多少可用的表面积使得气凝胶，其表面的反应是很重要的应用程序非常有用的。当化学或生物化学功能气凝胶纳米结构内组装时，它已被证明的物理孔隙率和气凝胶的增强表面积有助于提高传感器，以及用于电池电极，燃料电池，以及超级电容器应用11,15-23 。以干燥在留下不变多孔固体基质的方式气凝胶，这是典型的以去除后通过超临界溶剂提取的溶胶 - 凝胶合成保留在孔中的溶剂。可以由表面张力为从凝胶的溶剂蒸发引起的任何孔隙崩溃，因为在超临界干燥，液 - 汽界面从未形式被最小化。 。c将要包封在已保持湿润或已ambiently 24-30干燥溶胶-凝胶。在溶胶 - 凝胶包封的生物分子所再超临界干燥的报告，以形成气凝胶由于必要的处理，可以是有害的许多蛋白质​​的结构是罕见。在细胞色素c。将的情况下，一定的条件下使得能够保留的细胞色素c。要检测和气凝胶内气相氧化氮反应的能力。一旦在气凝胶稳定，气凝胶的高品质的孔结构有利于细胞色素。c和一氧化氮4,8,9之间的反应。细胞色素。C可气凝胶内首先它围绕金或银纳米粒子多层溶液4-8关联进行封装。这些多层上层建筑用于保护气凝胶基质中的蛋白质。在最近approac我们开发小时，当该蛋白质浓度和缓冲力与其它合成条件一起控制，细胞色素。Ç保留了气凝胶内完整性，即使没有金属纳米颗粒初始关联9。 的合成开始尽可能多的气凝胶合成开始通过混合二氧化硅溶胶 - 凝胶前体的一段时间内。它是一组混合该细胞色素。c为加入作为缓冲溶液到该混合物中的时间之后。凝胶化则发生以形成其中的孔填充有水，甲醇，剩余的反应物和副产物的多孔硅固体的结构。该液体填充的孔可以用各种溶剂，通过一系列溶剂交换的冲洗出来，用液体二氧化碳服用临界点干燥装置内发生的最后的交流保持在低的温度。将上述的二氧化碳的临界温度（31.1℃）的凝胶便于作为形成可排放到形成干的，高度多孔气凝胶加压装置内upercritical流体。相对于其他的溶剂所需的二氧化碳以形成超临界流体的相对低的温度是有利的，因为它保持在低于它可能变性的温度下的蛋白质。 我们的无金属纳米颗粒的方法来在气凝胶包封细胞色素。c是有利的，因为它是一个简单的过程，可能会导致更普遍适用的协议的发展用于封装其他蛋白质为好。许多蛋白质可能不与在细胞色素相同的方式的金属纳米颗粒相互作用。Ç确实与金属纳米颗粒合成或购买增加了额外的时间和费用的程序。在气凝胶封装蛋白的报道很少使这个过程的发展向前迈进了一步显著找到一个更一般的程序中，可能会帮助我气凝胶封装其他蛋白质ñ未来潜在的生物分析设备。 这个手稿的协议部分概述了如何合成硅溶胶-凝胶，封装细胞色素。ç到这些溶胶-凝胶，干燥这些复合溶胶-凝胶形成气凝胶，采用紫外可见和圆二色光谱表征这些bioaerogels和检测存在与这些bioaerogels气相一氧化氮。色素。C已被成功地包封在气凝胶首先溶解在磷酸盐缓冲液中的4.4至70毫水溶液时。然而，在气凝胶优化蛋白质的结构，已经发现包封的40mM磷酸盐缓冲细胞色素的解决方案时，所造成的。ç制备负载气凝胶细胞色素。在5范围C的浓度至15μM9。因此，下面给出的协议是使用导致加载细胞色素细胞色素c。将40 mM磷酸盐缓冲溶液合成气凝胶。在15微米的气凝胶C的浓度。&lt;/ P&gt;

