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>

<ol>
	<li>Klinman, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+copper-enzyme+family+of+dopamine+beta-monooxygenase+and+peptidylglycine+alpha-hydroxylating+monooxygenase:+resolving+the+chemical+pathway+for+substrate+hydroxylation.">The copper-enzyme family of dopamine beta-monooxygenase and peptidylglycine alpha-hydroxylating monooxygenase: resolving the chemical pathway for substrate hydroxylation.</a> The Journal of biological chemistry. 281, 3013-3016 (2006).</li><li>Uauy, R., Olivares, M., Gonzalez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essentiality+of+copper+in+humans.">Essentiality of copper in humans.</a> The American journal of clinical nutrition. 67, 952S-959S (1998).</li><li>Karlsson, H. L., Cronholm, P., Gustafsson, J., Copper Moller, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=oxide+nanoparticles+are+highly+toxic:+a+comparison+between+metal+oxide+nanoparticles+and+carbon+nanotubes.">oxide nanoparticles are highly toxic: a comparison between metal oxide nanoparticles and carbon nanotubes.</a> Chemical research in toxicology. 21, 1726-1732 (2008).</li><li>Parekh, G., 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=Layer-by-layer+nanoencapsulation+of+camptothecin+with+improved+activity.">Layer-by-layer nanoencapsulation of camptothecin with improved activity.</a> International journal of pharmaceutics. 465, 218-227 (2014).</li><li>Harrington, M. J., Masic, A., Holten-Andersen, N., Waite, J. H., Fratzl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron-clad+fibers:+a+metal-based+biological+strategy+for+hard+flexible+coatings.">Iron-clad fibers: a metal-based biological strategy for hard flexible coatings.</a> Science. 328, 216-220 (2010).</li><li>Keyson, D., 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=CuO+urchin-nanostructures+synthesized+from+a+domestic+hydrothermal+microwave+method.">CuO urchin-nanostructures synthesized from a domestic hydrothermal microwave method.</a> Materials Research Bulletin. 43, 771-775 (2008).</li><li>Liu, B., Zeng, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesoscale+organization+of+CuO+nanoribbons:+formation+of+'dandelions'.">Mesoscale organization of CuO nanoribbons: formation of 'dandelions'.</a> J Am Chem Soc. 126, 8124-8125 (2004).</li><li>Peng, 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=Controllable+synthesis+of+self-assembled+Cu2S+nanostructures+through+a+template-free+polyol+process+for+the+degradation+of+organic+pollutant+under+visible+light.">Controllable synthesis of self-assembled Cu2S nanostructures through a template-free polyol process for the degradation of organic pollutant under visible light.</a> Materials Research Bulletin. 44, 1834-1841 (2009).</li><li>Deodhar, S., Huckaby, J., Delahoussaye, M., DeCoster, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Aspect+Ratio+Bio-Metallic+Nanocomposites+for+Cellular+Interactions.">High-Aspect Ratio Bio-Metallic Nanocomposites for Cellular Interactions.</a> IOP Conference Series: Materials Science and Engineering. 64, 012014 (2014).</li><li>Montes-Burgos, I., 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=Characterisation+of+nanoparticle+size+and+state+prior+to+nanotoxicological+studies.">Characterisation of nanoparticle size and state prior to nanotoxicological studies.</a> Journal of Nanoparticle Research. 12, 47-53 (2010).</li><li>Wiogo, H. T., Lim, M., Bulmus, V., Yun, J., Amal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilization+of+magnetic+iron+oxide+nanoparticles+in+biological+media+by+fetal+bovine+serum+(FBS).">Stabilization of magnetic iron oxide nanoparticles in biological media by fetal bovine serum (FBS).</a> Langmuir. 27, 843-850 (2011).</li><li>Yunker, P. J., Still, T., Lohr, M. A., Yodh, 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=Suppression+of+the+coffee-ring+effect+by+shape-dependent+capillary+interactions.">Suppression of the coffee-ring effect by shape-dependent capillary interactions.</a> Nature. 476, 308-311 (2011).</li><li>Kahler, H., Lloyd Jr, B., Eden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+Microscopic+and+Other+Studies+on+a+Copper&#8211;Cystine+Complex.">Electron Microscopic and Other Studies on a Copper&#8211;Cystine Complex.</a> The Journal of Physical Chemistry. 56, 768-770 (1952).</li><li>Furia, E., Sindona, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexation+of+L-cystine+with+metal+cations.">Complexation of L-cystine with metal cations.</a> Journal of Chemical &amp; Engineering Data. 55, 2985-2989 (2010).</li><li>Hawkins, C., Perrin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polynuclear+Complex+Formation.+II.+Copper+(II)+with+Cystine+and+Related+Ligands.">Polynuclear Complex Formation. II. Copper (II) with Cystine and Related Ligands.</a> Inorganic Chemistry. 2, 843-849 (1963).</li><li>Hallman, P., Perrin, D., Watt, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+computed+distribution+of+copper+(II)+and+zinc+(II)+ions+among+seventeen+amino+acids+present+in+human+blood+plasma.">The computed distribution of copper (II) and zinc (II) ions among seventeen amino acids present in human blood plasma.</a> Biochem. J. 121, 549-555 (1971).</li><li>Jensen, L. S., Maurice, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+sulfur+amino+acids+on+copper+toxicity+in+chicks.">Influence of sulfur amino acids on copper toxicity in chicks.</a> The Journal of nutrition. 109, 91-97 (1979).</li><li>Lee, Y., Choi, J. R., Lee, K. J., Stott, N. E., Kim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+synthesis+of+copper+nanoparticles+by+chemically+controlled+reduction+for+applications+of+inkjet-printed+electronics.">Large-scale synthesis of copper nanoparticles by chemically controlled reduction for applications of inkjet-printed electronics.</a> Nanotechnology. 19, 415604 (2008).</li><li>Hume, J., 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=Engineered+coiled-coil+protein+microfibers.">Engineered coiled-coil protein microfibers.</a> Biomacromolecules. 15, 3503-3510 (2014).</li></ol>

