The authors would like to acknowledge the technical assistance of Alfred Gunasekaran in electron microscopy studies at the Institute of Micromanufacturing at Louisiana Tech University, and Dr. Jim McNamara for assistance with additional microscopy studies. The work described was supported in part by Louisiana board of Regents PKSFI Contract No. LEQSF (2007-12)-ENH-PKSFI-PRS-04 and the James E. Wyche III Endowed Professorship from Louisiana Tech University (to M.D.).

1.规划实验<ol><li>确定所需的合成铜纳米复合材料的体积。在此基础上，选择一些体积小烧瓶（25 平方厘米），或在准备材料如下所示大瓶。 </li><li>对于此合成中，使用一个37℃培养箱，用5％的CO 2和至少40％的湿度。确保这种培养箱可用，并且，它不会被重复干扰比合成的周期（大约24小时）。 注意：反复开闭的保育箱的肯定会引起温度的波动，这可能会导致该纳米复合材料结构的改变的合成。 </li></ol> 2.准备材料<ol><li>制备实验开始前新鲜的所有材料，通过加入固体材料对溶剂权之前合成是开始。保持原液的胱氨酸和铜的原料在液体长时间b乘积安伏的实验，不推荐并可能导致不同的结果。一旦从供应商处打开，一直原料干燥通过包装容器用Parafilm的顶部。 注意：下面的协议被用作用于在使用7微升胱氨酸，6643微升无菌水，350微升CNPS的的25cm 2细胞培养烧瓶中反应的一个例子。 </li><li>制备2毫克/毫升溶液的铜纳米颗粒的通过称出至少2毫克CNPS的。在这一步戴一次性手套，以防止CNPS与皮肤接触的可能。将纳米粒子在一个空的无菌16毫升玻璃管形瓶中。 <ol><li>向含有CNPS小瓶，在适当的体积加无菌去离子水，使2毫克/毫升溶液，并旋涡振荡溶液，持续20秒，以提供合成开始之前的纳米颗粒的分散体（至少1毫升总体积建议）。不填小瓶超过一半的水，因为这会抑制由涡旋混合。 CNPS WIL升快速沉降到小瓶的底部，并会出现暗的颜色（灰色到黑色）。 </li><li>超声清洗在室温下17分钟的CNP溶液合成开始之前提供的CNPS最大分散。定期检查，以确保CNPS是由于混合，超声。一个成功的超声处理后，CNPS保持在溶液中至少30分钟，悬浮，溶液颜色深。 </li></ol></li><li>称出胱氨酸的足够的质量以使72.9毫克/毫升溶液用于合成。因为胱氨酸不直接溶于水，放置称量胱氨酸在抗静电计量容器中。 <ol><li>称重含有胱氨酸容器，添加无菌，1M的NaOH足够体积，以使胱氨酸完全溶解。例如，完全溶解7.29毫克胱氨酸在100μl的1M氢氧化钠，使一个72.9毫克/毫升溶液。 </li><li>为了保持无菌的条件下，进行这一步在无菌流组织培养罩。 注意：氢氧化钠在1M的浓度是苛性，因此在此步骤中，以防止浓NaOH的皮肤接触穿一次性手套</li></ol></li><li>工作在无菌组织培养罩，与6643微升无菌水加7微升胱氨酸到无菌合成烧瓶第一，让孵育在孵化30分钟，在37℃下用该烧瓶帽排气（疏松的），以提供有效的混合。重悬2毫克/毫升的CNP溶液通过涡旋30秒，因为CNPS将已在超声步骤后结算。 <ol><li>添加足够的CNP溶液到合成烧瓶（使用无菌技术），以保持下列组分比率：将1份胱氨酸，50份CNPS，和949份无菌水中的25cm 2细胞培养烧瓶，开始合成。例如，对于一个7毫升合成量，结合7微升胱氨酸原液，350微升CNPS的，和6643微升无菌水。更换盖在烧瓶并拧紧，以便它是SECUR即</li><li>结合所有成分的合成后，轻轻在烧瓶中纷飞的4-5倍混合。放置烧瓶中的CO 2培养箱中并通过松开帽，以便将有进出合成在烧瓶的气体交换泄烧瓶中。 </li></ol></li><li>允许合成在孵化大约24小时运行。在合成过程中，人们可以观察到，用显微镜和眼睛，形成高度线性的复合材料。 注意：形成的结构的方法，可以在这个意义上，结构是最初难以检测突然发生，那么外观快速前进到增加密度。可形成24小时之前发生的，因此。