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>

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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 border="0" cellpadding="0" cellspacing="0" width="661">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Potassium phosphate, monobasic</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">P285-500</td>
			<td style="width:167px;">Certified ACS (also possible to use sodium phosphate monobasic)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">Potassium phosphate dibasic anhydrous</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">P288-500</td>
			<td>Certified ACS (also possible to use sodium phosphate dibasic)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Water</td>
			<td style="width:127px;">Millipore Direct-Q</td>
			<td style="width:119px;"></td>
			<td>18 M&#937; cm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">pH meter and electrode</td>
			<td style="width:127px;">Denver Instrument</td>
			<td style="width:119px;">UB-10</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:249px;">Cytochrome c from equine heart</td>
			<td style="width:127px;">Sigma Aldrich</td>
			<td>C7752-100MG&#160;</td>
			<td>&#8805;95% based on Mol. Wt. 12,384, used as received and stored at -20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Glass scintillation vials</td>
			<td style="width:127px;">Wheaton</td>
			<td>03-341-25J</td>
			<td>20 mL, O.D. x height (with cap):&#160; 28 mm x 61 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Disposable cuvette</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td>14-955-126</td>
			<td>methacrylate, 10 mm x 10 mm x 45 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">Ultraviolet Visible Spectrophotometer</td>
			<td style="width:127px;">Shimadzu</td>
			<td style="width:119px;">UV-1800</td>
			<td>Uses UVProbe v 2.33 software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">Circular dichroism spectrometer (or spectropolarimeter)</td>
			<td style="width:127px;">JASCO</td>
			<td style="width:119px;">J-810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Isotemp Laboratory Refrigerator</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Polypropylene disposable beakers</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">01-291-10</td>
			<td>50 mL</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:249px;">Tetramethylorthosilicate (also known as tetramethoxysilane, TMOS)</td>
			<td style="width:127px;">Sigma Aldrich</td>
			<td style="width:119px;">218472-500G</td>
			<td>98% purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Methanol</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">A457-4</td>
			<td>GC Resolv grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Ammonium hydroxide solution</td>
			<td style="width:127px;">Sigma Aldrich</td>
			<td style="width:119px;">221228-25ML-A</td>
			<td>ACS reagent, 28.0-30.0%</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">General purpose polypropylene scintillation vials</td>
			<td style="width:127px;">Sigma Aldrich</td>
			<td style="width:119px;">Z376825-1PAK</td>
			<td>16 mm x 57 mm, volume size 6.5 mL, slice off bottom with sharp knife or razor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">generic plastic wrap</td>
			<td style="width:127px;">various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Parafilm M laboratory wrapping film</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">S37440</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Plastic syringe plunger</td>
			<td style="width:127px;">various</td>
			<td style="width:119px;"></td>
			<td>use syringe plunger from 3 mL syringe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Ethyl alcohol</td>
			<td style="width:127px;">Acros</td>
			<td style="width:119px;">61509-0040</td>
			<td>Absolute, 200 proof, 99.5% A.C.S. reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Acetone</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">A949-4</td>
			<td>HPLC grade</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">Critical point drying apparatus</td>
			<td style="width:127px;">Quorum Technologies</td>
			<td style="width:119px;"></td>
			<td>E3000 Series</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Circulator</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">Isotemp 3016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Carbon dioxide cylinder</td>
			<td style="width:127px;">Tech Air</td>
			<td style="width:119px;"></td>
			<td>siphon tube</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">Micrometer</td>
			<td style="width:127px;">Central Tool Company</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">GRAMS/AI 8.0 software</td>
			<td style="width:127px;">Thermo Electron Corporation</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Nitrogen cylinder</td>
			<td style="width:127px;">Tech Air</td>
			<td style="width:119px;"></td>
			<td>Another inert gas could be substituted</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">10% nitric oxide/90% nitrogen cylinder</td>
			<td style="width:127px;">Airgas</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Tygon tubing</td>
			<td style="width:127px;">various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">T-switch valve</td>
			<td style="width:127px;">various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">syringe needles</td>
			<td style="width:127px;">various</td>
			<td style="width:119px;"></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