<table><tbody><tr><td>Potassium phosphate, monobasic</td><td>Fisher Scientific</td><td>P285-500</td><td>Certified ACS (also possible to use sodium phosphate monobasic)</td></tr><tr><td>Potassium phosphate dibasic anhydrous</td><td>Fisher Scientific</td><td>P288-500</td><td>Certified ACS (also possible to use sodium phosphate dibasic)</td></tr><tr><td>Water</td><td>Millipore Direct-Q</td><td></td><td>18 M&amp;Omega; cm</td></tr><tr><td>pH meter and electrode</td><td>Denver Instrument</td><td>UB-10</td><td></td></tr><tr><td>Cytochrome c from equine heart</td><td>Sigma Aldrich</td><td>C7752-100MG&amp;nbsp;</td><td>&amp;ge;95% based on Mol. Wt. 12,384, used as received and stored at -20&amp;nbsp;&amp;deg;C</td></tr><tr><td>Glass scintillation vials</td><td>Wheaton</td><td>03-341-25J</td><td>20 ml, O.D. x height (with cap): 28&amp;nbsp;mm x 61&amp;nbsp;mm</td></tr><tr><td>Disposable cuvette</td><td>Fisher Scientific</td><td>14-955-126</td><td>methacrylate, 10 mm x 10 mm x 45&amp;nbsp;mm</td></tr><tr><td>Ultraviolet Visible Spectrophotometer</td><td>Shimadzu</td><td>UV-1800</td><td>Uses UVProbe v 2.33 software</td></tr><tr><td>Circular dichroism spectrometer (or spectropolarimeter)</td><td>JASCO</td><td>J-810</td><td></td></tr><tr><td>Isotemp Laboratory Refrigerator</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Polypropylene disposable beakers</td><td>Fisher Scientific</td><td>01-291-10</td><td>50 ml</td></tr><tr><td>Tetramethylorthosilicate (also known as tetramethoxysilane, TMOS)</td><td>Sigma Aldrich</td><td>218472-500G</td><td>98% purity</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A457-4</td><td>GC Resolv grade</td></tr><tr><td>Ammonium hydroxide solution</td><td>Sigma Aldrich</td><td>221228-25ML-A</td><td>ACS reagent, 28.0%-30.0%</td></tr><tr><td>General purpose polypropylene scintillation vials</td><td>Sigma Aldrich</td><td>Z376825-1PAK</td><td>16 mm x 57 mm, volume size 6.5&amp;nbsp;ml, slice off bottom with sharp knife or razor</td></tr><tr><td>generic plastic wrap</td><td>various</td><td></td><td></td></tr><tr><td>Parafilm M laboratory wrapping film</td><td>Fisher Scientific</td><td>S37440</td><td></td></tr><tr><td>Plastic syringe plunger</td><td>various</td><td></td><td>use syringe plunger from 3 ml syringe</td></tr><tr><td>Ethyl alcohol</td><td>Acros</td><td>61509-0040</td><td>Absolute, 200 proof, 99.5% A.C.S. reagent</td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>A949-4</td><td>HPLC grade</td></tr><tr><td>Critical point drying apparatus</td><td>Quorum Technologies</td><td></td><td>E3000 Series</td></tr><tr><td>Circulator</td><td>Fisher Scientific</td><td>Isotemp 3016</td><td></td></tr><tr><td>Carbon dioxide cylinder</td><td>Tech Air</td><td></td><td>siphon tube</td></tr><tr><td>Micrometer</td><td>Central Tool Company</td><td></td><td></td></tr><tr><td>GRAMS/AI 8.0 software</td><td>Thermo Electron Corporation</td><td></td><td></td></tr><tr><td>Nitrogen cylinder</td><td>Tech Air</td><td></td><td>Another inert gas could be substituted</td></tr><tr><td>10% nitric oxide/90% nitrogen cylinder</td><td>Airgas</td><td></td><td></td></tr><tr><td>Tygon tubing</td><td>various</td><td></td><td></td></tr><tr><td>T-switch valve</td><td>various</td><td></td><td></td></tr><tr><td>syringe needles</td><td>various</td><td></td><td></td></tr></tbody></table>

encapsulating cytochrome c in silica aerogel nanoarchitectures without metal nanoparticles while retaining gas-phase bioactivity