该过程也可以通过眼睛观察到，一旦结构变得更大，它们的密度增加而增加。而产生的结构，可以随着时间的推移，在显微镜下，并用肉眼在稍后的时间点观察到的，连续中断的合成条件和温度会导致不良的综合结果。 </li><li>通过紧紧盖住合成烧瓶中，并存储该容器在冰箱（4℃）终止合成生物复合材料的。结构，一旦产生，在这种形式至少一年保持稳定。标签与合成的条件，包括部件利用，合成的日期和终止前的合成的孵育时间的烧瓶中。 </li></ol> 3.合成使用硫酸铜<ol><li>通过用硫酸铜盐代替CNPS进行自组装的合成。使用无菌技术，溶解至少2毫克硫酸铜在无菌去离子水足量，使一个2毫克/毫升溶液。的硫酸铜晶体容易进入溶液在该浓度，但是涡如果需要的小瓶中，并通过眼睛检查，以确保所有的晶体溶解。 </li><li>制备硫酸铜后，进行合成如先前描述的，使用硫酸铜代替CNPS。 注意：在使用硫酸铜作为起始材料的自组装纳米复合材料被认为是许多在比从CNPS合成结构最终形状更加一致。 </li><li>终止的硫酸铜生物复合材料的合成，作为CNP复合材料（步骤2.6），并将其储存长期在4℃。 </li></ol> 4.表征和处理生物材料的后期合成的<ol><li>从定性和CNPS从硫酸铜由白色光镜9和电子显微镜得出9生物复合材料。 <ol><li>为表征和生物复合材料的合成后通过白光显微镜检查，使用倒置显微镜作为复合材料会沉淀到烧瓶的底部表面铺设烧瓶平坦的几分钟之内，并且然后可以带入焦点。使用显微镜上的明场设置，最大限度生物复合材料和液体介质之间的对比。从CNPS和警察衍生复合材料每硫酸都将出现明显的不透明的颜色，但反应的CNP聚集会出现颜色很深。 <ol><li>使用连接到显微镜的数码相机来捕获复合材料的图像。的长度为各个构造A范围将被观察到。 </li></ol></li><li>为表征和生物复合材料的合成后的检查和储存在4℃后，让烧瓶来室温至少15分钟的烧瓶将初步形成在除去从冰箱，这将掩盖有效聚焦而进行显微成像缩合。允许平衡至室温后，擦拭烧瓶的顶面和底面，用干净的纸巾最大化显微成像质量。 </li><li>当30秒的工作或成像已经存储长期复合材料，涡流烧瓶解离形成而在冰箱复合材料的团块。混匀后，检查结构，倒置MICroscope确保已经聚集分离，并在必要时重复涡旋。 </li><li>使用倒置白光显微图像，以评估合成的功效，使用CNPS一个给定的实验。例如，记录的未反应CNPS中用于从烧瓶带有不同参数的CNP衍生生物复合材料，如合成的时间合成烧瓶存在或不存在。 注意：个别CNPS太小观察用光学显微镜，但未反应的CNP聚合将显示为圆形和暗的物体，而相比之下，成功地合成了CNP-复合材料将具有高的纵横比，线性形式，并将具有一系列不同的长度。避免进行合成过长的一段时间终止之前的，因为这将导致在高度支化的“海胆”型结构，这是难以分散成一旦形成单个结构。 </li><li>使用白色倒光镜评估疗效的合成使用硫酸铜一个给定的实验。由于硫酸铜变为完全进入溶液使用此协议，该解决方案会出现比从合成使用CNPS溶液少暗。通过比较烧瓶用不同的合成条件，如合成的终止时间之前文件的硫酸铜复合材料的大小和程度。 注意：成功合成复合材料将表现出一系列不同的长度。避免进行合成过长的一段时间终止之前的，因为这将导致在复合材料中，其中一些将在结构“海胆状”的高度支化的聚集体，并且其很难分散成一旦形成单独的结构。 </li></ol></li><li>专心生物复合合成后，在离心管中的复合材料的离心分离机的解决方案。添加6毫升任CNP衍生结构或硫酸衍生的铜结构的至15ml离心管中。离心10分钟在500×g离心在室温形成沉淀。对于较小的体积，加入500μl结构的解决方案，以0.6毫升大小的试管。离心机在2000×g离心在RT至少10分钟，以形成颗粒。 <ol><li>离心足够长的时间（至少10分钟为microfuges）后，保存可观察沉淀在管所在的建筑物集中通过仔细除去上清液沉淀上面的底部。从硫酸铜衍生生物复合材料结构出现蓝色的颜色和CNPS衍生结构较深（灰色至黑色）。 </li><li>添加更多的复合材料，以该管并重复该过程以相同的管，如果需要集中的结构。驱散浓缩粒料，加入溶液到管所需体积，并涡旋为10-30秒。 </li></ol></li><li>声处理结构一旦形成，要移动的组织的平均群体大小（长度），以较低的值。结构发生在无菌去离子水，超声在乐AST 10分钟。使用该方法，随着时间的推移，结构变得支离破碎和平均长度（见文本图6）小。用倒置的白色光镜，数码相机复合尺寸不同时代超声文档更改。 </li></ol>