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在培...

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，线性生?...

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。 

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

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

科研

生物工程

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

环境控制机械自适应聚合物纳米复合材料的微拉伸测试<em&gt;体外</em&gt;表征

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

就地 液体生物组件的 TEM

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

基于角蛋白纳米纤维生物医学工程合成

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

交变磁场敏感的混合明胶微凝胶药物控释

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and as soft robots. Here, we demonstrate the preparation of shape-responsive liquid crystal elastomers (LCEs) and LCE nanocomposites along with characterization of their shape-responsiveness, mechanical properties, and microstructure. Two types of LCEs &#8212; polysiloxane-based and epoxy-based &#8212; are synthesized, aligned, and characterized. Polysiloxane-based LCEs are prepared through two crosslinking steps, the second under an applied load, resulting in monodomain LCEs. Polysiloxane LCE nanocomposites are prepared through the addition of conductive carbon black nanoparticles, both throughout the bulk of the LCE and to the LCE surface. Epoxy-based LCEs are prepared through a reversible esterification reaction. Epoxy-based LCEs are aligned through the application of a uniaxial load at elevated (160 &#176;C) temperatures. Aligned LCEs and LCE nanocomposites are characterized with respect to reversible strain, mechanical stiffness, and liquid crystal ordering using a combination of imaging, two-dimensional X-ray diffraction measurements, differential scanning calorimetry, and dynamic mechanical analysis. LCEs and LCE nanocomposites can be stimulated with heat and/or electrical potential to controllably generate strains in cell culture media, and we demonstrate the application of LCEs as shape-responsive substrates for cell culture using a custom-made apparatus.

We demonstrate the preparation of siloxane-based and epoxy-based liquid crystal elastomers (LCEs) and LCE nanocomposites. The LCEs are characterized with respect to reversible strain, liquid crystal ordering, and stiffness. As a potential application, we demonstrate their use as shape-responsive substrates in a custom device for active cell culture.

单域液晶的弹性体和液晶橡胶纳米复合材料的制备

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

模块化 DNA 设备的自动机器人液体处理组件

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

生物相容的液晶弹性体泡沫的合成中用作细胞支架的三维空间细胞培养

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

从癌细胞培养物中收集的介质光谱分析的选定金核素的同步量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

人类多能干细胞和免疫损伤的成熟小脑有机体可伸缩生成

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...