应用，例如传感器，电池和燃料电池已经通过使用高度多孔气凝胶当被封装的气凝胶内官​​能化合物得到改善。然而，存在被处理以形成气凝胶上的溶胶 - 凝胶内包封蛋白的报道很少。一种用于在硅石包封色素c（cyt的。c）议事（SiO 2）的被超临界处理以形成与一氧化氮气相活性bioaerogels溶胶-凝胶（NO）的呈现。色素。c为加入到在控制的蛋白质浓度的混合硅溶胶和缓冲力的条件。然后将溶胶混合物凝胶化和填充凝胶孔中的液体是通过一系列与液体二氧化碳溶剂交换的替代。二氧化碳被带到其临界点，并放空，以形成具有细胞色素干的气凝胶。Ç包封内。这些bioaerogels的特征在于用紫外 - 可见光谱的ð圆二色光谱，并且可以用来检测气相氧化氮的存在。这个过程的成功依赖于调节细胞色素。C的浓度和缓冲液浓度，并且不需要其他部件，例如金属纳米颗粒。它可能会封装用类似的方法使潜在的未来的生物分析设备开发此过程中重要的其他蛋白质。

所描述的过程导致含有活细胞色素。Ç气凝胶。截至年底出台规定，细胞色素。C可从缓冲溶液，范围从4.4到70毫米的磷酸盐进行封装。细胞色素的例子。Ç -二氧化硅（细胞色素。Ç-SiO 2）气凝胶从含有不同的缓冲浓度的溶液制成示于图4中 ，所有的凝胶是相对半透明的，与来自70制成凝胶毫缓冲最不透明。 的细胞色素。在不同条件下c中的光谱的比较示于图5。一个典型谱（ 图5c）示出了周围408处的大的Soret峰为细胞色素。Ç-SiO 2气凝胶和非常相似细胞色素的光谱c。在溶液（ 图5a）。此外，细胞色素的光谱。Ç气凝胶内封装有金属纳米颗粒也显示（ 图5b）和细胞色素。Ç-SiO 2气凝胶光谱类似于该频谱为好。当细胞色素。Ç-SiO 2气凝胶被暴露于一氧化氮，所述的Soret峰的典型换档观察（ 图5d）。 用于从细胞色素制成凝胶的UV-vis光谱。在不同缓冲液浓度C溶液的示于图6中 ，所有这些凝胶的显示特性紫外可见光谱特征表明细胞色素。c不是在凝胶内的变性的状态。然而，凝胶的降低半透明度较低信噪比为这些光谱从70毫缓冲结果制成。 细胞色素的CD光谱。ç-SiO 2气凝胶相似的细胞色素的光谱。Ç </eM&gt;用的金属纳米粒子气凝胶内包封，而这两种类型的气凝胶光谱从在缓冲溶液中细胞色素c。将一个光谱差异（ 图7）。 图8示出了用于细胞色素一个典型的一氧化氮的监控响应。Ç-SiO 2气凝胶并且还含有除细胞色素。C金属纳米颗粒相应的气凝胶。在414处的吸光度，并且在408纳米之间的差被看作是增加，然后当该凝胶相继分别暴露于一氧化氮，然后氮降低。 如果超临界二氧化碳没有以足够慢速率，细胞色素的生存能力释放c。将形成的气凝胶内将受到损害。这是通过在不同的释放二氧化碳形成凝胶后比较所得紫外可见光谱显示速率（ 图9）。 <img alt="图1" src="/files/ftp_upload/53802/53802fig1.jpg" /> 图1:临界点干燥装置从上述（A）前和（B）中所示的临界点干燥装置背面与旁边显示装置的背面的转印船和设备的门。 <img alt="图2" src="/files/ftp_upload/53802/53802fig2.jpg" /> 图2:纸板平台组装纸板平台用于在仪器的光束的路径保持气凝胶。 <img alt="图3" src="/files/ftp_upload/53802/53802fig3.jpg" /> 图3:一氧化氮传感设置的一氧化氮传感设置示出包括（A）中的通风橱括10％的一氧化氮。