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Authors have nothing to disclose.

而评估纳米材料包括CNPS的潜在毒性作用，有人指出，在长期，CNPS被从初始较为分散的颗粒分布到较大 ​​的聚集形式（ 图2）转化。在某些情况下，这些中生产在细胞培养皿，生物条件下高度聚集的地层，形成了从中央骨料含有“海胆”高度线性突起，令人联想起先前所述铜的6。应当指出的是，这里所示的条件下，CNPS的浓度加入到细胞中是次最大，因此不杀死所有的细胞?...

Copper is a highly reactive metal that in the biological world is essential in some enzyme functions 1,2, but in higher concentrations is potently toxic including in the nanoparticulate form 3,4. Concern over copper toxicity has become more relevant as CNPs and other copper-based nanomaterials are utilized, due to the increased surface area/mass for nanostructures. Thus, even a small mass of copper, in nanoparticle form, could cause local toxicity due to its ability to penetrate the cell and be broken down into reactive forms. Some biological species can complex with and chelate metal ions, and even incorporate them into biological structures as has been described in marine mussels 5. In studying the potential toxic effects of nanomaterials 4, it was discovered that over time, and under biological conditions used for typical cell culturing (37 &#176;C and 5% CO2), stable copper biocomposites could be formed with a high-aspect ratio (linear) structure.
By a process of elimination, the initial discovery of these linear biocomposites, which occurred in complete cell culture media, was simplified to a defined protocol of essential elements needed for the biocomposites to self-assemble. Self-assembly of two types of highly linear biocomposites was discovered to be possible with two starting metal components: 1) CNPs and 2) copper sulfate, with the common biological component being cystine. Although more complex, so called &#8220;urchin&#8221; and &#8220;nanoflower&#8221; type copper-containing structures with nanoscale and microscale features have been previously reported, these were produced under non-biological conditions, such as temperatures of 100 &#176;C or greater 6-8. To our knowledge, synthesis of individual, linear copper-containing nanostructures that are scalable in liquid phase under biological conditions has not been previously described. 
One of the starting materials utilized for synthesis of nanocomposites, namely CNPs, has been reported previously to be very toxic to cells 4. It has recently been reported that after the nanocomposites are formed, these structures are less toxic on a per mass basis than the starting NPs 9. Thus, the synthesis described here may be derived from a biological and biochemical reaction that has utility in stabilizing reactive copper species, both in the sense of transforming the NP form into larger structures and in producing composites less toxic to cells. 
In contrast to many other nanomaterial forms which are known to aggregate or clump upon interaction with biological liquid media 10,11, once formed, the highly linear composites described here avoid aggregation, possibly due to a redistribution of charge which has been previously reported 9. As detailed in the current work, this avoidance of aggregation is convenient for the purposes of working with the structures once formed for at least 3 reasons: 1) composite structures once formed may be concentrated using centrifugation and then easily dispersed again using vortex mixing; 2) formed structures can be decreased in average size by sonication for different periods of time; and 3) the formed linear structures may provide an additional tool for avoiding the recently described &#8220;coffee ring effect&#8221; 12 and thus provide a dopant for creating more evenly distributed coatings of materials, especially those containing spherical particulates.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Mini Vortexer</td>
			<td>VWR (https://us.vwr.com)</td>
			<td>58816-121</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator Model # 2425-2</td>
			<td>VWR (https://us.vwr.com)</td>
			<td>Contact vendor</td>
			<td>Current model calalog # 98000-360</td>
		</tr>
		<tr>
			<td>Eppendorf Centrifuge (Refrigerated Microcentrifuge)</td>
			<td>Labnet (http://labnetinternational.com/)</td>
			<td>C2500-R</td>
			<td>Model Prism R</td>
		</tr>
		<tr>
			<td>Cell Culture Centrifuge Model Z323K</td>
			<td>Labnet (http://labnetinternational.com/)</td>
			<td>Contact vendor</td>
			<td>Current model Z206A catalog # C0206-A</td>
		</tr>
		<tr>
			<td>Sonicator (Ultrasonic Cleaner)</td>
			<td>Branson Ultrasonics Corporation (http://www.bransonic.com/)</td>
			<td>1510R-MTH</td>
			<td></td>
		</tr>
		<tr>
			<td>Balance</td>
			<td>Sartorius (http://dataweigh.com)</td>
			<td></td>
			<td>Model CP225D similar model CPA225D</td>
		</tr>
		<tr>
			<td>Olympus IX51 Inverted Light Microscope</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>Contact vendor</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus DP71 microscope digital camera</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>Contact vendor</td>
			<td></td>
		</tr>
		<tr>
			<td>external power supply unit- white light for Olympus microscope</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>TH4-100</td>
			<td></td>
		</tr>
		<tr>
			<td>10x, 20, and 40x microscope objectives</td>
			<td>Olympus (http://olympusamerica.com</td>
			<td>Contact vendor</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope</td>
			<td>Hitachi (http://hitachi-hitec.com/global/em/sem/sem_index.html)</td>
			<td>model S-4800</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>Zeiss (http://zeiss.com/microscopy/en_de/products.html)</td>
			<td>model Libra 120</td>
			<td></td>
		</tr>
		<tr>
			<td>Table Top Work Station Unidirectional Flow Clean Bench</td>
			<td>Envirco (http://envirco-hvac.com)</td>
			<td>model PNG62675</td>
			<td>Used for sterile cell culture technique</td>
		</tr>
	</tbody>
</table>