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

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

在生物系统中的离子浓度测量荧光纳米粒子

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

科研

生物工程

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

制定通过Nanoprecipitation技术嵌段聚合物纳米粒子

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12. Although tobacco tars and related products in the particle phase of cigarette smoke are major causes of carcinogenic and mutagenic related diseases, cigarette smoke contains significant amounts of free radicals that are also considered as an important group of carcinogens9,10. Free radicals attack cell constituents by damaging protein structure, lipids and DNA sequences and increase the risks of developing various types of cancers. Inhaled radicals produce adducts that contribute to many of the negative health effects of tobacco smoke in the lung3. Studies have been conducted to reduce free radicals in cigarette smoke to decrease risks of the smoking-induced damage. It has been reported that haemoglobin and heme-containing compounds could partially scavenge nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke4. A 'bio-filter' consisted of haemoglobin and activated carbon was used to scavenge the free radicals and to remove up to 90% of the free radicals from cigarette smoke14. However, due to the cost-ineffectiveness, it has not been successfully commercialized. Another study showed good scavenging efficiency of shikonin, a component of Chinese herbal medicine8. In the present study, we report a protocol for introducing common natural antioxidant extracts into the cigarette filter for scavenging gas phase free radicals in cigarette smoke and measurement of the scavenge effect on gas phase free radicals in mainstream cigarette smoke (MCS) using spin-trapping Electron Spin Resonance (ESR) Spectroscopy1,2,14. We showed high scavenging capacity of lycopene and grape seed extract which could point to their future application in cigarette filters. An important advantage of these prospective scavengers is that they can be obtained in large quantities from byproducts of tomato or wine industry respectively11,13

Spin-trapping ESR spectroscopy was used to study the effect of plant antioxidants lycopene, pycnogenol and grape seed extract on scavenging gas-phase free radicals in cigarette smoke.

卷烟主流烟气中检测和清除气相自由基议定书

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12.  ...

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

生物标志物发现的介孔二氧化硅薄膜的低分子量蛋白质富集

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

病毒粒子为<em在体内</em&gt;肿瘤显像

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

纳米谐波再生研究

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Identification of Metal Oxide Nanoparticles in Histological Samples by Enhanced Darkfield Microscopy and Hyperspectral Mapping

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the identification, localization, and characterization of nanoscale materials in biological samples is on the rise. Currently available methods, such as electron microscopy, tend to be resource-intensive, making their use prohibitive for much of the research community. Enhanced darkfield microscopy complemented with a hyperspectral imaging system may provide a solution to this bottleneck by enabling rapid and less expensive characterization of nanoparticles in histological samples. This method allows for high-contrast nanoscale imaging as well as nanomaterial identification. For this technique, histological tissue samples are prepared as they would be for light-based microscopy. First, positive control samples are analyzed to generate the reference spectra that will enable the detection of a material of interest in the sample. Negative controls without the material of interest are also analyzed in order to improve specificity (reduce false positives). Samples can then be imaged and analyzed using methods and software for hyperspectral microscopy or matched against these reference spectra in order to provide maps of the location of materials of interest in a sample. The technique is particularly well-suited for materials with highly unique reflectance spectra, such as noble metals, but is also applicable to other materials, such as semi-metallic oxides. This technique provides information that is difficult to acquire from histological samples without the use of electron microscopy techniques, which may provide higher sensitivity and resolution, but are vastly more resource-intensive and time-consuming than light microscopy.

Enhanced darkfield microscopy and hyperspectral imaging with spectral mapping enable screening, localization, and identification of nanoscale materials in histological samples with improved speed and accuracy over traditional methods. The goal of this paper is to provide methods for darkfield imaging and hyperspectral mapping of metal oxide nanoparticles in histological samples.

金属氧化物纳米颗粒的组织学样品鉴定增强暗场显微镜和高光​​谱遥感制图

Nanomaterials are increasingly prevalent throughout industry, manufacturing, and biomedical research. The need for tools and techniques that aid in the ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

液体中脉冲激光烧蚀制备金属纳米粒子:研究纳米粒子抗菌性能的工具

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a cell-penetrating peptide, TAT, for intracellular delivery. However, TAT has shown non-specific biological activities. Gold nanoparticles (GNPs) can be used as drug delivery carriers without recognized toxicity. Therefore, we have used a GNP/peptide hybrid to deliver PKC&#948;i. Two short peptides (P2: CAAAAE and P4: CAAAAW), at a 95:5 ratio, were used to modify the surface properties of GNP. GNPs conjugated with PKC&#948;i (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and phosphate-buffered saline (PBS) containing bovine serum albumin or fetal bovine serum. Intravenous injection of GNP-PKCi was previously shown to prevent ischemia-reperfusion injury of the lung. This article outlines a protocol to formulate GNP/PKCi and assess the physiochemical properties of GNP/PKCi. We have used similar methods to formulate other peptide-based drugs with GNP. This article will hopefully draw more attention to this novel intracellular drug delivery technology and its applications in vivo.

We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly.

金纳米粒子的蛋白质激酶 c-三角洲抑制剂肽配方

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a ...