，90％的氮气气缸，油管，和T型阀，和（B）具有插入针的杯中。 <img alt="图4" src="/files/ftp_upload/53802/53802fig4.jpg" /> 图4:样品色素 C -SiO 2 气凝胶气凝胶在4.4毫米，40毫米，和70 mM磷酸钾缓冲液包封15μM色素C在比较一毛示从左至右。这些气凝胶大约0.2-0.5厘米高。转载许可9。 <img alt="图5" src="/files/ftp_upload/53802/53802fig5.jpg" /> 图5:色素 C -SiO 2 气凝胶光谱中的（a）50mM磷酸盐缓冲溶胶15微米的细胞色素c可见光谱。ution; （ 二 ）金（5纳米）〜色素C -SiO 2气凝胶。 （ 三 ）色素C -SiO 2气凝胶（暴露在空气中）。 （ 四 ）CYT。ç-SiO 2气凝胶（暴露一氧化氮3.5分钟）。每种凝胶的这些代表性光谱为了清楚偏移，而虚线表示的细胞色素c。在缓冲的索瑞峰的位置。而每个光谱是15μM细胞色素c。将，凝胶厚度（或高度）相比，1厘米比色皿溶液导致更高的溶液的吸光度仅0.2-0.5厘米。转载许可9。 <img alt="图6" src="/files/ftp_upload/53802/53802fig6.jpg" /> 图6:气凝胶光谱法封装缓冲液浓度而变化场均紫外可见气凝胶凝胶路径长度凝胶包裹15分的光谱吸收_。6，M色素C在70毫（黑色）（平均4光谱的），40毫摩尔（红，虚线）（平均8光谱），和4.4毫米（绿色，虚线）（平均9光谱的）磷酸钾缓冲液。 。转载许可9。 <img alt="图7" src="/files/ftp_upload/53802/53802fig7.jpg" /> 图7:。气凝胶圆二色光谱的磷酸盐缓冲钠溶液色素C（固体）的圆二色谱，细胞色素氧化两个具有代表性的光谱Ç-SiO 2气凝胶（虚线）和Au的两个有代表性的光谱（5纳米） 〜CYT。çSiO- 2气凝胶（点）。转载许可9。 <img alt="图8" src="/files/ftp_upload/53802/53802fig8.jpg" /> 图8:一氧化氮检测与色素 C -SiO 2。 </。子&gt; 气凝胶监测移（ΔA= A 414纳米 - 408纳米 ）。在细胞色素的索瑞强度C（纯红色）和Au〜细胞色素C（蓝色虚线）封装在SiO 2复合气凝胶nanoarchitectures的气流。在氮之间切换（其中索瑞峰最大值是在约408纳米）和一氧化氮（其中索瑞峰最大值是在约414纳米）。每条曲线是3-4试验的平均，有两个细胞色素氧化Ç-SiO 2项试验在监控？A = A 414纳米 -从最初的索瑞峰最大值是在对这些试验407纳米为407nm。转载许可9。 <img alt="图9" src="/files/ftp_upload/53802/53802fig9.jpg" /> 图9:超临界流体释放时间效应场均紫外-可见吸收光谱用凝胶路径长度除以CYTç-SiO 2气凝胶ENCA。psulating 10微米的细胞色素c。在50毫米，其中超临界干燥气凝胶透过以下方式超临界二氧化碳超过45分钟（黑色实（平均9谱））或7分钟（红色虚线（平均4制成磷酸盐缓冲液谱））。转载许可9。