generation of scalable, metallic high-aspect ratio nanocomposites in a biological liquid medium

该协议的目的是描述两种新型生物复合材料具有高纵横比结构的合成。该生物复合材料包括铜和胱氨酸，无论是与铜纳米颗粒（CNPS）或硫酸铜贡献的金属部件。合成是在生物条件下（37℃）和24小时后的自组装的复合材料的形式下是液体。一旦形成，这些复合材料是在两种液体介质和干燥形式高度稳定。该复合物由纳米规模向微型范围在长度和从几微米到直径25纳米。能量色散X射线光谱仪（EDX）的场发射扫描电子显微镜表明，硫存在于NP衍生线性结构，而这是从起始CNP材料不存在，从而证实胱氨酸作为硫在最终纳米复合材料的源。在综合这些线性纳米和微观复合材料，STR的长度多种多样uctures形成在合成容器。合成后的液体混合物的超声处理被证明有助于通过用超声处理的时间增加减少的平均长度控制的结构的平均尺寸。因为所形成的结构是高度稳定的，不结块，并形成有在液相，离心也可用于帮助浓缩和分离形成的复合材料。

图1示出的合成步骤，以形成在这项工作中描述的线性生物复合材料的流程图示意图。 CNPS或硫酸铜作为起始原料相结合，与无菌水，以形成2毫克/毫升溶液，该溶液混合并超声处理以提供更混合物，使该铜溶液然后在下面的比例混合用于合成：949份无菌水：50份混合铜：1份胱氨酸原液。实际体积可以根据这些比率来放大或缩小最后合成收率而增加或减少。孵育至少2小时所指示的，线?...

Watch this Scientific Journal Video about Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium at JoVE.com

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

在这里，我们提出了一个协议，以合成下生物条件和在液体介质新颖的，高宽比生物复合材料。的生物复合材料从纳米扩展到微米直径和长度。铜纳米粒子（CNPS）和硫酸铜结合胱氨酸是关键部件的合成。

可扩展的，金属的高宽比纳米复合材料在生物液体中产生

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

generation-scalable-metallic-high-aspect-ratio-nanocomposites

Research

JoVE Journal

Bioengineering

9.0K 次观看.  Centenary College of Louisiana. 在这里，我们提出了一个协议，以合成下生物条件和在液体介质新颖的，高宽比生物复合材料。的生物复合材料从纳米扩展到微米直径和长度。铜纳米粒子（CNPS）和硫酸铜结合胱氨酸是关键部件的合成。 