Watch this Scientific Journal Video about Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity at JoVE.com

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

细胞色素封装<em&gt;ç</em&gt;在二氧化硅气凝胶Nanoarchitectures没有金属纳米粒子，同时保持气相生物活性

此过程描述如何封装在硅细胞色素C（CYT; C）（SiO 2）的溶胶-凝胶过程中，这些凝胶形成bioaerogels，并使用这些bioaerogels通过气相反应迅速承认一氧化氮（NO）。这种类型的协议可能会在生物传感器或其他生物分析设备的未来发展提供帮助。

Applications such as sensors, batteries, and fuel cells have been improved through the use of highly porous aerogels when functional compounds are ...

encapsulating-cytochrome-c-silica-aerogel-nanoarchitectures-without

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

10.3K 次观看.  Fairfield University. 此过程描述如何封装在硅细胞色素C（CYT; C）（SiO 2）的溶胶-凝胶过程中，这些凝胶形成bioaerogels，并使用这些bioaerogels通过气相反应迅速承认一氧化氮（NO）。这种类型的协议可能会在生物传感器或其他生物分析设备的未来发展提供帮助。 

视频: Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle diameters ranging from 30-250 nm by varying the reaction conditions. Through the selection of a microwave compatible solvent, silicic acid precursor, catalyst, and microwave irradiation time, these microwave-assisted methods were capable of overcoming the previously reported shortcomings associated with synthesis of silica nanoparticles using microwave reactors. The siloxane precursor was hydrolyzed using the acid catalyst, HCl. Acetone, a low-tan &#948; solvent, mediates the condensation reactions and has minimal interaction with the electromagnetic field. Condensation reactions begin when the silicic acid precursor couples with the microwave radiation, leading to silica nanoparticle sol formation. The silica nanoparticles were characterized by dynamic light scattering data and scanning electron microscopy, which show the materials&#39; morphology and size to be dependent on the reaction conditions. Microwave-assisted reactions produce silica nanoparticles with roughened textured surfaces that are atypical for silica sols produced by St&#246;ber&#39;s methods, which have smooth surfaces.

Silica nanoparticles were prepared using acid-catalysis of a siloxane precursor and microwave-assisted synthetic techniques resulting in the controlled growth of nanomaterials ranging from 30-250 nm in diameter. The growth dynamics can be controlled by varying the initial silicic acid concentration, time of the reaction, and temperature of reaction.

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle ...

科研

化学

Preparing Silica Aerogel Monoliths via a Rapid Supercritical Extraction Method

A procedure for the fabrication of monolithic silica aerogels in eight hours or less via a rapid supercritical extraction process is described. The procedure requires 15-20 min of preparation time, during which a liquid precursor mixture is prepared and poured into wells of a metal mold that is placed between the platens of a hydraulic hot press, followed by several hours of processing within the hot press. The precursor solution consists of a 1.0:12.0:3.6:3.5 x 10-3 molar ratio of tetramethylorthosilicate (TMOS):methanol:water:ammonia. In each well of the mold, a porous silica sol-gel matrix forms. As the temperature of the mold and its contents is increased, the pressure within the mold rises. After the temperature/pressure conditions surpass the supercritical point for the solvent within the pores of the matrix (in this case, a methanol/water mixture), the supercritical fluid is released, and monolithic aerogel remains within the wells of the mold. With the mold used in this procedure, cylindrical monoliths of 2.2 cm diameter and 1.9 cm height are produced. Aerogels formed by this rapid method have comparable properties (low bulk and skeletal density, high surface area, mesoporous morphology) to those prepared by other methods that involve either additional reaction steps or solvent extractions (lengthier processes that generate more chemical waste).The rapid supercritical extraction method can also be applied to the fabrication of aerogels based on other precursor recipes.

This article describes a rapid supercritical extraction method for fabricating silica aerogels. By utilizing a confined mold and hydraulic hot press, monolithic aerogels can be made in eight hours or less.

通过快速超临界萃取法制备二氧化硅气凝胶巨石

A procedure for the fabrication of monolithic silica aerogels in eight hours or less via a rapid supercritical extraction process is described. The ...

Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) technique), make use of various surface modification techniques, that can, for example, prevent surface fouling, tune the hydrophobicity / hydrophilicity of the surface, adapt to a variety of electronic environments, and most frequently, induce specificity towards a target of interest.1-5 These techniques extend the functionality of otherwise highly sensitive biosensors to real-world applications in complex environments, such as blood, urine, and wastewater analysis.2,6-7 While commercial biosensing platforms, such as Biacore, have well-understood, standard techniques for performing such surface modifications, these techniques have not been translated in a standardized fashion to other label-free biosensing platforms, such as Whispering Gallery Mode (WGM) optical resonators.8-9
WGM optical resonators represent a promising technology for performing label-free detection of a wide variety of species at ultra-low concentrations.6,10-12 The high sensitivity of these platforms is a result of their unique geometric optics: WGM optical resonators confine circulating light at specific, integral resonance frequencies.13 Like the SPR platforms, the optical field is not totally confined to the sensor device, but evanesces; this "evanescent tail" can then interact with species in the surrounding environment. This interaction causes the effective refractive index of the optical field to change, resulting in a slight, but detectable, shift in the resonance frequency of the device. Because the optical field circulates, it can interact many times with the environment, resulting in an inherent amplification of the signal, and very high sensitivities to minor changes in the environment.2,14-15
To perform targeted detection in complex environments, these platforms must be paired with a probe molecule (usually one half of a binding pair, e.g. antibodies / antigens) through surface modification.2 Although WGM optical resonators can be fabricated in several geometries from a variety of material systems, the silica microsphere is the most common. These microspheres are generally fabricated on the end of an optical fiber, which provides a "stem" by which the microspheres can be handled during functionalization and detection experiments. Silica surface chemistries may be applied to attach probe molecules to their surfaces; however, traditional techniques generated for planar substrates are often not adequate for these three-dimensional structures, as any changes to the surface of the microspheres (dust, contamination, surface defects, and uneven coatings) can have severe, negative consequences on their detection capabilities. Here, we demonstrate a facile approach for the surface functionalization of silica microsphere WGM optical resonators using silane coupling agents to bridge the inorganic surface and the biological environment, by attaching biotin to the silica surface.8,16 Although we use silica microsphere WGM resonators as the sensor system in this report, the protocols are general and can be used to functionalize the surface of any silica device with biotin.

Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.

将生物探针使用硅烷偶联剂的硅光学生物传感器

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) ...

生物工程

Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst immobilization for improved production yield and sustained synthesis. Described herewith is the experimental procedure for the development of such a system consisting of two E. coli strains that express functionally complementary enzymes. Together, these two enzymes can function co-operatively to mediate the regeneration of expensive cofactors for improving the product yield of the bioreaction. In addition, the method of synthesizing an immobilized form of the coupled biocatalytic system by encapsulation of whole cells in calcium alginate beads is reported. As an example, we present the improved biosynthesis of L-xylulose from L-arabinitol by coupling E. coli cells expressing the enzymes L-arabinitol dehydrogenase or NADH oxidase. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, which is higher than those reported in the literature. The immobilized form of the coupled whole-cell biocatalysts demonstrated good operational stability, maintaining 65% of the yield obtained in the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost the catalytic activity. Therefore, the methods reported here provides two strategies that could help improve the industrial production of L-xylulose, as well as other value-added compounds requiring the use of cofactors in general.

Presented is the protocol for co-immobilizing whole-cell biocatalysts for cofactor regeneration and improved reusability, using the production of L-xylulose as an example. The cofactor regeneration is achieved by coupling two Escherichia coli strains expressing functionally complementary enzymes; the whole-cell biocatalyst immobilization is achieved by cell encapsulation in calcium alginate beads.

藻酸盐珠多生物催化剂辅因子再生和可重用性改进的固定化

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst ...

Fabrication and Testing of Catalytic Aerogels Prepared Via Rapid Supercritical Extraction

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three preparation methods are described: (a) the incorporation of metal salts into silica or alumina wet gels using an impregnation method; (b) the incorporation of metal salts into alumina wet gels using a co-precursor method; and (c) the addition of metal nanoparticles directly into a silica aerogel precursor mixture. The methods utilize a hydraulic hot press, which allows for rapid (&#60;6 h) supercritical extraction and results in aerogels of low density (0.10 g/mL) and high surface area (200-800 m2/g). While the work presented here focuses on the use of copper salts and copper nanoparticles, the approach can be implemented using other metal salts and nanoparticles. A protocol for testing the three-way catalytic ability of these aerogels for automotive pollution mitigation is also presented. This technique uses custom-built equipment, the Union Catalytic Testbed (UCAT), in which a simulated exhaust mixture is passed over an aerogel sample at a controlled temperature and flow rate. The system is capable of measuring the ability of the catalytic aerogels, under both oxidizing and reducing conditions, to convert CO, NO and unburned hydrocarbons (HCs) to less harmful species (CO2, H2O and N2). Example catalytic results are presented for the aerogels described.

Here we present protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms. Methods for preparing materials using copper salts and copper-containing nanoparticles are featured. Catalytic testing protocols demonstrate the effectiveness of these aerogels for three-way catalysis applications.

快速超临界萃取制备催化气凝胶的制备与试验研究

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three ...

Preparation of Functional Silica Using a Bioinspired Method

The goal of the protocols described herein is to synthesize bioinspired silica materials, perform enzyme encapsulation therein, and partially or totally purify the same by acid elution. By combining sodium silicate with a polyfunctional bioinspired additive, silica is rapidly formed at ambient conditions upon neutralization. 
The effect of neutralization rate and biomolecule addition point on silica yield are investigated, and biomolecule immobilization efficiency is reported for varying addition point. In contrast to other porous silica synthesis methods, it is shown that the mild conditions required for bioinspired silica synthesis are fully compatible with the encapsulation of delicate biomolecules. Additionally, mild conditions are used across all synthesis and modification steps, making bioinspired silica a promising target for the scale-up and commercialization as both a bare material and active support medium.
The synthesis is shown to be highly sensitive to conditions, i.e., the neutralization rate and final synthesis pH, however tight control over these parameters is demonstrated through the use of auto titration methods, leading to high reproducibility in reaction progression pathway and yield. 
Therefore, bioinspired silica is an excellent active material support choice, showing versatility towards many current applications, not limited to those demonstrated here, and potency in future applications.

Here, we present a protocol to synthesize bioinspired silica materials and immobilize enzymes therein. Silica is synthesized by combining sodium silicate and an amine &#39;additive&#39;, which neutralize at a controlled rate. Material properties and function can be altered either by in situ enzyme immobilization or post-synthetic acid elution of encapsulated additives.

用 Bioinspired 法制备功能性二氧化硅

The goal of the protocols described herein is to synthesize bioinspired silica materials, perform enzyme encapsulation therein, and partially or totally ...

A Rapid Synthesis Method for Au, Pd, and Pt Aerogels Via Direct Solution-Based Reduction

Here, a method to synthesize gold, palladium, and platinum aerogels via a rapid, direct solution-based reduction is presented. The combination of various precursor noble metal ions with reducing agents in a 1:1 (v/v) ratio results in the formation of metal gels within seconds to minutes compared to much longer synthesis times for other techniques such as sol-gel. Conducting the reduction step in a microcentrifuge tube or small volume conical tube facilitates a proposed nucleation, growth, densification, fusion, equilibration model for gel formation, with final gel geometry smaller than the initial reaction volume. This method takes advantage of the vigorous hydrogen gas evolution as a by-product of the reduction step, and as a consequence of reagent concentrations. The solvent accessible specific surface area is determined with both electrochemical impedance spectroscopy and cyclic voltammetry. After rinsing and freeze drying, the resulting aerogel structure is examined with scanning electron microscopy, X-ray diffractometry, and nitrogen gas adsorption. The synthesis method and characterization techniques result in a close correspondence of aerogel ligament sizes. This synthesis method for noble metal aerogels demonstrates that high specific surface area monoliths may be achieved with a rapid and direct reduction approach.

A rapid, direct solution-based reduction synthesis method to obtain Au, Pd, and Pt aerogels is presented.

直接溶液还原法快速合成金、钯、铂气凝胶的研究

Here, a method to synthesize gold, palladium, and platinum aerogels via a rapid, direct solution-based reduction is presented. The combination of various ...

Synthesis Method for Cellulose Nanofiber Biotemplated Palladium Composite Aerogels

Here, a method to synthesize cellulose nanofiber biotemplated palladium composite aerogels is presented. Noble metal aerogel synthesis methods often result in fragile aerogels with poor shape control. The use of carboxymethylated cellulose nanofibers (CNFs) to form a covalently bonded hydrogel allows for the reduction of metal ions such as palladium on the CNFs with control over both nanostructure and macroscopic aerogel monolith shape after supercritical drying. Crosslinking the carboxymethylated cellulose nanofibers is achieved using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in the presence of ethylenediamine. The CNF hydrogels maintain their shape throughout synthesis steps including covalent crosslinking, equilibration with precursor ions, metal reduction with high concentration reducing agent, rinsing in water, ethanol solvent exchange, and CO2 supercritical drying. Varying the precursor palladium ion concentration allows for control over the metal content in the final aerogel composite through a direct ion chemical reduction rather than relying on the relatively slow coalescence of pre-formed nanoparticles used in other sol-gel techniques. With diffusion as the basis to introduce and remove chemical species into and out of the hydrogel, this method is suitable for smaller bulk geometries and thin films. Characterization of the cellulose nanofiber-palladium composite aerogels with scanning electron microscopy, X-ray diffractometry, thermal gravimetric analysis, nitrogen gas adsorption, electrochemical impedance spectroscopy, and cyclic voltammetry indicates a high surface area, metallized palladium porous structure.

A synthesis method for cellulose nanofiber biotemplated palladium composite aerogels is presented.The resulting composite aerogel materials offer potential for catalysis, sensing, and hydrogen gas storage applications.

纤维素纳米纤维生物模板复合气凝胶的合成方法

Here, a method to synthesize cellulose nanofiber biotemplated palladium composite aerogels is presented. Noble metal aerogel synthesis methods often ...

Aesthetically Enhanced Silica Aerogel Via Incorporation of Laser Etching and Dyes

A procedure for aesthetically enhancing silica aerogel monoliths by laser etching and incorporation of dyes is described in this manuscript. Using a rapid supercritical extraction method, large silica aerogel monolith (10 cm x 11 cm x 1.5 cm) can be fabricated in about 10 h. Dyes incorporated into the precursor mixture result in yellow-, pink- and orange-tinged aerogels. Text, patterns, and images can be etched onto the surface (or surfaces) of the aerogel monolith without damaging the bulk structure. The laser engraver can be used to cut shapes from the aerogel and form colorful mosaics.

This protocol describes a method for etching text, patterns, and images onto the surface of silica aerogel monoliths in native and dyed form and assembling the aerogels into mosaic designs.

通过将激光蚀刻和染料结合，美学增强硅气凝胶

A procedure for aesthetically enhancing silica aerogel monoliths by laser etching and incorporation of dyes is described in this manuscript. Using a rapid ...

工程学

Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope

Gas reactions studied by in situ electron microscopy can be used to capture the real-time morphological and microchemical transformations of materials at length scales down to the atomic level. In situ closed-cell gas reaction (CCGR) studies performed using (scanning) transmission electron microscopy (STEM) can separate and identify localized dynamic reactions, which are extremely challenging to capture using other characterization techniques. For these experiments, we used a CCGR holder that utilizes microelectromechanical systems (MEMS)-based heating microchips (hereafter referred to as &#34;E-chips&#34;). The experimental protocol described here details the method for performing in situ gas reactions in dry and wet gases in an aberration-corrected STEM. This method finds relevance in many different materials systems, such as catalysis and high-temperature oxidation of structural materials at atmospheric pressure and in the presence of various gases with or without water vapor. Here, several sample preparation methods are described for various material form factors. During the reaction, mass spectra obtained with a residual gas analyzer (RGA) system with and without water vapor further validates gas exposure conditions during reactions. Integrating an RGA with an in situ CCGR-STEM system can, therefore, provide critical insight to correlate gas composition with the dynamic surface evolution of materials during reactions. In situ/operando studies using this approach allow&#160;for detailed investigation of the fundamental reaction mechanisms and kinetics that occur at specific environmental conditions (time, temperature, gas, pressure), in real-time, and at high spatial resolution.

Performing In Situ Closed-Cell Gas Reactions in the Transmission Electron Microscope

Here, we present a protocol for performing in situ TEM closed-cell gas reaction experiments while detailing several commonly used sample preparation methods.

在传输电子显微镜中执行 原位 闭电池气体反应

Gas reactions studied by in situ electron microscopy can be used to capture the real-time morphological and microchemical transformations of materials at ...

细胞色素封装

摘要

探索更多视频

此视频中的章节

Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis

通过快速超临界萃取法制备二氧化硅气凝胶巨石

将生物探针使用硅烷偶联剂的硅光学生物传感器

藻酸盐珠多生物催化剂辅因子再生和可重用性改进的固定化

快速超临界萃取制备催化气凝胶的制备与试验研究

用 Bioinspired 法制备功能性二氧化硅

直接溶液还原法快速合成金、钯、铂气凝胶的研究

纤维素纳米纤维生物模板复合气凝胶的合成方法

通过将激光蚀刻和染料结合，美学增强硅气凝胶

在传输电子显微镜中执行原位闭电池气体反应

细胞色素封装

摘要

探索更多视频

此视频中的章节

Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis

通过快速超临界萃取法制备二氧化硅气凝胶巨石

将生物探针使用硅烷偶联剂的硅光学生物传感器

藻酸盐珠多生物催化剂辅因子再生和可重用性改进的固定化

快速超临界萃取制备催化气凝胶的制备与试验研究

用 Bioinspired 法制备功能性二氧化硅

直接溶液还原法快速合成金、钯、铂气凝胶的研究

纤维素纳米纤维生物模板复合气凝胶的合成方法

通过将激光蚀刻和染料结合，美学增强硅气凝胶

在传输电子显微镜中执行 原位 闭电池气体反应

在传输电子显微镜中执行原位闭电池气体反应